專利名稱：具有降低吸附增強(qiáng)水解作用的枯草桿菌蛋白酶dy變異體的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及用于各種清洗組合物的新的酶變異體，以及編碼這些酶變異體的DNA序列。
背景技術(shù)：
酶構(gòu)成了最大一類的天然存在蛋白質(zhì)。每種類型的酶通常催化(促進(jìn)反應(yīng)而本身不被消耗)不同種類的化學(xué)反應(yīng)。已知一類稱為蛋白酶的酶能水解(將化合物分解為兩個(gè)或多個(gè)簡(jiǎn)單化合物，并在裂解的化學(xué)鍵的每一端攝取水分子的H和OH部分)其它蛋白質(zhì)。已通過將天然存在的和蛋白質(zhì)工程化的蛋白酶作為添加劑攙入到洗衣洗滌劑中對(duì)這種水解蛋白質(zhì)的能力加以利用。衣物上所許多污漬是蛋白質(zhì)性的污漬，特異性廣的蛋白酶能基本上除去這些污漬。
遺憾的是，通常這些蛋白在它們天然細(xì)菌環(huán)境中的效力水平不能轉(zhuǎn)移到相對(duì)非天然的洗滌環(huán)境中。特別是當(dāng)不在這些酶的天然環(huán)境中使用時(shí)，蛋白酶的性質(zhì)如熱穩(wěn)定性、pH穩(wěn)定性、氧化穩(wěn)定性和底物特異性不是需要的最佳狀態(tài)。
蛋白酶的氨基酸序列決定了蛋白酶的性質(zhì)。蛋白酶氨基酸序列的改變可以不同程度地改變酶的性質(zhì)、甚至可使酶失活，這取決于氨基酸序列中改變的位置、性質(zhì)和/或程度。已采取了一些措施改變野生型蛋白酶的氨基酸序列以試圖改善它們的性質(zhì)，目標(biāo)在于增強(qiáng)蛋白酶在洗滌環(huán)境中的效力。這些措施包括改變氨基酸序列以在非常多樣的環(huán)境中增強(qiáng)熱穩(wěn)定性和改善氧化穩(wěn)定性。
盡管現(xiàn)有技術(shù)中描述了各種方法，仍繼續(xù)需要新的有效的蛋白酶變異體，用于清洗各種表面。
發(fā)明目的本發(fā)明的一個(gè)目的是提供枯草桿菌蛋白酶DY酶變異體，它們與野生型酶相比具有改善的水解作用。
本發(fā)明的另一個(gè)目的是提供包含這些枯草桿菌蛋白酶變異體的清洗組合物。
概述本發(fā)明涉及枯草桿菌蛋白酶DY變異體，它們是對(duì)野生型枯草桿菌蛋白酶DY氨基酸序列中某些氨基酸序列進(jìn)行改良，野生型氨基酸序列包含第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)和第五環(huán)區(qū)；其中改良的氨基酸序列是在一個(gè)或多個(gè)環(huán)區(qū)內(nèi)特定識(shí)別位置上包括與野生型枯草桿菌蛋白酶DY不同的氨基酸(即取代)，由此，枯草桿菌蛋白酶DY變異體與野生型枯草桿菌蛋白酶DY相比對(duì)不溶性底物具有降低的吸附作用和增強(qiáng)的水解作用。本發(fā)明還涉及編碼這種枯草桿菌蛋白酶DY變異體的DNA序列。本發(fā)明也涉及包含該枯草桿菌蛋白酶DY變異體的用于清洗各種表面的組合物。
詳述I.枯草桿菌蛋白酶DY變異體本發(fā)明涉及枯草桿菌蛋白酶，特別是枯草桿菌蛋白酶DY，其通過突變編碼該酶的各種核苷酸序列改變酶的氨基酸序列而已得到改良。本發(fā)明改良的枯草桿菌蛋白酶(下文即“枯草桿菌蛋白酶DY變異體”)與野生型枯草桿菌蛋白酶相比對(duì)不溶性底物具有降低的吸附作用和增強(qiáng)的水解作用。本發(fā)明還涉及編碼這些枯草桿菌蛋白酶DY變異體的DNA序列。
本發(fā)明的枯草桿菌蛋白酶屬于蛋白酶類。蛋白酶是裂解肽鍵的催化劑。蛋白酶中的一類是絲氨酸蛋白酶。絲氨酸蛋白酶的特征是在活性位點(diǎn)有一個(gè)必需的絲氨酸殘基。
酶對(duì)可溶性底物的水解速率隨酶濃度而增加，有許多文件資料證明了這個(gè)觀察結(jié)果。因此看來，對(duì)于如在許多清洗情況中遇到的表面結(jié)合的底物而言，水解速率會(huì)隨表面濃度的增加而增加似乎是合理的。已經(jīng)證明事實(shí)是這樣的。(Brode，P.F.III和D.S.Rauch，LANGMUIR，“枯草桿菌蛋白酶BPN′對(duì)固定化底物的活性(Subtilisin BPN′Activity onan immobilized Substrate)”，第8卷，1325-1329頁(yè)(1992))。事實(shí)上，對(duì)于不溶性底物來說，當(dāng)酶的表面濃度改變時(shí)發(fā)現(xiàn)水解速率與表面濃度呈線性依賴關(guān)系。(Rubingh，D.N.和M.D.Bauer，“蛋白酶在蛋白-溶液界面的水解催化作用(Catalysis of Hydrolysis by Proteases atthe Protein-Solution Inerface)”，聚合物溶液，混合物與界面(InPolymer Solutions，Blends and Interfaces)，I.Noda和D.N.Rubingh，Elsevier編，464頁(yè)(1992))。令人驚訝的是，在試圖應(yīng)用這一原理尋找具有更好清洗效果的變異蛋白酶時(shí)，我們沒有發(fā)現(xiàn)吸附越好性能越好的酶。事實(shí)上，我們意外地測(cè)定到了相反的情況酶對(duì)底物吸附作用降低導(dǎo)致底物的水解作用增加(即清洗效果更好)。
當(dāng)不擬受理論的限制時(shí)，認(rèn)為當(dāng)一種變異體與另一種比較時(shí)，改善的性能是源于這樣一個(gè)事實(shí)，即酶吸附作用越小結(jié)合緊密性也越小，由此在不溶性蛋白底物要被除去的表面上的流動(dòng)性更大。在相當(dāng)?shù)拿溉芤簼舛认?，這種增強(qiáng)的流動(dòng)性足以勝過任何通過對(duì)表面施加更高的酶濃度而賦予的優(yōu)越性。
此處所述的突變被指定為改變(即降低)酶對(duì)表面結(jié)合污物的吸附作用。在枯草桿菌蛋白酶DY中，某些氨基酸形成了酶分子的外部環(huán)。為便于討論，這些環(huán)被稱為第一、第二、第三、第四和第五環(huán)區(qū)。具體而言，位置58-65形成第一環(huán)區(qū)；位置94-106形成第二環(huán)區(qū)；位置125-132形成第三環(huán)區(qū)；位置153-166形成第四環(huán)區(qū)；位置186-190形成第五環(huán)區(qū)；位置199-219形成第六環(huán)區(qū)(位置編號(hào)與野生型枯草桿菌蛋白酶DY的氨基酸序列(SEQ ID NO1)中的位置相同)。
據(jù)信，這些環(huán)區(qū)在酶分子對(duì)表面結(jié)合肽的吸附中起重要作用，在一個(gè)或多個(gè)這些環(huán)區(qū)中的特定突變對(duì)這種吸附作用會(huì)有重要影響。當(dāng)不擬受理論限制時(shí)，認(rèn)為環(huán)區(qū)對(duì)枯草桿菌蛋白酶DY分子的吸附作用是重要的，至少有兩個(gè)原因第一，構(gòu)成環(huán)區(qū)的氨基酸可以與酶分子作用的任何表面緊密接觸。第二，環(huán)區(qū)與枯草桿菌蛋白酶DY分子的活性位點(diǎn)和結(jié)合空穴間的鄰近使得它們?cè)诿笇?duì)表面結(jié)合底物(肽/蛋白質(zhì)污物)的催化產(chǎn)生的吸附中起作用。
這里所用的“變異體”指具有與野生型不同的氨基酸序列的酶。
這里所用的“突變型枯草桿菌蛋白酶DY DNA”指編碼枯草桿菌蛋白酶DY變異體的DNA序列。
這里所用的“野生型枯草桿菌蛋白酶DY”指SEQ ID NO1表示的酶?？莶輻U菌蛋白酶DY的氨基酸序列由Nedkov，P.，Oberthur，W.和Braunitzer，G.，在生物化學(xué)第366卷第421-430頁(yè)(BIOL.CHEM.，Vol.366，pp.421-430(1985))作了進(jìn)一步描述，在此引作參考。
這里所用的“枯草桿菌蛋白酶DY野生型氨基酸序列”包括SEQ IDNO1以及除下列任一位置之外具有改變的氨基酸序列的SEQ IDNO1，所指位置包括58、59、60、61、62、64、65、94、95、96、97、98、99、100、101、102、103、104、105、106、125、126、127、128、129、130、131、132、153、154、155、156、157、158、159、160、161、162、163、164、165、166、186、187、188、189、190、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219。
這里所用的“親水性更強(qiáng)的氨基酸”指比目標(biāo)氨基酸具有更強(qiáng)親水性的任一其他氨基酸，參考下表所示的親水性。下面親水性表(表1)中以親水性增強(qiáng)的次序由上向下列出氨基酸(參見Hopp，T.P.，和Woods，K.R.，“由氨基酸序列推測(cè)蛋白抗原決定簇(Prediction of ProteinAntigenic Determinants from Amino Acid Sequences)”，美國(guó)國(guó)家科學(xué)院匯編(Proceedings of The National Academy of Science USA)，第78卷，3824-3828頁(yè)，1981，在此引為參考)。表1氨基酸 親水性值Trp -3.4Phe -2.5Tyr -2.3Leu，Ile -1.8Val -1.5Met -1.3Cys -1.0Ala，His -0.5Thr -0.4Pro，Gly -0.0Gln，Asn 0.2Ser 0.3Arg+，Lys+，Glu-，Asp- 3.0
表1還指明了哪一個(gè)氨基酸帶電荷(該特征基于pH為約8-9)。帶正電荷的氨基酸為Arg和Lys，帶負(fù)電荷的氨基酸為Glu和Asp，剩下的氨基酸均為中性。在本發(fā)明優(yōu)選實(shí)施方案中，取代的氨基酸是中性的或是帶負(fù)電荷的，更優(yōu)選帶負(fù)電荷的(即Glu或Asp)。
因此，例如“用中性或帶負(fù)電荷的親水性相同或更強(qiáng)的氨基酸取代Gln”這句話的意思是Gln會(huì)被Asn(親水性與Gln相同)或Ser、Glu或Asp(親水性比Gln強(qiáng))取代；其中每種氨基酸均為中性或帶負(fù)電荷，并且與Gln相比具有更高的親水性值。與此相似，“用中性或帶負(fù)電荷的親水性更強(qiáng)的氨基酸取代Pro”這句話的意思是Pro會(huì)被Gln、Asn、Ser、Glu或Asp取代。
在本發(fā)明一個(gè)優(yōu)選實(shí)施方案中，枯草桿菌蛋白酶DY變異體具有對(duì)枯草桿菌蛋白酶DY野生型氨基酸序列進(jìn)行改良的序列，其中，在野生型氨基酸序列的第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)、第五環(huán)區(qū)或第六環(huán)區(qū)中的一個(gè)或多個(gè)環(huán)區(qū)中的一個(gè)或多個(gè)位置處包括一個(gè)取代；由此枯草桿菌蛋白酶DY變異體與野生型枯草桿菌蛋白酶DY相比對(duì)不溶性底物具有降低的吸附作用和增強(qiáng)的水解作用。
在本發(fā)明優(yōu)選實(shí)施方案中，參照表1對(duì)一個(gè)或多個(gè)環(huán)區(qū)中的一個(gè)或多個(gè)位置取代的氨基酸是中性的或帶負(fù)電荷的，與野生型氨基酸序列的目標(biāo)位置上的氨基酸相比親水性相同或更強(qiáng)，優(yōu)選親水性更強(qiáng)。
A.在第一環(huán)區(qū)的取代當(dāng)取代發(fā)生在第一環(huán)區(qū)時(shí)，在第58、59、60、61、62、64或65位的一個(gè)或多個(gè)位置上發(fā)生取代。
當(dāng)取代發(fā)生在58位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)取代發(fā)生在59位時(shí)，取代氨基酸是Glu。
當(dāng)取代發(fā)生在60位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在61位時(shí)，取代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)取代發(fā)生在62位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在64位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在65位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
B.在第二環(huán)區(qū)的取代當(dāng)取代發(fā)生在第二環(huán)區(qū)時(shí)，在第94、95、96、97、98、99、100、101、102、103、104、105或106位的一個(gè)或多個(gè)位置上發(fā)生取代。
當(dāng)取代發(fā)生在94位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
當(dāng)取代發(fā)生在95位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在96位時(shí)，取代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)取代發(fā)生在97位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在98位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在99位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在100位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在101位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在102位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)取代發(fā)生在103位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在104位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在105位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
當(dāng)取代發(fā)生在106位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
C.在第三環(huán)區(qū)的取代當(dāng)取代發(fā)生在第三環(huán)區(qū)時(shí)，在第125、126、127、128、129、130、131或132位的一個(gè)或多個(gè)位置上發(fā)生取代。
當(dāng)取代發(fā)生在125位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在126位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在127位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在128位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
當(dāng)取代發(fā)生在129位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在130位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在131位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在132位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
D.在第四環(huán)區(qū)的取代當(dāng)取代發(fā)生在第四環(huán)區(qū)時(shí)，在第153、154、155、156、157、158、159、160、161、162、163、164、165或166位的一個(gè)或多個(gè)位置上發(fā)生取代。
當(dāng)取代發(fā)生在153位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在154位時(shí)，取代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)取代發(fā)生在155位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在156位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在157位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在158位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在159位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在160位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在161位時(shí)，取代氨基酸是Asn、Asp、Glu、或Ser。
當(dāng)取代發(fā)生在162位時(shí)，取代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)取代發(fā)生在163位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)取代發(fā)生在164位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在165位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在166位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
E.在第五環(huán)區(qū)的取代當(dāng)取代發(fā)生在第五環(huán)區(qū)時(shí)，在第186、187、188、189或190位的一個(gè)或多個(gè)位置上發(fā)生取代。
當(dāng)取代發(fā)生在186位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
當(dāng)取代發(fā)生在187位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在188位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Ile、Met、Pro、Ser、Thr、Tyr或Val。
當(dāng)取代發(fā)生在189位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在190位時(shí)，取代氨基酸是Asp或Glu。
F.在第六環(huán)區(qū)的取代當(dāng)取代發(fā)生在第六環(huán)區(qū)時(shí)，在第199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219位的一個(gè)或多個(gè)位置上發(fā)生取代。
當(dāng)取代發(fā)生在199位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
當(dāng)取代發(fā)生在200位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
當(dāng)取代發(fā)生在201位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在202位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
當(dāng)取代發(fā)生在203位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在204位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
當(dāng)取代發(fā)生在205位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在206位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在207位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)取代發(fā)生在208位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Phe、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在209位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
當(dāng)取代發(fā)生在210位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在211位時(shí)，取代氨基酸是Asp、Gln、Glu或Ser。
當(dāng)取代發(fā)生在212位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)取代發(fā)生在213位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在214位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
當(dāng)取代發(fā)生在215位時(shí)，取代氨基酸是Asp或Glu。
當(dāng)取代發(fā)生在216位時(shí)，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
當(dāng)取代發(fā)生在217位時(shí)，取代氨基酸是Asp、Gln、Glu、或Ser。
當(dāng)取代發(fā)生在218位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
當(dāng)取代發(fā)生在219位時(shí)，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
G.酶變異體的制備實(shí)施例1突變型枯草桿菌蛋白酶DY DNA構(gòu)建含野生型枯草桿菌蛋白酶DY基因的噬菌粒(“DYP”)。質(zhì)粒PUC119(Vieira，J.和Messing，J.，“單鏈質(zhì)粒DNA的生產(chǎn)(Prodution of Single-Stranded Plasmid DNA)”，153METHODS INENZYMOLOGY3-11(1989))的2.8 Kbp PvuII限制酶片段被克隆入質(zhì)粒pUB110(芽孢桿菌基因存儲(chǔ)中心(Bacliius Genetic Stock Center)，Columbus，OH1E9)的PvuII位點(diǎn)。此pUC119-pUB110雜交質(zhì)粒稱為pJMA601。在pJMA601的BamHI限制位點(diǎn)克隆入聚合酶鏈反應(yīng)擴(kuò)增的來自染色體DNA的枯草桿菌蛋白酶DY基因得到噬粒DYP。噬粒DYP被轉(zhuǎn)化入大腸桿菌(Escherichia coli)Ung-株CJ236中，并且使用VCSM13輔助噬菌體制備含尿嘧啶的單鏈DNA模板(Kunkel，T.A.，J.D.Roberts和R.A.Zakour，“無需表型選擇的快速有效的位點(diǎn)特異誘變(Rapid ang efficient site-specific mutagenesiswithout phenotypic selection)”，METHODS IN ENZYMOLOGY，Vol.54，pp367-382(1987)；如Yuckenberg，P，D.，F(xiàn).Witney，J.Geisselsoder和J.McClary所改進(jìn)的，“使用含尿嘧啶的DNA及噬菌粒載體體外定位誘變(Site-directed in vitro mutagenesis using uracil-containingDNA and phagemid vectors)”，DIRECTED MUTAGENESIS-APRATICAL APPROCH，M.J.McPherson編，pp27-48，(1991)；這兩篇文獻(xiàn)在此均引作參考)。Zoller和Smith的單鏈引物定位誘變修飾方法(Zoller，M.j.，和M.Smith，“使用M13-衍生的載體進(jìn)行寡核苷酸定向誘變?cè)贒NA任何片段中產(chǎn)生點(diǎn)突變的有效和通用方法(Oligonucleotide-directed mutagenesis using M13-derivedvectorsan efficient and general procedure for the production of pointmutations in any fragment of DNA)”，NUCLEIC ACIDSRESEARCH，VOL.10，pp6487-6500，(1982)，在此引為參考)被用于生產(chǎn)所有的突變體(基本上與Yuckenberg等人在1991年上述文獻(xiàn)中所述相同)。使用AplliedBiosystem Inc.380B DNA合成儀制備寡核苷酸。誘變反應(yīng)產(chǎn)物被轉(zhuǎn)化到大腸桿菌株MM294(American Type Culture CollectionE.coli.33625)中。所有的突變體由DNA測(cè)序來確證，分離的DNA被轉(zhuǎn)化入枯草芽孢桿菌(Bacillus subtilis)表達(dá)型菌株BG2036中(Yang，M.Y.，E.Ferrari和D.J.Henner，(1984)，“枯草芽孢桿菌中性蛋白酶基因的克隆及使用克隆基因產(chǎn)生體外衍生的缺失突變(Cloning ofthe Neutral Protease Gene of Bacillus subtilis and the Use of ClonedGene to Create an In Vitro-derived Deletion Mutation)”，JOUNALOF BACTERIOLOGY，Vol.160，pp15-21)。對(duì)于一些環(huán)區(qū)突變體使用在相應(yīng)環(huán)中具有移碼終止子突變的經(jīng)修飾的DYP生產(chǎn)尿嘧啶模板。設(shè)計(jì)寡核苷酸用于恢復(fù)合適的閱讀框及編碼以下位置的隨機(jī)取代氨基酸58、59、60、61、62、64、65、94、95、96、97、98、99、100、101、102、103、104、105、106、125、126、127、128、129、130、131、132、153、154、155、156、157、158、159、160、161、162、163、164、165、166、186、187、188、189、190、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219(這些密碼子的三個(gè)堿基均使用等摩爾和/或不同量的四種核苷酸的混合物)。校正移碼終止并產(chǎn)生功能酶的突變通過消化酪蛋白的能力進(jìn)行鑒別。通過DNA測(cè)序確定隨機(jī)取代。
實(shí)施例2發(fā)酵含目的枯草桿菌蛋白酶突變體的枯草芽孢桿菌細(xì)胞(BG2036)在一升LB-葡萄糖肉湯中生長(zhǎng)至中對(duì)數(shù)期，并接種于Biostat ED發(fā)酵罐(B.Braun Biotech，Inc.，Allentown.Pennsylvania)，總體積為10升。發(fā)酵培養(yǎng)基含酵母提取物(Yeast Extract)、淀粉、消泡劑、緩沖劑和痕量礦物質(zhì)(參見FERMENTATIONAPRACTICAL APPROACH，Ed.B.McNeil和L.M.Harvey，1990)。在進(jìn)行發(fā)酵的過程中，肉湯保持恒定的PH值為7。加入氯霉素用于誘變質(zhì)粒的抗生素選擇。細(xì)胞在37℃培養(yǎng)一晝夜至A600約60，收集細(xì)胞。
實(shí)施例3純化將發(fā)酵肉湯通過以下步驟獲得純化的酶。離心使肉湯不含枯草桿菌細(xì)胞，并使用100K截留膜除去細(xì)小的顆粒使肉湯澄清。然后在一10K的截留膜上濃縮，流動(dòng)透析以降低離子強(qiáng)度，并用0.025M的MES緩沖液(2-(N-嗎啉代)乙烷磺酸)調(diào)pH至5.5。將酶加樣于陽(yáng)離子交換色譜柱或親和吸附色譜柱上并用NaCl或丙二醇梯度從柱中洗脫(參見Scopes，R.K.，蛋白質(zhì)純化原理及實(shí)踐(PROTEIN PURIFICATIONPRINCIPLES AND PRACTICE)，Springer-Verlag，New York(1984)，此處引為參考)使酶進(jìn)一步純化。
使用pNA分析(DelMar，E.G.，C.Largman，J.W.Brodrick和M.C.Geokas，ANAL.BIOCHEM.，Vol.99，pp.316-320，(1979)，此處引為參考)測(cè)定梯度洗脫中收集級(jí)分的活性酶濃度。此分析測(cè)定當(dāng)酶水解可溶性合成底物琥珀酰-丙氨酰-丙氨酰-脯氨酰-苯丙氨酰-對(duì)硝基苯胺(sAAPF-pNA)時(shí)對(duì)硝基苯胺的釋放速率。在分光光度計(jì)上410nm下測(cè)量水解反應(yīng)中黃色物質(zhì)的產(chǎn)生速率，此速率與活性酶的濃度成比例。另外，在280nm下測(cè)量的吸光度被用于確定總蛋白濃度。由活性酶/總蛋白的比率得出酶的純度，并用來鑒別要合并作為存儲(chǔ)溶液的級(jí)分。
為避免存儲(chǔ)中酶的自身水解，向從色譜柱獲得的收集級(jí)分中加入等重量的丙二醇。在純化步驟完成時(shí)，用SDS-PAGE(十二烷基硫酸鈉聚丙烯酰胺凝膠電泳)檢測(cè)存儲(chǔ)酶溶液的純度，并且使用胰蛋白酶抑制劑II-T型購(gòu)自Sigma Chemical Company(St.Louis，Missouri)的火雞卵白通過活性位點(diǎn)滴定法確定絕對(duì)酶濃度。測(cè)得的轉(zhuǎn)化系數(shù)將表明在酶分子不同位置上進(jìn)行的哪些變化會(huì)導(dǎo)致酶變異體與野生型相比對(duì)可溶性底物pNA具有增強(qiáng)的活性。
準(zhǔn)備使用時(shí)，將酶存儲(chǔ)溶液通過一個(gè)Sephadex-G25(Pharmacia，Piscataway，New Jersey)分子排阻柱洗脫以除去丙二醇并交換緩沖液。將酶存儲(chǔ)溶液中的MES緩沖液換為含0.01MCaCl2并用HCl調(diào)節(jié)pH為8.6的0.1MTris緩沖劑(三(羥甲基-氨基甲烷))。所有實(shí)驗(yàn)均在pH8.6恒溫于25℃的Tris緩沖液中進(jìn)行。
H.酶變異體的特征實(shí)施例4典型表面制備購(gòu)自CPG Inc.(Fairfield，New Jersey)的氨丙基可控多孔玻璃(CPG)被用作共價(jià)連接sAAPF-pNA底物的載體，此底物購(gòu)自Bachem，Inc.(Torrence，California)。反應(yīng)在二甲基亞砜中進(jìn)行，(1-乙基-3-〔3-(二甲基氨基)丙基〕碳二亞胺鹽酸鹽)(EDC)用作偶聯(lián)劑。完成(用pNA分析監(jiān)測(cè))時(shí)，除去過量的溶劑，并用二甲基亞砜(DMSO)和雙蒸水洗滌CPGsAAPF-pNA。然后，在約70℃下的干燥箱中用N2吹干。反應(yīng)方案和固定化底物的制備根據(jù)Brode，P.F.III和D.S.Rauch“Subtilisin BPN’Activity on anImmobilized Substrate，”LANGMUIR，Vol.8，p.1325-1329，(1992)(此處引為參考文獻(xiàn))中描述的方法進(jìn)行。
CPG表面將有62,000±7,000pNA分子/μm2。其表面積與CPG公司為我們所購(gòu)的CPG提供的值50.0m2/g相比沒有改變。這表明將sAAPF-pNA加入到CPG的方法不會(huì)損壞多孔結(jié)構(gòu)(平均直徑為486)。
實(shí)施例5表面水解作用分析使用CPGsAAPF-pNA，可在一項(xiàng)實(shí)驗(yàn)中測(cè)量酶變異體的吸附作用和CPG-結(jié)合肽的水解。向含Tris緩沖液和已抽氣的CPGsAAPF-pNA的燒瓶中加入少量的酶變異體存儲(chǔ)溶液。在作曲形運(yùn)動(dòng)的搖床上搖動(dòng)燒瓶90分鐘，在此過程中搖床在不同的時(shí)間間隔內(nèi)停機(jī)(例如，在吸附水解作用早期如起初的20分鐘內(nèi)每2分鐘為一間隔，至實(shí)驗(yàn)結(jié)束時(shí)每10分鐘為一間隔)。使CPGsAAPF-pNA固定并由溶液取樣。實(shí)驗(yàn)方法及吸附作用和水解的計(jì)算方法均按照上述Brode等人于1992年描述的方法進(jìn)行。
監(jiān)測(cè)所有酶抵抗自身水解的穩(wěn)定性，在實(shí)驗(yàn)過程中應(yīng)沒有明顯的自身水解造成的損失。因此，可通過測(cè)量溶液的消耗量確定酶的吸附作用。初始酶變異體濃度與每一個(gè)時(shí)間點(diǎn)測(cè)量的濃度間的差給出酶變異體吸附的量。通過在410nm下讀取等分樣品的吸光度測(cè)量pNA從表面的水解量。將取樣量和存在于燒瓶中的量相加計(jì)算水解的pNA總量。通過減去酶不存在時(shí)由pH 8.6的Tris緩沖液水解的pNA的量校正該值。根據(jù)酶的效力，這個(gè)本底水解作用占總水解作用的7-29％。
實(shí)施例6可溶性底物動(dòng)力學(xué)分析通過在DU-70分光光度計(jì)上于410nm下測(cè)量吸收升高與時(shí)間的函數(shù)來監(jiān)測(cè)可溶性底物sAAPF-pNA的水解速率。酶濃度保持不變，且對(duì)于每一次動(dòng)力學(xué)測(cè)定底物濃度在90-700μM sAAPF-pNA變化，同時(shí)制備的酶濃度范圍為6-10nmol。在900秒的期間內(nèi)每秒測(cè)一個(gè)吸光度數(shù)據(jù)，且將該數(shù)據(jù)轉(zhuǎn)移至LOTUSTM座標(biāo)紙(LotusDevelopment corporation，Cambridge，Massachusetts)。動(dòng)力學(xué)參數(shù)分析通過標(biāo)準(zhǔn)Lineweaver Burk分析進(jìn)行，其中過程的初始部分(通常第一分鐘)數(shù)據(jù)與線性回歸曲線吻合給出V0。以標(biāo)準(zhǔn)倒數(shù)形式將V0和S0作圖，給出KM和kcat。
I.枯草桿菌蛋白酶DY變異體的例子下表2-36例舉了本發(fā)明的對(duì)表面結(jié)合底物具有降低吸附作用和增強(qiáng)水解作用的枯草桿菌蛋白酶DY變異體。在描述特定的突變體時(shí)，野生型中存在的原始氨基酸寫在第一位，位置編號(hào)寫在第二位，取代氨基酸寫在第三位。
表2環(huán)1-單一突變的變異體Thr58AsnThr58AspThr58GlnThr58GluThr58GlyThr58ProThr58SerAsp59GluGly60AsnGly60AspGly60GlnGly60GluGly60ProGly60SerAsn61AspAsn61GlnAsn61GluAsn61SerGly62AsnGly62AspGly62GlnGly62GluGly62ProGly62SerGly64AsnGly64AspGly64GlnGly64GluGly64ProGly64SerThr65AsnThr65AspThr65GlnThr65GluThr65GlyThr65ProThr65Ser表3環(huán)1-雙點(diǎn)突變的變異體Thr58Gln + Asn61GluThr58Asn + Asn61AspAsp59Glu + Gly64ProThr58Glu + Gly62ProAsp59Glu + Gly60SerGly60Asn + Gly64AsnAsn61Asp + Thr65GlnThr58Asp + Asn61SerThr58Asp + Thr65ProGly64Glu + Thr65GlyGly64Asp + Thr65AsnThr58Glu + Gly62GlnGly60Asn + Asn61GluThr58Asn + Gly64SerThr58Pro + Asp59GluGly64Glu + Thr65GlnThr58Pro + Gly62SerThr58Gly + Gly64AsnAsn61Gln + Gly64GlnThr58Gly + Asn61AspGly60Pro + Asn61AspGly62Asn + Gly64ProGly62Asp + Thr65AsnThr58Asn + Gly62AsnThr58Pro + Gly62AsnGly64Glu + Thr65SerGly60Pro + Gly62GluGly60Glu + Thr65GlnGly60Pro + Gly62SerThr58Asp + Asn61GlnGly60Asn + Asn61AspThr58Ser + Gly62GluGly60Glu + Gly64SerGly60Pro + Asn61GluThr58Gln + Asn61SerGly60Asp + Thr65ProAsn61Asp + Thr65SerAsn61Ser + Gly64Asp表4環(huán)1-三點(diǎn)突變的變異體Thr58Asn + Gly60Gln + Gly64ProThr58Gly + Asn61Ser + Thr65SerAsp59Glu + Gly60Pro + Thr65SerAsn61Glu + Gly62Pro + Gly64AsnThr58Asp + Gly60Asn + Thr65GlnThr58Ser + Gly62Ser + Thr65GlyThr58Pro + Asp59Glu + Gly62ProGly60Glu + Asn61Gln + Thr65GlnGly60Glu + Gly64Asn + Thr65ProGly60Asn + Gly62Asp + Thr65SerThr58Asn + Asn61Glu + Thr65GlnThr58Asn + Asp59Glu + Gly62ProGly60Gln + Gly62Gln + Gly64GluAsn61Gln + Gly62Pro + Thr65SerAsn61Gln + Gly62Asn + Thr65GlnAsn61Ser + Gly62Gln + Thr65GlyGly60Ser + Gly64Gln + Thr65GlyThr58Pro + Gly60Pro + Gly64GlnThr58Asn + Gly60Asn + Thr65ProAsp59Glu + Gly62Gln + Thr65AsnThr58Ser + Gly60Gln + Gly62AspThr58Asn + Gly64Ser + Thr65AsnGly60Pro + Gly62Asp + Thr65GlnAsn61Glu + Gly64Ser + Thr65GlyThr58Pro + Asn61Asp + Gly64GlnAsp59Glu + Asn61Ser + Gly62SerThr58Asn + Gly60Asp + Thr65AsnAsp59Glu + Asn61Gln + Gly62AsnThr58Asn + Gly60Asp + Asn61AspThr58Asn + Gly60Glu + Asn61Glu表5環(huán)1-四點(diǎn)突變的變異體Thr58Asn + Gly60Glu + Gly64Pro + Thr65GlnAsp59Glu + Gly60Ser + Gly64Pro + Thr65ProThr58Ser + Gly60Asn + Asn61Gln + Thr65ProAsp59Glu + Gly60Ser + Gly64Ser + Thr65GlyAsp59Glu + Gly60Asn + Asn61Ser + Gly62SerAsp59Glu + Gly60Asn + Asn61Ser + Gly64AsnThr58Gln + Asn61Gln + Gly62Glu + Gly64AsnThr58Asn + Asp59Glu + Gly60Gln + Thr65ProThr58Gly + Gly60Gln + Gly62Pro + Gly64GluGly60Asp + Asn61Asp + Gly62Pro + Gly64GlnThr58Pro + Gly60Glu + Asn61Asp + Gly64AsnThr58Asp + Asp59Glu + Gly62Asn + Thr65GlnThr58Asp + Asp59Glu + Gly60Ser + Gly64AsnThr58Glu + Asp59Glu + Gly62Asn + Thr65SerThr58Asp + Asp59Glu + Gly60Pro + Asn61SerThr58Asn + Gly60Gln + Asn61Glu + Gly62AspAsp59Glu + Gly60Glu + Asn61Glu + Gly62SerThr58Gln + Asp59Glu + Gly60Glu + Asn61GluThr58Pro + Asp59Glu + Gly60Glu + Asn61GluThr58Gln + Asp59Glu + Gly60Asp + Asn61AspThr58Pro + Gly60Glu + Asn61Asp + Gly62GluGly60Glu+Asn61Glu+Gly62Glu+Thr65GlyGly60Asp+Asn61Asp+Gly62Asp+Thr65GlnThr58Asp+Asp59Glu+Gly60Glu+Thr65SerAsp59Glu+Asn61Asp+Gly62Asp+Thr65GlnAsp59Glu+Asn61Asp+Gly62Glu+Thr65GlyAsp59Glu+Gly60Asp+Gly62Asp+Thr65GlnThr58Ser+Asp59Glu+Gly60Asp+Gly62Asp表6環(huán)2-單一突變的變異體Val 94AlaVal 94AsnVal 94AspVal 94CysVal 94GlnVal 94GluVal 94GlyVal 94HisVal 94MetVal 94ProVal 94SerVal 94ThrLeu 95AlaLeu 95AsnLeu 95AspLeu 95CysLeu 95GlnLeu 95GluLeu 95GlyLeu 95HisLeu 95IleLeu 95MetLeu 95ProLeu 95SerLeu 95ThrLeu 95ValAsn 96AspAsn 96GlnAsn 96GluAsn 96SerSer 97AspSer 97GluSer 98AspSer 98GluGly 99AsnGly 99AspGly 99GlnGly 99GluGly 99ProGly 99SerSer100AspSer100GluGly101AsnGly101AspGly101GlnGly101GluGly101ProGly101SerThr102AsnThr102AspThr102GlnThr102GluThr102GlyThr102ProThr102SerTyr103AlaTyr103AsnTyr103AspTyr103CysTyr103GlnTyr103GluTyr103GlyTyr103HisTyr103IleTyr103LeuTyr103MetTyr103ProTyr103SerTyr103ThrTyr103ValSer104AspSer104GluAla105AsnAla105AspAla105GlnAla105GluAla105GlyAla105HisAla105ProAla105SerAla105ThrIle106AlaIle106AsnIle106AspIle106CysIle106GlnIle106GluIle106GlyIle106HisIle106LeuIle106MetIle106ProIle106SerIle106ThrIle106Val表7環(huán)2-雙點(diǎn)突變的變異體Val 94Gln+Ser100GluAsn 96Ser+Gly 99GlnVal 94Asp+Thr102GlnVal 94His+Ala105ProVal 94Pro+Ser 97GluAsn 96Ser+Ile106AspSer 97Glu+Gly101SerLeu 95Gly+Thr102GluSer100Glu+Ile106ThrAsn 96Ser+Gly 99SerAla105Ser+Ile106ProAsn 96Ser+Gly101SerThr102Asn+Ile106GlnVal 94His+Asn 96GlnLeu 95Cys+Ala105AspLeu 95Gly+Asn 96AspLeu 95Cys+Asn 96AspThr102Ser+Ile106CysAsn 96Gln+Thr102GlnGly101Ser+Tyr103GlyAsn 96Ser+Ile106ThrSer 98Asp+Gly 99GlnLeu 95Ser+Ser100GluLeu 95Met+Thr102GlyLeu 95His+Thr102GluAsn 96Asp+Tyr103HisTyr103Gln+Ile106AspSer104Asp+Ile106GlnThr102Asn+Tyr103AspVal 94Asn+Ser 97GluVal 94Gly+Gly101SerLeu 95Gln+Ile106AspAsn 96Ser+Ser104AspTyr103Glu+Ile106HisThr102Ser+Tyr103GluVal 94Cys+Ser104AspLeu 95Gln+Thr102AspAsn 96Glu+Gly101SerLeu 95Glu+Ala105SerVal 94Met+Leu 95ProVal 94Thr+Gly101GlnTyr103Gly+Ile106HisThr102Asn+Ile106GlySer104Asp+Ile106ThrGly 99Ser+Ser100AspVal 94Glu+Gly 99AsnGly 99Gln+Gly101SerVal 94Thr+Asn 96AspSer 97Glu+Ile106LeuThr102Asp+Tyr103MetSer 97Asp+Ala105HisAsn 96Gln+Ser100AspGly101Asn+Ile106GlnAsn 96Glu+Tyr103SerTyr103Thr+Ile106ProSer100Asp+Tyr103IleGly101Glu+TyrI03LeuSer100Asp+Thr102AsnVal 94His+Leu 95ThrSer104Asp+Ala105Asn表8環(huán)2-三點(diǎn)突變的變異體Val 94Gln+Leu 95Thr+Ser100GluSer 98Asp+Ala105Ser+Ile106AsnVal 94Gly+Gly 99Pro+Ile106ValVal 94Ser+Leu 95Gln+Ser104GluVal 94His+Tyr103Glu+Ile106GlyVal 94Pro+Asn 96Gln+Ile106CysVal 94Thr+Ser 98Asp+Ile106CysVal 94Met+Ser100Asp+Gly101SerVal 94Ala+Ser 97Asp+Gly 99GlnLeu 95Gly+Gly 99Pro+Ala105GlyVal 94Cys+Ser100Asp+Tyr103AlaGly101Asn+Thr102Asp+Ile106AlaGly 99Ser+Ser104Glu+Ile106ValAsn 96Gln+Ser100Glu+Tyr103ThrLeu 95Met+Thr102Pro+Ile106LeuAsn 96Glu+Thr102Ser+Ala105HisSer100Glu+Thr102Asn+Tyr103ValSer 98Glu+Gly 99Gln+Ile106MetAsn 96Asp+Gly 99Ser+Ala105ThrGly101Asp+Thr102Asn+Tyr103ThrVal 94Ala+Gly 99Gln+Tyr103HisThr102Gly+Tyr103Glu+Ala105GlySer 97Glu+Gly101Ser+Thr102GlnVal 94Pro+Gly 99Ser+Gly101AspSer 97Asp+Gly 99Ser+Ile106GlyVal 94Cys+Leu 95Val+Gly101GlnSer100Asp+Gly101Asn+Tyr103GlnThr102Asp+Tyr103Leu+Ile106ValVal 94Cys+Ser104Glu+Ala105AsnVal 94Ser+Thr102Asn+Ala105ThrSer 98Glu+Gly 99Asn+Tyr103ValLeu 95Val+Gly101Gln+Ala105AspSer 97Glu+Gly 99Pro+Thr102SerVal 94Ser+Gly101Glu+Ile106AlaGly 99Asn+Gly101Asp+Thr102GluGly 99AsP+Ser100Glu+Thr102AsnVal 94Thr+Gly 99Glu+Ser100AspGly 99Asp+Ser100Asp+Tyr103ProGly 99Glu+Ser100Glu+Gly101GlnAsn 96Glu+Ser 97Glu+Ala105AsnAsn 96Glu+Ser 97Asp+Gly101ProLeu 95Met+Asn 96Asp+Ser 97GluGly 99Ser+Ser104Asp+Ala105GluSer 97Asp+Ser 98Glu+Gly101SerAsn 96Asp+Ser 98Asp+Gly 99GluAsn 96Glu+Ser 97Glu+Gly 99GluLeu 95Asp+Ser100Glu+Ile106LeuAsn 96Asp+Ser100Glu+Gly101GluSer104Asp+Ala105Thr+Ile106GluAsn 96Glu+Ser 98Asp+Tyr103AlaLeu 95Asn+Ser 98Asp+Ser100GluLeu 95Gly+Ser 98Glu+Ser100GluSer 98Asp+Ser100Glu+Ala105SerSer 98Glu+Ser100Asp+Thr102AsnVal 94Asn+Ser 97Glu+Gly 99GluAsn 96Glu+Ser100Glu+Thr102ProVal 94Asp+Gly101Asp+Thr102ProGly101Glu+Thr102Asp+Ser104AspSer 98Glu+Ser100Asp+Gly101AspLeu 95Glu+Gly101Asp+Ile106Glu表9環(huán)2-四點(diǎn)突變的變異體Val 94Asr+Gly101Asp+Tyr103Ile+Ala105ThrVal 94Asn+Gly 99Ser+Ser100Glu+Thr102SerLeu 95Cys+Ser 98Asp+Gly101Pro+Ile106AlaLeu 95His+Gly101Ser+Tyr103Ile+Ala105SerVal 94Ser+Leu 95Val+Gly 99Pro+Ser100GluGly 99Ser+Ser100Glu+Tyr103Ile+Ala105SerVal 94Ala+Ser 97Glu+Thr102Ser+Ala105ProVal 94Thr+Tyr103Gly+Ser104Asp+Ala105GlnVal 94Ala+Leu 95Pro+Ser100Glu+Ile106SerLeu 95Asn+Ser 98Asp+Tyr103Gly+Ala105ProAsn 96Ser+Gly101Glu+Tyr103Leu+Ala105ProLeu 95Met+Ser 98Glu+Gly 99Asn+Tyr103ThrVal 94Ala+Leu 95Ala+Ser 97Asp+Thr102AsnGly 99Gln+Ser100Glu+Thr102Ser+Ile106LeuAsn 96Glu+Gly 99Gln+Ala105Pro+Ile106ThrVal 94Gly+Gly101Glu+Tyr103Gln+Ile106HisVal 94Thr+Ser 97Asp+Gly101Gln+Ala105ThrVal 94Gly+Leu 95Val+Asn 96Gln+Ser104GluLeu 95Gln+Gly 99Glu+Ser100Glu+Thr102ProLeu 95Met+Ser100Asp+Gly101Asp+Tyr103SerLeu 95Ile+Thr102Pro+Ser104Asp+Ala105AspSer 97Asp+Ser 98Asp+Tyr103Met+Ala105ProLeu 95Ser+Ser 97Asp+Ser 98Asp+Thr102SerSer 97Glu+Ser 98Glu+Ala105Pro+Ile106AsnSer 97Glu+Ser 98Glu+Gly101Ser+Ile106CysSer 97Asp+Ser 98Asp+Gly101Pro+Thr102ProLeu 95Glu+Ser100Glu+Gly101Asp+Thr102SerVal 94Met+Leu 95Glu+Asn 96Glu+Gly101AspLeu 95His+Gly 99Glu+Ser100Glu+Gly101GluAsn 96Glu+Gly 99Glu+Gly101Ser+Ile106AsnLeu 95Val+Asn 96Asp+Ser 98Glu+Tyr103GlnAsn 96Asp+Ser 98Glu+Gly 99Ser+Thr102AsnAsn 96Glu+Ser 98Glu+Gly101Ser+Thr102ProLeu 95Val+Asn 96Glu+Ser 98Asp+Ile106AlaVal 94Gly+Asn 96Glu+Ser 98Asp+Tyr103ThrSer 98Glu+Gly 99Gln+Ser100Asp+Gly101ProSer 98Glu+Gly 99Gln+Ser100Asp+Thr102GlnAsn 96Glu+Ser100Glu+Tyr103Gln+Ile106ProAsn 96Asp+Gly 99Asn+Ser100Glu+Tyr103IleLeu 95Glu+Asn 96Gln+Ser 98Glu+Gly 99GluSer 97Asp+Ser 98Glu+Ser100Asp+Ile106ValSer 97Asp+Ser 98Asp+Ser100Glu+Thr102SerSer 97Asp+Ser 98Asp+Ser100Glu+Gly101GlnLeu 95Thr+Ser 97Asp+Ser 98Glu+Ser100AspVal 94Met+Asn 96Glu+Gly 99Asp+Gly101AspSer 98Asp+Ser100Glu+Gly101Glu+Ala105SerVal 94Cys+Ser 98Asp+Ser100Asp+Gly101AspVal 94Glu+Ser100Glu+Gly101Glu+Tyr103IleLeu 95Glu+Ser 97Asp+Ser 98Asp+Ala105SerLeu 95Ala+Asn 96Asp+Gly101Asp+Thr102AspVal 94Cys+Gly 99Asp+Gly101Asp+Ile106ProLeu 95Ser+Ser100Glu+Gly101Gln+Thr102GluLeu 95Met+Ser100Asp+Thr102Glu+Ile106GlnAsn 96Ser+Gly101Asp+Tyr103Asn+Ile106AspLeu 95Cys+Gly101Glu+Ala105Pro+Ile106GluVal 94Asp+Gly 99Glu+Ser100Asp+Ala105SerSer 97Glu+Ser 98Glu+Gly101Asp+Tyr103SerSer 97Asp+Ser100Glu+Thr102Gln+Tyr103ThrSer 97Glu+Ser100Asp+Tyr103Ser+Ala105Gly表10環(huán)3-單一突變的變異體Leu125AlaLeu125AsnLeu125AspLeu125CysLeu125GlnLeu125GluLeu125GlyLeu125HisLeu125IleLeu125MetLeu125ProLeu125SerLeu125ThrLeu125ValGly126AsnGly126AspGly126GlnGly126GluGly126ProGly126SerGly127AsnGly127AspGly127GlnGly127GluGly127ProGly127SerPro128AsnPro128AspPro128GlnPro128GluPro128GlyPro128SerSer129AspSer129GluGly130AsnGly130AspGly130GlnGly130GluGly130ProGly130SerSer131AspSer131GluThr132AsnThr132AspThr132GlnThr132GluThr132GlyThr132ProThr132Ser表11環(huán)3-雙點(diǎn)突變的變異體Leu125Gln+Ser129GluLeu125Pro+Gly126SerGly127Gln+Ser131AspGly130Ser+Thr132GlyLeu125Met+Gly130AspLeu125Pro+Pro128AspSer129Asp+Gly130AsnLeu125Asp+Pro128AsnGly127Ser+Ser131GluLeu125Ile+Gly130SerLeu125Thr+Thr132AspLeu125Thr+Thr132AsnLeu125Pro+Gly127GluLeu125Glu+Thr132AsnPro128Asn+Thr132ProLeu125Thr+Gly130AspGly127Pro+Thr132SerGly127Asn+Thr132AsnGly126Gln+Ser131AspLeu125Val+Thr132GlyGly126Gln+Gly130SerSer129Asp+Thr132GlyGly126Asp+Gly130ProLeu125Cys+Pro128GlnGly127Gln+Thr132GlyLeu125His+Gly126AspLeu125Val+Ser129GluGly126Ser+Pro128AspLeu125Val+Gly126GluGly127Glu+Pro128SerLeu125Asn+Thr132ProLeu125Met+Gly126AspLeu125Ser+Ser129GluLeu125Ser+Gly126AsnLeu125Val+Thr132GlnLeu125Val+Gly130AspSer129Glu+Thr132SerPro128Gly+Ser129AspGly126Pro+Thr132AspGly126Asp+Thr132SerPro128Asp+Thr132GlnGly126Pro+Ser129AspGly130Ser+Thr132GluGly127Glu+Pro128GlnGly126Asn+Gly130ProPro128Asn+Ser129GluGly130Asp+Thr132GlyGly126Gln+Ser131GluGly127Glu+Gly130SerGly126Asn+Ser129GluPro128Asp+Gly130AsnPro128Asp+Thr132GlyGly126Gln+Pro128AspSer131Glu+Thr132GlyGly130Glu+Thr132AsnLeu125Val+Pro128GlyLeu125Ile+Gly130ProPro128Asn+Ser131AspGly130Asn+Thr132AspGly127Ser+Pro128Glu表12環(huán)3-三點(diǎn)突變的變異體Gly127Asp+Gly130Gln+Thr132SerPro128Asp+Gly130Pro+Thr132SerGly127Asn+Ser131Asp+Thr132ProLeu125Ser+Ser131Asp+Thr132ProLeu125Met+Pro128Glu+Thr132ProLeu125Asp+Gly126Ser+Thr132GlnGly127Asn+Pro128Glu+Gly130AsnGly127Ser+Pro128Gln+Ser129GluGly126Pro+Gly127Pro+Ser131GluLeu125Ile+Gly127Asp+Thr132GlnGly126Asn+Gly130Ser+Thr132ProGly126Pro+Gly127Gln+Thr132AspGly126Asp+Gly130Asn+Thr132GlnLeu125Ser+Pro128Gln+Thr132AsnGly126Pro+Gly127Pro+Pro128GluLeu125Met+Gly126Glu+Gly130GlnLeu125Met+Ser129Asp+Thr132GlnLeu125His+Ser129Asp+Gly130SerGly127Pro+Ser129Glu+Gly130SerPro128Gln+Gly130Asn+Thr132GlnGly126Asn+Gly127Pro+Gly130GlnLeu125Pro+Gly126Ser+Gly127GlnLeu125His+Gly127Ser+Ser131GluLeu125His+Gly127Asn+Thr132GluGly126Glu+Gly130Pro+Thr132GlnLeu125His+Gly127Gln+Gly130GluGly127Pro+Pro128Gly+Thr132AsnLeu125Gly+Pro128Gly+Gly130AspGly126Asn+Pro128Asn+Ser129GluGly126Pro+Gly130Ser+Thr132GlyLeu125Ser+Gly126Pro+Ser131GluLeu125Val+Gly126Glu+Thr132GlyPro128Asn+Ser131Asp+Thr132ProLeu125Gly+Pro128Asn+Thr132ProGly127Glu+Pro128Ser+Thr132ProGIy127Gln+Ser129Glu+Thr132GlnLeu125Ile+Gly127Pro+Gly130AspGly126Ser+Gly127Asp+Gly130AsnPro128Glu+Gly130Pro+Thr132GlnLeu125Val+Gly126Asp+Gly127AspPro128Asn+Gly130Asp+Ser131AspGly130Glu+Ser131Asp+Thr132GlnGly126Pro+Gly130Glu+Ser131AspGly127Glu+Pro128Glu+Thr132SerGly130Gln+Ser131Glu+Thr132AspLeu125Gln+Ser131Asp+Thr132AspGly130Ser+Ser131Glu+Thr132GluGly130Pro+Ser131Asp+Thr132AspGly127Pro+Ser131Glu+Thr132AspLeu125Asn+Ser131Asp+Thr132AspPro128Gln+Ser131Asp+Thr132GluPro128Gly+Ser131Asp+Thr132AspGly126Gln+Ser129Asp+Gly130AspLeu125Ile+Ser129Asp+Gly130GluGly127Glu+Pro128Asp+Ser129GluSer129Asp+Gly130Asp+Ser131AspSer129Glu+Gly130Asp+Ser131GluSer129Glu+Gly130Glu+Ser131AspGly126Glu+Gly127Glu+Pro128GluGly130Glu+Ser131Asp+Thr132Asp表13環(huán)3-四點(diǎn)突變的變異體Gly126Ser+Pro128Glu+Gly130Ser+Thr132SerLeu125Cys+Gly127Gln+Ser129Glu+Thr132ProLeu125Gln+Gly126Pro+Pro128Ser+Ser129GluLeu125Thr+Ser129Glu+Gly130Ser+Thr132ProGly127Gln+Pro128Gln+Ser129Glu+Gly130ProGly127Asn+Pro128Asn+Ser131Glu+Thr132GlyLeu125Met+Gly127Glu+Gly130Asn+Thr132GlyLeu125Met+Gly127Asn+Pro128Asn+Ser129GluLeu125Met+Gly126Ser+Gly127Gln+Ser131AspLeu125Asn+Gly126Asp+Gly127Gln+Thr132SerLeu125Gln+Gly127Gln+Gly130Glu+Thr132ProLeu125Ala+Gly130Asn+Ser131Asp+Thr132ProLeu125Met+Gly126Pro+Gly127Ser+Ser131AspLeu125Cys+Gly126Pro+Ser129Glu+Thr132AsnLeu125Gly+Pro128Gly+Ser131Asp+Thr132GlnLeu125Met+Gly130Asn+Ser131Asp+Thr132GlnLeu125Val+Gly126Pro+Gly130Gln+Thr132AspGly126Asn+Gly127Pro+Pro128Gln+Ser129GluLeu125Gln+Gly126Glu+Gly127Glu+Gly130AsnLeu125Val+Gly126Asp+Gly127Glu+Gly130ProLeu125Asp+Gly126Glu+Gly127Pro+Thr132ProLeu125Glu+Gly126Glu+Gly127Asn+Thr132SerLeu125Gln+Gly127Pro+Gly130Glu+Ser131AspLeu125Cys+Gly130Glu+Ser131Asp+Thr132GlnGly126Gln+Gly127Glu+Pro128Glu+Gly130AsnGly126Ser+Gly127Asp+Pro128Asp+Gly130SerLeu125Thr+Gly127Ser+Ser131Glu+Thr132AspPro128Asn+Gly130Asn+Ser131Asp+Thr132GluLeu125Met+Gly126Ser+Ser129Asp+Gly130AspGly126Gln+Pro128Gln+Ser129Glu+Gly130GluGly127Asn+Pro128Asn+Ser129Glu+Gly130GluGly127Ser+Ser129Glu+Gly130Asp+Thr132GlnGly127Asp+Pro128Glu+Ser129Asp+Gly130AsnGly127Asp+Pro128Glu+Ser129Asp+Thr132SerLeu125Gln+Ser129Glu+Gly130Asp+Ser131AspLeu125Cys+Ser129Glu+Gly130Asp+Ser131GluGly127Gln+Pro128Glu+Ser129Glu+Gly130AspLeu125Met+Gly126Asp+Gly127Glu+Pro128GluGly127Glu+Pro128Gln+Ser129Glu+Thr132SerLeu125Pro+Gly127Asp+Pro128Ser+Ser129AspGly126Pro+Gly127Glu+Pro128Gly+Ser129AspLeu125Pro+Gly127Asp+Ser129Glu+Gly130GluGly127Asp+Pro128Ser+Ser129Glu+Gly130AspGly127Asp+Ser129Glu+Gly130Glu+Thr132ProLeu125Ile+Gly127Glu+Ser129Asp+Gly130AspGly126Glu+Gly127Asp+Ser129Glu+Gly130ProGly126Asp+Gly127Glu+Ser129Asp+Thr132ProLeu125Met+Gly126Asp+Gly127Asp+Ser129AspGly126Ser+Ser129Asp+Ser131Glu+Thr132GlnGly127Gln+Pro128Asn+Ser129Asp+Ser131AspPro128Gln+Ser129Glu+Ser131Asp+Thr132SerGly126Pro+Ser129Asp+Ser131Glu+Thr132SerLeu125Ala+Ser129Glu+Gly130Pro+Ser131AspGly127Gln+Ser129Glu+Gly130Ser+Ser131AspGly127Ser+Ser129Glu+Ser131Glu+Thr132GlnGly127Gln+Pro128Gln+Ser129Glu+Ser131AspLeu125Ala+Gly126Ser+Ser129Asp+Ser131GluGly126Pro+Pro128Gln+Ser129Asp+Ser131AspGly126Ser+Pro128Ser+Ser129Asp+Ser131GluGly126Glu+Pro128Asp+Ser129Asp+Thr132Asn表14環(huán)4-單一突變的變異體Gly153AsnGly153AspGly153GlnGly153GluGly153ProGly153SerAsn154AspAsn154GlnAsn154GluAsn154SerSer155AspSer155GluGly156AsnGly156AspGly156GlnGly156GluGly156ProGly156SerSer157AspSer157GluSer158AspSer158GluGly159AsnGly159AspGly159GlnGly159GluGly159ProGly159SerSer160AspSer160GluGln161AsnGln161AspGln161GluGln161SerAsn162AspAsn162GlnAsn162GluAsn162SerThr163AsnThr163AspThr163GlnThr163GluThr163GlyThr163ProThr163SerIle164AlaIle164AsnIle164AspIle164CysIle164GlnIle164GluIle164GlyIle164HisIle164LeuIle164MetIle164ProIle164SerIle164ThrIle164ValGly165AsnGly165AspGly165GlnGly165GluGly165ProGly165SerTyr166AlaTyr166AsnTyr166AspTyr166CysTyr166GlnTyr166GluTyr166GlyTyr166HisTyr166IleTyr166LeuTyr166MetTyr166ProTyr166SerTyr166ThrTyr166Val表15環(huán)4-雙點(diǎn)突變的變異體Asn154Ser+Ser155GluGly165Glu+Tyr166CysSer158Asp+Gly165ProSer157Asp+Thr163AsnGly156Pro+Thr163SerAsn154Glu+Ile164CysGly153Asn+Thr163AsnThr163Glu+Ile164GlyGly156Gln+Ile164ThrSer157Asp+Ile164HisSer158Glu+Tyr166HisGly153Pro+Tyr166AspGly153Pro+Gly165GlnSer157Glu+Gly159AsnAsn154Ser+Gly159ProGly156Glu+Asn162GlnGln161Asp+Ile164MetSer157Asp+Thr163SerSer157Glu+Tyr166IleGln161Glu+Ile164HisSer157Glu+Asn162GlnGly159Pro+Tyr166AspAsn162Asp+Ile164CysGln161Ser+Asn162AspAsn154Asp+Asn162GlnSer158Glu+Gly165AsnIle164Met+Tyr166ValGln161Asn+Gly165ProSer155Asp+Gly159ProSer160Asp+Asn162SerGly156Glu+Ile164CysThr163Asp+Gly165ProGly153Asn+Ser155GluSer158Asp+Thr163GlySer160Asp+Ile164MetGly159Asp+Thr163GlnSer155Glu+Tyr166CysGln161Ser+Tyr166GlySer160Asp+Ile164AlaSer157Asp+Gln161SerSer160Glu+Asn162GlnGly153Asn+Gly156GlnSer157Asp+Ile164ValAsn162Gln+Thr163GluSer155Asp+Gln161SerGly159Glu+Gly165GlnSer160Glu+Ile164HisGly153Ser+Ser160AspGly159Gln+Gly165GluGly153Glu+Gly159GlnGly156Glu+Thr163GlyGly156Ser+Tyr166AsnGly153Asp+Thr163AsnAsn154Ser+Ser157GluThr163Ser+Ile164ProSer155Glu+Asn162SerGly156Pro+Ile164GlyThr163Glu+Gly165AsnGly153Gln+Ser155GluIle164Ser+Tyr166Cys表16環(huán)4-三點(diǎn)突變的變異體Gly153Gln+Asn154Ser+Ser155GluSer155Asp+Thr163Pro+Tyr166GlnGly156Gln+Thr163Glu+Ile164GlyGly159Gln+Gln161Ser+Gly165SerAsn154Gln+Thr163Asp+Tyr166GlnAsn154Gln+Gly159Pro+Ile164ThrAsn162Gln+Ile164Gln+Gly165GluGly153Asp+Gln161Ser+Thr163ProGly153Gln+Asn154Gln+Tyr166IleAsn154Gln+Gln161Asn+Tyr166ProGly153Gln+Ser158Glu+Tyr166SerSer157Asp+Ile164Val+Tyr166AlaThr163Gly+Gly165Asp+Tyr166ProGIn161Glu+Asn162Gln+Tyr166ThrGly156Pro+Gln161Glu+Thr163SerSer158Glu+Gly159Pro+Gln161AsnSer158Glu+Gln161Asn+Asn162SerAsn154Glu+Gln161Ser+Gly165AsnGly153Asn+Asn154Gln+Ser158AspSer157Glu+Ile164Val+Tyr166ValSer155Asp+Gln161Asn+Tyr166IleGly153Glu+Gln161Asn+Gly165SerGly156Pro+Asn162Asp+Gly165ProGly156Asn+Asn162Asp+Ile164LeuAsn154Gln+Thr163Asn+Gly165SerSer158Asp+Gly159Asn+Gln161AsnSer155Glu+Gln161Asn+Tyr166LeuSer158Glu+Asn162Ser+Tyr166HisAsn154Asp+Thr163Gln+Ile164MetGly156Asp+Ser157Asp+Gly159SerGly159Asp+Ser160Glu+Thr163GlnSer158Asp+Gly159Asp+Tyr166CysAsn154Glu+Ser155Glu+Gly156SerSer155Asp+Gly156Asp+Asn162SerSer157Asp+Ser158Glu+Gly159ProSer160Asp+Gln161Asp+Ile164AlaGly159Gln+Ser160Asp+Gln161AspGly156Asp+Ser157Asp+Ser158AspGly159Asp+Gln161Glu+Gly165GlnGly153Pro+Gly159Glu+Gln161GluSer157Glu+Ser158Asp+Gln161GluSer157Glu+Gly159Glu+Gly165SerSer157Glu+Gly159Glu+Ser160GluSer158Asp+Ser160Asp+Ile164ThrSer158Asp+Ser160Glu+Tyr166ValAsn154Gln+Ser158Asp+Ser160AspSer158Glu+Ser160Asp+Tyr166HisGly153Asp+Asn154Glu+Gly165GluSer155Asp+Ser157Asp+Tyr166IleSer155Glu+Ser157Asp+Gly159ProGly153Asn+Ser157Glu+Thr163GluSer155Asp+Ser157Asp+Ser158GluSer157Asp+Gly159Asp+Gln161GluSer155Asp+Gly156Glu+Ser158AspAsn154Gln+Ser160Glu+Asn162AspSer158Glu+Ser160Glu+Asn162GluSer157Glu+Gln161Asp+Thr163AspAsn154Glu+Ser155Glu+Ser157GluSer157Asp+Ser160Glu+Tyr166GlyGly153Asn+Ser157Glu+Ser160Asp表17環(huán)4-四點(diǎn)突變的變異體Asn154Ser+Gly159Asp+Ile164Met+Gly165ProSer155Glu+Gly156Ser+Asn162Gln+Thr163AsnGly156Gln+Ser157Asp+Gly159Ser+Asn162SerSer155Asp+Gly159Pro+Gln161Ser+Ile164CysAsn154Gln+Gly156Asp+Thr163Gly+Tyr166MetAsn154Gln+Ser155Asp+Thr163Asn+Gly165SerGln161Ser+Asn162Gln+Thr163Gly+Tyr166GlySer155Asp+Gly159Pro+Gly165Pro+Tyr166GlnAsn154Ser+Ser155Asp+Thr163Gly+Ile164ValGly159Asp+GIn161Ser+Thr163Gln+Ile164ThrGly153Pro+Ser158Asp+Asn162Gln+Ile164CysGly153Pro+Gly156Asp+Ile164Ser+Tyr166LeuGly153Glu+Gly156Pro+Gln161Ser+Tyr166MetSer155Glu+Thr163Ser+Gly165Pro+Tyr166LeuSer157Asp+Thr163Gly+Ile164Gln+Tyr166GlyGly153Ser+Ser155Asp+Thr163Pro+Ile164ProAsn154Ser+Gly159Gln+Gln161Glu+Tyr166MetAsn154Ser+Ser160Asp+Ile164Gln+Gly165SerGly159Asn+Asn162Glu+Gly165Gln+Tyr166CysGly156Pro+Gly159Pro+Ile164Gln+Gly165GlnGly153Ser+Gly156Asn+Asn162Gln+Gly165AspSer158Asp+Gly159Glu+Gln161Ser+Ile164ThrGly153Gln+Asn154Glu+Ser155Glu+Tyr166GlyGly153Ser+Ser155Asp+Gly156Asp+Ile164GlnAsn154Ser+Ser155Glu+Gly156Asp+Tyr166ValSer155Glu+Gly156Glu+Thr163Asn+Ile164LeuSer157Glu+Ser158Asp+Gln161Asn+Ile164CysSer160Glu+Gln161Asp+Thr163Gln+Gly165ProSer157Asp+Ser158Glu+Gly159Asp+Ile164CysAsn154Gln+Ser160Glu+Gln161Glu+Asn162AspGly153Glu+Gly159Gln+Gln161Asn+Gly165GluAsn154Ser+Ser158Asp+Gly159Asn+Gln161GluAsn154Glu+Gly156Asp+Thr163Ser+Tyr166AsnSer158Glu+Ser160Asp+Gln161Asn+Thr163ProAsn154Glu+Ser155Asp+Gly156Gln+Thr163AspSer158Glu+Gln161Glu+Thr163Asp+Ile164HisSer155Glu+Ser157Asp+Asn162Ser+Gly165ProSer155Asp+Ser157Asp+Gly159Pro+Ile164CysGly159Glu+Ser160Asp+Asn162Glu+Ile164AsnSer157Glu+Gln161Asp+Thr163Asp+Gly165GlnAsn154Glu+Ser155Glu+Ser157Asp+Gly159ProAsn154Gln+Ser160Asp+Gln161Asp+Thr163AspSer160Asp+Gln161Asp+Asn162Gln+Thr163AspSer155Asp+Thr163Asp+Ile164Gly+Gly165AspGly156Ser+Ser158Asp+Thr163Asp+Ile164LeuSer155Glu+Ser158Glu+Thr163Glu+Ile164MetGly153Glu+Gly156Pro+Thr163Asp+Tyr166HisGly156Ser+Ser157Asp+Ser160Glu+Asn162AspGly153Asn+Ser157Glu+Asn162Asp+Ile164LeuGly153Glu+Ser155Glu+Ser157Asp+Ile164MetGly156Pro+Ser157Asp+Gly159Gln+Ser160GluGly153Pro+Ser155Glu+Gly156Glu+Gly165AspSer155Asp+Ser158Glu+Gln161Asn+Gly165ProSer155Asp+Ser158Glu+Gly159Gln+Ile164GlyGly153Asp+Ser157Glu+Gly159Pro+Thr163GluSer155Asp+Gly156Pro+Gln161Ser+Gly165AspGly156Glu+Ser157Glu+Ser160Asp+Ile164SerAsn154Ser+Gly156Asp+Ser158Asp+Ser160AspGly153Gln+Ser155Asp+Ser158Asp+Gly159GluAsn154Asp+Gly156Glu+Ser158Glu+Ile164Ala表18環(huán)5-單一突變的變異體Ala186AsnAla186AspAla186GlnAla186GluAla186GlyAla186HisAla186ProAla186SerAla186ThrSer187AspSer187GluPhe188AlaPhe188AsnPhe188AspPhe188CysPhe188GlnPhe188GluPhe188GlyPhe188HisPhe188IlePhe188LeuPhe188MetPhe188ProPhe188SerPhe188ThrPhe188TyrPhe188ValSer189AspSer189GluSer190AspSer190Glu表19環(huán)5-雙點(diǎn)突變的變異體Phe188Met+Ser190GluAla186Ser+Ser187GluPhe188Ser+Ser190AspAla186His+Ser187GluSer187Asp+Phe188CysAla186Ser+Ser190GluAla186Pro+Ser190AspPhe188Ser+Ser190GluAla186Gln+Ser187GluPhe188Asn+Ser190AspAla186Glu+Phe188MetAla186Ser+Phe188AlaSer187Glu+Phe188ThrAla186Thr+Phe188AspSer187Asp+Phe188LeuAla186Gly+Phe188CysPhe188Val+Ser190GluAla186Asp+Phe188ProAla186Asn+Ser187AspPhe188Leu+Ser190GluSer187Asp+Phe188AsnAla186Ser+Ser187AspPhe188Ala+Ser190GluAla186Gly+Ser187GluAla186Glu+Phe188HisAla186Gly+Phe188GluSer187Glu+Phe188GlyAla186Glu+Phe188AsnSer187Glu+Phe188HisAla186Ser+Phe188AspAla186Gly+Ser187AspPhe188Ile+Ser190GluAla186Thr+Ser190GluAla186Gly+Ser190GluPhe188His+Ser190AspPhe188Leu+Ser190AspAla186Ser+Ser190AspSer187Glu+Phe188IleAla186Thr+Phe188HisPhe188Gln+Ser190GluAla186His+Ser187AspPhe188Tyr+Ser190GluAla186Pro+Phe188ThrAla186Gln+Ser187AspSer187Glu+Phe188AlaAla186Asp+Phe188IleAla186Ser+Phe188ProAla186Asn+Ser187GluPhe188His+Ser190GluAla186Glu+Phe188SerAla186Ser+Phe188LeuPhe188Pro+Ser190GluSer187Asp+Phe188ThrAla186His+Phe188AlaAla186Asn+Phe188AspPhe188Ile+Ser190AspAla186Thr+Phe188MetAla186Asp+Phe188HisSer187Asp+Phe188AlaAla186Pro+Ser190Glu表20環(huán)5-三點(diǎn)突變的變異體Ala186His+Phe188Ala+Ser190AspAla186Asn+Ser187Asp+Phe188AlaAla186Gly+Phe188Asn+Ser190AspAla186Pro+Phe188Met+Ser190AspAla186Gly+Phe188Leu+Ser190GluAla186Gln+Ser187Asp+Phe188IleAla186Thr+Ser187Glu+Phe188CysAla186Ser+Phe188Ala+Ser190GluAla186Gln+Ser187Asp+Phe188SerAla186Gln+Ser187Glu+Phe188IleAla186Ser+Phe188Gly+Ser190AspAla186Ser+Ser187Asp+Phe188MetAla186Gly+Ser187Glu+Phe188GlyAla186Gln+Phe188Pro+Ser190AspAla186Asn+Phe188Ala+Ser190GluAla186Ser+Phe188Pro+Ser190GluAla186Asn+Ser187Glu+Phe188ProAla186Gln+Phe188Gly+Ser190GluAla186Asn+Ser187Glu+Phe188IleAla186His+Ser187Glu+Phe188GlyAla186His+Phe188Cys+Ser190AspAla186Thr+Ser187Glu+Phe188ValAla186Gln+Ser187Glu+Phe188MetAla186His+Phe188Ala+Ser190GluAla186Gly+Phe188Met+Ser190GluAla186Ser+Ser187Glu+Phe188HisAla186Asn+Phe188Gly+Ser190GluAla186Asn+Phe188Pro+Ser190AspAla186Asn+Phe188Tyr+Ser190AspAla186Gly+Phe188Thr+Ser190GluAla186His+Ser187Asp+Phe188LeuAla186Ser+Phe188Pro+Ser190AspAla186Thr+Ser187Glu+Phe188ProAla186Pro+Phe188Pro+Ser190GluAla186Thr+Phe188Asn+Ser190AspAla186Pro+Ser187Glu+Phe188HisAla186Asp+Ser187Glu+Phe188MetAla186Asp+Ser187Glu+Phe188GlnAla186Asp+Ser187Glu+Phe188CysAla186Glu+Ser187Asp+Phe188ProAla186Asp+Ser187Glu+Phe188TyrAla186Glu+Ser187Asp+Phe188ThrAla186Asp+Ser187Asp+Phe188ValAla186Glu+Ser187Glu+Phe188AsnAla186Asp+Ser187Asp+Phe188HisAla186Asp+Ser187Glu+Phe188AlaAla186His+Ser187Asp+Phe188AspAla186Gly+Ser187Asp+Phe188AspAla186Gln+Ser187Glu+Phe188GluAla186Asn+Ser187Asp+Phe188GluAla186Pro+Ser187Glu+Phe188AspAla186Asp+Ser187Glu+Phe188AspAla186Glu+Ser187Glu+Phe188GluAla186Asp+Ser187Asp+Phe188GluAla186Thr+Phe188Asp+Ser190GluAla186Ser+Phe188Glu+Ser190GluAla186Thr+Phe188Glu+Ser190AspAla186Pro+Phe188Glu+Ser190GluSer187Glu+Phe188Glu+Ser190GluSer187Asp+Phe188Asp+Ser190Glu表21環(huán)5-四點(diǎn)突變的變異體Ala186Gly+Ser187Glu+Phe188Asp+Ser190GluAla186Gly+Ser187Asp+Phe188Asp+Ser190AspAla186Gln + Ser187Asp + Phe188Asp + Ser190AspAla186Gln + Ser187Glu + Phe188Asp + Ser190GluAla186Ser + Ser187Asp + Phe188Glu + Ser190GluAla186Pro + Ser187Glu + Phe188Asp + Ser190AspAla186Thr + Ser187Glu + Phe188Asp + Ser190AspAla186Thr + Ser187Glu + Phe188Asp + Ser190GluAla186His + Ser187Asp + Phe188Glu + Ser190AspAla186Asn + Ser187Glu + Phe188Asp + Ser190AspAla186Gly + Ser187Glu + Phe188Glu + Ser190AspAla186Gln + Ser187Asp + Phe188Glu + Ser190GluAla186Gln + Ser187Asp + Phe188Glu + Ser190AspAla186Gly + Ser187Glu + Phe188Asp + Ser190AspAla186Asp + Ser187Glu + Phe188Gly + Ser190AspAla186Asp + Ser187Asp + Phe188Gln + Ser190AspAla186Glu + Ser187Glu + Phe188Val + Ser190AspAla186Asp + Ser187Glu + Phe188Pro + Ser190GluAla186Glu + Ser187Asp + Phe188Met + Ser190GluAla186Glu + Ser187Asp + Phe188Ala + Ser190GluAla186Asp + Ser187Asp + Phe188His + Ser190AspAla186Glu + Ser187Glu + Phe188Met + Ser190GluAla186Asp + Ser187Asp + Phe188Ala + Ser190GluAla186Glu + Ser187Glu + Phe188Cys + Ser190AspAla186Glu + Ser187Glu + Phe188Thr + Ser190GluAla186Asp + Ser187Asp + Phe188His + Ser190GluAla186Asp + Ser187Asp + Phe188Gln + Ser190GluAla186Asp + Ser187Asp + Phe188Tyr + Ser190AspAla186Glu + Ser187Asp + Phe188Ser + Ser190AspAla186Glu + Ser187Asp + Phe188Gly + Ser190AspAla186Asp + Ser187Asp + Phe188Asn + Ser190AspAla186Glu + Ser187Glu + Phe188Cys + Ser190GluAla186Glu + Ser187Asp + Phe188Ile + Ser190GluAla186Glu + Ser187Glu + Phe188Val + Ser190GluAla186Asp + Ser187Asp + Phe188Cys + Ser190AspAla186Asp + Ser187Glu + Phe188Val + Ser190GluAla186Asp + Ser187Glu + Phe188Ser + Ser190AspAla186Asp + Ser187Asp + Phe188Ala + Ser190AspAla186Asp + Ser187Asp + Phe188Gly + Ser190GluAla186Asp + Ser187Asp + Phe188Thr + Ser190GluAla186Glu + Ser187Glu + Phe188His + Ser190GluAla186Glu + Ser187Asp + Phe188Pro + Ser190GluAla186Glu + Ser187Glu + Phe188Ile + Ser190GluAla186Glu + Ser187Glu + Phe188Pro + Ser190GluAla186Glu + Ser187Glu + Phe188Gly + Ser190AspAla186Glu + Ser187Glu + Phe188Pro + Ser190AspAla186Glu + Ser187Asp + Phe188Ser + Ser190GluAla186Asp + Ser187Asp + Phe188Tyr + Ser190GluAla186Asp + Ser187Glu + Phe188His + Ser190GluAla186Ser + Ser187Glu + Phe188Ser + Ser190GluAla186His + Ser187Glu + Phe188Ser + Ser190GluAla186Ser + Ser187Asp + Phe188Cys + Ser190GluAla186Ser + Ser187Glu + Phe188Asn + Ser190GluAla186Gln + Ser187Glu + Phe188Ala + Ser190AspAla186Gln + Ser187Asp + Phe188Met + Ser190GluAla186Asn + Ser187Glu + Phe188His + Ser190AspAla186Thr + Ser187Glu + Phe188Ala + Ser190GluAla186Pro + Ser187Asp + Phe188Cys + Ser190GluAla186Pro + Ser187Asp + Phe188Leu + Ser190AspAla186Asn + Ser187Asp + Phe188Gln + Ser190Glu表22環(huán)6-單一突變的變異體Ala199AsnAla199AspAla199GlnAla199GluAla199GlyAla199HisAla199ProAla199SerAla199ThrPro200AsnPro200AspPro200GlnPro200GluPro200GlyPro200SerGly201AsnGly201AspGly201GlnGly201GluGly201ProGly201SerVal202AlaVal202AsnVal202AspVal202CysVal202GlnVal202GluVal202GlyVal202HisVal202MetVal202ProVal202SerVa1202ThrSer203AspSer203GluVal204AlaVa1204AsnVal204AspVal204CysVal204GlnVal204GluVal204GlyVal204HisVal204MetVal204ProVal204SerVal204ThrTyr205AlaTyr205AsnTyr205AspTyr205CysTyr205GlnTyr205GluTyr205GlyTyr205HisTyr205IleTyr205LeuTyr205MetTyr205ProTyr205SerTyr205ThrTyr205ValSer206AspSer206GluThr207AsnThr207AspThr207GlnThr207GluThr207GlyThr207ProThr207SerTyr208AlaTyr208AsnTyr208AspTyr208CysTyr208GlnTyr208GluTyr208GlyTyr208HisTyr208IleTyr208LeuTyr208MetTyr208ProTyr208SerTyr208ThrTyr208ValPro209AsnPro209AspPro209GlnPro209GluPro209GlyPro209SerSer210AspSer210GluAsn211AspAsn211GlnAsn211GluAsn211SerThr212AsnThr212AspThr212GlnThr212GluThr212GlyThr212ProThr212SerTyr213AlaTyr213AsnTyr213AspTyr213CysTyr213GlnTyr213GluTyr213GlyTyr213HisTyr213IleTyr213LeuTyr213MetTyr213ProTyr213SerTyr213ThrTyr213ValThr214AsnThr214AspThr214GlnThr214GluThr214GlyThr214ProThr214SerSer215AspSer215GluLeu216AlaLeu216AsnLeu216AspLeu216CysLeu216GlnLeu216GluLeu216GlyLeu216HisLeu216IleLeu216MetLeu216ProLeu216SerLeu216ThrLeu216ValAsn217AspAsn217GlnAsn217GluAsn217SerGly218AsnGly218AspGly218GlnGly218GluGly218proGly218SerThr219AsnThr219AspThr219GlnThr219GluThr219GlyThr219ProThr219Ser表23環(huán)6-雙點(diǎn)突變的變異體Val204Ser+Ser210GluPro200Gly+Thr214GlyThr212Asn+Thr219GlyThr207Ser+Asn211GluVal204Asn+Tyr208ThrSer215Asp+Asn217GlnPro200Gly+Ser215GluTyr208Gln+Tyr213GluPro200Gln+Leu216GluThr207Gln+Ser215AspTyr205Gly+Thr219GlyTyr208Gly+Asn211GluVal202Cys+Gly218AsnTyr205Ser+Tyr213LeuAla199Asn+Gly218ProThr207Gln+Thr214GluAsn211Gln+Ser215AspAla199Gly+Gly218GluPro200Asn+Thr214GluVal204Thr+Pro209AspPro209Gly+Tyr213CysThr214Gln+Asn217GluPro200Gln+Pro209GluAsn211Asp+Thr219GlySer203Asp+Val204GlnTyr205Val+Gly218GluTyr205Gln+Thr219GluSer203Asp+Leu216AlaSer210Asp+Gly218ProVal202Ala+Val204SerAla199Pro+Val204AlaTyr205Gln+Tyr213GlnTyr205Leu+Pro209AspVal204Ser+Tyr208MetThr212Asp+Tyr213MetVal202Ser+Asn211AspVal202Asp+Tyr213SerSer203Asp + Tyr205SerTyr213His + Thr219GluVal202Pro + Asn217GluVal202Ser + Ser210AspVal202Thr + Ser215AspTyr205Ser + Tyr208ProVal204Gly + Ser210GluThr212Glu + Thr219GlnTyr208Ala + Gly218GluPro200Gly + Tyr213AspThr207Pro + Asn211AspPro209Gly + Tyr213AlaVal204Met + Ser210AspThr214Pro + Asn217SerVal204Gln + Gly218ProTyr205Pro + Thr214GluAla199Gln + Ser215AspAla199Pro + Ser203AspGly201Pro + Thr212SerTyr205Pro + Tyr213HisVal204Gln + Thr207SerThr207Gly + Thr219AsnPro200Gln + Thr214ProVal202Cys + Ser215AspGly201Pro + Thr214SerVal204Ser + Pro209GluSer203Asp + Asn211GlnVal202Met + Thr212ProTyr205Gln + Thr207AsnAla199Gln + Tyr213MetGly201Ser + Ser210AspGly201Ser + Thr212AsnTyr208His + Pro209GlyPro209Gln + Ser215GluTyr208Pro + Leu216ThrTyr213Asp + Thr219GlnPro209Gln + Thr212GlyTyr208Met + Pro209GlyPro209Gly + Asn211AspAsn211Asp + Tyr213SerVal202Gln + Ser210GluSer203Glu + Thr207GlnPro200Gln + Val204ThrAsn211Asp + Thr214SerSer210Asp + Thr212GlnVal204Gln + Thr219GluVal202Gln + Tyr205HisThr214Glu + Gly218ProPro209Asn + Asn211SerAla199Gln + Asn217GluPro200Gly + Asn211SerGly201Pro + Tyr208ValVal202Asn + Tyr208CysVal202Gln + Ser215AspAla199Pro + Thr219SerGly218Pro + Thr219GluAla199Gln + Pro200SerThr207Pro + Asn217AspVal204Ser + Asn217GlnSer203Asp + Val204MetSer203Asp + Thr219ProThr212Pro + Thr219ProTyr205Ser + Ser210GluAla199His + Gly201GlnPro209Gly + Ser210GluTyr208Gln + Leu216AsnGly201Ser + Tyr205LeuVal202Met + Thr214GluTyr205Ser + Pro209AspPro209Ser + Thr212GluTyr205Gly + Asn217AspPro200Ser + Leu216SerAla199His + Tyr208HisAsn211Glu + Thr219AsnAla199Asn + Ser210GluTyr205Val + Tyr208HisThr207Ser + Asn211AspGly201Ser + Thr207SerThr207Asn + Tyr208ValTyr205Glu + Asn217GlnTyr208Pro + Thr212GluVal204Thr + Asn211GluTyr205Cys + Asn217AspLeu216Glu + Thr219SerTyr208Ala + Thr212ProAla199Ser + Ser203GluVal204His + Thr212SerVal204Gln + Gly218SerAla199Asn + Ser203GluPro200Gln + Val202MetAla199Asn + Ser203AspTyr205Pro + Leu216AspTyr205Gln + Ser210AspSer210Asp + Asn211GlnAla199Pro + Asn217AspAla199Gly + Asn211AspTyr205Ile + Leu216MetTyr205Gly + Thr214AsnPro209Glu + Thr219SerPro200Gln + Ser210GluAla199Gly + Pro200AsnTyr205Asp + Tyr213ValSer203Asp + Asn217GlnVal204Met + Thr219SerVal202Thr + Gly218SerPro200Gly + Tyr205CysThr207Gly+Thr219AspSer215Glu+Gly218ProGly201Pro+Tyr205MetTyr205Ser+Asn217AspVal202Ser+Pro209GlnPro200Gly+Gly218AsnTyr208Ser+Pro209AspPro209Gln+Ser215AspAla199Thr+Asn217GluThr214Asn+Leu216AspSer210Asp+Tyr213ValThr207Asn+Gly218AspThr212Pro+Thr219GluTyr213Asn+Leu216GluGly201Pro+Thr212AspGly201Gln+Asn211SerVal202Met+Leu216AlaSer215Asp+Leu216GlyGly201Gln+Leu216CysLeu216Gly+Thr219SerVal204Gln+Thr214GlnThr207Ser+Ser215GluThr207Gly+Asn217GluPro200Asn+Tyr213AlaThr212Asp+Thr219ProTyr205Glu+Leu216IleVal202Asp+Tyr208GlyTyr208Cys+Pro209AspThr207Asn+Tyr208IlePro200Gly+Ser210AspAsn211Glu+Thr212ProAla199His+Leu216SerVal204Thr+Asn211AspGly218Ser+Thr219GlySer203Glu+Val204AlaVal202Thr+Thr219GlyThr212Asp+Asn217Gln表24環(huán)6-三點(diǎn)突變的變異體Thr207Ser+Thr212Asn+Thr219GlyAla199Asn+Thr207Ser+Asn211GluPro200Gln+Leu216Glu+Gly218AsnTyr205Gly+Thr207Gln+Ser215AspTyr208Gly+Asn211Glu+Thr219GlyVal204Thr+Pro209Asp+Tyr213CysVal204Asn+Thr212Gly+Asn217GluPro200Ser+Tyr208Ser+Thr214GlyAla199Ser+Gly201Ser+Thr214SerAla199Gly+Val202Met+Ser210GluGly201Asn+Tyr205His+Thr214GlnAla199Thr + Asn211Asp + Gly218SerThr207Gln + Tyr213Gln + Leu216MetAla199Gly + Val202Cys + Ser210AspVal204Gln + Tyr213Ala + Thr214ProTyr213Leu + Asn217Ser + Thr219GluPro200Gly + Thr214Pro + Asn217AspPro200Gly + Leu216Val + Thr219GlnPro200Gly + Gly201Ser + Ser210AspGly201Gln + Val202His + Thr212GluTyr208His + Asn211Ser + Asn217AspThr207Ser + Ser210Asp + Gly218SerThr212Asn + Leu216Asn + Thr219GluVal202Pro + Val204Met + Pro209AspVal202Asn + Thr212Pro + Thr214GluTyr208Ile + Thr212Asp + Tyr213HisSer210Asp + Thr212Ser + Leu216AlaPro200Gln + Thr214Pro + Leu216HisTyr208Met + Pro209Asp + Leu216IleVal202Met + Leu216Gly + Thr219GlyPro200Ser + Tyr205Val + Leu216CysVal204Thr + Tyr205Glu + Asn217GlnThr212Glu + Leu216Ile + Asn217SerVal204Thr + Tyr208Pro + Asn211GluThr207Gly + Leu216Glu + Thr219SerSer203Glu + Tyr208Ala + Thr212ProAla199Ser + Val204His + Thr212SerAla199Asn + Val204Gln + Gly218SerPro200Gln + Thr214Pro + Gly218AspAla199Asn + Val202Met + Ser203AspPro200Gln + Ser210Asp + Asn211GlnTyr205Ile + Thr214Asn + Leu216MetPro200Gln + Pro209Glu + Thr219SerAla199Gly + Pro200Asn + Ser210GluVal204Met + Asn217Gln + Thr219SerVal202Thr + Thr214Asp + Gly218SerGly201Pro + Tyr205Met + Gly218ProVal202Ser + Tyr205Ser + Asn217AspPro200Gly + Pro209Gln + Gly218AsnAla199Thr + Thr214Asn + Asn217GluThr207Asn + Thr212Pro + Gly218AspGly201Pro + Asn211Ser + Thr212AspGly201Gln + Ser215Asp + Leu216GlyVal204Gln + Leu216Cys + Thr219SerThr207Ser + Thr214Gln + Ser215GluPro200Asn + Thr207Gly + Asn217GluThr212Asp + Tyr213Ala + Thr219ProThr207Asn + Tyr208Ile + Pro209GluAla199His + Asn211Glu + Thr212ProVal204Thr + Asn211Asp + Leu216SerSer203Glu + Val204Ala + Thr219GlyThr207Gly + Thr212Gly + Leu216HisGly201Asn + Thr207Gly + Tyr208GlyGly201Asn + Val202Glu + Thr207SerThr207Gln + Leu216Glu + Gly218SerTyr205His + Ser210Asp + Thr219ProPro200Gly + Tyr208Val + Ser210AspAla199Ser + Ser203Asp + Tyr208GlnGly201Ser + Asn211Glu + Tyr213ProAla199Gln + Tyr205His + Thr219GluPro200Ser + Thr207Pro + Thr214SerPro200Gln + Tyr205Met + Thr212ProAla199Gly + Val204His + Asn211GluThr207Asn + Thr212Ser + Thr219AsnTyr205Pro + Thr207Gln + Ser215GluVal204His + Gly218Glu + Thr219GlyTyr205Pro + Tyr208Ile + Ser215GluAla199Asn + Pro200Ser + Ser203GluTyr208Gln + Tyr213Met + Thr219GluVal202Glu + Val204Thr + Asn217SerVal204Gln + Pro209Gln + Thr214GlyThr214Gln + Ser215Asp + Leu216GlyVal204Thr + Tyr213Ile + Ser215GluVal204Gly + Asn211Gln + Ser215GluPro200Ser + Val204Gly + Tyr213IlePro200Gly + Thr212Asp + Leu216HisAsn211Glu + Tyr213Thr + Leu216ProLeu216Asn + Asn217Asp + Gly218SerGly201Gln + Pro209Asn + Ser215GluGly201Asn + Ser203Glu + Pro209AsnGly201Asn + Val202Ser + Leu216GlyAsn211Asp + Tyr213His + Thr219GlnThr207Gln + Asn217Asp + Gly218SerTyr208Gly + Ser210Glu + Thr214GlyThr207Gln + Tyr208Gln + Tyr213AspAla199Asn + Tyr205Ile + Gly218ProPro200Asn + Val202Gln + Val204SerVal204Asn + Ser215Glu + Leu216ProPro200Ser + Gly201Ser + Tyr208AlaTyr208Thr + Tyr213Val + Leu216AspThr207Gly + Thr212Ser + Tyr213AlaGly201Asn + Gly218Asp + Thr219AsnAla199Gly + Tyr205Cys + Tyr208ThrGly201Gln + Tyr213Cys + Thr214GlyAla199Gln + Pro200Ser + Thr214AspAla199Ser + Gly201Asn + Thr219ProVal204Gly + Leu216Val + Thr219GlnGly201Asn + Ser203Glu + Thr212GlyGly201Ser + Ser203Glu + Asn217GlnPro200Gly + Thr207Gln + Asn217AspAla199Thr + Tyr208Gln + Thr219ProThr207Ser + Thr212Ser + Gly218AspPro200Ser + Tyr208Ile + Asn217SerVal204Pro + Tyr205Asp + Thr214ProGly201Ser + Tyr205Glu + Thr207AsnPro200Ser + Thr207Gln + Asn217GlnGly201Asn + Val202Glu + Leu216CysAla199Ser + Pro209Ser + Gly218AspGly201Pro + Tyr205Met + Asn211AspGly201Ser + Thr214Pro + Ser215AspVal204Pro + Thr212Gly + Tyr213ValGly201Pro + Tyr208Ser + Thr212GluAla199Gln + Ser203Asp + Leu216SerVal202Gly + Asn211Glu + Tyr213LeuSer203Asp + Thr212Gly + Thr214GlnGly201Pro + Tyr205Gln + Thr207GlyGly201Asn + Tyr208Asn + Tyr213ProPro200Ser + Tyr213Asp + Gly218ProAla199Asn + Pro200Gly + Thr214SerSer210Glu + Tyr213Gln + Gly218AsnAla199Pro + Tyr213Thr + Ser215AspVal202Pro + Tyr208Cys + Asn211GlnAla199Gln + Ser203Glu + Asn211SerThr207Gln + Pro209Ser + Ser210GluPro200Ser + Val202Asn + Gly218SerTyr205Val + Tyr213Asn + Gly218GluVal202Glu + Val204His + Tyr205GlnAla199Gly + Thr207Pro + Asn211GluVal202Glu + Tyr208Met + Pro209GlyVal204Ser + Tyr208Ile + Leu216MetTyr205Thr + Tyr208Ser + Asn217SerThr207Ser + Pro209Ser + Leu216AsnGly201Asn + Pro209Asp + Tyr213MetGly201Asn + Pro209Gln + Thr214AsnPro200Asn + Val202Gln + Thr207GlnAla199Asn + Ser203Glu + Val204GlnSer203Asp + Pro209Gly + Leu216AlaAla199Thr + Pro200Asn + Ser210GluAla199Thr + Tyr205Leu + Asn217GluGly201Asn + Val202Met + Thr214AspThr212Gln + Thr214Glu + Thr219SerTyr205Glu + Tyr208Gly + Tyr213LeuPro200Ser + Asn217Glu + Gly218GlnSer210Glu + Thr214Pro + Leu216AsnTyr208Asn + Pro209Asn + Tyr213SerAla199Pro + Gly201Pro + Ser210AspGly201Gln + Thr207Gly + Gly218GluPro200Gln + Tyr208Gly + Ser215AspPro200Ser + Thr214Pro + Leu216GluAla199Gln + Asn217Asp + Gly218ProPro200Asn + Val202Asp + Pro209SerThr212Gly + Tyr213Asn + Ser215AspGly201Gln + Tyr205Met + Thr219GluPro200Gly + Tyr213Gly + Thr219GluPro200Gly + Ser203Glu + Thr214GlnTyr208Thr + Ser215Glu + Gly218SerSer210Asp + Asn211Ser + Leu216AsnAsn211Gln + Thr214Glu + Ser215AspPro209Glu + Ser210Asp + Leu216CysTyr208Pro + Asn217Asp + Gly218AspTyr213Cys+Ser215Asp+Leu216AspThr212Asp+Tyr213Asp+Gly218SerSer210Asp+Asn211Glu+Tyr213GlnVal204Cys+Ser210Glu+Asn211GluTyr205Glu+Tyr208Ala+Ser215GluVal202Glu+Ser203Glu+Asn217AspPro200Gln+Ser203Glu+Ser215AspAlal99Asn+Ser203Glu+Asn217GluSer203Asp+Tyr208Cys+Asn217GluTyr205Asp+Asn211Gln+Thr214Glu表25環(huán)6-四點(diǎn)突變的變異體Gly201Asn+Tyr205Gly+Thr207Gln+Tyr213IleVal202Cys+Tyr205Ser+Thr212Pro+Gly218AsnAlal99Ser+Tyr213His+Thr214Gln+Leu216AsnAlal99Ser+Pro200Ser+Tyr208Ser+Thr214SerAlal99Gly+Gly201Ser+Val202Met+Ser210GluGly201Asn+Tyr205His+Thr207Gln+Thr214GlnVal202Cys+Thr207Gln+Tyr213Gln+Leu216MetVal204Gln+Tyr20SAsp+Tyr213Ala+Thr214ProPro200Gly+Tyr213Leu+Thr214Pro+Asn217SerPro200Gly+Leu216Val+Asn217Asp+Thr219GlnAlal99Thr+Val204Ala+Asn217Gln+Gly218GlnGly201Gln+Val204Gln+Thr207Gln+Leu216Hispro200Asn+Val202Pro+Thr207Gly+Ser215AspAlal99Ser+Gly201Pro+Ser215Asp+Leu216SerAlal99Thr+Tyr205Gly+Ser215Glu+Asn217SerAlal99Gly+Thr207Gln+Tyr208Thr+Pro209GlnAla199His+Pro200Gly+Gly218Gln+Thr219GlnAlal99Gly+Thr207Asn+Asn211Ser+Thr212SerPro200Ser+Val202Gln+Tyr208Ile+Thr219ProPro200Gln+Tyr208Met+Pro209Asp+Leu216HisSer203Asp+Asn211Gln+Leu216Thr+Gly218GlnAla199Gly+Gly201Ser+Tyr205Asp+Leu216GlyAla199Gln+Gly201Asn+Tyr205Gln+Thr214AspPro200Ser+Gly201Pro+Val204Asn+Tyr208ProGly201Pro+Val202Gly+Ser210Asp+Leu216MetPro200Gly+Val202Cys+Ser203Asp+Leu216ProTyr205Gly+Thr207Pro+Asn211Gln+Asn217SerGly201Pro+Thr207Gln+Thr212Ser+Asn217AspPro200Gln+Tyr208Leu+Tyr213Met+Leu216GlnTyr205Met+Asn211Gln+Tyr213Met+Thr219AspVal202Met+Tyr205Leu+Tyr208Thr+Tyr213GlySer203Asp+Val204Ser+Tyr213Gly+Gly218ProGly201Gln+Val202Met+Ser215Asp+Leu216AlaVal204Gln+Thr214Gln+Leu216Cys+Thr219SerPro200Asn+Thr212Asp+Tyr213Ala+Thr219ProAlal99His+Val204Thr+Asn211Asp+Leu216SerVal202Thr+Ser203Glu+Val204Ala+Thr219AsnThr207Gly+Tyr208Gly+Thr212Gly+Leu216HisGly201Asn + Ser203Glu + Thr207Gly + Asn211GlnTyr205His + Thr207Gln + Leu216Gly + Gly218SerAla199Gln + Tyr205His + Tyr213Pro + Thr219GluAla199Gly + Tyr205Met + Asn211Glu + Thr212ProGly201Pro + Thr207Asn + Thr212Gln + Thr219AsnPro200Gln + Leu216Thr + Asn217Glu + Thr219ProGly201Ser + Tyr205Cys + Thr214Asn + Leu216ThrGly201Gln + Val204Pro + Asn211Asp + Gly218ProSer203Asp + Tyr205Val + Thr207Ser + Leu216CysVal202His + Pro209Gln + Ser215Asp + Leu216GlyPro200Asn + Gly201Gln + Val202Pro + Ser203GluPro200Ser + Gly201Gln + Val202His + Leu216ThrPro200Asn + Gly201Asn + Leu216Ala + Gly218ProAla199Pro + Val202Ser + Val204His + Thr214GluGly201Gln + Ser203Glu + Tyr213Ile + Gly218AsnAla199His + Ser203Asp + Val204Gly + Asn211GlnPro200Gln + Val202Ala + Val204Pro + Thr207ProGly201Asn + Thr212Ser + Tyr213Ala + Gly218AspGly201Gln + Tyr213Cys + Thr214Gly + Gly218GluAla199Gln + Pro200Ser + Gly201Asn + Thr214AspAla199Ser + Val204Gly + Leu216Val + Thr219ProPro200Gly + Val202His + Thr207Gln + Asn217AspAla199Thr + Tyr208Gln + Thr212Ser + Thr219ProPro200Ser + Thr207Ser + Tyr208Ile + Gly218AspVal204Pro + Tyr205Asp + Thr214Pro + Asn217SerGly201Pro + Tyr205Met + Asn211Asp + Thr214ProGly201Ser + Tyr213Val + Thr214Pro + Ser215AspGly201Pro + Tyr208Ser + Thr212Gly + Tyr213AlaVal202Asn + Asn211Glu + Tyr213Leu + Thr219ProGly201Pro + Tyr205Gln + Thr207Gly + Thr212GlyPro200Gln + Tyr208Cys + Ser215Glu + Thr219GlyGly201Asn + Tyr208Asn + Tyr213Pro + Ser215GluPro200Ser + Gly201Pro + Tyr213Asp + Gly218ProAla199Pro + Pro200Ser + Thr212Asp + Thr219SerPro200Asn + Ser203Asp + Thr207Gln + Thr212GlyVal204Cys + Pro209Ser + Ser215Asp + Gly218GlnVal202Gln + Val204Ser + Thr212Asp + Asn217GlnAla199Asn + Pro209Gly + Asn211Glu + Thr214ProAla199Asn + Val204Thr + Ser210Asp + Tyr213LeuPro200Asn + Val204Met + Thr207Gln + Tyr213AspVal204Gln + Pro209Gly + Thr212Gly + Tyr213CysPro209Asn + Tyr213Ile + Thr214Gly + Leu216MetPro200Gly + Ser203Asp + Leu216Cys + Asn217SerVal204Ser + Tyr208Ile + Asn211Asp + Leu216MetGly201Asn + Tyr205Ile + Pro209Gln + Leu216ValSer203Glu + Val204Gln + Tyr213Leu + Gly218AsnPro200Asn + Ser210Asp + Thr214Ser + Leu216CysAla199Thr + Pro200Asn + Ser210Glu + Leu216CysAla199Thr + Val202Met + Tyr205Leu + Thr214AspGly201Asn + Thr212Gln + Thr214Glu + Thr219SerGly201Pro + Tyr208Asn + Pro209Asn + Tyr213SerAla199Pro + Gly201Gln + Thr207Gly + Ser210AspAla199Gly + Pro200Gln + Tyr208Asn + Leu216ThrPro200Asn + Val202Asp + Pro209Ser + Gly218ProPro200Gly + Thr207Asn + Leu216Asn + Thr219AspAla199Ser + Pro200Gly + Val202Asp + Thr207SerVal204Ala + Thr207Asn + Ser210Glu + Tyr213MetPro200Gln + Gly201Ser + Tyr205Thr + Ser215GluAla199His + Gly201Asn + Tyr208Ala + Ser210AspPro200Gln + Val204Met + Tyr205Ala + Pro209GluVal202Ala + Tyr205Leu + Thr214Glu + Thr219GlnThr207Gln + Pro209Asn + Ser210Glu + Thr214ProGly201Asn + Ser203Asp + Tyr208Val + Asn211GlnAla199Ser + Pro200Gly + Leu216Val + Gly218ProGly201Gln + Asn211Gln + Thr212Glu + Leu216AlaVal202Cys + Thr207Asn + Tyr213Pro + Leu216AspAla199Gly + Val202His + Val204Ala + Ser215GluAla199Gln + Thr212Pro + Tyr213Glu + Leu216AlaPro200Gln + Val202Gln + Thr214Gln + Gly218GluGly201Gln + Thr207Pro + Tyr208Met + Thr219ProSer203Asp + Val204Gln + Tyr208Ser + Leu216ProAla199Asn + Ser203Glu + Val204Asn + Tyr208ProPro200Gly + Tyr213Met + Leu216Val + Gly218AsnTyr205Ile + Tyr213Ile + Thr214Asn + Thr219ProPro200Gln + Thr207Gly + Ser215Glu + Leu216MetAla199Ser + Tyr208Ala + Asn211Gln + Thr219AspGly201Pro + Val204Ser + Tyr205His + Thr219AspVal202Met + Thr207Pro + Thr212Gly + Asn217GluPro200Gln + Gly201Pro + Tyr208Thr + Ser215GluVal204Thr + Thr212Gly + Thr214Glu + Gly218SerPro200Gln + Tyr208Ala + Tyr213Glu + Asn217SerTyr213Met + Thr214Ser + Ser215Asp + Leu216ProThr207Ser + Pro209Asp + Thr212Gly + Thr219GlnThr207Gln + Asn211Gln + Thr214Glu + Ser215AspTyr205Ala + Tyr213Met + Thr214Glu + Ser215AspTyr205Met + Thr214Asp + Ser215Glu + Asn217GlnThr207Asn + Thr214Gly + Leu216Glu + Asn217AspVal202Thr + Val204His + Asn211Glu + Thr212AspPro200Gln + Asn211Asp + Thr212Glu + Thr214SerAsn211Glu + Thr212Glu + Tyr213Ile + Thr214AsnGly201Asn + Val202Glu + Ser203Glu + Thr212GlyThr212Asp + Tyr213Asp + Gly218Ser + Thr219GlyVal204Ala + Thr212Glu + Tyr213Glu + Thr214AsnPro200Gly + Ser210Asp + Asn211Glu + Thr212ProPro209Gln + Ser210Asp + Asn211Glu + Thr219ProPro200Ser + Val204Met + Ser210Asp + Asn211GluAla199Pro + Val202Ala + Ser210Glu + Asn211GluPro209Asp + Ser210Asp + Asn211Glu + Thr212ProVal202Asp + Ser203Asp + Tyr213Gln + Asn217GluSer203Asp + Ser215Asp + Leu216Glu + Thr219AsnPro200Ser + Ser203Glu + Leu216Glu + Asn217GluSer203Asp + Pro209Gly + Ser215Glu + Leu216AlaSer203Glu + Thr214Gln + Ser215Glu + Gly218SerGly201Asn + Ser203Asp + Tyr213Ala + Ser215GluSer203Glu + Thr207Ser + Tyr213Gly + Ser215AspAla199Gln + Ser203Glu + Thr214Pro + Asn217AspSer203Glu+Val204Asn+Thr212Gly+Asn217GluSer203Asp+Thr207Gln+Tyr208Cys+Asn217GluGly201Ser+Ser203Glu+Thr214Pro+Asn217AspAsn211Glu+Thr212Asp+Tyr213Glu+Thr219GlyAla199Pro+Ser203Asp+Val204Pro+Leu216GluAlal99Asn+Pro209Asp+Asn211Glu+Tyr213IlePro200Gln+Tyr205Leu+Pro209Glu+Thr212AspSer203Asp+Tyr205Glu+Tyr208Gly+Tyr213LeuAlal99Ser+Val202Glu+Leu216Cys+Gly218AspPro200Ser+Thr212Pro+Thr214Glu+Leu216GluAlal99Asn+Val202Pro+Ser215Glu+Asn217AspPro200Gly+Thr207Ser+Ser215Glu+Asn217GluGly201Asn+Val202Glu+Thr207Ser+Leu216GluAlal99Asn+Ser203Glu+Tyr205Glu+Asn217GluTyr205Glu+Thr214Pro+Ser215Asp+Asn217GluGly201Gln+Thr212Ser+Tyr213Glu+Ser215GluAlal99Ser+Tyr213Asp+Thr214Pro+Ser215GluAsn211Ser+Ser215Asp+Asn217Glu+Gly218AspSer203Asp+Thr212Gln+Ser215Asp+Gly218AspVal202Glu+Thr212Gly+Ser215Asp+Leu216GlnVal202Asp+Val204Cys+Tyr208Pro+Thr219GluSer203Asp+Pro209Gln+Tyr213Thr+Thr214AspVal202Asp+Tyr205Glu+Tyr208Gly+Leu216IleTyr208Gly+Thr214Asp+Asn217Glu+Gly218AspPro200Gln+Tyr208Gln+Tyr213Glu+Leu216GluGly201Gln+Tyr205Leu+Thr214Glu+Asn217AspSer203Asp+Val204Gly+Thr212Glu+Ser215AspGly201Gln+Val202Pro+Asn2l1Glu+Thr214GluThr207Gln+Pro209Ser+Asn211Asp+Thr214GluTyr205Ala+Ser210Glu+Thr212Asp+Ser215AspAlal99Gln+Thr214Glu+Leu216Glu+Thr219GluThr212Asp+Thr214Glu+Leu216Asn+Asn217GluGly201Asn+Asn211Glu+Thr214Asp+Leu216GluPro209Asp+Ser215Asp+Leu216Asp+Gly218GlnGly201Pro+Val202Cys+Ser203Asp+Thr219AspSer203Glu+Thr212Gln+Gly218Gln+Thr219Glu表26環(huán)6-五點(diǎn)取代的變異體Gly201Asn+Thr207Gln+Ser210Glu+Tyr213Ile+Leu216IleVal202Cys+Tyr205Ser+Thr212Pro+Tyr213Leu+Gly218AsnAlal99Ser+Pro200Ser+Tyr213His+Thr214Gln+Leu216AsnAla199Ser+Gly201Ser+Tyr208Ser+Ser210Glu+Thr214SerAla199Gly+Gly201Asn+Val202Met+Tyr205His+Thr214GlnPro200Gly+Tyr213Leu+Thr214Pro+Asn217Ser+Thr219GluAla199Thr + Tyr205Gln + Thr207Gln + Thr212Asp +Gly218GlnVal204Gly + Tyr205Ala + Tyr208Gly + Asn211Gln +Gly218AspAla199Ser + Gly201Pro + Tyr205Gly + Ser215Glu +Leu216SerAla199His + Thr207Gln + Tyr208Thr + Gly218Gln +Thr219GlnAla199Gly + Pro209Gly + Asn211Ser + Thr212Ser +Tyr213SerPro200Ser + Val202Gln + Thr207Asn + Tyr208Ile +Thr219ProPro200Ser + Gly201Ser + Tyr208Gln + Ser210Glu +Asn217GlnAla199Gly + Gly201Ser + Val202Gly + Tyr205Asp +Leu216GlyAla199Gln + Pro200Ser + Gly201Ser + Tyr205Gln +Thr214AspAla199Pro + Gly201Pro + Val204Asn + Tyr208Pro +Thr214GlyPro200Gly + Val202Gly + Ser203Asp + Asn211Ser +Leu216ProTyr205Gly + Thr207Pro + Pro209Asp + Asn211Gln +Asn217SerPro200Gln + Val204Ala + Tyr208Leu + Thr212Glu +Tyr213MetTyr205Met + Asn211Gln + Tyr213Met + Thr214Asn +Thr219AspSer203Asp + Val204Ser + Tyr213Gly + Thr214Asn +Gly218ProGly201Gln + Val204Gln + Ser215Asp + Leu216Ala +Thr219SerGly201Asn + Thr207Gly + Tyr208Gly + Thr212Gly +Leu216HisGly201Asn + Tyr205His + Thr207Ser + Leu216Glu +Gly218SerAla199Gln + Pro200Ser + Tyr205His + Thr214Ser +Thr219GluPro200Gln + Gly201Gln + Leu216His + Asn217Asp +Gly218GlnAla199Gly + Val204His + Tyr205Met + Asn211Glu +Thr212ProVal204His + Tyr205Pro + Tyr208Ile + Gly218Glu +Thr219GlyPro200Ser + Val202Gln + Val204Gly + Tyr213Ile +Ser215GluAla199Ser + Pro200Asn + Asn211Glu + Tyr213Thr +Leu216ProSer203Asp + Val204Gly + Asn211Gln + Tyr213Ile +Gly218AsnAla199Asn + Pro200Gln + Val202Ala + Val204Pro +Thr207ProAla199Thr + Gly201Gln + Val202Gly + Ser215Asp +
Leu216AlaThr212Gln + Tyr213Val + Leu216His + Gly218Gln +Thr219GluAla199Gln + Pro200Ser + Gly201Asn + Tyr205Ala +Thr214GlyGly201Asn + Val204Gly + Thr212Gly + Leu216Val +Thr219ProPro200Gly + Val202His + Thr207Gln + Tyr208Gln +Asn217AspAla199Thr + Thr207Ser + Thr212Ser + Gly218Asp +Thr219ProPro200Ser + Val204Pro + Tyr208Ile + Thr214Pro +Asn217SerPro200Gln + Val202Ser + Tyr205Ser + Asn211Ser +Ser215AspVal202Thr + Thr212Gln + Ser215Asp + Asn217Ser +Thr219AsnVal204Pro + Thr212Gly + Tyr213Val + Thr214Pro +Ser215AspGly201Ser + Val204His + Tyr208Leu + Thr212Ser +Gly218GluSer203Asp + Tyr205Gln + Thr207Gly + Thr212Gly +Thr214GlnPro200Gln + Tyr208Cys + Thr212Gly + Ser215Glu +Thr219GlyPro200Ser + Gly201Pro + Val202Ala + Tyr213Asp +Gly218ProVal204Gly + Ser210Asp + Thr212Asn + Thr214Pro +Thr219SerPro200Gly + Ser203Asp + Pro209Asn + Leu216Cys +Asn217SerTyr205Thr + Thr207Ser + Tyr208Ser + Pro209Ser +Asn217SerGly201Asn + Tyr205Ile + Pro209Gln + Thr214Asn +Leu216ValAla199Asn + Ser203Glu + Val204Gln + Tyr213Leu +Gly218AsnAla199Thr + Val202Met + Tyr205Leu + Asn211Ser +Asn217GluAla199Pro + Gly201Pro + Pro209Asn + Ser210Asp +Tyr213SerAla199Ser + Gly201Pro + Pro209Gly + Tyr213Pro +Gly218ProPro200Ser + Pro209Gly + Thr212Pro + Thr214Pro +Leu216GluGly201Ser + Thr207Gly + Ser210Asp + Asn211Ser +Leu216IleAla199Ser + Gly201Gln + Val204Asn + Tyr205Leu +Thr219AspThr207Pro + Tyr208Thr + Ser210Glu + Thr212Gly +Asn217SerGly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp +Thr212GlyPro200Asn + Tyr205Glu + Tyr208Met + Tyr213Val +Leu216IleTyr205Ala + Tyr213His + Thr214Ser + Asn217Gln +Thr219GlnAla199Gln + Pro200Gly + Val202Met + Leu216Met +Gly218ProPro200Ser + Val204Ala + Ser210Glu + Asn211Gln +Gly218SerAla199Ser + Pro200Ser + Val202Asn + Leu216Val +Gly218ProPro200Gln + Val202Gln + Tyr213Leu + Thr214Pro +Gly218GluPro200Ser + Gly201Gln + Thr207Pro + Gly218Pro +Thr219ProAla199Asn + Ser203Asp + Val204Asn + Tyr208Pro +Leu216ProTyr205Ile + Tyr213Ile + Thr214Asn + Ser215Glu +Thr219ProGly201Ser + Val204His + Asn211Gln + Thr212Gln +Leu216MetAla199Ser + Gly201Pro + Asn211Glu + Gly218Gln +Thr219SerAla199Ser + Thr207Ser + Thr214Gln + Ser215Glu +Leu216AsnAla199His + Val204Cys + Asn211Ser + Thr214Gly +Thr219GluAla199Asn + Val202His + Tyr208Asn + Leu216Thr +Thr219GlySer203Glu + Thr207Pro + Tyr208Leu + Thr212Ser +Asn217GlnPro200Ser + Val202Asp + Val204Asn + Tyr208Met +Thr212GlnGly201Ser + Thr207Asn + Tyr208Leu + Ser210Asp +Asn217SerVal202Asp + Val204Gln + Tyr208Cys + Pro209Gln +Tyr213ValAla199Gly + Val202Thr + Thr207Gln + Thr212Glu +Thr214GlnGly201Pro + Val202Cys + Ser210Asp + Thr212Pro +Tyr213CysAla199Asn + Gly201Ser + Val204Met + Pro209Asn +Thr214ProGly201Asn + Ser203Asp + Val204Thr + Thr207Ser +Leu216AlaVal202Thr + Tyr205Gly + Thr207Gln + Asn211Asp +Leu216AlaAla199Gln + Va1204Gly + Thr207Pro + Asn211Asp +Thr214GlyGly201Pro + Val202Asn + Tyr205Ser + Asn211Glu +Gly218AsnVal204Gly + Pro209Asn + Asn211Asp + Asn217Gln +Thr219GlyVal202Gly + Val204Gly + Ser210Glu + Gly218Ser +
Thr219SerAla199Ser + Asn211Ser + Tyr213Met + Thr214Asp +Thr219ProAla199Asn + Gly201Asn + Ser203Glu + Tyr213His +Leu216IleAla199Gln + Pro200Asn + Thr207Ser + Pro209Ser +Asn217SerTyr205Met + Thr214Asp + Ser215Glu + Leu216Cys +Asn217GlnAla199Pro + Val202His + Tyr208Met + Thr214Glu +Ser215AspPro200Asn + Pro209Glu + Ser210Asp + Thr214Ser +Leu216CysVal204Ser + Tyr208Pro + Leu216Met + Asn217Asp +Gly218AspTyr205Pro + Thr207Pro + Leu216Asn + Asn217Asp +Gly218AspPro200Gly + Tyr205Asn + Tyr208Gly + Asn217Glu +Gly218GluVal202Thr + Val204His + Pro209Gly + Asn211Glu +Thr212AspGly201Gln + Val202Asn + Thr207Asn + Ser215Glu +Leu216AspAla199His + Pro200Gly + Ser210Asp + Asn211Glu +Thr212ProPro209Gln + Ser210Asp + Asn211Glu + Asn217Gln +Thr219ProAla199Pro + Val202Ala + Ser210Glu + Asn211Asp +Gly218AsnAla199Asn + Thr207Asn + Ser210Asp + Asn211Asp +Thr212GlyVal204Ala + Tyr205Glu + Thr212Ser + Ser215Glu +Asn217GlnGly201Pro + Val202Asp + Thr207Gln + Thr212Ser +Asn217AspThr207Ser + Pro209Asp + Ser210Glu + Asn211Asp +Tyr213AsnGly201Ser + Val202Glu + Ser203Glu + Thr214Pro +Asn217AspSer203Glu + Thr207Asn + Thr214Gly + Leu216Glu +Asn217AspGly201Ser + Ser203Asp + Tyr208Ile + Leu216Asp +Asn217AspGly201Pro + Tyr208Gly + Leu216Asp + Asn217Glu +Gly218AspSer203Glu + Val204Pro + Thr207Gly + Tyr213Leu +Ser215AspGly201Asn + Ser203Asp + Tyr208Val + Asn211Gln +Ser215AspAla199Asn + Pro200Ser + Ser203Glu + Tyr213Met +Ser215GluAla199Ser + Ser203Glu + Asn211Gln + Ser215Glu +Leu216CysSer203Glu + Val204Gln + Thr214Gln + Ser215Asp +Leu216GlyAla199Pro + Gly201Pro + Ser203Asp + Tyr205Val +Ser215GluAla199Thr + Ser203Asp + Val204Gln + Tyr213Gly +Ser215AspVal202Cys + Ser203Asp + Tyr205Met + Tyr213Gln +Asn217GluSer203Glu + Val204Asn + Thr212Gly + Asn217Glu +Thr219GlnGly201Asn + Ser203Asp + Tyr208Val + Pro209Ser +Asn217GluSer203Asp + Thr212Pro + Tyr213Gly + Asn217Glu +Gly218SerVal204Thr + Asn211Asp + Thr212Asp + Tyr213Asp +Leu216SerVal202Ala + Ser203Asp + Val204Gln + Tyr205Met +Leu216GluSer203Glu + Val204Ser + Thr207Gly + Tyr208Val +Leu216AspPro200Gln + Pro209Gln + Ser210Glu + Thr212Glu +Thr214GlyPro209Glu + Asn211Asp + Asn217Gln + Gly218Gln +Thr219ProPro200Ser + Tyr205Pro + Pro209Asp + Asn211Glu +Tyr213IleAla199Asn + Ser203Asp + Tyr205Glu + Thr207Asn +Leu216GluSer203Glu + Tyr205Glu + Tyr213Met + Leu216Ile +Asn217GlnVal202Glu + Ser203Glu + Asn211Gln + Leu216Met +Gly218GluAla199Ser + Val202Ser + Tyr205Asp + Tyr208Gly +Tyr213AspPro200Ser + Thr207Pro + Thr212Asp + Thr214Asp +Thr219SerPro200Gly + Thr207Ser + Thr214Gln + Ser215Glu +Asn217GluPro200Gln + Thr207Gly + Ser215Asp + Leu216Gln +Asn217AspAla199Asn + Tyr205Glu + Tyr208Ala + Ser215Glu +Asn217GluGly201Pro + Val204Ser + Tyr205His + Asn217Glu +Thr219AspAla199Ser + Tyr208Gln + Tyr213Asp + Thr214Pro +Ser215GluThr207Asn + Tyr208Gln + Thr212Ser + Tyr213Glu +Ser215GluGly201Gln + Tyr205Gly + Pro209Glu + Thr212Pro +Tyr213GluSer203Asp + Pro209Ser + Thr212Gln + Ser215Asp +Gly218AspSer203Asp + Pro209Gly + Ser215Glu + Leu216Ala +
Gly218AspPro200Ser + Ser203Asp + Thr207Gln + Leu216Gln +Gly218GluPro200Asn + Gly201Pro + Ser203Glu + Thr207Gly +Gly218GluVal202Asp + Val204Cys + Tyr208Ala + Gly218Gln +Thr219GluVal202Glu + Val204Thr + Tyr208Gln + Asn217Ser +Thr219GluPro200Ser + Gly201Pro + Val202Asp + Asn211Gln +Thr219GluGly201Asn + Ser203Glu + Thr212Gln + Thr214Asp +Thr219SerGly201Asn + Ser203Asp + Thr214Glu + Leu216Cys +Thr219GlyGly201Asn + Val202Cys + Ser203Asp + Thr214Asp +Thr219ProVal202Cys + Ser203Asp + Tyr208Thr + Thr214Asp +Thr219ProVal204Ser + Tyr208Leu + Ser210Asp + Tyr213Asp +Thr214AspAla199His + Ser203Asp + Tyr208Ser + Tyr213Glu +Thr214AspVal204Cys + Ser210Asp + Asn211Ser + Tyr213Asp +Thr219GlnAla199Asn + Ser210Glu + Thr212Asn + Tyr213Glu +Thr214GlnSer203Asp + Tyr205Glu + Tyr208Gly + Tyr213Leu +Gly218AspVal202Glu + Tyr205Glu + Pro209Asn + Leu216Thr +Gly218GluTyr208Val + Ser215Asp + Asn217Ser + Gly218Asp +Thr219GluTyr205Leu + Thr214Asp + Ser215Glu + Gly218Glu +Thr219AsnAla199Pro + Pro209Asp + Thr212Asp + Ser215Asp +Gly218SerSer203Glu + Thr212Glu + Tyr213Ser + Thr214Glu +Leu216CysSer203Asp + Tyr208Cys + Thr212Asp + Tyr213Ile +Thr214AspVal202Met + Val204Thr + Asn211Asp + Thr214Asp +Ser215AspPro209Asn + Asn211Asp + Thr214Glu + Ser215Glu +Leu216GlyPro200Asn + Tyr205Glu + Thr212Asp + Tyr213Ala +Thr219ProPro200Asn + Ser203Asp + Tyr205His + Thr212Asp +Ser215AspSer203Asp + Val204Thr + Thr212Glu + Tyr213Ile +Ser215GluTyr205Gly + Thr207Gln + Tyr208Ile + Thr212Glu +Ser215GluAla199Asn + Tyr205Asp + Ser210Glu + Thr212Glu +Thr214GlyVal202Met- Tyr205Ala + Ser210Glu + Thr212Asp +Ser215AspThr207Gln + Tyr208Leu + Asn211Glu - Tyr213Glu +Leu216GluVal204Met + Pro209Ser + Asn211Asp + Thr214Glu +Leu216AspPro200Ser - Ser203Glu + Tyr213Glu + Leu216Gly +Asn217GluAla199Gly + Tyr213Pro + Thr214Asp + Asn217Asp +Thr219AspAla199Gln + Tyr208Ala + Thr214Glu + Asn217Glu +Thr219AspAla199His + Val202His + Ser203Glu + Thr207Gly +Thr219AspGly201Ser + Val202Gly + Ser203Glu + Tyr205Thr +Thr219AspGly201Pro + Pro209Glu + Ser210Asp + Ser215Glu +Leu216MetTyr205Glu + Ser215Glu + Leu216Cys + Gly218Gln +Thr219GluSer203Asp + Pro209Ser + Asn211Glu + Thr214Asp +Leu216AsnAla199Thr + Ser203Glu + Thr207Gln + Asn211Asp +Thr214GluGly201Ser + Ser203Asp + Tyr205Gly + Pro209Asp +Ser215AspPro209Glu + Thr214Asn + Leu216Glu + Asn217Asp +Thr219Asn表27環(huán)6-六點(diǎn)取代的變異體Gly201Asn + Tyr205Gly + Thr207Gln + Ser210Glu +Tyr213Ile + Thr219GlyPro200Asn + Gly201Ser + Tyr208Gly + Asn211Asp +Thr212Ser + Thr219AsnGly201Pro + Ser203Asp + Val204Gln + Tyr205Pro +Thr212Ser + Tyr213HisPro200Gln + Val204His + Thr207Ser + Thr214Pro +Ser215Asp + Thr219AsnAla199Pro + Pro200Ser + Gly201Pro + Val204Asn +Tyr208Pro + Thr214GlyPro200Gln + Val204Ala + Tyr208Leu + Thr212Glu +Tyr213Met + Leu216GlnAla199Pro + Val202Met + Ser203Glu + Tyr205Leu +Asn211Gln + Tyr213GlyGly201Gln + Val204Gln + Thr214Gln + Ser215Asp +Leu216Gly + Thr219SerVal204Thr + Tyr208Met + Pro209Glu + Tyr213Gly +Thr214Pro + Gly218SerSer203Asp + Val204Met + Thr207Gln + Pro209Gln +Asn217Ser + Thr219AsnPro200Ser + Val202Met + Val204Gly + Pro209Gln +Tyr213Ile + Ser215GluAla199His + Ser203Asp + Val204Gly + Thr207Gly +Asn211Gln + Thr214SerAla199Gln + Pro200Gln + Gly201Gln + Pro209Glu +Leu216Ile + Asn217SerPro200Gln + Tyr208Ile + Pro209Gln + Thr212Gly +Ser215Glu + Thr219GlyPro200Asn + Ser203Asp + Val204Cys + Thr207Gln +Pro209Ser + Thr214AsnAla199Asn + Val202Gln + Val204Ser + Pro209Gly +Thr212Asp + Asn217GlnPro200Asn + Val204Met + Thr207Gln + Pro209Gly +Thr212Gly + Tyr213AspPro200Gly + Val204Gln + Pro209Asn + Tyr213Ile +Thr214Gly + Leu216MetAla199Asn + Tyr205Thr + Tyr208Ser + Pro209Ser +Thr214Asp + Asn217SerTyr205Ser + Thr207Gln + Tyr208Cys + Pro209Asn +Thr212Asn + Leu216GluAla199Asn + Pro200Ser + Ser203Asp + Tyr205Cys +Tyr208Asn + Thr214GlnThr207Pro + Tyr213Ser + Thr214Asn + Asn217Ser +Gly218Ser + Thr219GluAla199Pro + Gly201Pro + Thr207Gly + Pro209Asn +Ser210Asp + Tyr213SerAla199Gln + Pro200Gly + Val202Met + Thr212Asp +Leu216Met + Gly218ProAla199Ser + Pro200Gly + Val202Asn + Asn211Gln +Leu216Val + Gly218ProAla199His + Pro200Gln + Val202Gln + Thr214Gln +Leu216Thr + Gly218GluPro200Ser + Gly201Gln + Thr207Pro + Tyr208Thr +Gly218Pro + Thr219ProPro200Gln + Val204Met + Thr207Gly + Ser215Glu +Leu216Met + Thr219ProThr207Ser + Pro209Asp + Thr212Gly + Thr214Ser +Leu216Pro + Thr219GlnPro200Asn + Gly201Ser + Thr207Asn + Tyr208Leu +Ser210Asp + Asn217SerAla199Asn + Val202Ser + Tyr205Cys + Thr207Gln +Tyr213Leu + Asn217SerVal202Ser + Pro209Gln + Tyr213Asp + Leu216Met +Asn217Ser + Thr219GlyGly201Gln + Val204Gly + Pro209Asn + Asn211Asp +Asn217Gln + Thr219GlyVal202Gly + Ser203Glu + Val204Gly + Tyr213Met +Gly218Ser + Thr219SerAla199Gln + Pro200Asn + Tyr205Thr + Thr207Ser +Thr214Gln + Asn217SerAla199Ser + Gly201Asn + Val202Met + Val204Asn +Asn211Gln + Gly218GluPro200Asn + Val202Pro + Val204Gln + Tyr208Pro +Gly218Gln + Thr219GluThr207Gly + Tyr208Ile + Pro209Gly + Tyr213Gly +Thr214Pro + Leu216AsnVal202Met + Ser203Asp + Val204Met + Thr212Asn +Thr214Asn + Leu216GlyAla199Ser + Val202Met + Val204Ala + Thr212Gly +Tyr213Thr + Thr214ProTyr208Val + Pro209Gln + Thr214Ser + Leu216Cys +Asn217Glu + Thr219AsnPro200Gln + Gly201Gln + Tyr205Ala + Pro209Gln +Leu216Pro + Thr219GluAla199Gly + Pro200Gln + Gly201Ser + Tyr208Gln +Ser215Asp + Gly218AsnAla199Asn + Pro200Asn + Val202Asn + Val204Gln +Thr214Gly + Asn217GluPro200Gly + Gly201Gln + Tyr213Gly + Thr214Asn +Leu216Gln + Thr219SerGly201Gln + Val202Gly + Thr207Gly + Asn211Gln +Thr214Gly + Ser215AspAla199Thr + Pro200Ser + Tyr208Ala + Thr212Ser +Ser215Asp + Asn217SerAla199His + Val202Asn + Ser203Glu + Val204His +Tyr213Ile + Asn217GlnGly201Asn + Val202Cys + Tyr205Cys + Tyr213Ile +Leu216Pro + Asn217AspAla199Thr + Gly201Ser + Val202Gly + Tyr205Pro +Leu216Thr + Gly218GluAla199Ser + Val204Cys + Tyr208Cys + Pro209Gly +Tyr213Ala + Leu216AspAla199Gly + Gly201Asn + Val204Met + Thr207Pro +Thr214Glu + Leu216MetPro200Gly + Tyr205Met + Tyr208Val + Thr212Gly +Tyr213Ser + Leu216ValGly201Pro + Tyr208Val + Pro209Glu + Asn211Gln +Tyr213Ala + Thr219GlyAla199Ser + Pro200Ser + Val202Thr + Tyr205Gln +Gly218Asp + Thr219ProAla199Gln + Ser203Glu + Val204Pro + Thr214Ser +Gly218Gln + Thr219ProAla199Thr + Pro200Gly + Gly201Gln + Thr212Ser +Leu216Val + Gly218SerPro200Ser + Val204Gln + Thr207Pro + Thr214Gly +Ser215Glu + Gly218GlnAla199Gly + Gly201Gln + Val204His + Tyr205Asp +Pro209Gln + Thr212SerPro200Gln + Tyr208Asn + Asn211Ser + Thr212Ser +Tyr213Thr + Leu216AspAla199His + Tyr208Leu + Thr212Pro + Ser215Glu +Leu216Ser + Gly218GlnGly201Pro + Val204Cys + Pro209Gln + Thr212Ser +Tyr213Cys + Thr214GlnVel204Ser + Tyr205Ser + Thr207Asn + Tyr208Cys +Ser215Asp + Asn217SerPro200Asn + Tyr208Als + Tyr213Pro + Thr214Glu +Ser215Asp + Thr219GlyVal202His + Tyr208Met + Pro209Asn + Thr212Asn +Thr214Glu + Ser215AspPro200Gln + Gly201Ser + Val202Pro + Val204Ser +Thr214Asp + Ser215GluAla199Gln + Gly201Ser + Val204Gly + Thr207Ser +Thr214Asp + Ser215GluVal202Asn + Tyr205Gly + Pro209Gln + Thr212Ser +Thr214Glu + Ser215GluPro200Asn + Pro209Glu + Ser210Asp + Asn211Ser +Thr214Ser + Leu216CysPro200Ser + Val204Pro + Tyr208Ile + Thr214Pro +Asn217Asp + Gly218AspGly201Gln + Tyr205Asn + Tyr208Gly + Thr212Gly +Asn217Glu + Gly218GluVal202His + Thr207Asn + Tyr208Gln + Tyr213Pro +Ser215Glu + Leu216AspAla199Asn + Gly201Ser + Val202Pro + Tyr205Thr +Ser215Asp + Leu216AspPro200Gly + Val202Ala + Val204Ala + Ser210Glu +Asn211Glu + Gly218SerVal204Asn + Pro209Ser + Ser210Asp + Asn211Asp +Tyr213Asn + Leu216MetAla199Asn + Val204Thr + Ser210Asp + Asn211Glu +Tyr213Leu + Thr214ProVal202Cys + Val204Ala + Tyr205Glu + Thr212Ser +Ser215Glu + Asn217GlnThr207Pro + Ser210Asp + Asn211Glu + Thr212Glu +Asn217Ser + Thr219SerPro200Asn + Val202Asp + Tyr205Ala + Pro209Ser +Asn217Asp + Gly218ProVal202Gln + Pro209Glu + Ser210Glu + Asn211Glu +Thr212Gln + Asn217GlnVal202Asp + Ser203Asp + Thr207Gln + Thr212Ser +Tyr213Gln + Asn217GluAla199Asn + Ser203Glu + Val204Asn + Tyr208Pro +Leu216Glu + Asn217AspVal204Thr + Thr212Gly + Thr214Glu + Ser215Asp +Leu216Glu + Gly218SerAla199Gln + Ser203Asp + Val204Cys + Thr207Gly +Ser215Glu + Leu216GlnAla199Pro + Pro200Gln + Ser203Asp + Asn211Ser +Ser215Asp + Leu216AsnSer203Glu + Tyr208Thr + Tyr213Cys + Thr214Gln +Ser215Glu + Gly218SerAla199Thr + Pro200Gln + Gly201Gln + Ser203Asp +Ser215Asp + Gly218SerPro200Gly + Val202Cys + Ser203Asp + Asn211Ser +Ser215Glu + Leu216ProAla199Gln + Ser203Glu + Tyr205Gly + Tyr213Met +Ser215Glu + Asn217SerPro200Ser + Gly201Asn + Ser203Asp + Tyr208Val +Pro209Ser + Asn217GluPro200Ser + Gly201Ser + Val202His + Ser203Asp +Asn217Glu + Gly218ProVal202Asn + Tyr205Glu - Thr207Gln + Tyr208Leu +Thr214Asp + Gly218ProSer203Asp + Val204Thr + Tyr205Glu + Thr212Ser +Tyr213Ile + Ser215GluAla199Asn + Pro200Ser + Tyr205Pro + Pro209Asp +Asn211Glu + Tyr213IleGly201Pro + Val202Cys + Val204Ser + Pro209Glu +Thr212Glu + Thr214SerAla199Asn + Val202Asp + Ser203Asp + Tyr208Cys +Ser215Asp + Leu216AsnAla199Asn + Ser203Glu + Tyr205Cys + Thr212Gly +Thr214Glu + Ser215AspAla199Thr + Pro200Ser + Tyr208Met + Thr214Ser +Ser215Glu + Asn217AspAla199Pro + Tyr208Gly + Ser215Asp + Leu216Val +Asn217Asp + Thr219SerPro200Asn + Pro209Gly + Tyr213Pro + Ser215Asp +Leu216Gly + Asn217AspPro200Ser + Gly201Pro + Va1202Ala + Asn211Glu +Tyr213Asp + Gly218ProVal202Met + Val204Ser + Tyr205His + Thr212Gly +Asn217Glu + Thr219AspTyr205Val + Tyr208Pro + Ser215Glu + Leu216Met +Asn217Asp + Gly218AspVal202Ser + Tyr208Ala + Asn211Ser + Thr214Glu +Leu216Asp + Asn217AspGly201Pro + Tyr208Gln + Thr212Asp + Tyr213Asp +Ser215Asp + Asn217SerAla199Gln + Gly201Gln + Tyr205Gly + Pro209Glu +Thr212Pro + Tyr213GluSer203Asp + Pro209Ser + Asn211Gln + Thr212Gln +Ser215Asp + Gly218AspSer203Asp + Pro209Gly + Tyr213Leu + Ser215Glu +Leu216Ala + Gly218AspVal202Glu + Val204Cys + Thr212Gly + Ser215Asp +Leu216Gln + Gly218GlnPro200Gly + Gly201Gln + Val202Glu + Val204Ala +Thr207Pro + Ser215AspAla199Gln + Pro200Ser + Gly201Ser + Ser203Asp +Gly218Glu + Thr219AspAla199Gln + Gly201Ser + Tyr205Gln + Ser210Asp +Thr212Glu + Thr214AspGly201Asn + Val202Pro + Ser210Glu + Thr212Glu +Tyr213Thr + Thr214GluPro200Gln + Val202Met + Ser203Asp + Val204Gly +Tyr208Ile + Gly218AspPro200Ser + Gly201Pro + Val202Asp + Tyr208Cys +Asn211Gln + Thr219GluSer203Asp + Tyr208Pro + Pro209Ser + Asn211Ser +Thr214Asp + Thr219ProAla199His + Gly201Asn + Ser203Glu + Tyr213His +Thr214Glu + Leu216IleSer203Glu + Tyr208Pro + Tyr213His + Thr214Asp +Leu216Cys + Asn217SerVal202Asp + Tyr205Met + Thr214Asp + Ser215Glu +Leu216Cys + Asn217GlnVal202Cys + Val204Ser + Tyr208Leu + Ser210Asp +Tyr213Asp + Thr214AspGly201Asn + Tyr205Gln + Pro209Asn + Ser210Glu+Tyr213Glu + Leu216AlaVal202Cys + Pro209Asp + Tyr213Asp + Thr214Asn +Ser215Glu + Asn217GlnAla199Gly + Gly201Gln + Ser215Asp + Leu216Asp +Gly218Asn + Thr219GluAla199His + Thr207Asn + Asn211Ser + Leu216Glu +Asn217Gln + Thr219AspVal202Met + Thr207Gln + Pro209Gly + Thr214Glu +Ser215Asp + Gly218AspPro200Gln + Val202Ala + Val204Pro + Thr207Gln +Pro209Glu + Thr214GluGly201Pro + Tyr208Gly + Thr214Asp + Leu216Cys +Asn217Glu + Gly218AspAla199Gln + Thr207Pro + Tyr208Cys + Pro209Gln +Tyr213Asp + Leu216AspSer203Asp + Thr214Gly + Ser215Glu + Leu216Ala +Asn217Ser + Thr219GluPro200Gln + Ser203Glu + Val204His + Thr212Asp +Thr214Asp + Gly218GlnAla199His + Gly201Ser + Ser203Glu + Tyr205Ser +Thr214Glu + Gly218GluGly201Pro + Ser203Glu + Thr207Gly + Tyr208Cys +Thr214Asp + Gly218GluSer203Glu + Tyr205Leu + Thr207Asn + Thr212Asp +Tyr213Met + Ser215GluAla199Pro + Ser203Asp + Pro209Ser + Thr212Glu +Ser215Asp + Gly218GlnSer203Glu + Tyr205His + Thr212Asp + Tyr213Met +Ser215Asp + Thr219SerSer203Asp + Val204Gly + Thr212Glu + Thr214Pro +Ser215Asp + Asn217SerGly201Pro + Tyr205Gln + Thr207Gly + Asn211Glu +Thr212Gly + Thr214GluPro200Asn + Val202Gln + Thr207Gln + Pro209Ser +Asn211Asp + Thr214GluGly201Gln + Thr207Gly + Pro209Gln + Thr212Glu +Ser215Glu + Gly218GlnVal202Met + Tyr205Ala + Ser210Glu + Thr212Asp +Ser215Asp + Leu216AlaGly201Gln + Pro209Gly + Asn211Asp + Tyr213Asp +Thr214Gly + Leu216AspAla199Gly + Val202Glu + Ser203Glu + Tyr213Glu +Thr214Pro + Thr219SerGly201Ser + Val202Cys + Ser203Asp + Tyr205Asp +Leu216Gly + Thr219AsPPro200Ser + Ser203Glu + Tyr213Glu + Leu216Gly +Asn217Glu + Thr219GlyTyr205Asn + Pro209Asn - Thr212Glu + Tyr213Met +Ser215Glu + Asn217AspAla199Gln + Tyr208Ala + Asn211Gln + Thr214Glu +Asn217Glu + Thr219AspGly201Pro + Val202Gly + Ser203Glu + Tyr205Thr +Tyr208Ala + Thr219AspPro200Asn + Gly201Pro + Ser203Asp + Thr207Gln +Gly218Ser + Thr219GluGly201Asn + Ser203Asp + Val204Gly + Tyr208Val +Asn211Gln + Thr219AspAla199His + Pro200Asn + Ser203Asp + Tyr205Thr +Pro209Asp + Thr214GluGly201Pro + Val202Gly + Pro209Glu + Ser210Asp +Ser215Glu + Leu216MetAla199Gly + Val202His + Val204Ala + Pro209Asp +Ser210Glu + Ser215GluGly201Asn + Val204Gln + Tyr208Leu + Thr214Glu +Ser215Glu + Thr219GluSer203Glu + Tyr205Gln + Tyr208Gly + Asn211Glu +Thr214Glu + Thr219SerGly201Ser + Ser203Asp + Tyr205Ala + Pro209Asp +Tyr213Met + Ser215AspVal202Asn + Pro209Asp + Asn211Glu + Thr212Ser +Tyr213Leu + Ser215GluAla199Pro + Val204Thr + Tyr205Glu + Ser210Glu +Asn211Asp + Tyr213LeuVal202Ala + Ser203Asp + Val204Gln + Tyr205Met +Thr212Glu + Leu216GluGly201Pro + Ser203Asp + Tyr208Thr + Thr214Asp +Asn217Gln + Thr219AspPro200Gln + Gly201Asn + Thr207Gln + Tyr213Glu +Asn217Asp + Gly218AspPro200Gln + Thr207Gly + Tyr208Ala + Thr212Glu +Tyr213Glu + Asn217GluThr207Ser + Tyr208Ser + Pro209Glu + Thr214Glu +Asn217Glu + Thr219AsnAla199Gly + Tyr205Pro + Pro209Glu + Thr214Glu +Leu216Asn + Asn217AspAla199His + Gly201Asn + Tyr205Pro + Pro209Glu +Ser215Glu + Asn217GluPro200Gly + Pro209Asp + Tyr213Gln + Ser215Glu +Leu216Ile + Asn217AspAla199His + Val204Ser + Thr207Gln + Pro209Asp +Ser210Asp + Thr219AspAla199Thr + Thr207Pro + Tyr208Val + Pro209Glu +Ser210Asp + Thr219AspTyr208His + Pro209Glu + Ser210Glu + Tyr213Val +Leu216Cys + Thr219AspTyr205Met + Thr207Gly + Ser210Asp + Thr212Ser +Asn217Glu + Gly218AspSer210Glu + Thr212Pro + Thr214Gln + Asn217Asp +Gly218Glu + Thr219SerPro200Gln + Val204Thr + Tyr208Asn + Asn211Asp +Ser215Glu + Leu216GluGly201Pro + Tyr208Gln + Asn211Glu + Thr212Asp +Asn217Glu + Thr219SerVal202Asp + Ser203Asp + Asn211Glu + Thr212Pro +Tyr213Leu + Thr214AsnVal202Glu + Ser203Asp + Tyr205Leu + Pro209Gly +Thr212Asp + Thr219SerAla199Gly + Pro200Asn + Tyr208Leu + Ser210Asp +Asn211Asp + Asn217AspPro200Ser + Gly201Ser + Val202Asp + Tyr208Gln +Ser210Glu + Asn211AspVal202Asp + Thr207Gln + Tyr208Thr + Ser210Glu +Thr214Gln + Asn217GluThr207Gln + Asn211Asp + Thr214Glu + Leu216His +Asn217Asp + Gly218GlnSer203Glu + Thr207Ser + Pro209Gly + Asn211Asp +Tyr213Gly + Ser215AspSer203Glu + Tyr205Val + Pro209Gly + Asn211Asp +Ser215Asp + Thr219Gly表28環(huán)6-七點(diǎn)取代突變變異體Ala199Ser + Pro200Ser + Val204Met + Tyr208Ser +Tyr213His + Thr214Gln + Leu216AlaAla199Ser + Gly201Ser + Val202Met + Thr207Gln +Ser210Glu + Thr214Ser + Asn217GlnGly201Gln + Val204Gly + Tyr205Ala + Thr207Gln +Tyr208Gly + Asn211Gln + Gly218AspAla199Gly + Pro200Gly + Thr207Gln + Tyr208Thr +Pro209Gln + Gly218Gln + Thr219GlnAla199Gly + Pro200Ser + Val202Gln + Thr207Asn +Asn211Ser + Thr212Ser + Thr219ProPro200Gln + Val204Ala + Tyr208Leu + Thr212Glu +Tyr213Met + Thr214Asn + Leu216GlnSer203Asp + Val204Met + Thr207Gln + Tyr208Met +Pro209Gln + Asn217Ser + Thr219AsnAla199Gln+ Pro200Ser + Gly201Gln + Tyr205Ala +Tyr213Cys + Thr214Gly + Gly218GluAla199Thr + Pro200Ser + Thr207Gln + Tyr208Gln +Thr212Ser + Gly218Asp + Thr219ProGly201Ser + Val204Pro + Tyr208Ser + Thr212Gly +Tyr213Val + Thr214Pro + Ser215AspPro200Gln + Tyr208Cys + Pro209Gln + Thr212Gly +Tyr213Pro + Ser215Glu + Thr219GlyVal204Met + Thr207Gln + Pro209Gly + Thr212Gly +Tyr213Cys + Thr214Gly + Leu216MetPro200Gly + Ser203Asp + Thr207Asn + Pro209Asn +Tyr213Ile + Leu216Cys + Asn217SerPro200Asn + Gly201Asn + Tyr205Ile + Pro209Asp +Tyr213Met + Thr214Asn + Leu216ValAla199Asn + Ser203Glu + Val204Met + Pro209Gly +Tyr213Leu + Leu216Ala + Gly218AsnAla199His + Thr207Pro + Tyr213Ser + Thr214Asn +Asn217Ser + Gly218Ser + Thr219GluAla199Ser + Gly201Pro + Val204Gln + Thr207Ser +Thr212Asn + Tyr213Pro + Gly218GluAla199Ser + Pro200Asn + Tyr205Met + Tyr208Met +Thr212Asn + Thr214Gly + Leu216MetGly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp +Thr212Gly + Tyr213Val + Leu216IlePro200Asn + Tyr205Glu + Tyr208Met + Tyr213His +Thr214Ser + Asn217Gln + Thr219GlnAla199Gln + Pro200Gly + Val202Met + Tyr208Ile +Thr212Asp + Leu216Met + Gly218ProAla199Ser + Pro200Gly + Gly201Gln + Val202Asn +Asn211Gln + Leu216Val + Gly218ProAla199Asn + Val202Ser + Tyr205Cys + Thr207Gly +Tyr213Leu + Thr214Asp + Asn217SerGly201Pro + Val202Asn + Val204Asn + Tyr205Ser +Asn211Glu + Thr212Ser + Gly218AsnAla199Ser + Val204Cys + Thr207Ser + Asn211Ser +Thr214Gln + Ser215Glu + Asn217GlnPro200Ser + Val202Met + Tyr205Gly + Tyr208Pro +Ser210Glu + Thr212Ser + Tyr213GlyPro200Ser + Val202Gln + Ser203Glu + Tyr205Cys +Thr207Gln + Tyr208Ile + Asn211SerAla199Pro + Gly201Ser + Val204Ala + Thr207Ser +Pro209Gln + Tyr213Asp + Leu216HisGly201Ser + Ser203Asp + Val204Gln + Tyr208Thr +Asn211Gln + Leu216Ser + Thr219SerAla199Thr + Gly201Ser + Tyr205Pro + Tyr213Pro +Thr214Gly +Leu216Thr + Gly218GluPro200Asn + Gly201Gln + Tyr208Val + Thr214Gln +Ser215Asp + Gly218Ser + Thr219AsnPro200Gln + Val202Met + Val204Gly + Tyr208Gly +Asn211Ser + Tyr213Pro + Gly218AspAla199Asn + Pro200Gly + Tyr205Thr + Tyr208Val +Thr212Gly + Tyr213Ser + Leu216ValAla199Ser + Gly201Ser + Val202Gly + Tyr205Leu +Thr214Asn + Leu216Ala + Gly218GluAla199Ser + Gly201Gln + Val204Ser + Tyr208Asn +Pro209Ser + Thr214Asn + Ser215GluAla199Pro + Gly201Asn + Tyr208Cys + Tyr213Gly +Thr214Glu + Asn217Ser + Gly218GlnAla199Pro + Val202His + Tyr208Met + Pro209Asn +Thr212Asn + Thr214Glu + Ser215AspAla199His + Val202Gly + Val204Ser + Thr207Gln +Pro209Asp + Ser210Asp + Asn211SerAla199Gly + Pro209Glu + Ser210Glu + Thr212Gln +Tyr213Ala + Leu216Pro + Thr219AsnAla199His + Gly201Gln + Val202Met + Val204His +Thr207Gly + Pro209Glu + Ser210AspAla199His + Val202Gln + Thr207Gly + Thr214Gln +Leu216Thr + Asn217Asp + Gly218GluGly201Gln + Val204Thr + Tyr205Pro + Thr207Gly +Leu216Asn + Asn217Asp + Gly218AspGly201Ser + Tyr208Thr + Pro209Gln + Tyr213Ser +Asn217Asp + Gly218Glu + Thr219GlyGly201Ser + Val202Cys + Tyr205Val + Thr207Gln +Tyr213Ile + Leu216Asp + Asn217AspGly201Asn + Val202Glu + Ser203Glu + Val204Gly +Thr212Gly + Leu216Val + Thr219ProVal202Asp + Ser203Glu + Val204Asn + Thr207Pro +Tyr208Leu + Thr212Ser + Asn217GlnGly201Gln + Tyr208Ser + Pro209Asn + Asn211Gln +Tyr213Asp + Thr214Asp + Asn217GlnAla199Asn + Pro200Asn + Val204Thr + Ser210Asp +Asn211Glu + Tyr213Leu + Thr214ProGly201Ser + Val202Ala + Tyr208Met + Ser210Asp +Asn211Asp + Thr212Asp + Asn217SerAla199Ser + Pro200Gly + Gly201Asn + Val202Glu +Leu216Cys + Asn217Asp + Gly218AsnAla199Asn + Pro200Ser + Ser203Asp + Tyr205Cys +Tyr208Asn + Thr214Gln + Ser215GluAla199Gln + Val202Gly + Ser203Glu + Tyr205Gly +Tyr213Met +Ser215Glu + Asn217SerGly201Asn + Val202Gly + Ser203Asp + Tyr208Ile +Tyr213Pro + Ser215Glu + Asn217SerVal202Met + Ser203Glu + Thr207Asn + Thr214Gly +Ser215Asp + Leu216Glu + Asn217AspAla199Gln + Ser203Glu + Val204Cys + Asn211Ser +Thr214Gly + Asn217Glu + Thr219ProAla199Gln + Ser203Asp + Tyr205Thr + Thr207Ser +Thr212Asn + Asn217Asp + Gly218AsnPro200Ser + Val202Ser + Ser203Asp + Val204Gly +Thr212Asn + Tyr213Met + Asn217GluSer203Glu + Tyr205Met + Tyr208Thr + Pro209Gly +Asn217Glu + Gly218Asn + Thr219SerAla199Gln + Tyr205Leu + Tyr208Pro + Asn211Glu +Thr212Asp +Tyr213Asp + Thr214GlyGly201Pro + Val202Thr + Val204His + Tyr205Met +Asn211Glu + Thr212Asp + Tyr213AspGly201Pro + Ser203Glu + Tyr205Asp + Thr207Ser +Ser215Asp +Leu216His + Thr219SerAla199Asn + Val202Gln + Val204Ser + Pro209Gly +Ser210Glu + Thr212Asp + Asn217GlnPro200Gly + Gly201Pro + Tyr208Val + Pro209Glu +Asn211Asp+Thr212Gln + Thr219SerAla199Asn + Pro200Gln + Val202Ala + Val204Pro +Thr207Asn + Pro209Glu + Asn211GluAla199Asn + Ser203Asp + Thr207Gly + Asn211Ser +Ser215Glu + Asn217Asp + Gly218GlnPro200Gln + Ser203Glu + Val204Gly + Tyr213Pro +Ser215Glu + Asn217Glu + Thr219GlnPro200Gly + Val202Asp + Ser203Asp + Thr207Ser +Thr212Gly + Ser215Glu + Gly218SerVal202Glu + Ser203Asp + Tyr208Ser + Asn211Gln +Tyr213Ala + Ser215Asp + Thr219SerAla199Gln + Ser203Asp + Tyr205Met + Thr214Asp +Ser215Glu + Leu216Cys + Asn217GlnGly201Gln + Ser203Glu + Tyr205Glu + Pro209Gln +Asn211Ser + Thr214Glu + Leu216GluGly201Asn + Val202Cys + Ser203Asp + Tyr205Asp +Thr214Asp + Leu216Gly + Thr219ProAla199Gly + Val202Met + Ser203Glu + Val204Pro +Tyr205Glu + Thr207Ser + Thr214AspGly201Ser + Val204Met + Pro209Gly + Asn211Ser +Tyr213Cys + Thr214Glu + Leu216AspGly201Pro + Val202Asp + Thr207Gln + Thr212Ser +Asn217Asp + Gly218Asn + Thr219AspPro200Ser + Val204Gly + Tyr205Gly + Tyr208Cys +Thr214Ser + Ser215Glu + Asn217GluGly201Asn + Tyr205Ile + Tyr213Ile + Thr214Asn +Ser215Glu + Asn217Asp + Thr219ProPro200Ser + Val202Gln + Tyr208His + Thr212Pro +Thr214Ser + Ser215Glu + Asn217GluPro200Gly + Val204Gln + Thr207Ser + Tyr213Leu +Thr214Gln + Ser215Glu + Asn217GluThr207Gly + Ser210Asp + Asn211Asp + Thr212Gly +Tyr213Asp + Asn217Gln + Thr219GlnSer203Glu + Thr207Asn + Tyr208His + Asn211Gln +Thr214Asp + Leu216Glu + Asn217GluGly201Pro + Val202Met + Val204Ser + Tyr205His +Thr212Gly + Asn217Glu + Thr219AspAla199Asn + Val204Cys + Tyr208Cys + Tyr213Ala +Thr214Glu + Leu216Asp + Asn217GluPro200Gln + Thr207Gly + Tyr208Cys + Thr214Asp +Ser215Asp + Leu216Gln + Asn217AspAla199Gln + Val204His + Asn211Ser + Thr212Ser +Thr214Glu + Ser215Glu+ Asn217GluVal202Asp + Ser203Asp + Tyr205Ala + Thr207Gln +Tyr213Met + Thr214Glu+ Ser215AspAla199Gly + Pro200Gln + Val204Cys + Thr212Glu +Thr214Glu + Ser215Glu +Leu216GlnVal204Ala + Thr207Gly + Ser210Asp + Asn211Asp +Thr212Glu + Thr214Glu + Asn217SerGly201Pro + Pro209Ser + Ser215Glu + Leu216Glu +Asn217Asp + Gly218Ser + Thr219AspPro200Asn + Gly201Pro + Ser203Asp + Thr207Gln +Asn217Asp + Gly218Ser + Thr219GluAla199Pro + Val202Gly + Ser203Asp + Pro209Gln +Thr212Asn + Ser215Glu + Gly218AspAla199Gln + Pro200Ser + Gly201Ser + Tyr205Gln +Ser210Asp + Thr212Glu + Thr214AspAla199Pro + Pro200Ser + Thr207Gln + Ser210Glu +Thr212Asp + Thr214Asp + Thr219SerAla199His + Ser203Asp + Val204Gly + Thr207Gly +Asn211Gln + Thr214Ser + Gly218GluGly201Asn + Val202Asp + Val204Gly + Tyr208Val +Asn211Gln + Asn217Ser + Thr219AspVal202Asn + Tyr205Glu + Thr207Gln + Tyr208Leu +Thr214Asp + Asn217Asp + Gly218ProAla199Asn + Gly201Asn + Ser203Asp + Tyr208Asn +Thr214Glu + Leu216Cys + Thr219GlyVal202Cys + Ser203Asp + Val204Ser + Tyr208Thr +Thr214Asp + Asn217Gln + Thr219ProSer203Asp + Tyr208Pro + Pro209Ser + Asn211Ser +Tyr213Met + Thr214Asp + Thr219ProAla199Asn + Pro200Gln + Ser203Asp + Thr212Glu +Thr214Asp + Ser215Glu + Asn217SerPro200Asn + Gly201Asn + Val202Glu + Ser203Asp +Thr212Gly + Ser215Glu + Thr219AspAla199Gln + Gly201Gln + Pro209Gly + Ser210Glu +Thr212Pro + Tyr213Glu + Leu216AlaPro200Ser + Val204Ser + Thr207Asn + Tyr208Leu +Ser210Asp + Asn211Ser + Tyr213AspAla199His + Ser203Asp + Tyr205Asp + Thr207Asn +Tyr208Ile + Leu216Ser + Gly218AspAla199Gln + Gly201Pro + Val202Glu + Tyr205Glu +Pro209Gly + Thr214Asp + Leu216GlnGly201Asn + Val204Pro + Tyr208Cys + Ser210Glu +Asn211Glu + Thr212Pro + Thr214AspAla199His + Pro200Asn + Tyr208Val + Thr214Glu +Ser215Glu + Leu216Glu + Thr219AspPro200Gly + Val202Glu + Tyr205Pro + Tyr208Ala +Thr214Glu + Asn217Asp + Gly218AsnVal202Glu + Tyr205His + Pro209Gln + Thr214Glu +Ser215Asp + Asn217Ser + Gly218AspPro200Ser + Tyr205Leu + Thr212Gln + Thr214Asp +Ser215Glu + Gly218Glu + Thr219AsnSer203Asp + Val204Gly + Tyr205Ser + Thr212Glu +Thr214Pro + Ser215Asp + Asn217GluPro200Asn + Val204Gln + Pro209Glu + Ser210Glu +Asn211Glu + Thr212Asn + Ser215AspAla199Gln + Thr207Pro + Tyr208Cys + Pro209Gln +Tyr213Asp + Thr214Asn + Leu216AspAla199Gly + Pro209Glu + Ser210Asp + Thr214Glu +Ser215Glu + Gly218Pro + Thr219GlyAla199His + Thr207Ser + Tyr208Thr + Pro209Gly +Ser215Asp + Asn217Gln + Gly218GluAla199Ser + Ser203Glu + Tyr208Ala + Asn211Gln +Ser215Glu + Leu216Cys + Thr219AspGly201Pro + Val202Gly + Ser203Glu + Tyr205Thr +Tyr208Ala + Ser215Asp + Thr219AspPro200Gln + Val202His + Ser203Glu + Asn211Gln +Thr212Asp + Thr214Asp + Thr219GlyGly201Pro + Ser203Asp + Thr207Gln + Ser210Glu +Thr214Glu + Ser215Asp + Thr219SerAla1995er + Tyr205Gly + Thr207Ser + Ser210Asp +Tyr213Glu + Ser215Glu + Leu216GluVal204Thr + Thr207Pro + Ser210Glu + Asn211Glu +Thr212Glu + Gly218Asp + Thr219SerVal202Asp + Ser203Asp + Thr207Gly + Pro209Asn +Asn211Glu + Asn217Asp + Gly218SerPro200Ser + Ser203Asp + Val204Pro + Tyr208Ile +Thr214Asp + Asn217Ser + Gly218AspPro200Ser + Gly201Pro + Val202Gly + Ser203Glu +Tyr205Glu + Pro209Asp + Tyr213GluAla199Gly + Gly201Gln + Val202Ser + Asn211Asp +Ser215Glu + Leu216Asp + Asn217GluAla199Thr + Tyr205Gln + Thr207Gln + Pro209Gln +Thr212Asp + Ser215Glu + Gly218GlnAla199Gln + Val204Ser + Tyr205Glu + Thr207Asn +Ser210Glu + Thr212Glu + Asn217GlnTyr205Val + Tyr208Pro + Pro209Gln + Ser210Asp +Thr212Glu + Ser215Glu + Leu216GlyAla199Pro + Gly201Ser + Thr214Glu + Ser215Glu +Asn217Gln + Gly218Glu + Thr219AspSer203Glu + Ser210Asp + Asn211Gln + Thr212Glu +Tyr213Ser + Thr214Glu + Leu216CysAla199His + Pro200Gln + Pro209Asn + Tyr213Gln +Thr214Asp + Leu216Glu + Thr219AspAla199Asn + Ser203Asp + Tyr205Glu + Thr207Asn +Pro209Asn + Asn211Glu + Leu216GluAla199Thr + Gly201Pro + Ser210Glu + Thr214Asp +Ser215Asp + Asn217Ser + Thr219SerSer203Glu + Thr207Pro + Tyr208Ala + Ser210Asp +Ser215Glu + Asn217Asp + Thr219ProVal202Asp + Val204Cys + Tyr205Asp + Tyr208Ala +Pro209Gly + Gly218Gln + Thr219GluVal202Ala + Val204Thr + Tyr208Leu + Asn211Glu +Thr212Glu + Ser215Asp + Leu216GluPro200Asn + Val202Gln + Thr207Gln + Pro209Ser +Asn211Asp + Thr214Glu + Leu216AspAla199Asn + Val202Met + Thr207Asn + Pro209Glu +Tyr213GIu + Ser215Glu + Asn217AspAla199Gly + Val202Cys + Val204Gln + Tyr205Asp +Ser210Asp + Asn211Gln + Thr214GluAla199His + Tyr208Thr + Pro209Asp + Ser215Asp +Leu216Met + Asn217Asp + Gly218AspAla199Asn + Pro200Asn + Gly201Gln + Pro209Asp +Thr214Ser + Ser215Asp + Leu216AspAla199Asn + Pro200Gln + Gly201Ser + Val202Asp +Asn211Asp + Ser215Glu + Leu216AspAla199Thr + Val202Asp + Val204Cys + Ser210Asp +Tyr213Ile + Ser215Asp + Leu216AspGly201Ser + Val202Glu + Tyr205Gly + Tyr208Leu +Ser210Asp + Ser215Glu + Asn217AspGly201Gln + Thr207Ser + Tyr208His + Pro209Glu +Asn211Glu + Tyr213Asp + Asn217GluPro200Gly + Gly201Asn + Ser203Glu + Tyr208Cys +Thr212Asn + Asn217Gln + Thr219GluAla199Gly + Gly201Asn + Val202His + Val204Ala +Pro209Asp + Ser210Glu + Ser215GluGly201Asn + Val202Glu + Pro209Gly + Ser210Glu +Thr212Glu + Tyr213Thr + Thr214GluVal202Cys + Val204Ala + Tyr205Glu + Thr212Ser +Ser215Glu + Asn217Gln + Thr219AspGly201Ser + Ser203Glu + Ser210Glu + Asn211Ser +Thr212Glu + Ser215Glu + Leu216ProAla199His + Ser203Glu + Thr207Gln + Ser210Asp +Thr212Asp + Ser215Asp + Gly218GlnAla199Gln + Pro200Asn + Gly201Gln + Val204Asn +Thr214Asp + Ser215Glu + Thr219GluAla199Pro + Gly201Pro + Ser203Asp + Tyr205Val +Pro209Glu + Ser215Glu + Thr219ProPro200Gly + Ser203Asp + Thr207Gln + Asn211Glu +Tyr213Glu + Leu216Thr + Asn217GlnVal202Asp + Tyr205Glu + Tyr208Gly + Thr212Asp +Tyr213Ala + Leu216Ile + Thr219ProPro200Ser + Val202Glu + Tyr205Asp + Asn211Glu +Thr212Asp + Tyr213Cys + Thr219GlyVal204Pro + Tyr205Asp + Asn211Asp + Thr214Pro +Ser215Asp + Asn217Ser + Thr219ProGly201Asn + Ser203Asp + Pro209Gln + Tyr213Thr +Thr214Asp + Gly218Asn + Thr219GluPro200Gln + Val204Thr + Thr212Asn + Tyr213Glu +Thr214Pro + Asn217Asp + Gly218AspAla199Gln + Gly201Gln + Val204Asn + Tyr208Met +Ser210Glu + Thr214Glu + Asn217SerGly201Pro + Val204Thr + Tyr208Gly + Asn211Asp +Thr214Asp + Asn217Glu + Gly218AspPro200Asn + Pro209Glu + Ser210Asp + Asn211Ser +Thr214Ser + Leu216Cys + Thr219AspSer203Asp + Thr207Ser + Tyr208Asn + Pro209Asp +Ser210Asp + Thr214Gln + Asn217GlnVal204Ser + Tyr208Pro + Pro209Ser + Asn211Asp +Leu216Met + Asn217Asp + Gly218AspTyr205Ser + Tyr208Cys + Pro209Asn + Asn211Asp +Thr212Asr + Leu216Glu + Asn217AspPro200Gly + Ser210Asp + Thr212Pro + Tyr213Val +Asn2I7Ser + Gly218Asp + Thr219AspVal202Cys + Tyr205Ala + Thr207Asn + Asn211Asp +Tyr213Val + Ser215Asp + Leu216AspAla199Gln + Val202Glu + Ser203Asp + Val204Thr +Pro209Gln + Asn211Asp + Tyr213ProGly201Pro + Val202Glu + Ser203Glu + Val204Gly +Pro209Gln + Thr212Glu + Leu216AlaPro200Gln + Val202Asp + Ser203Asp + Tyr208Ala +Thr212Glu + Leu216Val + Gly218GlnAla199Thr + Gly201Gln + Val202Gly + Ser210Glu +Asn211Asp + Ser215Asp + Leu216AlaAla199Gly + Gly201Gln + Val204Asn + Ser210Asp +Asn211Asp + Ser215Glu + Thr219AsnAla199Asn + Val202Ala + Ser203Asp + Val204Gln +Ser210Glu + Thr212Glu + Leu216GluVal202Asp + Val204Asn + Pro209Ser + Ser210Asp +Asn211Asp + Tyr213Asn + Leu216MetVal202Pro + Ser203Asp + Val204Ser + Thr212Glu +Tyr213Asp + Thr214Asn + Gly218AspSer203Glu + Val204Pro + Tyr205Asp + Pro209Glu +Thr212Gln + Tyr213Asn + Gly218AspPro200Ser + Thr207Gly + Ser210Glu + Thr212Gly +Thr214Glu + Ser215Asp + Gly218GluVal202Met + Val204His + Ser210Glu + Thr212Asp +Tyr213Ile + Leu216Asp + Gly218GluSer203Glu + Tyr208Ser + Asn211Asp + Thr212Pro +Thr214Asn + Ser215Glu + Gly218ProAla199Thr + Gly201Pro + Ser203Asp + Tyr205Gly +Asn211Asp + Ser215Glu + Asn217SerPro200Gln + Gly201Asn + Ser203Glu + Pro209Asn +Asn211Asp + Thr212Gly + Ser215Glu表29多環(huán)雙點(diǎn)突變變異體Leu 95Gly+Ser203GluThr 58Gln+Asp 59GluTyr103Cys+Leu216GlnVal202Gln+Thr212GlyGly126Gln+Gly156GluThr214Gly+Ser215GluAsn 96Gln+Ile106AlaGly101Asp+Tyr205ProAsn 61Ser+Gly165GlnGly 64Gln+Thr132GlyAsp 59Glu+Tyr205AsnSer100Glu+Tyr205HisGln161Glu+Tyr205ProThr 58Asp+Tyr205IleAsn 61Asp+Asn162SerGln161Glu+Pro209GlnGly 62Asp+Pro209AsnIle106Leu+Asn211AspTyr166Pro+Ser215GluIle106Leu+Pro209GluThr163Pro+Val202CysThr 65Pro+Leu216SerThr132Gly+Phe188ValGly156Glu+Ala186ThrPro128Ser+Ser160AspGly 62Gln+Gly165AspLeu 95Gln+Pro128GluSer 97Glu+Asn211SerLeu 95Asn+Phe188LeuLeu125Gln+Gly159GluSer187Glu + Tyr213AlaTyr205Cys + Asn211AspAsn162Gln + Tyr213ProSer158Asp + Leu216GlnGly156Asp + Val202AlaSer100Asp + Tyr205IleGly 99Ash + Thr214GluThr 58Asp + Gly130GlnGly156Glu + Tyr208ProAla105Pro + Leu216IleSer187Asp + Tyr205GlnLeu125Asn + Ser187GluThr 65Gln + Thr102GluTyr103Ile + Tyr205GluThr132Asp + Gly218GlnAsn211Ser + Thr212AspThr212Gln + Ser215GluThr132Pro + Tyr205GlyTyr103Glu + Thr132SerGly165Gln + Ser203GluGly 60Pro + Thr 65ProGly 99Asn + Gly153AsnPro128Asn + Val202HisAla105Glu + Gly130SerThr212Gly + Gly218GluPhe188Val + Tyr205SerAsn217Glu + Thr219GlyLeu 95Gln + Ser215AspVal 94Gln + Thr212GluAsn 61Ser + Ser203AspSer129Glu + Asn154GlnGly 62Glu + Thr219AsnSer 97Glu + Tyr213HisGly 99Asp + Ile164ThrGly101Pro + Ser215AspThr 58Pro + Tyr205GlnGly101Gln + Tyr208GlnAsn 61Gln + Thr212AspThr212Asp + Leu216IleAsn211Glu + Leu216HisGly153Glu + Thr163GlnThr132Asn + Tyr205AspAsn 61Ser + Ser203GluSer215Asp + Leu216SerGly 60Asp + Met198GlnSer158Glu + Tyr205MetPhe188Leu + Thr212GluGly130Pro + Ser190GluGly101Asp + Gly126SerAla105Gly + Ser157AspAsn 61Ser + Ser131AspAsn 61Ser + Phe188SerSer100Glu + Asn162GlnThr 58Gly + Pro128AspSer100Glu + Tyr205AlaGly127Glu + Tyr213ThrAsn154Glu + Leu216GlnSer 97Asp + Ala199ProSer203Glu + Leu216SerGly126Gln + Ser215GluGly 60Ser + Asn154GluThr 65Ser + Gly156AspPro128Ser + Thr163GlnSer215Asp + Leu216ValSer129Glu + Leu216AlaAsn211Asp + Tyr213LeuVal 94Ser + Phe188ValSer158Glu + Thr212GlnGly 99Pro + Asn154GlnVal202Ser + Tyr205GluTyr205Val + Asn217GluVal 94Gly + Ser215AspTyr205Glu + Tyr213ProAsn 96Ser + Gly127GluTyr205Thr + Thr214GlyGly 62Ser + Thr 65SerThr 65Glu + Pro128AsnSer215Asp + Leu216GlnAsp 59Glu + Gly 60ProVal 94Thr + Leu216MetSer 97Glu + Thr212GlnGly101Asp + Val202AsnSer129Asp + Val202GlyTyr103Ala + Thr132SerLeu 95Met + Leu216AspSer100Asp + Gly101ProSer100Asp + Thr219ProVal 94Asn + Ser215AspGly153Asn + Tyr205AspThr 58Glu + Ile106CysThr 65Glu + Tyr103ProVal 94Met + Ser 98GluSer210Glu + Gly218SerLeu125Pro + Ala199HisIle106Gly + Thr212GluTyr205Ile + Thr214ProGly130Glu + Tyr208HisSer187Asp + Leu216ValThr102Glu + Thr219GlnLeu125Ser + Gly153GluGly153Asn + Val202AspThr163Glu + Tyr205GlySer129Glu + Tyr166ProGly153Gln + Pro209GlnThr212Ser + Ser215GluThr163Asp + Tyr205HisAsn154Glu + Tyr166ThrAsn 96Ser + Ala105GlyGly101Ser + Phe188GlyGly153Glu + Gly218AsnSer100Asp + Thr212ProGly126Pro + Gly156AspLeu125Ser + Ser215GluAsp 59Glu + Thr 65GlyGly156Pro + Gly159AspVa1202Cys + Tyr213SerLeu 95Met + Ser131GluSer 97Asp + Gly 99SerAsn 61Gln + Leu 95AspGly126Asn + Tyr205GluGly159Pro + Gly218AsnLeu 95Thr + Leu216AlaAla105Glu + Leu216ThrThr102Glu + Gly130ProGly 64Gln + Ser215GluTyr103Asp + Gly126AsnGly101Glu + Gly127GlnThr132Asn + Ser160AspThr 58Pro + Gly153AsnSer203Glu + Thr214SerTyr103His + Ser129AspSer 98Asp + Pro209SerAsp 59Glu + Tyr205SerGly 60Gln + Ser215AspThr132Asp + Val202AsnGly218Ser + Thr219GlySer190Asp + Val202ThrAsp 59Glu + Gln161AsnLeu216Asn + Asn217AspVal 94Asp + Tyr205AsnTyr103Ser + Ser203AspGly 99Pro + Phe188GlnSer 97Asp + Leu216HisThr163Asp + Leu216AsnAsn 61Asp + Thr132SerLeu 95Asn + Ser215AspSer100Asp + Thr132GlyTyr166Thr + Asn211GlnThr207Pro + Leu216ThrIle106Ala + Gly165AspTyr205Asn + Ser215GluLeu 95Asn + Val202GlnSer160Asp + Phe188GlyGly 64Ser + Asn 96GlnGly101Asp + Asn154GlnGly 64Asn + Ser157GluPhe188Asp + Tyr213AsnSer215Glu + Leu216IleTyr103His + Ser187GluAsn 96Ser + Thr214AsnGly 62Asn + Ser 98AspIle106Ser + Ser157GluThr 65Glu + Thr212GlyGly127Ser + Ser215AspGly156Ser + Asn217GluSer 98Asp + Tyr103ThrGly127Asn + Gly165ProGly153Glu + Leu216MetSer129Asp + Gly165ProAsn 61Asp + Thr163GlnGly126Asn + Tyr205MetGly165Gln + Ser215GluThr 65Glu + Gly101ProVal 94Gln + Gly165GluTyr103Ile + Ser190GluAsn217Gln + Gly218SerSer 98Glu + Gly 99AsnThr102Asp + Thr212SerThr212Ser + Ser215AspGly130Glu + Pro209AsnPro128Glu + Gly165ProThr 58Asn + Leu125GluLeu125His + Thr163SerThr212Asp + Tyr213AlaThr 65Pro + Ser210GluVal 94Thr + Thr132GlyTyr205Cys + Thr214GlnGly127Gln + Ala186GlnGly127Glu + Tyr205CysSer 98Glu + Gly130SerGly127Glu + Phe188TyrPhe188Asp + Asn211SerGly 64Asn + Leu216AspSer203Glu + Thr212SerTyr166Val + Ser210AspGly165Glu + Tyr205MetAsn 96Gln + Thr132GluSer 98Glu + Gly101ProLeul25Cys + Ser215AspLeu125Cys + Gly126SerGly 99Gln + Ser210AspSer160Asp + Phe188LeuVal204Gln + Ser215GluAsn 61Gln + Leu216AspLeu125Gly + Ser155AspAla186Pro + Val204GlnIle106Met + Ser160AspLeu125Ile + Leu216SerGly 60Ser + Ser 97GluGly165Asp + Tyr213GlnPro128Glu + Asn154GlnGly 60Asp + Gly126ProSer203Glu + Leu216AlaGly126Glu + Val202GlyThr132Asp + Tyr213SerSer157Asp + Thr212SerGly130Ser + Ser203GluGly 99Asn + Gly130SerAsp 59Glu + Gly159AsnGly153Asn + Thr212AspGly153Asp + Tyr205SerSer100Glu + Tyr205ThrGly126Asn + Asn154GluSer190Asp + Tyr205ValThr102Glu + Tyr166ThrSer157Glu + Pro209GlySer100Asp + Tyr205LeuSer131Glu + Thr132AsnGly127Asp + Leu216ThrSer210Glu + Asn217GlnAsn 61Asp + Ile106GlnAla105Pro + Tyr205ProSer190Asp + Leu216CysSer203Glu + Tyr205ThrLeu 95Asp + Asn 96SerTyr205Ala + Thr212GluThr163Gln + Phe188GluTyr103Ala + Thr212AspSer155Glu + Gly218AsnVal 94Thr + Thr163SerThr102Glu + Asn217GlnGly1595er + Val202GluAsn154Ser + Tyr166AlaAsp 59Glu + Phe188GlyThr163Gln + Gly218SerGly153Asn + Tyr205ValSer129Glu + Leu216ProGly126Ser + Thr212GluSer203Asp + Leu216AsnGly126Glu + Thr212ProVal 94Pro + Ala186GluSer129Asp + Gly153AsnGly165Ser + Ser215AspGly 60Gln + Ser187GluSer203Asp + Leu216HisGly101Glu + Ile106MetVal 94Glu + Gly127GlnVal202Gly + Leu216HisAsp 59Glu + Thr132AsnVal202Asn + Tyr205ProLeu 95Ile + Ser215GluPro128Glu + Leu216ProSer 97Asp + Tyr213MetSer187Glu + Leu216CysAsn154Ser + Gly156GlnLeu125Gly + Gly126SerAsn154Ser + Leu216AsnGly126Asn + Val202AsnAsn 96Gln + Gly127SerGly 64Ser + Thr212GlySer160Asp + Asn211SerSer210Glu + Gly218GlnTyr205Ala + Thr212AspSer158Asp + Leu216SerThr212Gly + Ser215GluAla199Ser + Va1202AlaSer129Glu + Tyr205AsnAsn154Gln + Gly201GlnSer190Glu + Thr212SerThr 58Pro + Ser160AspGly127Glu + Gly130SerAsn154Glu + Thr212GlyGly153Ser + Gln161AsnTyr205Thr + Ser210GluLeu125Gly + Pro200GlnTyr103Val + Thr132GlyGly126Asp + Leu216AlaSer100Glu + Gly127AsnSer129Asp + Ala186SerSer160Glu + Tyr205SerThr163Glu + Ile164HisGly159Ser + Gln161GluAsp 59Glu + Gly159ProGly101Asp + Tyr166CysThr 58Asn + Ser129GluAsn 61Glu + Thr214SerGly127Ser + Ser203GluGly127Pro + Gly130AspGly127Glu + Val202GlyPhe188Met + Ser203GluSer 97Glu + Tyr166AsnGly 60Glu + Gly 99AsnAsn162Glu + Asn217SerThr102Asp + Leu216GlyGly 99Gln + Ala105ThrGly153Ser + Ser203GluTyr103Pro + Ser158GluSer187Glu + Tyr205ProSer160Asp + Val204ThrSer100Asp + Leu216HisGly101Asn + Ser203AspAsn 96Ser + Tyr205ProThr132Gly + Thr212ProIle106Pro + Val202CysGly 62Ser + Pro209GluPhe188Cys + Leu216AspLeu 95Cys + Ser104GluSer104Asp + Tyr205MetTyr205Glu + Thr214GlyGly153Glu + Leu216AsnVal 94Thr + Ser100GluThr 65Asp + Val202HisVal202Gln + Ser215AspVal 94Ser + Ala105GluThr 58Pro + Gly153AspAla105Asn + Ser203GluSer104Asp + Ala105GlyGly126Gln + Ile164ValGly153Ser + Ser215GluPro200Gly + Ser215Glu表33多環(huán)三點(diǎn)突變變異體Thr 58Gln + Leu 95Gly + Ser203GluAsp 59Glu + Tyr103Cys + Leu216GlnGly156Glu + Val202Gln + Thr212GlyGly126Gln + Thr214Gly + Ser215GluAsn 96Gln + Ile106Ala + Gly156GluAsn 61Ser + Gly1o1Asp + Tyr205ProGly 64Gln + Thr132Gly + Gly165GlnThr 65Pro + Thr163Pro + Val202CysThr132Gly + Phe188Val + Leu216SerLeu 95Asn + Ser 97Glu + Asn211SerAsn162Gln + Ser187Asp + Tyr213ProSer100Asp + Val202Ala + Tyr205IleThr 58Asp + Gly 99Asn + Gly130GlnGly156Glu + Tyr208Pro + Leu216IleThr 65Gln + Leu125Asn + Ser187GluAsn211Ser + Thr212Asp + Gly218GlnTyr205Gly + Thr212Gln + Ser215GluThr 58Glu + Gly 60Pro + Thr 65ProAsn 96Gln + Gly153Pro + Leu216ValGly 99Asn + Gly153Asn + Pro209GluAla105Glu + Pro128Asn + Val202HisTyr205Ser + Thr212Gly + Gly218GluGly 99Gln + Asn217Glu + Thr219GlyLeu 95Gln + Val202Thr + Ser215AspGly101Pro + Ile164Thr + Ser215AspThr 58Pro + Gly130Asp + Tyr205GlnGly101Gln + Ser190Glu + Tyr208GlnAsn 61Gln + Thr212Asp + Leu216IleAsn 61Ser + Thr132Asn + Tyr205AspGly130Pro + Phe188Leu + Thr212GluAsn 61Ser + Ala105Gly + Ser131AspAsn 61Ser + Asn162Gln + Phe188SerGly126Gln + Ser203Glu + Leu216SerThr 65Ser + Gly156Asp + Thr163GlnPro128Ser + Ser215Asp + Leu216ValVal 94Ser + Ser100Asp +P he188ValGly 99Pro + Asn154Gln + Ser203GluTyr166Asp + Tyr205Thr + Thr214GlyGly 62Ser + Thr 65Ser + Asn211GluThr 65Glu + Pro128Asn + Leu216GlnVal 94Thr + Thr212Gln + Leu216MetSer129Asp + Thr132Ser + Val202GlyLeu 95Met + Tyr103Ala + Leu216AspVal 94Met + Ser 93Glu + Tyr103ProLeu125Pro + Ala199His + Ser203GluIle106Gly + Tyr205Ile + Thr214ProSer129Glu + Tyr166Pro + Tyr205GlyPro209Gln + Thr212Ser + Ser215GluAsn154Glu + Tyr166Thr + Tyr205HisAsn 96Ser + Gly101Ser + Ala105GlyGly153Glu + Phe188Gly + Gly218AsnGly126Pro + Gly156Asp + Thr212ProGly156Pro + Gly159Asp + Val202CysLeu 95Met + Ser 97Asp + Gly 99SerGly126Asn + Gly159Pro + Gly218AsnThr 65Gln + Leu 95Thr + Leu216AlaTyr103Asp + Gly126Asn + Gly127GlnThr 58Pro + Ala105Glu + Gly153AsnGly 60Gln + Tyr205Ser + Ser215AspThr132Asp + Gly218Ser + Thr219GlyTyr103Ser + Phe188Gln + Ser203AspSer 97Asp + Gly 99Pro + Leu216HisAsn 61Asp + Thr132Ser + Leu216AsnLeu 95Asn + Thr132Gly + Ser215AspTyr166Thr + Asn211Gln + Leu216ThrIle106Ala + Gly165Asp + Thr207ProLeu 95Asn + Val202Gln + Ser215GluGly 64Ser + Gly101Asp + Asn154GlnThr 58Asn + Gly 64Asn + Ser157GluGly 62Asn + Asn 96Ser + Thr214AsnGly130Asp + Phe188Asn + Gly218ProGly 99Ser + Pro128Glu + Leu216GlyVal 94Ala + Pro209Ser + Ser215AspLeu 95Thr + Gly126Gln + Leu216GlyAsp 59Glu + Gly159Ser + Tyr205ProVal202Gln + Ser203Glu + Tyr205AsnThr 58Ser + Ser190Glu + Tyr205ProLeu 95Asn + Tyr205Ala + Pro209GluThr 58Asn + Gly153Pro + Ser210GluSer131Glu + Thr132Asn + Gly165AsnGly127Ser + Thr212Asn + Ser215AspThr 65Gly + Thr102Asp + Gly127AsnGly 60Asn + Tyr2AsThr + Asn217GlnAla199Ser + Val202Ala + Thr212GlyThr 58Pro + Gly130Ser + Ser160AspGly153Ser + Asn154Glu + Thr212GlyLeu125Gly + Tyr205Thr + Ser210GluTyr103Val + Thr132Gly + Pro200GlnSer100Glu + Gly127Asn + Leu216AlaSer160Glu + Ala186Ser + Tyr205SerGly159Ser + Thr163Glu + Ile164HisPhe188Met + Val202Gly + Ser203GluThr102Asp + Ala105Thr + Leu216GlyThr 58Asp + Asn 96Ser + Tyr205ProSer 97Asp + Thr132Gly + Thr212ProIle106Pro + Val202Cys + Ser215AspLeu 95Cys + Phe188Cys + Leu216AspThr 65Asp + Val 94Thr + Val202HisThr 58Pro + Ala105Asn + Gly153AspSer104Asp + Ala105Gly + Ile164ValGly126Gln + Ser203Asp + Leu216HisSer160Asp + Gly218Gln + Thr219AsnGly 62Ser + Ala105Glu + Pro128SerThr 58Gly + Ile106Gly + Leu216AspSer 97Asp + Thr163Gln + Ala186SerThr 65Glu + Gly126Gln + Leu216HisPhe188Ile + Ser203Glu + Leu216ThrThr132Gln + Gly165Asp + Thr212SerSer129Asp + Asn211Ser + Leu216ThrLeu125Ile + Asn154Ser + Leu216MetThr 65Pro + Gly101Pro + Thr212AspThr102Asn + Thr212Gly + Leu216AspThr 65Gln + Tyr205Gln + Leu216MetLeu125Asn + Pro209Gly + Thr214GluLeu125Pro + Val202His + Ser215AspSer160Glu + Gln161Asn + Tyr205ValGly126Ser + Thr212Glu + Gly218AsnVal 94Thr + Ser155Asp + Thr207GlySer157Glu + Tyr208Gln + Thr212GlnTyr205Met + Thr212Gln + Ser215GluPro128Gln + Thr132Pro + Tyr213AsnGly 60Ser + Gly156Asp + Pro209AsnAsn162Asp + Thr163Asn + Ala199GlyGly165Ser + Tyr205Thr + Ser215GluSer100Asp + Gly156Pro + Tyr205GlnAla105His + Ser157Glu + Ala186AsnAsn162Gln + Ser190Glu + Val202SerGly 99Gln + Gly126Gln + Gln161AspGly126Asp + Gly159Ser + Thr212GlyGly 62Glu + Asn154Gln + Leu216GlnAsn154Asp + Tyr205Ser + Tyr213MetAsn 61Asp + Leu 95Ser + Val204GlnThr102Gln + Gly159Gln + Ser190GluPro128Asn + Gly159Asn + Asn211SerGly159Ser + Thr163Gly + Ala199ThrAsn211Gln + Tyr213Gln + Ser215GluGly 99Ser + Leu125Gly + Ser215GluThr 65Gly + Ser190Glu + Thr219SerVal 94Met + Asn162Ser + Leu216MetLeu 95Ser + Tyr103His + Gly156GluThr 58Gly + Asn211Glu + Leu216AlaAla105Gly + Thr212Pro + Gly218GluSer158Asp + Gly159Ser + Tyr205ProThr 58Gln + Ser158Glu + Thr163GlyThr 65Pro + Leu125Met + Thr163GlyGly 62Asn + Gly126Pro + Ser210GluLeu 95Met + Thr132Asp + Asn211SerTyr103Ile + Gly159Asp + Val202GlyGly101Gln + Tyr166Pro + Asn211GlnPro128Gly + Thr132Pro + Thr214GlnPro200Ser + Thr212Asp + Leu216GlyThr212Asp + Leu216Met + Gly218GlnThr163Gln + Tyr205Cys + Thr212ProThr 58Gln + Ser 98Glu + Thr212ProThr102Pro + Ser155Asp + Tyr205AsnGly 62Ser + Gly 99Glu + Val202SerLeu 95Asp + Ile106Gln + Gly126AsnSer100Glu + Gly101Gln + Val202AlaThr 58Ser + Ser100Glu + Ile164LeuGly101Ser + Phe188Asp + Tyr213GlySer104Glu + Gly156Gln + Thr212GlyThr 65Asp + Val 94Gly + Tyr205MetSer203Glu + Thr212Ser + Tyr213ValThr 65Ser + Leu125Glu + Gln161SerThr 65Gly + Tyr205Asp + Tyr213ThrGly101Pro + Ser104Asp + Thr163GlnAla105Gly + Asn154Asp + Pro209GlyGly126Gln + Gln161Ser + Leu216AlaGly153Gln + Tyr166Cys + Gly218AspGly 99Gln + Val204Pro + Thr219GluThr 58Ser + Ser 97Asp + Thr212ProAsn162Ser + Met198Gly + Val202ThrVal 94Ser + Leu125Asp + Tyr205ThrSer157Asp + Tyr205Ser + Leu216ThrGly165Asn + Ser215Glu + Leu216ProGly156Asn + Thr163Glu + Ala199SerPro128Ser + Gly156Glu + Thr219GlyAsn 96Gln + Gly126Gln + Ser203GluGly159Asp + Gly165Pro + Tyr213IleThr 58Gln + Gly153Ser + Asn217GlnPro128Ser + Thr132Glu + Thr212GlyGly156Gln + Tyr205Pro + Ser215GluGly 62Ser + Tyr205Ile + Leu216AspLeu125Pro + Gly159Glu + Gly165ProThr 58Gln + Ser203Asp + Thr212SerThr102Gln + Val202His + Thr214SerGly 99Glu + Ile106Gly + Gly218AsnSer 97Asp + Leu125Val + Tyr213IleIle106Leu + Phe188Glu + Thr212AsnThr 58Pro + Gly153Pro + Asn162GlnGly127Ser + Pro128Gln + Thr212ProTyr103Pro + Ser203Asp + Tyr205IleThr 58Gln + Thr163Ser + Tyr205SerVal 94Cys + Ser215Glu + Gly218ProThr102Pro + Gly165Pro + Val202ThrThr 58Glu + Tyr103Asn + Pro200GlnPhe188Thr + Val202Asn + Asn217SerGly 99Asn + Tyr205Ser + Ser215GluTnr102Gly + Gly165Asp + Gly218GlnGly 60Asp + Ala186Pro + Tyr205AsnThr102Asn + Ile106Asn + Ser158GluGly 62Ser + Ser 97Glu + Gly218AsnIle106His + Ser129Glu + Thr212AsnGly126Glu + Pro128Asn + Va1202AsnAsp 59Glu + Gly 64Asn + Tyr213SerThr163Pro + Val204Thr + Tyr205CysSer129Glu + Asn162Ser + Leu216CysGly156Asn + Phe188Ala + Tyr205HisVal 94Ser + Gly101Ser + Leu125AspAsn 96Glu + Tyr166Ala + Leu216CysTyr103Gln + Phe188Met + Ser210GluGly101Ser + Thr212Pro + Thr219ProSer104Glu + Gly159Gln + Thr212ProGly 62Asp + Gly126Ser + Leu216ProGly101Asp + Gly218Pro + Thr219AsnSer157Glu + Tyr205Pro + Thr212SerPro209Glu + Ser210Glu + Gly218SerAsn154Glu + Ser155Glu + Gly218ProAsn211Glu + Thr212Asp + Leu216HisTyr205Gly + Ser215Glu + Leu216AspVal202Ser + Ser215Asp + Leu216AspThr 58Asp + Asp 59Glu + Thr212SerTyr205Glu + Tyr213Pro + Ser215AspGly156Ser + Tyr205Asp + Ser215GluGly 60Glu + Ser 97Glu + Gly 99AsnSer155Glu + Asn162Ser + Ser190GluLeu 95Asn + Ser203Asp + Ser215AspSer203Asp + Ser215Asp + Thr219GlnGly 62Asn + Ser203Glu + Ser215GluSer203Glu + Tyr205Pro + Ser215AspPhe188Asn + Ser203Asp + Ser215AspGly 62Asn + Ser203Glu + Ser215AspSer203Glu + Ser215Asp + Leu216SerGly156Gln + Ser203Glu + Ser215AspSer203Asp + Tyr205Ala + Asn217GluTyr205Ala + Ser210Glu + Thr212AspIle164Cys + Ser210Glu + Thr212AspSer158Asp + Gln161Glu + Tyr205ProAsp 59Glu + Asn 61Glu + Gly130SerGly 64Gln + Ser 98Glu + Ser100AspSer 97Glu + Gly 99Asp + Tyr213HisAsn154Gln + Ser157Glu + Ser190AspSer158Asp + Ser160Glu + Tyr205CysGly201Pro + 5er203Glu + Tyr205AspGly 99Gln + Ser129Asp + Tyr166GluSer160Asp + Asn162Glu + Phe188GlySer129Asp + Ser131Asp + Gly218AsnAsp 59Glu + Gly165Asn + Pro209GluVal 94Gly + Ser215Asp + Asn217GluVal202Glu + Tyr205Gln + Leu216GluGly156Glu + Ser158Glu + Tyr205MetAsn 61Asp + Asn 96Glu + Gly127SerGly159Asn + Phe188Asp + Gly218AspTyr103His + Asn162Glu + Ser190AspSer 97Asp + Ser100Glu + Gly201AsnSer157Glu + Ser160Glu + Tyr205ProGly153Asp + Val202Ser + Gly218AspSer157Glu + Gly159Glu + Ser190AspAsp 59Glu + Asn 96Glu + Tyr205SerGly153Ser + Asn154Asp + Gly165AspAsn154Glu + Gly165Asp + Asn211SerSer155Asp + Gly156Asp + Thr219AspSer210Asp + Tyr213Asp + Thr214GluAsn 61Glu + Ala186Gly + Pro209AspAsn154Asp + Ser157Glu + Thr214SerSer100Glu + Leu125Glu + Tyr213HisGly 60Glu + Leu 95Glu + Ile106LeuGly 99Glu + Gly101Glu + Tyr103GluThr 58Glu + Thr 65Glu + Ile106CysSer 97Glu + Gly101Asp + Val202AsnGly 62Asp + Ser210Asp + Leu216AlaAsn 61Asp + Ser100Glu + Asn162SerGly127Glu + Asn162Glu + Gly165GluIle106Asn + Gly156Glu + Gly159GluSer100Glu + Gly126Glu + Ala186GlnGly 62Asp + Phe188Leu + Thr212GluIle106Gln + Ser215Glu + Gly218GluTyr103His + Ser155Glu + Gly218GluAla186Glu + Val202Glu + Ser215AspGly127Glu + Asn154Glu + Tyr213ThrSer157Glu + Ser160Asp + Ser187AspGly 62Asp + Leu216Glu + Thr219GlnThr 58Asp + Ser210Asp + Thr212GluSer155Glu + Ser157Asp + Gly218AspPro128Asp + Asn162Glu + Asn217SerGly156Asp + Gly165Glu + Tyr205AsnSer158Glu + Phe188Asp + Val202MetThr102Glu + Ser104Asp + Leu125GluSer 98Asp + Ser100Asp + Ser104GluGly 62Asp + Pro209Asp + Asn211AspSer160Glu + Ser187Asp + Ser190AspGly159Asp + Val202Asp + Ser203AspPro128Glu + Thr212Asp + Tyr213AspLeu 95Glu + Tyr205Asp + Ser215AspAsp 59Glu + Tyr205Glu + Ser215GluGly 60Ser + Thr 65Glu + Leu216AspGly101Ser + Asn154Glu + Ser158GluPro128Asp + Ser203Asp + Ser215AspSer203Asp + Ser210Glu + Ser215GluSer104Glu + Ser203Asp + Asn217GluSer160Asp + Ser203Asp + Leu216AspSer203Glu + Ser210Asp + Leu216AspGly127Glu + Ser129Glu + Ser215GluPro128Glu + Ala186Glu + Phe188AspAsp 59Glu + Asn 61Glu + Ser203AspSer 97Asp + Gly 99Asp + Ser210AspAsn 96Glu + Ser100Asp + Thr212GluThr 65Asp + Thr163Gln + Ser215AspSer 98Asp + Thr102Glu + Leu216GlnThr132Glu + Ser157Asp + Ser190GluSer129Glu + Ser157Asp + Ser190GluSer190Glu + Ser203Glu + Tyr205GluAsn 61Asp + Ser 98Asp + Tyr205AspSer129Asp + Phe188Glu + Ser190AspSer100Glu + Ser104Glu + Tyr205MetGly127Glu + Ser190Asp + Tyr205LeuAsp 59Glu + Ser203Glu + Pro209AspGln161Glu + Ser215Glu + Asn217AspAsp 59Glu + Ser 97Glu + Ser157AspLeu125Glu + Gly153Glu + Ser203GluSer104Glu + Ser157Asp + Ser160AspAsp 59Glu + Asn 96Glu + Ser187GluSer129Asp + Thr132Glu + Asn162GluSer131Glu + Ser203Glu + Gly218GluGly156Asp + Ser203Asp + Thr214AspSer160Glu + Thr163Asp + Ser215AspThr 58Asn + Gly 99Glu + Ala105GluAsp 59Glu + Phe188Leu + Thr212AspSer104Asp + Ser131Glu + Tyr205GluGly 62Asp + Ser104Asp + Ser131AspSer104Glu + Ser131Glu + Gly156GluGly 60Asp + Gly101Asp + Met198GlnSer 98Glu + Ser215Glu + Gly218AspTyr103Asp + Ser129Asp + Tyr205GluSer100Asp + Ile106Asp + Ser215AspThr 65Asp + Ser100Glu + Ala105GluAsp 59Glu + Ser100Asp + Tyr205IleGly153Glu + Asn162Asp + Ser203GluSer 98Glu + Thr212Glu + Ser215GluSer131Asp + Thr212Glu + Ser215AspAla105Asp + Thr212Glu + Ser215AspSer158Glu + Thr212Asp + Ser215AspSer157Asp + Ser203Glu + Thr219AspGly 62Gln + Thr212Glu + Leu216AspSer 97Glu + Ser187Glu + Asn217AspSer155Glu + Asn217Asp + Gly218AsnGly159Glu + Ser190Glu + Asn211GluPro128Asp + Ser190Glu + Ser215AspAsp 59Glu + Gly156Asp + Ser160GluGly 60Asp + Ser100Glu + Ser215GluGly130Glu + Gly153Glu + Tyr205AsnThr102Glu + Gly153Glu + Asn217AspSer129Glu + Ser155Glu + Gln161AsnTyr205Glu + Asn211Glu + Leu216CysGly153Glu + Gln161Asp + Ala199AsnGly 62Asn + Gly153Glu + Val202AspSer129Asp + Asn162Glu + Asn211GluGly153Glu + Ser158Asp + Leu216AspSer157Glu + Leu216Ile + Thr219AspSer158Glu + Ser215Aso + Thr219Asp表34多環(huán)四點(diǎn)突變變異體Tyr103Cys + Val202Gln + Thr212Gly + Leu216GlnAsn 61Gln + Gly101Asp + Ile106Ala + Tyr205proAsn 61Ser + Gly 64Gln + Thr132Gly + Gly165GlnLeu 95Ile + Tyr205Glu + Tyr213Ala + Thr219AsnGly126Ser + Val202Ser + Tyr205Cys + Asn217GluAsn 96Ser + Asn154Glu + Tyr166Thr + Tyr205HisGly101Ser + Ala105Gly + Phe188Gly + Gly218AsnGly165Ser + Val202Gln + Tyr205Thr + Thr212SerGly126Asn + Ser190Glu + Pro209Asn + Leu216ValIle106Glu + Gly127Ser + Asn154Ser + Tyr205LeuThr 58Pro + Ser203Asp + Tyr213Met + Leu216ValGly 62Gln + Asn 96Glu + Ile164Asn + Leu216IleSer 98Glu + Tyr166Gln + Thr212Asn + Leu216ValGly156Asp + Tyr166Ala + Tyr208Pro + Leu216IleThr 58Pro + Ser 98Asp + Gly130Pro + Tyr205ValGly130Asp + Phe188Asn + Leu216Gly + Gly218ProLeu 95Thr + Gly126Gln + Pro209Ser + Leu216GlyVal202Gln + Ser203Glu + Tyr205Asn + Gly218AsnThr 58Asn + Leu 95Asn + Gly153Pro + Ser210GluGly127Ser + Ser131Glu + Thr132Asn + Gly165AsnThr 65Gly + Gly127Asn + Thr212Gln + Thr214GluAsn 96Gln + Gly126Asn + Gly127Ser + Val202AsnGly 64Ser + Ser160Asp + Asn211Ser + Thr212GlyAsp 59Glu + Ala199Ser + Val202Ala + Tyr205SerLeu125Gly + Thr132Gly + Pro200Gln + Tyr205ThrGly159Ser + Thr163Glu + Ile164His + Tyr205SerGly127Pro + Phe188Met + Val202Gly + Ser215GluGly101Gln + Asn211Gln + Thr212Pro + Ser215AspPro128Gly + Pro200Ser + Thr214Gln + Leu216GlyTyr205Cys + Thr212Pro + Leu216Met + Gly218GlnThr102Pro + Val202Ser + Tyr205Asn + Gly218AspLeu 95Asp + Ile106Gln + Gly126Asn + Val202AlaTyr103Gln + Ser203Asp + Thr212Asn + Leu216AsnPhe188Met + Ser210Glu + Thr212Pro + Thr219ProSer104Glu + Gly159Gln + Thr212Pro + Leu216ProGly 60Gln + Ala186Gly + Thr212Gly + Ser215AspThr102Glu + Thr132Pro + Leu216Pro + Asn217GlnLeu 95Ala + Phe188Thr + Tyr205Gln + Ser215AspThr 58Gly + Gly 60Asp + Val202Cys + Thr212ProGly156Asn + Val202Asp + Thr212Ser + Thr214GlyThr 58Pro + Gly165Glu + Tyr205Ser + Tyr213HisGly 99Asn + Tyr166Asn + Ser203Asp + Leu216HisGly 62Glu + Leu 95Met + Ala105Ser + Gly126SerIle106Gln + Ser187Glu + Tyr205Gly + Thr219ProGer160Glu + Tyr205Ser + Thr212Pro + Leu216ThrGly130Gln + Ser131Glu + Asn162Gln + Leu216AsnGly156Glu + Thr163Ser + Asn211Ser + Thr212AsnGly165Gln + Phe188Leu + Val202His + Thr212GluVal 94Cys + Pro128Asp + Val202Thr + Tyr205AlaLeu 95Thr + Gly156Ser + Thr214Glu + Leu216AlaGly 99Pro + Tyr103Glu + Gly165Pro + Leu216CysGly 64Asn + Thr 65Glu + Tyr166Ala + Phe188TyrTyr103Gln + Gly156Pro + Tyr166Asn + Ser215GluGly126Gln + Pro128Gln + Val202Thr + Thr212GluGly 64Ser + Val 94Met + Gly130Asp + Tyr213GlyGly 99Asn + Val202Pro + Thr212Asn + Ser215GluGly101Pro + Asn154Ser + Leu216His + Asn217GluTyr103Asp + Gly153Pro + Ala186Asn + Val202SerLeu125Thr + Gln161Asn + Thr212Gly + Leu216AsnGly 64Pro + Val 94His + Ser210Asp + Thr212GlyAsn 61Ser + Thr 65Gln + Ser190Asp + Val202GlyAla105Thr + Gly153Gln + Gly156Gln + Asn211GlnAsn 61Gln + Tyr166Ile + Ala186Asn + Ser215AspGly 60Gln + Tyr103Gly + Gly130Asn + Asn217AspLeu 95Pro + Tyr205Leu + Asn211Gln + Ser215GluThr102Asp + Gly126Gln + Val202Asn + Thr212GlyVal 94Thr + Gly153Pro + Ala186Pro + Tyr205GluSer104Asp + Ala105Asn + Thr163Ser + Tyr205AlaGly 64Ser + Val 94Asn + Gly130Asp + Leu216CysGly101Asn + Leu125Asn + Gly201Pro + Leu216SerGly159Pro + Gly165Pro + Ala186Thr + Thr212ProPro128Gln + Ser129Asp + Ala186Ser + Asn217SerGly 60Asn + Gly159Ser + Phe188Val + Ser203GluSer 98Glu + Gly 99Asn + Thr102Asn + Ala186ProThr 65Asn + Gly 99Gln + Thr212Ser + Leu216AspVal202Gln + Tyr205Thr + Tyr213Val + Ser215GluThr132Ser + Gly156Ser + Phe188Thr + Asn211GluLeu125His + Gly156Ser + Thr212Glu + Leu216AsnGly126Ser + Tyr205Asp + Thr214Gln + Leu216AsnAla186Gly + Ser215Glu + Gly218Pro + Thr219GlnThr 58Gly + Gly 60Asn + Leu125His + Leu216GlyAsp 59Glu + Val 94Gly + Tyr205Gln + Tyr213IleSer155Glu + Met198Thr + Val202Met + Tyr205ThrGly127Asn + Tyr166Pro + Phe188Leu + Thr214GluLeu 95Cys + Gly130Gln + Thr212Ser + Leu216ValAsp 59Glu + Thr 65Gly + Leu 95Gly + Leu216AsnSer131Glu + Gly201Ser + Val202Gln + Tyr205GlyLeu125Val + Tyr205Gln + Ser215Asp + Gly218GlnVal 94Gly + Thr102Ser + Ser203Glu + Leu216AsnAsp 59Glu + Gly 60Gln + Thr212Gly + Leu216CysAla186Gln + Gly201Gln + Val202Pro + Ser203GluThr 65Ser + Ser 98Asp + Gln161Ser + Tyr205CysTyr166Ala + Ser187Glu + Tyr205Gly + Leu216ValGly 99Asp + Met198Cys + Pro209Gly + Thr212AsnThr 65Pro + Ile106Gly + Tyr205Gln + Leu216GluLeu 95Ser + Thr102Asn + Leu216Asp + Asn217SerThr 58Gly + Gly127Ser + Tyr166Glu + Val202AsnVal 94Pro + Tyr213Thr + Thr214Glu + Asn217GlnSer 97Glu + Gln161Asn + Tyr166Gly + Val202SerTyr103Met + Thr132Gly + Tyr166Met + Ser210AspLeu 95Met + Gly156Asn + Val202Glu + Leu216GlnSer 97Asp + Met198Ala + Val204Thr + Tyr205ThrGly101Ser + Gly153Pro + Asn217Glu + Thr219ProIle106Met + Thr163Glu + Tyr166Ile + Thr212SerGly165Ser + Ser190Asp + Pro200Ser + Val202MetGly 62Ser + Gly153Asp + Thr163Ser + Gly201GlnGly101Gln + Thr102Ser + Gly153Ser + Val202AspVal 94Ala + Leu 95Gly + Leu125Thr + Gly130AspAsn 96Asp + Tyr205Ala + Thr212Pro + Thr219GlyGly153Gln + Tyr205Ile + Thr212Gly + Ser215GluIle106Cys + Asn154Glu + Tyr205Ala + Leu216AlaAsn 96Asp + Gly 99Pro + Tyr205Val + Asn217GlnAla105Thr + Leu125Thr + Val204Ala + Tyr205MetThr102Pro + Gly156Pro + Tyr208Val + Thr212AsnGly 60Ser + Phe188Ile + Thr212Ser + Leu216AspSer100Asp + Tyr103Gln + Gly159Gln + Leu216ValPro128Gly + Gly165Gln + Ser203Glu + Leu216SerSer129Glu + Thr132Gln + Met198Thr + Thr219GlnVal 94Pro + Gly 99Asn + Thr132Gly + Gly159AspSer155Asp + Gly165Ser + Tyr205His + Thr212GlyGly 99Asp + Phe188Val + Thr207Asn + Leu216ProThr102Gln + Gly126Asn + Gly159Asn + Leu216AspAsp 59Glu + Thr132Pro + Thr163Pro + Phe188ThrGly101Ser + Ala105Glu + Ala186Pro + Thr212GlySer129Asp + Gly153Asn + Gly159Pro + Val202MetGly 60Pro + Asn 96Asp + Gly126Gln + Tyr166AlaThr 65Gln + Gly 99Pro + Tyr205Ala + Ser215AspVal 94Gln + Gln161Asp + Tyr205Pro + Leu216MetAsp 59Glu + Gly 60Glu + Ala186Gly + Gly218AsnSer 98Asp + Gly 99Glu + Phe188Met + Tyr213AlaSer160Asp + Gln161Asp + Tyr205Ser + Thr219GlnAla105Gly + Ile106Cys + Tyr205Asp + Ser215AspPro200Asn + Tyr205Asp + Asn211Gln + Ser215GluTyr205Asp + Thr207Ser + Thr214Ser + Ser215AspAsn 61Asp + Thr 65Gln + Ser 97Asp + Tyr205AlaLeu125His + Ser203Glu + Ser215Glu + Leu216AspThr 65Asn + Ser203Glu + Tyr205Cys + Ser215AspGly 64Pro + Ala186Ser + Ser203Asp + Ser215AspPro128Ser + Ser203Asp + Tyr213Ala + Asn217GluVal202His + Tyr205Val + Ser210Glu + Thr212AspGly 99Asn + Gly130Ser + Ser210Glu + Thr212AspTyr205Ala + Ser210Glu + Thr212Asp + Gly218GlnPhe188Ala + Ser203Asp + Tyr205Asp + Thr212SerAsn154Glu + Ser155Asp + Thr163Asp + Tyr213ThrGly 99Gln + Ser129Asp + Tyr166Glu + Leu216ThrThr102Gly + Ala105Thr + Ser155Glu + Ser157AspGly127Pro + Ser157Glu + Gly159Asp + Gln161AspGly101Asn + Asn154Asp + Thr163Gly + Gly218GluPro128Ser + Ser158Asp + Thr163Asp + Asn217SerIle106Val + Thr214Asn + Ser215Asp + Asn217AspThr 58Gly + Asn154Asp + Ser157Asp + Ser190GluLeu125Asn + Gly153Glu + Thr163Asp + Thr214ProGly 64Pro + Ser157Glu + Gln161Glu + Tyr205CysSer155Asp + Ser187Asp + Ser190Glu + Thr212GlnVal 94Pro + Ser155Asp + Ser157Asp + Ser187GluGly126Gln + Ser203Asp + Ser215Glu + Gly218AspGly156Ser + Ser187Asp + Ser190Glu + Thr212SerAsn154Asp + Ser187Glu + Val202Asn + Leu216SerAsn162Asp + Gly165Asp + Thr212Gln + Leu216CysSer155Glu + Ser187Asp + Pro200Ser + Tyr213ValAsn 96Glu + Thr102Asp + Ala105Pro + Tyr213ValAsn154Ser + Gly156Glu + Asn162Asp + Thr212SerAsn 61Glu + Ser100Asp + Tyr205Val + Leu216GlyThr 65Gly + Asn154Glu + Ser157Asp + Asn162GluTyr205Leu + Thr212Glu + Ser215Glu + Leu216GluAsn 61Gln + Gly 62Glu + Ser 98Asp + Thr214GlnGly 62Glu + Ser 98Glu + Gly130Ser + Val202ProThr 65Glu + Tyr205Met + Thr212Asp + Thr214GlyAla105His + Phe188Asp + Val202Asp + Asn211SerAsp 59Glu + Pro209Ser + Asn211Asp + Thr212GluLeu 95Gln + Gly126Gln + Gly127Asp + Ser155AspGly 60Asp + Val 94His + Asn154Ser + Ser210GluThr 58Gly + Gly126Glu + Ser155Asp + Gly201ProGly156Asn + Tyr166Pro + Thr212Glu + Ser215GluThr212Asp + Ser215Glu + Leu216Ala + Thr219SerThr 58Asp + Gly101Ser + Ser210Asp + Thr212GluIle106Glu + Gly126Asp + Gly127Asn + Leu216ProAsn 61Ser + Gly 99Asp + Thr102Asp + Ser104AspSer210Asp + Thr212Asp + Tyr213Pro + Ser215GluGly127Pro + Pro128Ser + Ala186Glu + Ser203AspGly101Glu + Thr102Asp + Ser129Asp + Leu216AsnGly 62Glu + Leu 95Asp + Gly159Asn + Leu216GlyAsn 61Asp + Ser 97Asp + Ser210Glu + Thr212GlnSer203Glu + Tyr205Pro + Thr212Ser + Thr219GluThr 65Glu + Tyr103Pro + Ser215Asp + Asn217AspGln161Asp + Thr163Glu + Ser187Glu + Asn211GlnGly153Glu + Tyr166Gly + Ser187Glu + Leu216MetAsp 59Glu + Gly159Glu + Ser160Asp + Pro209GlnGly 60Asn + Ser129Glu + Ser158Asp + Gly159AspGly130Glu + Ser131Asp + Ser155Glu + Thr212AsnGly130Asp + Asn154Glu + Ser155Asp + Gln161AsnSer129Asp + Gly130Glu + Gly156Glu + Gln161SerGly 99Glu + Ser100Asp + Ser203Glu + Leu216SerPro128Asp + Ser129Asp + Val202Asn + Ser215GluTyr103Asp + Ser104Glu + Thr212Pro + Ser215GluThr 65Pro + Ser157Glu + Ser158Asp + Ser215AspThr 58Asn + Asn 96Asp + Ser 97Asp + Ser187AspSer104Asp + Ile164Gly + Ser215Asp + Leu216AspSer157Asp + Ser215Asp + Leu216Glu + Gly218GlnGln161Ser + Ser187Asp + Ser215Glu + Leu216GluThr 58Pro + Ser104Asp + Thr212Glu + Tyr213GluThr102Glu + Ser160Glu + Gln161Asp + Leu216ValAsn 61Glu + Gly 62Glu + Ser203Glu + Thr212GlyTyr205Val + Ser210Asp + Asn211Glu + Leu216GluAsp 59Glu + Gly126Ser + Tyr205Glu + Ser215GluPro128Asn + Ser129Asp + Tyr205Asp + Ser215AspGly101Glu + Tyr205Glu + Ser215Asp + Leu216HisSer155Glu + Ile164Ser + Tyr205Asp + Ser215AspTyr103Ala + Tyr166Glu + Tyr205Glu + Ser215GluGly 60Glu + Ser 97Glu + Gly 99Asn + Asn162GluSer187Glu + Ser203Glu + Tyr205Asp + Tyr213ProSer157Glu + Ser203Asp + Tyr208Pro + Ser215GluSer 98Glu + Ser203Glu + Tyr205Val + Ser215GluGly127Gln + Ser160Glu + Ser203Asp + Ser215GluVal 94Glu + Ser203Asp + Ser215Asp + Leu216ProSer100Asp + Ser203Glu + Ser215Glu + Leu216ThrSer157Glu + Phe188Thr + Ser203Asp + Ser215AspVal 94Gly + Leu 95Glu + Ser203Glu + Ser215AspPhe188Val + Ser190Asp + Ser203Asp + Ser215GluSer158Asp + Ser203Asp + Ser215Asp + Asn217SerSer157Asp + Ser203Asp + Ser215Asp + Thr219GlnGly126Pro + Gly153Glu + Ser203Glu + Ser215GlnSer131Glu + Gly165Asn + Ser203Glu + Ser215AspTyr103Glu + Gly153Asp + Ser190Glu + Tyr205ValGly159Glu + Ser203Glu + Leu216Gln + Asn217GluSer100Asp + Gly153Gln + Thr163Asp + Ser190AApThr102Asp + Gly127Glu + Ser155Asp + Tyr213ProAsn 61Glu + Asn154Glu + Ser190Asp + Val204MetAsn154Asp + Gln161Ser + Ser190Asp + Ser203GluThr 65Gly + Gly127Asp + Gly165Asp + Ser203AspAsn 96Asp + Gly 99Glu + Gly101Asn + Ser187GluThr102ASP + Ala105Glu + Gln161Ser + Pro209AspGly127Glu + Ser129Glu + Gly130Ser + Ser160AspThr102Glu + Ser104Glu + Pro200Asn + Thr212GluSer 98Asp + Ser100Glu + Pro128Gly + Gly159AspGly159Asp + Gln161Asp + Val204Gly + Ser215GluSer155Glu + Phe188Glu + Tyr205Asn + Ser215GluSer129Glu + Thr132Asn + Gly165Asp + Gly218AspSer100Glu + Gly153Asp + Gly165Gln + Thr219AspGly126Asp + Ser131Glu + Thr207Gly + Gly218GlnAsn 61Asp + Gly 99Glu + Tyr103Thr + Ser131AspGly 99Asp + 5er203Asp + Tyr205Asp + Thr212ProSer190Glu + Ser203Glu + Tyr205Asp + Leu216HisGly 60Pro + Ser157Asp + Ser203Glu + Tyr205AspSer157Glu + Thr163Glu + Pro209Ser + Ser215GluGly 60Asn + Ser157Asp + Thr163Glu + Asn211AspThr102Glu + Gly127Asn + Gly130Glu + Tyr205GlnAsn 61Glu + Gly153Ser + Pro209Asp + Ser215GluAsp 59Glu + Asn 61Gln + Leu2I6Asp + Gly218AspLeu125His + Ser129Asp + Ser131Asp + Val202GluIle106Cys + Tyr205Asp + Ser210Glu + Leu216AspSer 97Glu + Gly156Asp + Thr163Asp + Thr212GlnSer131Asp + Asn154Gln + Ser215Glu + Asn217AspSer104Glu + Thr212Ser + Ser215Asp + Asn217GluSer157Asp + Ser187Asp + Ser203Asp + Thr212GlyAsn 61Asp + Asn 96Asp + Ala105His + Asn162AspAsn 61Asp + Asn 96Glu + Gly127Ser + Gly130AspThr 58Gly + Ser158Glu + Asn211Asp + Tyr213AspSer158Asp + Asn162Asp + Thr212Asp + Leu216ThrGly 99Ser + Ser157Asp + Asn162Glu + Leu216AspGly159Gln + Asn162Glu + Ser190Asp + Ser215GluSer 97Asp + Ser100Asp + Gly126Asn + Ser160AspGly 60Glu + Ser 98Glu + Gly153Gln + Ala186AspSer157Asp + Ser160Asp + Thr212Asn + Ser215GluGly 62Pro + Ser131Glu + Gly165Glu + Thr219AspSer190Asp + Val202Glu + Ser215Glu + Leu216AlaAsn 96Glu + Gly153Asp + Gly156Asp + Gln161AsnTyr103Glu + Ser131Asp + Ser203Asp + Leu216CysGly130Glu + Ser157Asp + Ser187Asp + Tyr208HisAsn 61Asp + Thr163Gln + Ser203Asp + Thr214GluSer155Asp + Ser158Asp + Ser203Asp + Tyr205ThrSer155Asp + Gly156Ser + Ser215Glu + Thr219AspGly 60Gln + Thr 65Gln + Asn 96Asp + Leu125GluAsp 59Glu + Asn 96Glu + Ser203Glu + Thr214AsnAsp 59Glu + Asn 96Asp + Ser215Asp + Leu216IleGly 62Asp + Ser 97Glu + Ala105His + Ser131GluLeu125Asp + Gly165Asp + Ser203Asp + Tyr213GlnLeu 95Glu + Tyr166Asn + Ser203Glu + Thr214GluGly101Asp + Asn162Asp + Gly165Glu + Leu216AsnGly 99Glu + Tyr103Glu + Ser129Glu + Tyr205AsnAsp 59Glu + Asn 96Ser + Thr212Glu + Ser215AspAsn 61Glu + Ser100Asp + Ala186Gly + Thr212GluThr 65Gly + Ser 97Glu + Gly101Glu + Gly130GluAla186Glu + Phe188Tyr + Thr212Asp + Asn217AspAsn 61Gln + Gly156Glu + Ser215Glu + Thr219GluGly 99Gln + Ser129Glu + Ser155Glu + Gly218GluSer160Glu + Thr163Asp + Thr212Ser + Ser215AspAsn 96Asp + Thr102Asp + Ser157Glu + Ala186HisGly 99Glu + Ala105Asp + Thr214Asn + Leu216ValAsn162Gln + Ala186Asp + Tyr205Ser + Leu216AspSer 97Glu + Gly156Glu + Gly159Asp + Val202AsnIle106Cys + Ser129Glu + Ser155Glu + Ala186GluSer100Glu + Leu125Gly + Gly127Glu + Ser203AspThr 65Asp + Ser131Asp + Ile164Asn + Thr212AspSer 97Glu + Thr102Glu + Gln161Ser + Tyr205LeuAsp 59Glu + Gly 99Glu + Ser203Asp + Tyr205AsnAsp 59Glu + Gly 99Asp + Gly126Ser + Thr214GluSer160Asp + Pro209Asn + Ser215Asp + Gly218GluSer155Glu + Gly159Asn + Pro209Glu + Gly218GluSer155Asp + Asn211Asp + Tyr213Met + Gly218AspSer129Asp + Thr132Glu + Ser160Glu + Ala186SerVal 94Glu + Ser 97Asp + Gly101Pro + Ser203GluGly101Asp + Gly126Asp + Thr132Asp + Thr212AsnSer 98Glu + Gly127Glu + Thr163Asp + Thr212AsnGly 99Asp + Gly101Pro + Thr102Glu + Ser203GluGly159Asp + Thr212Asp + Ser215Asp + Asn217SerSer158Glu + Asn162Gln + Thr212Glu + Ser215AspVal 94Pro + Ser155Asp + Thr212Glu + Ser215GluGly 99Gln + Ile106Asp + Thr212Glu + Ser215GluThr 58Asp + Tyr205Pro + Thr212Glu + Ser215AspAsp 59Glu + Ser 98Glu + Ser203Asp + Tyr205GlyAsp 59Glu + Ser100Asp + Ser104Asp + Ala105ProAsp 59Glu + Ser100Asp + Ser104Asp + Tyr205IlePro128Glu + Asn162Asp + Thr212Glu + Leu216CysAsn 96Gln + Thr102Asp + Ser129Glu + Asn162GluSer 97Glu + Gly130Asn + Ser155Asp + Ala186GluAla105Glu + Ser155Glu + Gln161Asp + Tyr205ThrGly 62Asp + Ser158Asp + Ser215Asp + Leu216AsnThr102Asp + Gly126Asp + Ser158Asp + Thr212SerThr 58Asn + Gly 64Pro + Thr 65Glu + Val 94AspSer190Glu + Ser203Glu + Tyr213Glu + Leu216HisGly159Asp + Ser203Asp + Tyr205Cys + Tyr213AspIle106Asp + Gly130Asp + Ser215Asp + Asn217GlnSer 98Glu + Thr102Asp + Ser203Asp + Thr214GlyGly 99Gln + Ser104Asp + Gly130Asp + Asn154GluSer104Glu + Gly126Ser + Gly130Asp + Asn217GluAsn 96Gln + Ser100Glu + Ser104Glu + Tyr205AspAsn 61Gln + Gly101Asp + Ser157Asp + Gly165GluThr102Ser + Val202Cys + Thr214Glu + Gly218GluAsn 61Gln + Ser104Glu + Gly127Glu + Gly165ProThr 65Gly + Tyr205Glu + Asn211Glu + Leu216CysGly 99Gln + Gly159Ser + Pro209Asp + Leu216AspAsp 59Glu + Ser158Asp + Tyr205Pro + Thr212GluAsp 59Glu + Ser131Glu + Thr212Glu + Leu216IleAla105Asp + Leu125Glu + Asn211Glu + Leu216IleThr 58Asp + Gly156Ser + Asn211Asp + Ser215GluSer157Glu + Gly159Ser + Gly165Pro + Thr219GluAsn162Asp + Tyr205Gln + Thr212Glu + Leu216AspSer100Glu + Met198Gln + Thr212Asp + Leu216GluGly 64Gln + Pro128Glu + Thr132Asp + Thr163GlyGly 62Gln + Pro128Asp + Thr132Asp + Leu216ThrGly101Asp + Ile106Met + Leu125Gly + Pro128GluThr 65Glu + Asn 96Glu + Gly156Asp + Leu216CysSer104Asp + Leu125Gln + Ser129Asp + Asn162GlnAsp 59Glu + Gly130Glu + Gly153Glu + Tyr205AsnSer129Asp + Ser155Asp + Tyr205Val + Leu216AspSer129Glu + Ser155Glu + Gln161Asn + Ser210GluGly 60Asp + Gly127Asp + Asn162Glu + Phe188AsnGly 62Gln + Ser 98Glu + Ser187Asp + Ser203GluSer104Asp + Gly130Gln + Ser187Asp + Ser203GluAsn 61Asp + Tyr103Ser + Thr212Asp + Thr219AspAsp 59Glu + Gly101Asp + Gly159Pro + Gln161GluGly 60Glu + Leu125Ser + Gly153Glu + Val202AspThr132Glu + Ser157Glu + Thr212Gly + Thr219AspGly101Glu + Leu125Cys + Gly130Glu + Thr219AspGly 60Asp + Pro128Glu + Ser157Asp + Tyr205CysPro128Glu + Asn154Gln + Ser157Asp + Ser203AspSer 97Asp + Ala105Asp + Ser203Asp + Thr212GlnSer100Asp + Ser158Glu + Tyr166Asp + Phe188ProTyr205Thr + Asn211Glu + Ser215Glu + Leu216Asn表35多環(huán)五點(diǎn)突變變異體Tyr103Cys + Gly126Gln + Gly156Glu + Val202Gln +Thr212GlyVal 94Met + Gly101Asn + Thr102Asn + Tyr205Leu +Gly218AspSer 98Asp + Tyr103His + Leu125Cys + Gly159Asn +Met198CysLeu 95Met + Gly101Pro + Tyr103Ala + Gly165Ser +Leu216AspThr 65Asp + Val 94Thr + Thr132Gly + Asn154Ser +Thr219GlyVal 94Thr + Thr132Gly + Ala186Gln + Tyr205Cys +Thr214GlnThr163Gln + Tyr205Cys + Thr212Pro + Leu216Met +Gly218GlnLeu 95Asp + Gly101Gln + Ile106Gln + Gly126Asn +Val202AlaGly 99Glu + Gly126Ser + Tyr166Ala + Asn211Gln +Leu216AsnGly 60Asn + Gly159Glu + Gln161Ser + Asn162Gln +Leu216SerAla186His + Val202His + Thr212Pro + Ser215Glu +Leu216ThrGly 99Gln + Leu125Ile + Gly127Ser + Tyr205Val +Thr212AspThr 58Gly + Val 94Gly + Tyr166Cys + Ser203Asp +Tyr205HisVal 94Asp + Gly 99Asn + Gln161Ser + Gly201Ser +Thr212AsnGly126Asn + Gly127Pro + Gly153Asp + Gly159Pro +Ile164AlaAsn 61Glu + Thr102Asn + Gly165Gln + Phel88Leu +Val202HisVal 94Cys + Gly156Ser + Val202Thr + Tyr205Ala +Leu216AlaTyr103Gln + Gly126Gln + Gly156Pro + Tyr166Asn +Ser215GluGly 99Asn + Val202Pro + Thr212Asn + Tyr213Gly +Ser215GluGly101Asp + Leu125Thr + Gln161Asn + Thr212Gly +Leu216AsnAsn 61Ser + Gly153Gln + Gly156Gln + Val202Gly +Asn211GlnAsn 61Gln + Ala105Thr + Tyr166Ile + Ala186Asn +Ser215AspAla105Gln + Leu125Gly + Thr163Ser + Val202Gln +Asn217AspPro200Ser + Val202Ser + Ser203Glu + Thr207Gly +Thr214GlyVal 94Asn + Gly130Pro + Tyr205Ser + Ser215Asp +Thr219ProThr163Asn + Ala199Gln + Tyr205Asn + Thr212Ser +Ser215GluLeu 95Met + Gly156Asn + Val202Cys + Leu216Gln +Gly218GluVal 94His + Gly 99Glu + Asn211Ser + Tyr213His +Leu216MetGly 64Ser + Val 94Gln + Ser157Asp + Tyr205Ser +Thr207ProIle106Ala + Gly165Asn + Ser190Glu + Ala199Asn +Tyr205AsnAsp 59Glu + Gly159Ser + Gly165Asn + Phe188Thr +Asn217GlnGly130Pro + Ser155Glu + Gly159Asn + Tyr205Pro +Thr212SerTyr103Gln + Ser155Glu + Val202Asn + Thr212Ser +Thr219AsnGly 60Pro + Val 94Ser + Ser100Glu + Tyr103Leu +Tyr205SerGly 60Gln + Leu 95Val + Pro128Gly + Ser158Asp +Tyr205ThrThr132Gln + Asn162Ser + Gly165Asp + Tyr205Gly +Tyr208CysGly 62Asn + Val 94Thr + Ala105His + Pro128Glu +Tyr205HisThr 58Pro + Gly156Pro + Val202Pro + Thr212Glu +Leu216ThrLeu 95Met + Gly 99Gln + Tyr103Cys + Asn154Gln +Thr212GlnSer 98Glu + Tyr205Asn + Pro209Gln + Tyr213His +Thr219SerGly 62Ser + Val 94His + Tyr103Gln + Ser187Glu +Phe188TyrGly 62Gln + Asn 96Ser + Gly165Glu + Val202Gly +Tyr205ThrGly 60Pro + Ile106Thr + Ser160Glu + Thr214Gln +Leu216IleSer100Asp + Ala105Thr + Tyr205Ile + Thr214Gly +Leu216CysLeu 95Pro + Ala105Asn + Phe188Leu + Thr212Pro +Leu216ValAsp 59Glu + Gly 60Ser + Gly127Pro + Tyr205Thr +Leu216AlaAla105Asn + Ser131Asp + Thr163Pro + Leu216His +Gly218AsnLeu 95Cys + Tyr103Met + Ile106Cys + Ala186Asp +Tyr205MetLeu125Cys + Tyr205His + Leu216Asn + Asn217Ser +Thr219AsnAsn 96Ser + Gly153Glu + Asn154Gln + Ala186Gln +Thr212AsnGly101Gln + Ala105Pro + Gly130Asp + Asn154Gln +
Gly156AsnThr 58Gly + Gly159Pro + Thr163Ser + Tyr205Gln +Thr212AsnTyr205Pro + Thr212Asn + Tyr213Gln + Ser215Asp +Leu216GlnGly130Pro + Gly153Pro + Gln161Glu + Tyr205Met +Thr212GlnAla105Gly + Ser157Asp + Tyr166Asn + Val202Gln +Thr212SerThr 58Gln + Asn 96Ser + Ser203Asp + Thr207Gly +Leu216SerSer158Glu + Val202Ser + Thr212Asn + Thr214Gln +Thr219AsnAsn 61Ser + Ser100Glu + Tyr205Leu + Thr212Ser +Asn217SerGly 60Gln + Gly 99Glu + Thr212Ser + Tyr213Gln +Leu216ThrLeu125Asn + Ser129Asp + Asn162Ser + Ala186His +Tyr205GlnGly 64Gln + Leu 95Glu + Ala199Asn + Val204His +Leu216MetLeu 95His + Gly101Pro + Val204His + Tyr205Asn +Leu216GlnThr163Ser + Gly165Asn + Pro209Gly + Tyr213His +Leu216AspGly127Gln + Ser131Asp + Thr132Pro + Tyr205Cys +Leu216AsnAsn 61Gln + Gly101Pro + Ser190Asp + Tyr205Pro +Thr214GlyGly126Asn + Pro200Gly + Val204Gly + Tyr205Asp +Thr212AsnAsn 61Gln + Val 94Gln + Gly126Asp + Tyr205Val +Thr212GlnGly 60Gln + Ile106Pro + Ser131Glu + Ile164Ala +Val202ProGly101Ser + Tyr103Glu + Ser104Asp + Gly165Asn +Pro209GlnThr 58Glu + Asp 59Glu + Tyr166His + Val202Cys +Thr219AsnGly126Pro + Ala186His + Tyr205Asp + Tyr213Val +Ser215AspGly 60Asp + Ser 97Glu + Ala105Pro + Leu125Cys +Gly153ProAsn154Ser + Pro209Ser + Thr214Glu + Ser215Asp +Leu216GluThr163Pro + Ser203Glu + Val204Thr + Tyr205Cys +Ser215AspGly 62Ser + Met198Gln + Ser203Glu + Ser215Asp +Leu216HisGly156Asn + Ser203Asp + Tyr205Met + Ser215Asp +Gly218ProGly 60Gln + Pro128Ser + Ser203Glu + Ser215Glu +Leu216ProTyr166Cys - Tyr205Glu + Pro209Asn + Ser215Glu +Leu216AspTyr103Leu + Leu125Gly + Gly153Glu + Ser190Glu +Leu216IleGly 99Pro + Ile106Thr + Gly153Asp + Ser155Asp +Ser190AspThr 65Pro + Leu125His + Ser210Glu + Thr212Asp +Tyr213AlaGly 64Ser + Thr 65Ser + Gly 99Asn + Pro209Glu +Asn211GluLeu 95Asn + Gly101Asn + Asn154Asp + Gly156Glu +Tyr166GlnSer155Glu + Thr163Asp + Tyr205Cys + Thr212Pro +Leu216ValGly159Pro + Thr163Ser + Ser203Asp + Tyr205Asp +Thr212SerThr102Gln + Gly126Asp + Gly153Asp + Phe188Thr +Tyr205HisAsn 61Ser + Val202Glu + Ser203Glu + Tyr205Asp +Thr212ProThr 58Gln + Thr102Pro + Tyr213Cys + Ser215Glu +Asn217AspGly 60Asp + Gly 62Glu + Thr 65Glu + Gly126Asn +Leu216ThrAla105Gly + Gly126Pro + Gly153Glu + Leu216Thr +Gly218AspTyr103Gln + Ala105Thr + Val202Asp + Tyr205His +Ser215GluVal202Glu + Tyr205Thr + Pro209Asn + Ser215Asp +Leu216MetVal 94Asn + Ala105Asn + Ile106Val + Ser203Glu +Gly218AspAsn 96Ser + Ser203Asp + Tyr205Ile + Thr212Gln +Gly218AspAsn 61Asp + Gly 62Gln + Thr 65Asp + Gly126Asn +Gly159ProGlyI53Gln + Ala186Glu + Tyr205Thr + Leu216Thr +Asn217AspLeu125Asp + Gly126Asp + Ala186Ser + Gly218Glu +Thr219ProThr 58Glu + Thr 65Glu + Val 94Gln + Gly101Pro +Gly165GlnGly153Glu + Ser158Glu + Thr163Glu + Gly165Pro +Leu216MetGly159Asp + Thr163Glu + Tyr166Thr + Ala186His +Pro200SerLeu125Thr + Gly130Ser + Asn154Asp + Ser157Asp +Ala186GluAsp 59Glu + Asn 96Glu + Gly101Glu + Pro128Ser +Thr212AsnAsp 59Glu + Asn 96Asp + Gly101Asp + Tyr166Asn +Thr219AsnGly 99Glu + Gly101Glu + Gly127Glu + Tyr166Leu +
Asn217GlnAla186Gln + Ser187Asp + Leu216Pro + Gly218Glu +Thr219GlnThr 58Pro + Leu 95Ile + Gly126Glu + Gly153Glu +Phe188AspThr 58Glu + Asn 96Glu + Gly 99Asp + Phe188Met +Gly218GlnGly 62Glu + Asn162Ser + Val202Met + Thr212Asp +Leu216MetGly 62Pro + Gly 99Gln + Ser104Glu + Ser131Asp +Tyr213ThrGly 62Glu + Gly130Ser + Tyr205Glu + Ser215Glu +Leu216AsnGly126Gln + Gly127Glu + Asn154Glu + Tyr213Pro +Leu216HisSer129Glu + Asn154Glu + Asn162Ser + Gly165Asp +Leu216CysThr 58Gly + Leu125Asn + Gly156Asp + Gln161Glu +Tyr205GlyGly126Ser + Gly153Gln + Gly156Glu + Gln161Asp +Tyr205ProThr163Gln + Val202His + Tyr205Asp + Pro209Gly +Thr212AspGly 62Gln + Val202Met + Asn211Asp + Thr212Asn +Thr214GluThr102Gln + Gly126Pro + Thr212Asp + Ser215Glu +Leu216AsnGly101Ser + Gln161Asn + Thr212Glu + Ser215Asp +Leu216GlnLeu125Gln + Ala186Glu + Val202Cys + Ser203Asp +Ser215AspSer129Glu + Ser155Glu + Thr163Asp + Ala186Ser +Tyr208SerThr 58Ash + Leu125Glu + Pro128Glu + Thr163Ser +Gly165ProAsn 61Glu + Gly 62Asp + Gly130Ser + Asn211Asp +Thr212GlyThr 65Gln + Asn154Asp + Ser215Glu + Leu216His +Asn217GluAsp 59Glu + Ser 97Glu + Thr102Gln + Ser210Asp +Leu216ThrGly153Glu + Ile164Thr + Val202Gln + Ser203Glu +Asn217GluVal 94His + Leu125Cys + Gly126Ser + Ser155Glu +Gln161GluSer187Glu + Tyr205Thr + Thr212Asn + Tyr213Gly +Asn217GluGly 99Ser + Gly130Glu + Asn162Asp + Thr163Asp +Tyr205AsnPro128Gln + Gly156Asp + Ala186Glu + Ser203Glu +Tyr213GlnGly 62Asp + Gly156Asn + Ala186Pro + Thr212Glu +Leu216AspGly126Ser + Tyr166Gly + Ser203Asp + Pro209Asp +Ser215AspTyr103Val + Pro128Asp + Gly153Asn + Asn162Ser +Ser190AspGly130Pro + Gly156Asn + Gln161Asp + Thr163Glu +Ser187GluLeu125Pro + Ser203Asp + Pro209Gln + Thr212Glu +Leu216AspLeu 95Asp + Gly126Glu + Pro128Gly + Gly159Gln +Ala186HisGly 62Ser + Ser190Asp + Thr214Gly + Ser215Asp +Gly218AspGly 64Asn + Tyr103Ile + Leu125Asp + Ser203Asp +Asn217GluGly126Ser + Gln161Asp + Asn162Ser + Thr214Glu +Ser215GluIle106Leu + Gly126Asp + Gly127Glu + Gly130Gln +Val202GluAsp 59Glu + Thr102Ser + Ala105Gly + Gly159Glu +Ser160GluLeu 95Asp + Asn 96Asp + Tyr166Leu + Val204Ala +Gly218GluGly130Glu + Ser131Asp + Gly153Gln + Ser158Glu +Ala199ProGly 60Glu + Asn 61Asp + Gln161Asn + Pro209Gln +Ser215GluThr 65Asn + Gly 99Gln + Asn154Asp + Pro209Asp +Ser210GluAsn154Glu + Ser155Asp + Tyr213Thr + Ser215Glu +Gly218ProSer100Asp + Asn154Glu + Ser155Asp + Pro209Ser +Thr214GlyGly 62Asn + Gly153Asp + Asn211Glu + Thr212Asp +Gly218GlnTyr103His + Ser129Glu + Tyr205Pro + Asn211Asp +Thr212GluSer129Asp + Asn162Gln + Tyr205Leu + Asn211Asp +Thr212AspSer100Glu + Gly153Pro + Thr163Pro + Val202Glu +Ser203GluThr 58Asp + Thr 65Pro + Val202Glu + Ser203Asp +Leu216AsnThr 65Pro + Gly126Asn + Ser157Glu + Ser158Asp +Ser215AspGly101Asn + Ser157Asp + Ser158Glu + Ser215Glu +Leu216IleAsn 96Glu + Ser 97Glu + Gly159Asn + Phe188His +Ser210GluThr 65Asn + Ser 98Asp + Gly159Asn + Ser215Asp +Leu216AspAsn 96Glu + Pro128Ser + Tyr205Cys + Ser215Glu +Leu216GluIle106Gly + Ser155Glu + Thr212Asn + Ser215Glu +
Leu216AspLeu125Glu + Thr163Asn + Val202Thr + Ser215Asp +Leu216GluGly101Gln + Gly130Glu + Tyr205Ala + Ser215Glu +Leu216GluPro128Gly + Ser210Asp + Thr212Ser + Ser215Glu +Leu216GluSer104Asp + Ala105Glu + Met198Pro + Ser203Glu +Leu216SerSer 97Glu + Ser 98Glu + Ala105Gly + Gly153Asp +Asn217GlnGly153Gln + Ser210Asp + Asn211Asp + Thr212Ser +Ser215AspThr 65Glu + Leu125Cys + Gly126Ser + Val202Glu +Ser215AspThr102Gln + Gly165Asn + Ser203Glu + Tyr205Glu +Pro209AspLeu 95Glu + Asn 96Gln + Tyr205Glu + Pro209Ser +Ser215GluGly126Asn + Tyr205Asp + Ser210Asp + Ser215Glu +Leu216MetGly 62Gln + Leu 95Pro + Ser158Asp + Tyr205Asp +Ser215GluAsn 61Glu + Gly101Glu + Gly126Ser + Leu216Met +Thr219SerGly156Asp + Ala186Gln + Ser190Glu + Ser203Glu +Thr212ProGly 60Glu + Ser 97Glu + Gly 99Asn + Asn162Glu +Asn217SerThr 58Pro + Ser160Asp + Ser190Asp + Val202Asn +Thr212AsnGln161Asn + Ser203Asp + Asn211Glu + Tyr213Cys +Ser215GluPro128Glu + Gly165Gln + Ala186His + Ser203Glu +Ser215GluGly101Pro + Thr132Ser + Ser158Glu + Ser203Glu +Ser215GluPro128Ser + Ala186Gly + Ser203Glu + Ser210Glu +Ser215AspAsn 61Gln + Ser158Asp + Ala186Ser + Ser203Asp +Ser215GluLeu 95Val + Ser100Asp + Tyr166Met + Ser203Glu +Ser215AspGly153Glu + Ala186Gln + Ser203Glu + Ser215Asp +Thr219GlySer160Asp + Ser203Glu + Val204Gln + Thr212Pro +Ser215GluThr 58Pro + Leu 95His + Ser155Asp + Ser203Asp +Ser215AspAsn154Gln + Ser157Asp + Ser203Glu + Thr212Ser +Ser215GluLeu 95Gln + Ser100Asp + Ser203Glu + Tyr205His +Ser215GluAsn154Asp + Tyr166His + Ser203Asp + Tyr205Ala +Ser215GluSer 98Asp + Ser203Glu + Asn211Ser + Ser215Asp +Leu216SerAsn162Glu + Ser203Asp + Tyr208Asn + Thr212Ser +Ser215AspGln161Asp + Thr163Gln + Ser203Glu + Ser215Glu +Leu216ThrLeu 95Cys + Ser129Glu + Ser203Asp + Asn211Gln +Ser215AspThr132Asn + Asn154Glu + Ser203Asp + Thr212Asn +Ser215AspPro128Gln + Ser131Asp + Gly156Asn + Ser203Glu +Ser215AspThr 58Glu + Asn 61Ser + Gly 99Pro + Ser203Glu +Ser215AspSer100Asp + Gly153Ser + Ser203Asp + Tyr205Val +Ser215GluAla105Glu + Ser203Asp + Thr212Asn + Ser215Glu +Gly218AsnThr102Asn + Ser104Asp + Gly127Asn + Ser203Asp +Ser215AspGly 62Asp + Leu125Thr + Gly153Glu + Gly165Ser +Ser190GluGly153Glu + Phe188His + Ser190Glu + Ser203Glu +Leu216GlyThr 58Asp + Phe188Asn + Ser203Glu + Tyr205Leu +Asn217AspThr 65Gly + Thr102Ser + Gly126Asp + Thr212Ser +Gly218AspAsp 59Glu + Thr163Glu + Ser190Glu + Thr212Gln +Leu216SerTyr166Val + Ser203Glu + Ser210Asp + Thr212Ser +Leu216AspGly159Asp + Ser203Glu + Thr207Pro + Thr212Asn +Leu216AspSer157Asp + Asn162Gln + Gly165Gln + Ser203Glu +Leu216AspLeu 95Thr + Ser131Glu + Val202Ser + Ser203Asp +Leu216GluSer100Asp + Gly127Asn + Ser203Asp + Tyr208Val +Leu216AspThr102Gly + Ser129Glu + Ser203Asp + Tyr205Ala +Leu216AspThr 65Gln + Ser 98Asp + Thr102Pro + Gly126Asp +Gly165AspThr 65Asn + Gly127Glu + Ser129Asp + Tyr205Ile +Ser215GluGly 60Asn + Ser203Glu + Tyr205Val + Ser210Asp +Thr212GluVal 94Cys + Ser 98Glu + Pro209Ser + Ser210Asp +Thr212GluGly 60Gln + Tyr103Asn + Pro128Glu + Ser157Asp +
Gly159AspSer 98Glu + Ser100Asp + Ser203Glu + Thr212Gln +Leu216ThrGly 60Glu + Thr 65Asp + Thr132Gln + Asn154Ser +Leu216AspAsn 96Asp + Ser100Asp + Thr102Pro + Tyr205Asp +Thr212GlnThr102Gly + Phe188His + Gly201Asn + Tyr205Glu +Gly218AspAsn154Gln + Ser155Asp + Tyr205Gln + Pro209Glu +Thr212AspTyr103Val + Gly153Asn + Pro209Asp + Thr212Asp +Gly218GluThr 65Glu + Ile106Met + Leu125His + Tyr205Leu +Ser215AspLeu125Met + Gly156Asp + Tyr166Ser + Ser187Glu +Ser215AspThr 58Glu + Gly 60Glu + Ser131Glu + Tyr205Val +Gly218ProThr 58Asp + Gly 60Asp + Asn 61Ser + Asn162Gln +Ser203AspSer157Glu + Ser203Asp + Tyr205Asp + Thr212Ser +Leu216CysThr 58Asn + Thr102Gln + Ser155Glu + Ser157Asp +Thr212GluAla105Gln + Ser155Asp + Ser157Asp + Ser203Asp +Thr212GlyThr163Glu + Val202Asp + Tyr205Cys + Leu216Pro +Gly218GluThr 58Gln + Asn 61Glu + Ile106Thr + Pro209Asp +Ser215AspGly 62Glu + Ser 97Glu + Asn154Ser + Tyr205Asp +Tyr213ValLeu125Gln + Gly130Asp + Leu216Glu + Gly218Asp +Thr219GlyGly126Ser + Ser131Glu + Tyr205Ala + Leu216Asp +Gly218GluIle106Ala + Ser160Glu + Asn162Glu + Ala186Gly +Ser203AspAsp 59Glu + Thr 65Asp + Asn162Asp + Leu216Ile +Asn217SerAsn 96Gln + Thr102Pro + Gly127Glu + Ala186Pro +Ser190AspGly 99Asn + Leu125Thr + Ser157Glu + Gly165Glu +Thr212GlnVal 94His + Gly126Asn + Ser157Glu + Gly165Glu +Tyr205ValAsp 59Glu + Asn 61Ser + Val202Asn + Thr207Pro +Asn211AspAsp 59Glu + Tyr103Ala + Ser155Glu + Pro209Glu +Thr212AsnGly101Gln + Gly127Glu + Gly153Ser + Asn154Glu +Ser158AspSer 97Glu + Thr212Ser + Ser215Asp + Leu216Cys +Asn217GluGly 99Gln + Ser157Glu + Ser215Asp + Leu216Ser +Asn217AspSer131Glu + Gly159Asn + Tyr205Met + Ser215Glu +Asn217GluVal 94Ala + Gly101Asp+ Gly156Gln + Ser215Asp +Asn217AspAsn 61Asp + Asn 96Glu + Pro209Ser + Ser215Glu +Leu216AlaAsn 61Glu + Asn 96Glu + Gly127Ser + Ser203Glu +Thr214SerGly 99Pro + Ser100Asp + Thr102Glu + Ala105Gly +Asn211GluSer155Glu + Ala186Pro + Tyr205Asp + Leu216Cys +Gly218GluPhe188Asp + Ser203Asp + Tyr213Ser + Thr214Glu +Gly218AsnSer129Asp + Thr163Gly + Gly165Glu + Ser203Asp +Tyr205IleSer104Asp + Ser158Glu + Asn162Glu + Val202Gln +Leu216ValSer 98Asp + Ser158Asp + Asn162Glu + Leu216Ile +Thr219GlyPro128Gly + Ser158Asp + Asn162Glu + Val202Gln +Asn217GluThr102Glu + Ala105Asn + Ser129Asp + Tyr208Thr +Leu216GlnThr 65Asn + Ser157Asp + Asn162Glu + Ser203Asp +Tyr205GlyAla105Gly + Ser157Asp + Ser160Glu + Ala186His +Ser210AspSer 98Glu + Gly153Asp + Val202Ser + Tyr205Asn +Gly218AspGly 62Asp + Gly 64Asn + Gly 99Pro + Gly127Asp +Gly153GluThr 58Ser + Val 94Met + Ser104Glu + Gly153Asp +Tyr166AspAsn 61Asp + Thr 65Gly + Ala186Gln + Ser210Asp +Thr214AspGly127Asn + Val202Asp + Ser210Glu + Ser215Glu +Asn217SerGly 62Asn + Gly101Asp + Tyr103Met + Ser104Glu +Ser203GluGly159Asp + Asn162Glu + Ile164Ala + Thr212Asn +Ser215AspSer 98Asp + Gly101Glu + Asn154Gln + Tyr205Gln +Ser210AspLeu 95Glu + Tyr166Asn + Phe188Met + Ser203Glu +Thr214GluGly101Pro + Ser129Glu + Ser203Asp + Thr214Glu +Gly218GlnAsn 96Gln + Asn162Glu + Gly165Glu + Val202Asp +
Tyr205MetThr 65Gln + Val 94Asp + Ser104Glu + Leu125Asn +Thr212AspThr102Pro + Ser155Asp + Ser187Glu + Tyr205Val +Ser215GluAsn 96Glu + Thr102Gln + Ala186Asp + Phe188Gln +Ser190AspThr 58Glu + Asn 61Glu + Gly 64Asn + Ala105Pro +Asn217GluVal 94Ala + Gly126Asp + Asn154Glu + Tyr166Pro +Thr212GluThr 58Asn + Asn 61Ser + Gly130Glu + Phe188Glu +Asn217AspThr 58Asp + Ser 97Asp + Ile106Leu + Gly153Gln +Gly156AspThr 58Asp + Ser 97Glu + Thr102Pro + Ala105Ser +Ser158AspTyr103Glu + Gly127Glu + Asn154Gln + Ser157Asp +Thr163SerAsn 96Gln + Pro128Glu + Gly130Gln + Ser131Glu +Ser157AspGly127Glu + Gly153Asn + Pro209Asp + Thr214Glu +Thr219SerSer100Asp + Ile106Met + Pro128Asn + Ser190Asp +Gly218GluThr 58Asp + Asn 96Asp + Gly127Asp + Tyr166Ala +Ala186SerAsn 96Glu + Thr163Pro + Phe188Asp + Gly201Gln +Val202GluSer 98Glu + Gly127Asp + Ser131Glu + Val202Ala +Thr214SerGly 99Asn + Gly127Glu + Ser131Glu + Gly156Gln +Asn211GluVal 94Asp + Pro128Asp + Ser131Asp + Tyr205Thr +Leu216SerGly101Glu + Gly126Ser + Gly127Asp + Tyr205His +Ser215AspSer160Asp + Tyr205Glu + Asn211Gln + Thr212Asp +Thr219GlyGly 62Asp + Gly127Gln + Gly153Pro + Ser187Glu +Leu216GluGly 60Asp + Gly127Asp + Gly159Pro + Ser210Asp +Gly218AsnAsn 61Ser + Gly 99Asp + Thr102Asp + Ser203Asp +Thr212GlnGly126Pro + Ser160Asp + Tyr205Met + Thr212Glu +Ser215AspThr 65Ser + Gly127Asp + Thr132Ser + Thr212Asp +Ser215AspAsn154Ser + Ser187Glu + Thr212Glu + Tyr213Gln +Ser215GluSer129Asp + Gly153Glu + Ser158Glu + Gly165Asn +Leu216ThrIle106His + Leu125Pro + Ser129Glu + Ala186Glu +Ser203GluGly 60Pro + Gly 62Gln + Tyr205Thr + Ser215Glu +Thr219GluAsp 59Glu + Val 94Pro + Gly130Gln + Ser187Asp +Thr219AspGly153Ser + Ser155Glu + Ser160Asp + Asn162Ser +Thr212ProSer155Asp + Gln161Glu + Ala186Thr + Thr214Gly +Ser215GluSer104Asp + Gly130Asn + Ser187Glu + Thr212Gln +Asn217GluAla105Asn + Ser203Glu + Thr212Glu + Asn217Ser +Thr219AspLeu125His + Gly165Gln + Phe188Asp + Ser203Glu +Tyr205LeuAsn 96Asp + Ser104Asp + Gly201Pro + Val202His +Leu216SerGly 62Asp + Tyr103Val + Ser190Glu + Asn211Glu +Thr219SerGly 62Asp + Ser190Glu + Val202Ser + Asn211Glu +Thr214GlnSer100Asp + Pro128Asp + Tyr205Ile + Thr212Gln +Leu216HisThr132Gly + Gly156Asn + Gly159Glu + Ser190Asp +Ser203GluAsn 96Ser + Ser 98Asp + Gly159Asp + Ser190Asp +Leu216MetVal 94Asp + Ser 98Glu + Thr132Asn + Phe188Cys +Thr219AspGly101Ser + Tyr103Glu + Gly153Glu + Tyr166His +Ser187AspGly 99Ser + Ser160Asp + Ser190Glu + Val202Glu +Tyr205SerAsn 61Asp + Gly 99Gln + Ala186Glu + Thr212Gln +Leu216AspAla186His + Phe188Gln + Ser190Glu + Pro200Ser +Asn217AspAsn 61Ser + Thr 65Asp + Ser129Glu + Tyr205Glu +Tyr213GlyAsn 61Asp + Gly 62Ser + Ser203Asp + Asn211Gln +Tyr213GluThr102Asn + Gly130Asp + Tyr166Ser + Tyr205Asp +Gly218AspAsn 61Glu + Asn154Gln + Gln161Asn + Tyr205Asp +Gly218GluGly 99Glu + Gly127Asp + Thr163Pro + Tyr213Ile +Asn217GluGly101Gln + Gly126Asp + Ser131Asp + Gly156Asn +Ser203GluThr102Asp + Gly130Glu + Pro209Asn + Thr212Ser +Ser215AspThr 58Ser + Ser100Glu + Ser104Glu + Ile164Leu +
Ser215GluAsp 59Glu + Ile106Val + Gly127Glu + Tyr166Asn +Ser190GluGly 99Ser + Pro128Asp + Gly156Asp + Tyr205Cys +Asn217AspThr 65Asp + Leu 95Glu + Gly 99Pro + Ser158Asp +Tyr205GlnSer 97Asp + Ala105His + Ser155Glu + Tyr205Asn +Pro209GluGly 62Asn + Thr 65Asp + Ser 97Glu + Ser131Glu +Leu216GlyGly130Pro + Gly153Glu + Asn154Gln + Gln161Asp +Pro209AsnGly159Ser + Tyr166Glu + Tyr205Asp + Thr212Pro +Thr219GluGly 62Asp + Leu 95Cys + Ser100Glu + Thr163Pro +Ser187AspThr 58Gly + Gly159Glu + Ser187Asp + Ser203Glu +Thr219AsnThr 58Pro + Tyr166Asp + Val202Met + Pro209Asp +Ser215AspLeu 95Asp + Thr102Pro + Ala105Gly + Gly127Glu +Leu216GluPro128Glu + Gln161Asp + Phe188Gln + Ser203Glu +Asn217GlnLeu125Pro + Pro128Glu + Thr132Asn + Gln161Glu +Leu216AspSer 97Glu + Thr102Asp + Val202Thr + Ser215Glu +Gly218SerAsp 59Glu + Ser100Asp + Phe188Gln + Thr212Ser +Ser215GluGly101Gln + Thr102Asn + Gly130Glu + Thr163Glu +Ser210AspAsn 61Glu + Gly101Asn + Ser129Glu + Tyr205Thr +Leu216GluLeu 95Glu + Ile164Val + Thr212Glu + Tyr213Gly +Leu216AspThr 58Gln + Gly126Glu + Gly156Gln + Thr212Asp +Leu216AspLeu125Met + Gly126Ser + Ser131Asp + Thr212Asp +Leu216AspGly 60Asn + Asn 96Gln + Ser104Glu + Thr212Asp +Leu216GluThr 65Asp + Gly126Gln + Ser158Asp + Gly165Asp +Leu216ValSer158Asp + Gly165Glu + Ser203Asp + Leu216Ser +Gly218GlnGly101Asp + Leu125Gly + Pro128Glu + Ala186Gly +Thr219GlnThr 65Glu + Asn 96Glu + Tyr103Val + Ser155Glu +Met198AlaThr 65Asp + Asn 96Glu + Leu125His + Asn162Glu +Tyr166HisAsn 61Ser + Ser100Asp + Phe188Asp + Ser203Glu +Gly218SerSer104Glu + Gly156Gln + Phe188Asp + Ser203Glu +Thr212GlyGly 99Asp - Tyr205Asp + Pro209Asp + Thr212Pro +Tyr213MetThr102Asp + Asn154Ser + Tyr166Glu + Ser203Asp +Gly218SerGly101Glu + Ser129Asp + Ser157Asp + Thr214Ser +Leu216AsnAsp 59Glu + Gly101Asp + Gly127Ser + Gly159Pro +Gln161GluGly 60Asn + Gly127Asp + Thr132Asp + Asn162Gln +Ser203GluThr 58Asn + Gly 60Asp + Gly130Glu + Val202Ser +Thr212GluSer100Asp + Tyr166Asp + Ala186His + Tyr205His +Leu216ThrSer 97Asp + Gly101Ser + Ala105Glu + Tyr205Met +Ser215GluSer 97Glu + Ala105Glu + Leu125Ala + Ser158Asp +Tyr205CysSer100Glu + Ile106His + Gly127Ser + Ser158Glu +Tyr166GluThr102Asn + Gly130Ser + Ser131Asp + Asn211Asp +Ser215GluGly127Gln + Ser131Glu + Asn211Asp + Tyr213His +Ser215GluSer129Glu + Ser158Glu + Val202Gly + Ser215Asp +Leu216GlnLeu125pro + Pro128Ser + Ser157Glu + Ser203Glu +Gly218SerThr 65Gln + Gln161Glu + Ser210Glu + Thr212Gln +Ser215GluVal 94Met + Gly101Glu + Ala105Asn + Ala186His +Thr212Asp表36多環(huán)六點(diǎn)突變變異體Asn 61Ser + Gly 64Gln + Gly101Asp + Thr132Gly +Gly165Gln + Tyr205ProAsn 96Ser + Gly101Ser + Ala105Gly + Asn154Glu +Tyr166Thr + Tyr205HisVal 94Ala + Leu 95Thr + Gly126Gln + Pro209Ser +Ser215Asp + Leu216GlyThr 65Gln + Leu125Asn + Asn162Asp + Tyr205Gln +Pro209Gly + Leu216MetThr163Gln + Tyr205Cys + Thr212Pro + Leu216Met +Asn217Gln + Gly218GlnGly 60Gln + Val 94Cys + Asn 96Ser + Gly127Pro +Gly159Asp + Asn162SerGly 99Ser - Gly126Asn + Gly127Pro + Gly153Asp +3Ly159Prp - Ile164AlaTal-94Cys - Pro126Asp - Gly156Ser + Val202Thr +Tyr205Ala + Leu216AlaAsn 61Gln - Ala105Thr + Gly153Gln + Gly156Gln +Tyr166Ile + Asn211GlnGly 60Asn + Ser104Asp + Ala105Asn + Gln161Ser +Thr163Ser + Tyr205AlaGly 64Ser + Val 94Asn + Gly101Asn + Leu125Asn +Gly130Asp + Leu216CysGly159Pro + Gly165Pro + Ala186Thr + Gly201Pro +Thr212Pro + Leu216SerGly 99Asn + Gly159Pro + Ser203Glu + Tyr205Leu +Asn211Gln + Thr212SerThr 58Gln + Leu125Pro + Thr132Asp + Gly153Asn +Tyr213Ala + Leu216GlyIle106Ala + Asn154Glu + Asn162Gln + Met198Thr +Ala199Asn + Tyr205AsnAsp 59Glu + Gly159Ser + Gly165Asn + Phe198Thr +Pro200Gln + Asn217GlnGly 99Ser + Gly126Asn + Thr132Gly + Ser187Asp +Val202Ser + Tyr205HisTyr103Gln + Gly130Pro + Ser155Glu + Gly159Asn +Tyr205Pro + Thr212SerGly 60Pro + Val 94Ser + Ser100Glu + Tyr103Leu +Tyr205Ser + Thr219ProThr 58Gly + Leu125Ala + Val202Cys + Tyr205Asn +Ser215Asp + Leu216AlaVal 94Thr + Ser104Glu + Gly130Pro + Thr212Pro +Leu216Asn + Gly218SerSer 97Glu + Thr132Gly + Pro200Gly + Tyr205Ala +Asn211Ser + Tyr213GlnGly 62Asn + Leu 95Asp + Tyr166Val + Tyr205Cys +Asn211Ser + Leu216ValGly156Asn + Gly165Pro + Gly201Asn + Tyr213Ala +Thr214Gln + Asn217GluGIy 62Ser + Ser 98Asp + Gly126Ser + Tyr205Pro +Thr212Ser + Tyr213ThrAla105Gln + Gly127Ser + Gln161Asp + Thr163Gly +Tyr205Cys + Leu216ProVal 94Ala + Gly130Gln + Ala186His + Ser203Asp +Tyr205Cys + Thr214GlnThr 65Gln + Leu125Gly + Ser187Asp + Phe188Gln +Val202Asn + Thr212AsnThr 58Ser + Ile106Pro + Thr132Gly + Gln161Asn +Tyr205Asn + Thr212AspSer100Asp + Ala105Thr + Leu125Pro + Tyr205Ile +Thr214Gly + Leu216CysThr102Ser + Gly127Pro + Pro128Gly + Ser203Glu +Tyr208His + Tyr213ThrThr102Ser + Gln161Glu + Asn162Asp + Thr163Gln +Ala186His + Phe188IleGly 62Pro + Pro128Asp + Ser129Asp + Gly156Asn +Tyr213Cys + Gly218AsnThr 58Gln + Gly127Pro + Ala186Gln + Val202Asp +Ser203Asp + Leu216ValGly156Ser + Phe188Gly + Ser203Glu + Tyr213Gln +Ser215Asp + Leu216AspGly101Gln + Ala105His + Asn162Gln + Ala186Gly +Ser203Glu + Ser215GluThr 58Pro + Thr163Asn + Ser203Glu + Tyr205Ile +Ser215Glu + Leu216GlnThr 65Asn + Gly127Glu + Gly156Asn + Tyr166Glu +Phe188Met + Thr212ProLeu 95Asp + Ser100Glu + Gly101Gln + Ile106Gln +Ile164Leu + Val202AlaTyr103Pro + Gly127Glu + Ser129Asp + Gly130Glu +Asn162Gln + Leu216HisGly 60Gln + Gly165Ser + Ser203Glu + Tyr205Asp +Thr214Glu + Ser215AspLeu 95Asn + Leu125Pro + Ser203Asp + Tyr205Thr +Thr214Glu + Ser215AspGly 62Asn + Tyr103Ile + Leu125Cys + Gly126Glu +Gly153Asp + Tyr166CysGly 99Ser + Pro128Glu + Gly130Asp + Phe188Asn +Leu216Gly + Gly218ProAsp 59Glu + Gly 60Asp + Thr 65Asp + Asn162Ser +Thr163Gln + Tyr166GlnPhe188Glu + Tyr205Thr + Thr212Asn + Tyr213Gly +Asn217Glu + Gly218AspGly 62Gln + Ser203Glu + Tyr205Ser + Thr212Ser +Ser215Asp + Gly218AspSer187Glu + Ser190Asp + Tyr205Val + Pro209Ser +Thr214Ser + Thr219SerThr 58Ser + Asp 59Glu + Asn 96Asp + Asn154Ser +Ala186His +Leu216SerThr 65Gly + Thr102Gly + Leu125Asp + Gly165Asp +Ala199Ser + Val202ThrLeu125Asn + Ser158Glu + Gly159Asp + Ser190Glu +Thr212Gly + Leu216ValThr 58Pro + Asn 61Asp + Val 94Glu + Asn 96Asp +Gly126Ser +Gly165ProAla186Glu + Val202Glu + Tyr205Ala + Tyr213Val +Ser215Asp + Asn217GluTyr103Val + Ser203Glu + Tyr205Asp + Thr212Glu +Ser215Asp + Leu216IleGly 62Ser + Ile106Leu + Pro128Ser + Asn154Asp +Ser157Glu + Thr214SerVal 94Asp + Gly 99Asp + Pro128Gln + Ala199Gln +Tyr205Gln + Leu216IleAsn 61Gln + Thr102Gln + Gly126Asp + Ser155Glu +Ser190Asp + Val202SerLeu125Asp + Gly126Asp + Ala186Ser + Tyr205Gln +Gly218Glu + Thr219ProAsn 61Glu + Ser100Asp + Thr102Asn + Gly165Gln +Phe188Leu + Val202HisTyr103Gly + Tyr205Leu + Thr212Glu + Ser215Glu +Leu216Glu + Asn217AspGly 99Pro + Ser104Glu + Ile106Thr + Ser131Asp +Gly201Gln - Thr214SerLeu 95Ile + Gly126Glu + Gly153Glu + Ser187Asp +Phe166Asp - Asn211GlnGly 62Asn + Leu 95Ser + Gly153Ser + Tyr205Asp +Thr212Glu + Leu216AspAla105Gly + Ile106Cys + Ser155Asp + Tyr205Asp +Ser215Asp + Leu216AspTyr103Ala + Ser155Glu + Tyr166Pro + Ser203Glu +Tyr205Glu + Ser215GluPro128Gly + Asn162Glu + Gly165Asn + Ser203Glu +Tyr205Glu + Ser215GluGly 99Pro + Leu125Gln + Gly130Asn + Ala186Asp +Phe188Ser - Ser203GluIle106Asn + Gly156Glu + Gly159Asn + Phe188Asp +Ser215Glu + Gly218AspGly156Pro + Asn162Asp + Ser203Glu + Tyr205Gln +Ser215Asp + Asn217GluGly153Asn + Ser155Glu + Ser157Asp + Ser190Glu +Thr212Asp + Leu216IleLeu 95Glu + Gly 99Glu + Leu125Glu + Pro128Gly +Asn154Glu + Thr219GlyGly 60Asp + Ile106Met + Asn162Ser + Ser203Glu +Tyr205Asp + Leu216AspSer 97Asp + Asn162Ser + Thr163Gln + Ser203Glu +Tyr205Asp + Leu216AspLeu125Ile + Gly159Glu + Ser203Glu + Thr214Glu +Ser215Glu + Leu216GlnGly156Ser + Ser158Glu + Tyr205Asp + Tyr213Glu +Ser215Asp + Leu216CysGly 62Gln + Asn154Asp + Ser155Asp + Ser160Asp +Gly165Glu + Tyr205MetGly 64Pro + Ala186Glu + Ser187Asp + Ser203Glu +Tyr205His + Ser215GluTyr103Asp + Tyr166Ser + Ala186Asn + Val202Glu +Ser203Asp + Tyr205GluLeu 95Gln + Gly126Gln + Gly127Asp + Pro128Ser +Ser155Asp + Ser158GluAsp 59Glu + Ile106His + Tyr205Ala + Tyr213Glu +Ser215Glu + Leu216AspSer158Asp + Ser160Glu + Thr163Asp + Pro209Gln +Thr212Ser + Ser215GluGly 62Asp + Tyr103Met + Val202Ser + Asn211Glu +Thr214Gln + Leu216AlaAsp 59Glu + Ser 97Asp + Ser 98Asp + Gly153Ser +Gln161Asn + Ser203GluLeu125Asn + Gly153Glu + Ser157Asp + Thr163Glu +Val202Asp + Thr214ProLeu 95Glu + Ser100Asp + Leu125Asp + Pro128Asn +Ser131Asp + Tyr205GlyGly126Gln + Ser158Glu + Ser203Asp + Ser215Glu +Leu216Thr + Gly218AspGly101Ser + Pro128Asp + Ser158Glu + Gly159Asp +Ala166Gly + Phe188ValSer167Glu + Ser203Asp + Tyr205Cys + Pro209Gln +Ser215Asp + Leu216SerGly 62Ser + Ser187Glu + Met198Gln + Ser203Glu +Ser215Asp + Leu216HisThr132Asn + Gly165Pro + Ser187Glu + Ser203Glu +Tyr205His + Leu216AspAsn 96Ser + Ser131Glu + Gly159Asp + Ser160Asp +Thr214Gln + Leu216GlyGly130Glu + Ser158Asp + Gly159Glu + Thr163Gln +Tyr205Thr + Thr207AsnGly130Asp + Ser131Glu + Gly165Pro + Ser190Glu +Val202Gly + Asn217SerLeu 95Thr + Gly130Glu + Ser131Glu + Tyr205Ala +Ser210Glu + Gly218GlnThr 58Gly + Ala105Glu + Ile106Gly + Tyr205Val +Ser215Glu + Leu216AspThr 58Asp + Asp 59Glu + Leu125Asn + Ser203Glu +Tyr205Cys + Thr212SerThr 58Glu + Asp 59Glu + Tyr103Asn + Pro209Gln +Thr212Asn + Ser215GluThr 65Ser + Ser 97Asp + Ser 98Glu + Ser104Glu +Thr163Gln + Leu216SerSer 97Glu + Ser 98Glu + Gly101Gln + Thr212Asn +Leu216His + Gly218GluGly101Asp + Thr102Glu + Ile106Val + Thr163Pro +Tyr205Glu + Ser215AspGly 60Pro + Ser129Glu + Val202Glu + Thr214Asp +Ser215Asp + Leu216IleGly 62Asp + Gly 99Glu + Tyr205Asn + Asn211Asp +Tyr213Met + Thr214AspThr 58Gly + Gly127Glu + Ser131Glu + Ser155Asp +Gly165Pro + Tyr205CysGly159Asp + Tyr166Gly + Tyr205Asp + Tyr213Asn +Ser215Asp + Leu216ThrGly 62Glu + Gly101Ser + Thr132Gly + Ser203Glu +Asn211Asp + Tyr213GluAsn 96Asp + Ser 97Asp + Leu125Pro + Gly127Gln +Ser203Asp + Ser215AspAsp 59Glu + Val 94Glu + Ser 98Asp + Ser104Glu +Leu125Asn + Tyr213GlySer155Asp + Gly156Gln + Ser190Asp + Tyr205Glu +Thr212Ser + Leu216AlaVal 94Met + Gly 99Pro + Ile106Thr + Ser155Asp +Ser190Asp + Thr212GluGln161Ser + Thr163Glu + Val202Ala + Ser203Glu +Asn211Ser + Ser215AspGly156Glu + Ala186Asn + Ser203Asp + Thr212Gln +Ser215Glu + Leu216GlnSer 98Glu + Ser203Asp + Val204Cys + Ser215Asp +Asn217Ser + Thr219GlnAsn 61Gln + Thr 65Ser + Ser131Asp + Ser203Asp +Ser215Glu + Leu216CysIle106Val + Thr132Ser + Ser158Glu + Ser203Glu +Thr214Ser + Ser215GluVal 94Thr + Gly153Glu + Ala186Gln + Ser203Glu +Ser215Asp + Thr219GlyVal 94Met + Thr102Asp + Pro128Glu + Ser131Asp +Gly165Asn + Thr219SerSer155Asp + Gln161Asp + Serl90Glu + Ser203Glu +Thr212Ser + Leu216CysAsn 61Glu + Ser100Asp + Thr132Pro + Ala186Gly +Ser210Glu + Thr212GluAla105Glu + Thr132Ser + Gly201Ser + Ser203Glu +Thr212Pro + Asn217AspThr 58Gly + Gly 62Pro + Ala105Asp + Ser203Asp +Tyr208Ile + Leu216GluAsn 96Asp + Gly 99Asp + Ile106Gly + Asn154Gln +Gln161Asn + Thr214GluAsn 96Glu + Gly 99Glu + Thr102Asn + Gly127Ser +Ser203Glu + Asn217GlnAsp 59Glu + Gly127Asp + Ser129Asp + Tyr213His +Leu216Val + Thr219GlyThr 65Gln + Tyr205Ser + Pro209Gln + Ser210Glu +Thr212Glu + Thr219AspThr 58Asp + Asn 96Ser + Ser158Glu + Gln161Asp +Gly201Gln + Tyr205ValAsp 59Glu + Asn 61Glu + Tyr166His + Phe188Val +Thr212Gly + Ser215AspThr102Glu + Gly126Pro + Gly127Asn + Val202Asn +Pro209Asp + Asn211GluAsn 61Gln + Gly159Gln + Ser187Asp + Pro209Asp +Asn211Asp + Gly218SerSer 97Asp + Ser 98Glu + Gly156Asp + Ser158Glu +Thr163Gln + Pro209GlnGly 60Gln + Gly130Asn + Ser155Glu + Phe188Glu +Tyr205Asn + Ser215GluVal 94Asp + Ile106Glu + Pro128Asp + Ser131Asp +Tyr205Thr + Leu216SerGly 60Pro + Ser 98Asp + Ser100Glu + Pro128Gly +Ser157Asp + Gly159AspThr 58Asn + Gly 99Asp + Thr102Gln + Pro209Asp +Thr212Glu + Tyr213MetThr 65Glu + Tyr103Gln + Gly156Pro + Tyr166Asn +Phe188Tyr + Ser215GluVal 94Asp + Gly101Glu + Ile164Gly + Ala186Pro +Ser203Asp + Leu216ProGly 60Asn + Asn 61Glu + Gly153Ser + Pro209Asp +Asn211Asp + Ser215GluThr132Gly + Ser157Glu + Ser187Asp + Ser203Asp +Tyr205Met + Ser215GluAsn 61Asp + Asn 96Glu + Gly156Asn + Ser203Glu +Thr212Gln + Ser215GluGly 99Pro + Ser158Glu + Ser160Glu + Tyr205His +Thr212Ser + Ser215GluGly101Pro + Ser157Glu + Ser203Asp + Tyr205Asp +Thr212Ser + Leu216CysVal 94Ala + Pro128Ser + Gly156Gln + Ser190Asp +Ser203Asp + Tyr205GluIle106Met + Leu125Ile + Ser160Asp + Ser203Asp +Tyr205Glu + Leu216SerLeu 95Glu + Ser 97Glu + Thr102Glu + Gly159Asp +Tyr205Leu + Leu216AlaLeu125Cys + Gly126Ser + Gly159Asp + Gln161Glu +Ser215Glu + Asn217GluSer100Glu + Thr102Gly + Ala105Thr + Ser155Glu +Ser157Asp + Asn162SerSer 98Glu + Gly127Asn + Ser157Asp + Gln161Asn +Asn162Asp + Gly165GluAsn 61Gln + Ser100Asp + Ser104Glu + Ile106Asp +Ser215Asp + Leu216AlaSerl31Glu + Gly159Asn + Val202Cys + Ser215Asp +Leu216Asp + Thr219GluLeu125Cys + Gln161Asp + Asn162Glu + Ser203Glu +Leu216Thr + Gly218GluGly101Asp + Ala105Asp + Gly126Asn + Gln161Ser +Ala186Pro + Thr214GluSer100Glu + Thr102Glu + Tyr103Thr + Gly126Asp +Ser160Glu + Leu216MetGly127Gln + Asn154Ser + Ser157Glu + Thr163Glu +Ser215Glu + Asn217GluLeu 95Ile + Asn 96Glu + Thr102Gly + Tyr103Glu +Gly126Glu + Gly165GluSer129Asp + Ser131Asp + Thr132Asn + Gly159Ser +Tyr213Gln + Thr214GluGly101Pro + Gly126Glu + Ser129Asp + Tyr205Glu +Ser215Glu + Leu216GlyLeu 95Met + Pro128Gly + Ser129Glu + Gly153Glu +Thr163Gly + Leu216SerGly 60Glu + Val202Met + Tyr205Ile + Thr212Glu +Ser215Glu + Leu216AspThr 58Ser + Ser158Asp + Gln161Asn + Thr163Asp +Leu216Asp + Gly218AspIle106Ser + Ser158Asp + Ser187Asp + Ser190Asp +Tyr205Met + Ser215AspSer187Glu + Val202Gln + Tyr205Asn + Thr214Glu +Leu216Glu + Gly218SerAsp 59Glu + Ser100Asp + Gly126Pro + Gly127Asp +Tyr166Glu + Thr212AsnAsp 59Glu + Gly 62Pro + Gly127Asn + Ala186Pro +Val202Glu + Pro209AspAsp 59Glu + Asn 96Glu + Gly101Glu + Thr102Gln +Pro128Ser + Leu216GluGly159Asp + Asn162Asp + Ala186Gln + Val202Gln +Ser203Glu + Ser215AspGly159Ser + Ala186Asn + Ser210Asp + Tyr213Pro +Ser215Asp + Asn217AspSerl04Glu + Ser131Asp + Thr132Gln + Asn162Ser +Gly165Asp + Tyr208CysGly156Glu + Ser158Asp + Thr163Gly + Ser203Asp +Thr212Asn + Leu216SerGly 64Pro + Thr 65Glu + Ala105Asp + Val202Thr +Ser215Asp + Leu216GluThr 58Pro + Gly 99Ser + Ser100Asp + Gly101Ser +Thr102Asp + Asn211GluGly 62Glu + Asn 96Ser + Ser100Asp + Gly101Ser +Ser210Asp + Tyr213AlaGly 60Pro + Gln161Asp + Gly165Asp + Phe188Val +Ser190Glu + Ser210GluGly153Gln + Ser155Asp + Gly159Asn + Pro209Glu +Thr212Asp + Ser215AspThr 58Gln + Thr102Glu + Ser129Glu + Gly130Asp +Tyr205Ala + Ser215AspTyr103Ser + Asn154Asp + Ser187Glu + Val202Asn +Ser203Glu + Leu216SerLeu125Pro + Pro128Ser + Ser157Glu + Gln161Glu +Ser203Glu + Gly218SerSer 97Glu + Gly 99Asp + Ala105Ser + Leu125Asp +Gly165Gln + Tyr205AspThr 65Asp + Leu 95Cys + Ser 97Asp + Asn162Ser +Thr212Glu + Ser215AspSer 97Glu + Ser100Glu + Gly156Asn + Ser157Glu +Ser160Glu + Tyr205ProLeu 95Asp + Ala105Asp + Ser157Glu + Val202His +Tyr205Ile +Leu216MetLeu 95His + Gly156Pro + Ser157Asp + Asn162Glu +Ser187Glu + Leu216AspSer100Glu + Leu125Gly + Gly127Glu + Ser203Asp +Tyr208Val + Leu216AspAsn154Ser + Gly165Asp + Ser190Glu + Thr212Gly +Ser215Asp + Gly218ProThr 58Gln + Ser 98Glu + Gly153Asp + Val202Ser +Tyr205Asn + Gly218AspAsn 61Gln + Thr132Gly + Gly156Glu + Ser158Glu +Ser215Asp + Thr219GluVal 94Glu + Ser 97Glu + Ala105Asp + Gly165Pro +Leu216Met + Thr219AspVal 94Glu + Ser 97Glu + Ala105Asp + Asn154Gln +Gln161Asp + Ala186AsnGly130Gln + Ser155Glu + Ser158Glu + Ala186Gln +Ser203Asp + Leu216CysAsn 61Gln + Ser160Asp + Ser203Glu + Val204Gln +Thr212Glu + Ser215GluThr102Glu + Ala105Asp + Ser131Glu + Ser203Glu +Tyr205Ala + Thr214AsnThr 58Pro + Ser155Glu + Gly159Asn + Pro209Glu +Thr212Glu + Gly218GluAsp 59Glu + Gly 99Asp + Gly126Ser + Met198Thr +Thr212Asp + Gly218ProThr132Ser + Asn154Ser + Ser157Asp + Val202Glu +Ser215Glu + Leu216GlyVal 94Asp + Gly 99Asn + Ser100Asp + Gly130Ser +Asn154Gln - Ser215AspPro128Glu + Gly156Ser + Ser160Glu + Ser187Asp +Ser190Glu + Thr212SerAsn 61Gln + Ser 98Glu + Ile106Cys + Ser203Asp +Tyr205Gly + Gly218AspAla105His + Ser158Glu + Ser187Glu + Ser203Glu +Tyr205Leu + Ser215GluAsn 96Ser + Gly 99Gln + Gly153Glu + Ala186Glu +Tyr205Glu + Ser215GluAsp 59Glu + Ser 98Glu + Gly153Glu + Ser155Glu +Phe188Pro + Leu216HisAsn162Asp + Gly165Asp + Val204Gln + Tyr205Thr +Ser210Asp + Thr212ProGly101Pro + Gly156Asp + Tyr166Asp + Ser190Glu +Tyr205Asp + Thr212AsnGly126Glu + Thr132Asp + Ser155Asp + Ser157Asp +Val202Gly + Tyr213SerGly 60Asp + Asn 61Asp + Asn154Glu + Ser158Glu +Gly165Gln + Tyr213GlnGly 62Glu + Leu125His + Ser131Asp + Tyr166Leu +Ser203Glu + Ser215AspAsp 59Glu + Val 94Asp + Thr102Gly + Ser203Glu +Tyr205Asp + Thr219GlySer104Glu + Tyr166Cys + Val202Thr + Thr212Asp +Ser215Asp + Asn217GluSer100Asp + Gly126Pro + Asn154Asp + Gly201Ser +Ser203Glu + Thr219AspGly101Asp + Pro128Asp + Ser155Asp + Ser187Asp +Thr207Gly + Thr212GlnIle106Ser + Ser160Glu + Ser190Asp + Ser203Asp +Ser215Glu + Leu216ValGly 99Gln + Gly153Asp + Ser160Asp + Phe188Leu +Ser190Asp + Ser210AspSer158Asp + Gly165Asn + Ser190Glu + Val202His +Tyr205Asp + Tyr213LeuThr 65Asp + Leu 95Thr + Ser131Glu + Val202Ser +Ser203Asp + Leu216GluAsn 61Glu + Gly165Ser + Met198Gln + Val202Glu +Tyr205Glu + Leu216AsnVal 94Ala + Gly126Asp + Asn154Glu + Tyr166Pro +Val202Gln + Thr212GluSer 97Glu + Leu125Glu + Ser155Glu + Gly159Asn +Tyr166Asp + Leu216ValAsn 96Glu + Ser 97Asp + Thr102Gln + Leu125Asp +Leu216Glu + Thr219GlnTyr103Cys + Ser160Glu + Thr163Asp + Tyr208Ser +Leu216Met + Gly218GluVal 94Cys + Leu125Glu + Ser155Asp + Gly156Asp +Gly201Gln + Ser215GluAsn 61Ser + Thr 65Glu + Val 94Asp + Tyr103Pro +Ser215Asp + Asn217AspGly 99Gln + Gly127Asp + Thr132Pro + Ser155Glu +Ser187Glu + Ser203AspSer 98Glu + Ser129Glu + Gly153Glu + Ser190Asp +Tyr213Val + Thr214ProAsn 61Ser + Gly130Glu + Phe188Glu + Tyr205Asp +Thr212Pro + Asn217AspSer158Glu + Gly159Glu + Val202Ser + Tyr205Met +Pro209Glu + Ser215GluAsn 61Asp + Gly 99Asn + Gly101Asp + Ser203Asp +Tyr205Asp + Thr212ProSer 98Asp + Ser100Asp + Ser104Glu + Gly153Glu +Phe188Gly + Gly218AsnLeu 95Val + Gly 99Asp + Tyr103Val + Ala105Asp +Tyr205Asp + Ser215AspAsp 59Glu + Asn 61Asp + Ser160Glu + Pro200Asn +Asn211Ser + Thr212AspGly 62Glu + Thr 65Gly + Ser 97Asp + Thr102Pro +Ser131Glu + Leu216AspGly126Pro + Pro128Glu + Gly156Glu + Ser158Asp +Tyr205Asn + Asn217AspAsp 59Glu + Gly130Asn + Ser155Asp + Ser210Asp +Tyr213Gly + Gly218AsnAsn 96Gln + Gly 99Glu + Ser100Asp + Pro128Asp +Tyr205Cys + Asn217GluPro128Gly + Ser129Asp + Asn162Glu + Gly165Asn +Ser203Asp + Ser215AspAsn 96Ser + Ser100Glu + Tyr103Glu + Tyr205His +Ser215Glu + Leu216ProAsn 96Glu + Ser 98Glu + Ala186Asp + Val202Asn +Tyr213Met + Leu216GluGly 99Asp + Ser158Asp + Asn162Asp + Thr163Asn +Ser187Glu + Ala199GlyLeu 95Ser + Ser 97Asp + Ser 98Glu + Gly165Glu +Leu216Ile + Gly218GluGly101Asp + Leu125Asp + Gln161Asn + Tyr205Asp +Thr212Gly + Leu216AsnSer160Asp + Val202His + Ser203Asp + Thr212Asp +Tyr213Val + Leu216AspLeu 95Val + Tyr103Glu + Ala105Glu + Thr163Ser +Thr212Asp + Leu216GluSer129Asp + Gly130Ser + Ser157Asp + Gly165Asp +Tyr205Glu + Leu216GlnThr132Asn + Gly156Glu + Tyr166Thr + Ser203Glu +Thr212Glu + Asn217GluAsn 61Gln + Ser 97Glu + Ser187Glu + Ser203Glu +Pro209Gln + Ser215GluGly101Gln + Ala105Asp + Ser187Glu + Ser203Asp +Ser215Glu + Leu216HisGly 99Asn + Ser104Asp + Thr132Gly + Ser187Asp +Ser203Glu + Ser215AspGly 62Asp + Asn 96Gln + Ser104Glu + Tyr205Gln +Thr212Asp + Tyr213ValVal 94Gly + Asn 96Asp + Ala105Glu + Asn162Asp +Asn211Gln + Thr212GlnSer 97Glu + Ser187Asp + Ser203Glu + Tyr213Gln +Leu216Glu + Asn217GlnGly 64Pro + Ser104Glu + Gly126Glu + Tyr166Ser +Tyr205Glu + Ser215GluGly130Asn + Thr132Asp + Ser187Glu + Pro209Gln +Thr214Glu + Ser215GluGly126Glu + Ser160Asp + Tyr166Met + Val202Thr +Thr214Asp + Ser215GluSer 98Asp + Gly101Ser + Gly130Asp + Ser158Glu +Gly159Asp + Leu216AlaThr 58Glu + Tyr103Pro + Gly130Glu + Ser131Asp +Ser158Glu + Ala199ProGly 60Asp + Asn 61Asp + Ser131Asp + Gly153Ser +Thr163Glu + Val202CysGly 99Asp + Pro128Asn + Asn162Glu + Thr212Ser +Leu216Glu + Asn217GluGly 60Ser + Ser 98Asp + Asn154Asp + Ser155Glu +Gly159Pro + Asn211GluTyr103Pro + Ser104Glu + Leu125Cys + Val202Glu +Ser203Glu + Pro209GluGly101Gln + Ser104Asp + Leu125Val + Val202Asp +Ser203Asp + Thr212AspThr 65Asn + Ser 98Asp + Gly159Asn + Gly165Asp +Ser215Asp + Leu216AspGly127Glu + Gly165Pro + Tyr205Cys + Ser210Asp +Ser215Glu + Leu216GluVal 94Ser + Ser 98Glu + Gly 99Asp + Gly156Asp +Phe188Leu + Ser203GluGly 62Asp + Gly101Glu + Ser160Glu + Gln161Asp +Tyr205Met + Thr212ProSer 97Asp + Ser 98Glu + Ala105His + Ser157Glu +Thr212Ser + Ser215AspGly 62Gln + Ser 97Glu + Ser 98Asp + Gly126Glu +Gly130Pro + Thr212GluVal 94Glu + Ser 97Asp + Leu125Asn + Thr163Ser +Ser210Asp + Leu216IleLeu 95Glu + Asn 96Gln + Gly159Glu + Tyr205Glu +Pro209Ser + Ser215GluSer129Glu + Asn154Ser + Thr212Glu + Ser215Asp +Gly218Asp + Thr219SerAsp 59Glu + Gly127Gln + Val202Glu + Asn211Glu +Thr212Ser + Ser215AspSer104Asp + Ala105Pro + Ser187Glu + Ser203Glu +Tyr205Asp + Tyr213ProThr 58Pro + Asn 96Asp + Gly127Glu + Ser155Glu +Asn162Ser + Thr212SerThr 58Gly + Leu125Asn + Gly156Asp + Gln161Glu +Tyr205Gly + Ser215AspGly 60Ser + Gly156Asp + Pro209Ser + Thr214Asp +Leu216Thr + Thr219AspLeu125His + Gly127Gln + Ser131Glu + Ser203Asp +Asn211Asp + Ser215GluThr 58Glu + Pro128Glu + Gly165Gln + Ala186His +Ser203Glu + Ser215GluSer131Asp + Ser158Glu + Ser203Asp + Tyr205His +Ser215Asp + Leu216GlnThr 65Asn + Gly130Asp + Ser158Asp + Ser203Asp +Pro209Ser + Ser215GluGly 60Gln + Thr102Glu + Ser157Asp + Ser203Asp +Ser215Asp + Thr219GlnGly 64Gln + Gly127Asp + Ser160Glu + Ser203Asp +Ser215Glu + Gly218GlnIle106Leu + Thr132Asp + Thr163Asp + Ser203Glu +Ser215Asp + Leu216IleGly126Ser + Ser160Asp + Thr212Glu + Ser215Glu +Leu216Val + Thr219SerGly 60Pro + Gly101Asn + Ser158Glu + Tyr205Ile +Thr212Asp + Ser215GluGly 62Glu + Gly 99Ser + Val202Ser + Tyr205Asp +Thr212Asn + Thr214SerVal 94His + Thr132Glu + Gly165Asp + Tyr166Cys +Ser203Asp + Leu216GluGly 62Asn + Ser131Asp + Ser157Glu + Gln161Asn +Ser203Glu + Leu216GluGly 60Pro + Val 94Asp + Ala105Glu + Gly127Pro +Gln161Asp + Ser210AspGly 62Gln + Ser 97Asp + Asn154Asp + Ser190Asp +Ser203Glu + Thr212SerSer 97Glu + Asn154Glu + Gly159Asn + Ser190Asp +Tyr205Thr + Asn211GluThr 65Glu + Ser 98Asp + Thr102Pro + Gly126Asp +Gly165Asp + Gly218GlnAsn 96Glu + Tyr103Asp + Gly159Asp + Asn162Asp +Tyr208Leu + Thr212ProGly 62Asp + Ser104Glu + Ile106Asp + Ser157Asp +Gly218Ser + Thr219ProAsn 96Glu + Gly 99Asp + Ser160Asp + Gly201Ser +Asn211Gln + Ser215GluIle106Pro + Gly153Asp + Gly165Glu + Tyr166Ser +Ser203Asp + Thr212AspSer 97Asp + Gly 99Asn + Ser158Asp + Ser210Glu +Thr212Glu + Leu216ThrAsp 59Glu + Ser 98Asp + Ser160Glu + Gly201Ser +Thr212Gly + Tyr213AsnVal 94Ser + Ser 97Asp + Ser187Glu + Ser190Asp +Pro209Asp + Leu216AsnGly101Gln + Gly126Asp + Ser131Asp + Asn154Glu +Gly156Asn + Ser203AspGly 64Ser + Ser 98Glu + Ser100Glu + Ala186Glu +Thr212Glu +Leu216ProSer 97Asp + Gly 99Asp + Val202Asp + Thr207Asn +Asn211Glu + Leu216MetThr 65Pro + Thr132Gly + Ala186Asp + Val202Ala +Ser203Glu + Thr212AspIle106Asp + Gly127Ser + Asn154Glu + Gly156Glu +Thr214Pro + Ser215GluThr 58Asp + Ser129Glu + Thr132Asn + Gly165Asp +Leu216Val + Gly218AspThr 58Pro + Gly 60Glu + Gly 62Glu + Thr102Asp +Ser158Glu + Leu216AlaPro128Asp + Asn154Glu + Phe188Val + Val202Cys +Ser203Asp + Tyr205AsnGly101Gln + Ala105His + Ser155Asp + Tyr166Val +Ala186Asp + Pro209GluLeu 95Val + Ser155Glu + Tyr166Ala + Ala186Glu +Ser210Asp + Leu216MetAsn 96Asp + Gly101Glu + Asn162Glu + Val202Thr +Ser210Asp + Leu216AsnSer129Glu + Ser157Asp + Phe188Ala + Ser190Glu +Val202Ser + Tyr205GluSer131Asp + Ser158Asp + Ser160Asp + Tyr205Ile +Ser215Asp + Leu216MetGly 62Glu + Leu 95Glu + Tyr103Pro + Ala186His +Ser203Glu + Thr207GlySer100Glu + Leu125Ala + Ser157Glu + Ser203Asp +Tyr205Asp + Leu216CysThr102Gln + Ser104Asp + Gly153Pro + Ser158Glu +Ser203Glu + Tyr205AspAsn 61Glu + Asn 96Gln + Ser 98Asp + Ser158Asp +Thr212Asn + Thr219AspVal 94Thr + Thr132Asp + Ser155Asp + Ser157Glu +Thr207Gly + Ser215AspPro128Gly + Gly130Pro + Ser158Glu + Ser187Glu +Phe188Cys + Ser203AspThr 65Glu + Gly130Pro + Gly159Glu + Gly165Asp +Phe188Met + Pro209AspThr163Glu + Val202Asp + Tyr205Cys + Thr212Asp +Leu216Pro + Gly218GluSer 97Glu + Gly101Asn + Gly130Glu + Asn154Asp +Thr163Gly + Gly218GluThr 65Gly + Ala105Glu + Ser155Asp + Gln161Glu +Phe188Val + Asn217GlnThr 65Gln + Tyr103Asp + Pro128Glu + Ser155Asp +Ser215Asp + Leu216SerThr132Glu + Ala186Gln + Ser187Glu + Val202Pro +Thr207Gly + Asn217AspGly 60Glu + Ser129Glu + Ser131Glu + Gly165Asn +Ser203Glu + Pro209GlnGly101Pro + Pro128Asp + Gly130Asp + Ser203Glu +Tyr205Ile + Thr212AspVal 94Glu + Asn 96Glu + Asn154Asp + Tyr205Ala +Thr212Glu + Leu216CysSer 97Glu + Thr102Ser + Ser157Asp + Ser215Asp +Leu216Ser + Asn217GluSer 98Asp + Gly156Glu + Ser158Glu + Gln161Ser +Ser203Asp + Gly218AsnGly130Ser + Gly156Asp + Ser158Glu + Ser210Asp +Thr212Gln + Ser215AspSer100Glu + Thr102Asp + Gly126Pro + Gly127Ser +Ser187Glu + Ser215GluAsn 96Glu + Gly127Ser + Pro128Gln + Ser190Asp +Tyr213Asp + Ser215GluPro128Glu + Gly153Ser + Ser158Glu + Gly165Ser +Ala186Gly + Ser187GluThr 65Glu + Gly153Asp + Thr163Asp + Gly165Asn +Met198His + Ser203GluLeu125Gly + Ser129Glu + Thr163Glu + Ala186His +Ser203Glu + Tyr205LeuThr 58Glu + Asn 61Ser + Thr 65Gly + Ser158Asp +Asn162Asp + Ser203GluThr 65Asn + Val 94Thr + Gly126Glu + Ser157Asp +Asn162Glu + Leu216AspAsp 59Glu + Ser 97Glu + Pro128Asn + Thr132Glu +Ser157Asp + Tyr205GlyThr 58Glu + Gly 60Gln + Leu 95Glu + Val202Gln +Tyr205Gln + Ser215AspAsp 59Glu + Ser157Asp + Asn162Asp + Tyr166Asn +Leu216Ile + Asn217GluSer157Glu + Asn162Glu + Phe188Gly + Ser203Asp +Asn211Asp + Thr212SerSer157Glu + Asn162Asp + Ser210Asp + Thr212Ser +Tyr213Leu + Leu216AspGly 60Pro + Ser 97Glu + Ser100Asp + Ala105His +Ser203Glu + Asn211AspAsn 61Asp + Leu125His + Gly156Asn + Pro200Gln +Thr212Glu + Leu216AspThr 65Asp + Ser 97Asp + Leu125Asn + Ser157Glu +Thr163Gly + Thr212AspGly 62Glu + Thr 65Asn + Asn162Glu + Thr163Ser +Asn211Asp + Ser215GluVal 94Glu + Asn 96Ser + Gly101Pro + Thr102Asp +Ser155Glu + Thr212AspGly 60Glu + Ser 98Glu + Gly 99Gln + Ser104Glu +Ala105Thr + Thr214AspSer155Asp + Ser187Asp + Ala199Gln + Ser203Asp +Tyr205Val + Thr212GluSer104Glu + Ile106Cys + Thr163Ser + Gly165Glu +Ser190Glu + Thr212AspVal 94Thr + Ile106Asp + Gly130Asn + Ser131Asp +Ser203Glu + Tyr205ProSer 98Glu + Pro128Glu + Ser155Asp + Ser160Glu +Ala186Ser + Pro209AsnThr 58Glu + Gly159Ser + Tyr166Glu + Ser190Glu +Thr212Pro + Leu216SerLeu 95Glu + Leu125His + Gly126Asp + Tyr205Gln +Ser215Asp + Thr219AsnAsp 59Glu + Gly 99Asp + Ala105Glu + Ala186Pro +Ser187Asp + Thr212ProAsn 96Gln + Ser 98Glu + Gly101Pro + Thr132Glu +Val202Asp + Ser215AspGly 99Asp + Pro128Asp + Thr132Asn + Val202Asp +Ser215Asp + Leu216HisVal 94Gln + Gly101Asp + Ser104Asp + Thr163Pro +Ser187Asp + Ser215AspLeu 95Met + Gly101Asp + Ser104Glu + Gly153Glu +Tyr205Ala + Ser215GluSly101Glu - Ser104Glu + Ser203Asp + Tyr205Ile +Ser210Glu + Thr212ProVal 94Glu + Ser100Glu + Ile106Met + Gly130Glu +Val202Glu + Thr214AsnSer 98Asp + Ile106Asp + Asn154Asp + Gly165Asp +Val202Met + Leu216GlnLeu 95Met + Thr102Ser + Gly127Asn + Ser190Glu +Val202Gly + Asn217AspGly 62Pro + Ile106Gln + Gly126Asp + Ser129Glu +Ser160Asp + Ser215AspGly 62Asp + Asn154Glu + Ser160Glu + Ala186Thr +Ser167Asp + Leu216ThrGly 62Ser + Thr 65Ser + Ser155Asp + Gly159Glu +Ser203Glu + Thr219Gln
II.清洗組合物在本發(fā)明另一實(shí)施方案中，在用于清洗各種需要除去蛋白質(zhì)污物表面的組合物中包括一種或多種有效量的酶變異體。這種清洗組合物包括清洗硬表面的洗滌劑組合物，形式不限(如液體和顆粒狀)；用于清洗織物的洗滌劑組合物，形式不限(如顆粒、液體和條塊制劑)；洗碟劑組合物(形式不限)；口腔清潔組合物，形式不限(如牙粉、牙膏和漱口制品)；假牙清洗組合物，形式不限(如液體、片劑)；和隱形眼鏡清潔組合物，形式不限(如液體、片劑)。
清洗組合物除包括上述枯草桿菌蛋白酶DY變異體外，還包括一種或多種與蛋白酶相容的清洗組合物材料。這里使用的“清洗組合物材料”是指被選擇用在所需特殊類型的清洗組合物和產(chǎn)品型式(如液體、顆粒、條塊、霧狀、棒狀、膏、膠)中的任意一種液體、固體或氣體材料，這些材料也與用于組合物的枯草桿菌蛋白酶DY變異體相容?？紤]所要清洗的表面材料、使用中清洗條件(如在洗滌劑使用的整個(gè)過程中)所需的組合物形式很容易作出清洗組合物材料的特定選擇。這里使用的術(shù)語(yǔ)“相容的”是指清洗組合物材料不會(huì)降低枯草桿菌蛋白酶DY變異體的蛋白水解活性至該蛋白酶失去正常使用條件中所期望的效果的程度。后面將詳細(xì)例舉特定的清洗組合物材料。
這里所用的“有效量的酶變異體”是指在特定清洗組合物中獲得所需酶活性而需要的酶變異體的量。本領(lǐng)域普通技術(shù)人員很容易確定這種有效量，其基于多種因素，如所用的特殊的酶變異體、清洗應(yīng)用范圍、清洗組合物的特定組成以及需要的是液體還是干燥的(如顆粒、條塊)組合物等等。優(yōu)選清洗組合物包括約0.0001％至約10％的一種或多種本發(fā)明的酶變異體，更優(yōu)選約0.001％至約1％，更優(yōu)選約0.01％至約0.1％。下面更為詳細(xì)地討論了使用酶變異體的各種清洗組合物的一些例子。除非另有說明，這里所用的所有的份、百分含量和比例均指重量。
這里所用的“非織物清洗組合物”包括硬表面清洗組合物、洗碟劑組合物、口腔清潔組合物、假牙清洗組合物和隱性眼鏡清洗組合物。
A.用于硬表面、碟子和織物的清洗組合物本發(fā)明的酶變異體可用于需要大量泡沫并能很好地去除不溶性底物的各種洗滌劑組合物中。因此，該酶變異體可與各種常規(guī)成分一起使用，提供全配制的硬表面洗滌劑、洗碟劑組合物、織物洗滌組合物等等。這種組合物可呈液體、顆粒、條塊等形式，可被配制成現(xiàn)代的“濃縮”洗滌劑，含有多達(dá)30％-60％(重量百分含量)的表面活性劑。
這里的清洗組合物可選擇性地但優(yōu)選含各種陰離子、非離子、兩性離子等表面活性劑。這種表面活性劑通常含量約為組合物的5％-35％。
這里所用的表面活性劑非限制性例子包括常規(guī)的C11-C18烷基苯磺酸鹽及伯烷基和無規(guī)則烷基硫酸鹽、C10-C18仲(2，3)烷基硫酸鹽、分子式為CH3(CH2)X(CHOSO3)-M+)CH3和CH3(CH2)y(CHOSO3)-M+)CH2CH3，其中x和(y+1)是至少為大約7的整數(shù)，優(yōu)選至少為大約9，M是水溶性陽(yáng)離子，特別是鈉；C10-C18的烷基烷氧基硫酸鹽(特別是EO1-5乙氧基硫酸鹽)，C10-C18的烷基烷氧基羧酸鹽(特別是EO1-5乙氧基羧酸鹽)，C10-C18烷基多苷、及其相應(yīng)的硫酸化多苷、C12-C18α-磺酸化脂肪酸酯、C12-C18烷基和烷基苯酚烷氧基化物(特別是乙氧基化物和混合的乙氧基/丙氧基)、C12-C18甜菜堿和磺基甜菜堿(“sultaines”)、C10-C18氧化胺等等。此處優(yōu)選烷基烷氧基硫酸鹽(AES)和烷基烷氧基羧酸鹽(AEC)。(根據(jù)配方設(shè)計(jì)的需要，還優(yōu)選這種表面活性劑與前述氧化胺和/或甜菜堿或磺基甜菜堿表面活性劑結(jié)合使用。)其他常規(guī)使用的表面活性劑列于標(biāo)準(zhǔn)書籍中。特別有用的表面活性劑包括由Connor等人的于1993年3月16日公開的美國(guó)專利US5194639中所述的C10-C18N-甲基萄萄糖酰胺，這里引為參考。
此處的組合物可包括洗滌劑清洗組合物中有用的其他多種成分，包括其他活性成分、載體、水溶助長(zhǎng)劑、加工助劑、染料或顏料、液體配方所需溶劑等。如果需要額外增加泡沫，可在組合物中摻入象C10-C16烷氧酰胺這樣的增泡劑，通常含量約為1％至10％。C10-C14的單乙醇和二乙醇酰胺代表了一類這種典型的增泡劑。將這種增泡劑與多泡的輔助表面活性劑如上述的氧化胺、甜菜堿和磺基甜菜堿一起使用也是有利的。如果需要，可加入如MgCl2、MgSO4等這樣的可溶性鎂鹽，通常加入量約為0.1％-2％，以提供更多的泡沫。
這里的液體洗滌劑組合物可包含作為載體的水及其他溶劑。低分子量的伯醇或仲醇例如甲醇、乙醇、丙醇和異丙醇是合適的。優(yōu)選一元醇用于使表面活性劑增溶，但也可使用那些約含2-6個(gè)碳原子和2-6個(gè)羥基的多元醇(如1，3-丙二醇、乙二醇、甘油和1，2-丙二醇)。組合物可含有約5％-90％的，通常為約10％-50％的這種載體。
優(yōu)選配制這里的洗滌劑組合物使其用于水溶液清洗操作中時(shí)，洗滌水的pH在約6.8-11.0。通常在此范圍內(nèi)配制成品。將pH值控制于推薦使用范圍的技術(shù)包括使用緩沖劑、堿、酸等等，這是本領(lǐng)域技術(shù)人員所熟知的技術(shù)。
當(dāng)配制本發(fā)明的硬表面清洗組合物和織物清洗組合物時(shí)，配方師可希望使用約5％-50％(重量百分比)的各種助洗劑。典型的助洗劑包括1-10微米的沸石、聚羧酸鹽如檸檬酸鹽和氧聯(lián)二琥珀酸鹽、層狀硅酸鹽、磷酸鹽等等。其他常規(guī)助洗劑列于標(biāo)準(zhǔn)配方中。
與上述情況相似，配方師可希望在這種組合物中使用各種其他酶，如纖維素酶、脂酶、淀粉酶和蛋白酶，通常重量百分含量約為0.001％-1％。在洗衣洗滌劑領(lǐng)域各種洗滌和保護(hù)織物的酶是熟知的。
可在這種組合物中使用各種漂白化合物，如過碳酸鹽、過硼酸鹽等，通常重量百分含量在約1％-15％。如果需要，這種組合物中還可含有如四乙酰乙二胺、壬酰氧基苯磺酸鹽等這樣的漂白活化劑，它們?cè)诒绢I(lǐng)域均是熟知的。用量通常約為1％-10％(重量百分比)。
各種污垢解脫劑特別是陰離子低聚酯型的污垢解脫劑、各種螯合劑特別是氨基膦酸鹽和乙二胺二琥珀酸鹽、各種粘土類去污垢劑特別是乙氧基化的四亞乙基五胺、各種分散劑特別是聚丙烯酸鹽和聚天冬氨酸鹽、各種增白劑特別是陰離子增白劑、各種抑泡劑特別是硅氧烷和仲醇、各種織物柔軟劑特別是綠土粘土等等均可用于這種組合物中，重量百分含量約為1％-35％。標(biāo)準(zhǔn)配方書和公開專利中有這些常規(guī)材料的多種、詳細(xì)說明。
在清洗組合物中還可使用酶穩(wěn)定劑。這種酶穩(wěn)定劑包括丙二醇(優(yōu)選從約1％至約10％)、甲酸鈉(優(yōu)選約0.1％至約1％)及甲酸鈣(優(yōu)選約0.1％至約1％)。
1.硬表面清洗組合物這里使用的“硬表面清洗組合物”是指用于清洗如地板、墻壁、浴室瓷磚等這類硬表面的液體和顆粒狀洗滌劑。本發(fā)明的硬表面清洗組合物包括有效量的一種或多種本發(fā)明的酶變異體，優(yōu)選組合物中活性酶的重量百分比為約0.001％至約10％，更優(yōu)選約0.01％至約5％，更優(yōu)選約0.05％至約1％。除包含一種或多種酶變異體外，通常這種硬表面清洗組合物還包括一種表面活性劑和一種水溶性螯合助洗劑。但是，在某些象窗戶噴霧清洗劑這種特定的產(chǎn)品中，有時(shí)不使用表面活性劑，因?yàn)樗鼈儠?huì)在玻璃表面產(chǎn)生一層薄膜狀/條紋殘留物。
當(dāng)存在表面活性劑成分時(shí)，它在本發(fā)明組合物中所占比例可少至0.1％，但通常組合物中含有約0.25％至約10％的表面活性劑，更優(yōu)選約1％至5％。
通常該組合物含有約0.5％至約50％的洗滌助洗劑，優(yōu)選約1％至10％。
優(yōu)選pH應(yīng)在約8至12的范圍內(nèi)。如果需要調(diào)節(jié)pH的話，可使用常規(guī)的pH調(diào)節(jié)劑，如氫氧化鈉、碳酸鈉或鹽酸。
在組合物中可包括溶劑。有用的溶劑包括但不限于二醇醚，如二甘醇一己基醚，二甘醇一丁基醚、乙二醇一丁基醚、乙二醇一己基醚、丙二醇一丁基醚、二丙二醇一丁基醚，和二醇如2，2，4 三甲基-1，3-戊二醇和2-乙基-1，3-己二醇。使用時(shí)，通常這種溶劑的含量為約0.5％至約15％，優(yōu)選約3％至約11％。
此外，可在本發(fā)明組合物中使用高揮發(fā)性溶劑，如異丙醇或乙醇，以便在本發(fā)明組合物完全應(yīng)用于表面后不漂洗表面時(shí)易于組合物的快速揮發(fā)。當(dāng)使用高揮發(fā)性溶劑時(shí)其在組合物中的含量約2％至約12％。
本發(fā)明的硬表面清洗組合物的實(shí)例描述在下面的實(shí)施例中。
實(shí)施例7-12硬表面清洗液體組合物實(shí)施例號(hào)組分 7 8 9 10 11 12Phe188Asp 0.050.500.02 0.030.100.03Val202Ala - - - - 0.200.02Na2DIDA*EDTA**- - 2.90 2.90- -檸檬酸鈉 - - - - 2.902.90C12烷基苯磺酸鈉 1.95- 1.95 - 1.95-C12烷基硫酸鈉- 2.20- 2.20- 2.20C12乙氧基硫酸鈉 - 2.20- 2.20- 2.20C12二甲胺氧化物 - 0.50- 0.50- 0.50枯烯磺酸鈉1.30- 1.30 - 1.30-己基卡必醇***6.306.306.30 6.306.306.30水****100％*N-二甘醇-N，N-亞氨基二乙酸二鈉**乙二胺二乙酸四鈉鹽***二甘醇一己醚****所有配方均調(diào)節(jié)至pH7在實(shí)施例7-10中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Phe188Asp，得到基本相似的結(jié)果。
在實(shí)施例11-12中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意一種組合代替Phe188Asp和Val202Ala，得到基本相似的結(jié)果。
實(shí)施例13-18清洗硬表面和家庭除霉噴霧組合物實(shí)施例號(hào)組分 131415161718Asn 96Asp+Tyr103His 0.50 0.05 0.60 0.30 0.20 0.30Pro200Gly+Gly201Ser - - - - 0.30 0.10+Ser210Asp辛基硫酸鈉2.00 2.00 2.00 2.00 2.00 2.00十二烷基硫酸鈉4.00 4.00 4.00 4.00 4.00 4.00氫氧化鈉 0.80 0.80 0.80 0.80 0.80 0.80硅酸鹽(Na)0.04 0.04 0.04 0.04 0.04 0.04香料 0.35 0.35 0.35 0.35 0.35 0.35水 平衡至100％產(chǎn)品pH約為7。
在實(shí)施例13-16中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Asn96Asp+Tyr103His，得到基本相似的結(jié)果。
在實(shí)施例17-18中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意一種組合代替Asn 96Asp+Tyr103His和Pro200Gly+Gly201Ser+Ser210Asp，得到基本相似的結(jié)果。
2.洗碟劑組合物在本發(fā)明另一實(shí)例中，洗碟劑組合物包括一種或多種本發(fā)明的酶變異體。這里使用的“洗碟劑組合物”是指洗滌盤碟的所有形式組合物，包括顆粒劑和液體形式，但不限于此。本發(fā)明洗碟劑組合物的實(shí)例用下面的實(shí)施例說明。
實(shí)施例19-24洗碟組合物實(shí)施例號(hào)組分192021222324Gly127Asn 0.05 0.50 0.02 0.40 0.10 0.03Gly201Asn+Tyr205Gly - - - - 0.40 0.02+Thr207Gln+Tyr213IleC12-C14N-甲基-葡糖酰胺 0.90 0.90 0.90 0.90 0.90 0.90C12乙氧基(1)硫酸鹽 12.00 12.00 12.00 12.00 12.00 12.002-甲基十一烷酸 4.50 4.50 4.50 4.50 4.50 4.50C12乙氧基(2)羧酸鹽 4.50 4.50 4.50 4.50 4.50 4.50C12醇乙氧基化物(4) 3.00 3.00 3.00 3.00 3.00 3.00C12氧化胺 3.00 3.00 3.00 3.00 3.00 3.00枯烯磺酸鈉 2.00 2.00 2.00 2.00 2.00 2.00乙醇4.00 4.00 4.00 4.00 4.00 4.00Mg++(為MgCl2)0.20 0.20 0.20 0.20 0.20 0.20Ca++(為CaCl2)0.40 0.40 0.40 0.40 0.40 0.40水 平衡至 100％產(chǎn)品pH被調(diào)節(jié)至7。
在實(shí)施例19-22中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gly127Asn，得到基本相似的結(jié)果。
在實(shí)施例23-24中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意一種組合代替Gly127Asn和Gly201Asn+Thr207Gln+Tyr205Gln+Tyr213Ile，得到基本相似的結(jié)果。
3.織物清洗組合物在本發(fā)明另一實(shí)例中，織物清洗組合物包括一種或多種本發(fā)明的酶變異體。這里使用的“織物清洗組合物”是指洗滌織物的所有形式的洗滌劑組合物，包括顆粒劑、液體和條塊形式，但不限于此。優(yōu)選織物清洗組合物呈液體形式。
a.顆粒狀織物清洗組合物本發(fā)明的顆粒狀織物清洗組合物包括有效量的一種或多種本發(fā)明的酶變異體，優(yōu)選組合物中活性酶的重量百分比從約0.001％至約10％，更優(yōu)選從約0.005％至約5％，更優(yōu)選從約0.01％至約1％。除包含一種或多種酶變異體外，通常這種顆粒狀織物清洗組合物還包括至少一種表面活性劑、一種或多種助洗劑，且在某些情況下包括一種漂白劑。
本發(fā)明顆粒狀織物清洗組合物的實(shí)例描述于下面的實(shí)施例中。
實(shí)施例25-28顆粒狀織物清洗組合物實(shí)施例號(hào)組分 25 262728Gly156Glu+Asn162Gln 0.10 0.20 0.03 0.05Leu95Glu - - 0.02 0.05C13直鏈烷基苯磺酸鹽 22.00 22.00 22.00 22.00磷酸鹽(為三磷酸鈉)23.00 23.00 23.00 23.00碳酸鈉23.00 23.00 23.00 23.00硅酸鈉14.00 14.00 14.00 14.00沸石 8.20 8.20 8.20 8.20螯合劑(二亞乙基三胺五乙酸)0.40 0.40 0.40 0.40硫酸鈉5.50 5.50 5.50 5.50水 平衡至100％在實(shí)施例25-26中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gly156Glu+Asn162Gln，得到基本相似的結(jié)果。
在實(shí)施例27-28中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意一種組合代替Gly156Glu+Asn162Gln和Leu95Glu，得到基本相似的結(jié)果。
實(shí)施例29-32顆粒狀織物清洗組合物實(shí)施例號(hào)組分29303132Gln161Glu 0.10 0.20 0.03 0.05Ala186Ser+Phe188Ala+Ser190Glu - - 0.02 0.05C12烷基苯磺酸鈉12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸鈉 5.00 5.00 5.00 5.00檸檬酸鈉5.00 5.00 5.00 5.00熒光增白劑 0.10 0.10 0.10 0.10硫酸鈉 17.00 17.00 17 00 17.00在實(shí)施例29-30中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gln161Glu，得到基本相似的結(jié)果。
在實(shí)施例31-32中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意一種組合代替Gln161Glu和Ala186Ser+Phe188Ala+Ser190Glu，得到基本相似的結(jié)果。
實(shí)施例33-36顆粒狀織物清洗組合物實(shí)施例號(hào)組分33 34 35 36Asn 96Glrn+Ile106Ala0.10 0.20 0.03 0.05Gly 60Asn+Asn 61Glu+Gly153Ser - - 0.02 0.05+Pro209Asp+Asn211Asp+Ser215GluC13直鏈烷基苯磺酸鹽22.00 22.00 22.00 22.00磷酸鹽(為三聚磷酸鈉)23.00 23.00 23.00 23.00碳酸鈉 23.00 23.00 23.00 23.00硅酸鈉 14.00 14.00 14.00 14.00沸石8.20 8.20 8.20 8.20螯合劑(二亞乙基三胺五乙酸) 0.40 0.40 0.40 0.40硫酸鈉 5.50 5.50 5.50 5.50水 平衡至 100％在實(shí)施例33-34中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Asn96Gln+Ile106Ala，得到基本相似的結(jié)果。
在實(shí)施例35-36中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意一種組合代替Asn96Gln+Ile106Ala和Gly60Asn+Asn61Glu+Gly153Ser+Pro209Asp+Asn211Asp+Ser215Glu，得到基本相似的結(jié)果。
實(shí)施例37-40顆粒狀織物清洗組合物實(shí)施例號(hào)組分 37 38 39 40Gly60Asn 0.10 0.20 0.03 0.05Ala186Asn+Ser187Asp+Phe188Gln- - 0.02 0.05+Ser190GluC12十二烷基苯磺酸鹽 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸鈉 5.00 5.00 5.00 5.00檸檬酸鈉 5.00 5.00 5.00 5.00熒光增白劑 0.10 0.10 0.10 0.10硫酸鈉 17.00 17.00 17.00 17.00水及次要組分 平衡至100％在實(shí)施例37-38中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gly60Asn，得到基本相似的結(jié)果。
在實(shí)施例39-40中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意一種組合代替Gly60Asn和Ala186Asn+Ser187Asp+Phe188Gln+Ser190Glu，得到基本相似的結(jié)果。
實(shí)施例41-42顆粒狀織物清洗組合物實(shí)施例號(hào)組分 41 42直鏈烷基苯磺酸鹽 11.4 10.70牛油烷基硫酸鹽 1.80 2.40C14-15烷基硫酸鹽 3.00 3.107倍乙氧基化C14-15烷醇4.00 4.0011倍乙氧基化牛油醇 1.80 1.80分散劑 0.07 0.1硅氧烷流體 0.80 0.80檸檬酸三鈉 14.00 15.00檸檬酸 3.00 2.50沸石 32.50 32.10馬來酸丙烯酸共聚物 5.00 5.00二亞乙基三胺五亞甲基膦酸 1.00 0.20Ser215Asp0.30 0.30脂酶 0.36 0.40淀粉酶 0.30 0.30硅酸鈉 2.00 2.50硫酸鈉 3.50 5.20聚乙烯吡咯烷酮 0.30 0.50過硼酸鹽 0.51苯酚磺酸鹽 0.10.2過氧化物酶 0.10.1次要組分 至100 至100
實(shí)施例43-44顆粒狀織物清洗組合物實(shí)施例號(hào)組分 43 44C12直鏈烷基苯磺酸鈉 6.5 8.0硫酸鈉15.018.0沸石A 26.022.0次氮基三乙酸鈉5.0 5.0聚乙烯吡咯烷酮0.5 0.7四乙酰乙二胺 3.0 3.0硼酸 4.0 -過硼酸0.5 1苯酚磺酸鹽0.1 0.2Val202Met+Thr212Pro 0.4 0.4填料(如硅酸鹽、碳酸鹽、香料、水) 至100 至100實(shí)施例45濃縮的顆粒狀織物清洗組合物組分重量％烷基硫酸鹽 8.0烷基乙氧基硫酸鹽2.03和7倍乙氧基化的C25和C45烷醇混合物6.0多羥基脂肪酸酰胺2.5沸石17.0多層硅酸鹽/檸檬酸鹽 16.0碳酸鹽 7.0馬來酸丙烯酸共聚物 5.0污垢解脫聚合物 0.4羧甲基纖維素0.4聚(4-乙烯吡啶)-N-氧化物 0.1乙烯咪唑和乙烯吡咯烷酮共聚物0.1PEG2000 0.2Thr 65Glu+Ile106Met+Leu125His+Tyr205Leu 0.5+Ser215Asp脂酶0.2纖維素酶0.2四乙酰乙二胺6.0過碳酸鹽22.0乙二胺二琥珀酸 0.3抑泡劑 3.54，4′-二(2-嗎啉代-4-苯胺基-S-三嗪-6- 0.25基氨基)茋-2，2′-二磺酸鈉4，4′-二(2-磺基styril)聯(lián)苯二鈉 0.05水、香料及次要組分 至100實(shí)施例46顆粒狀織物清洗組合物組分 重量％直鏈烷基苯磺酸鹽 7.6C16-C18烷基磺酸鹽 1.37倍乙氧基化的C14-15烷醇 4.0椰子油-烷基-二甲基羥乙基氯化銨 1.4分散劑 0.07硅氧烷流體 0.8檸檬酸三鈉 5.0沸石4A 15.0馬來酸丙烯酸共聚物 4.0二亞乙基三胺五亞甲基膦酸 0.4過硼酸鹽 15.0四乙酰乙二胺 5.0綠土粘土 10.0聚氧乙烯(MW300,000) 0.3Thr207Asn+Tyr208Gln+Thr212Ser+Tyr213Glu 0.4+Ser215Glu脂酶 0.2淀粉酶 0.3纖維素酶 0.2硅酸鈉 3.0碳酸鈉 10.0羧甲基纖維素 0.2增白劑 0.2水、香料和次要組分 至100
實(shí)施例47顆粒狀織物清洗組合物組分重量％直鏈烷基苯磺酸鹽6.92牛油烷基硫酸鹽 2.057倍乙氧基化的C14-15烷醇 4.43倍乙氧基化的C12-15烷基乙氧基硫酸鹽 0.16沸石20.2檸檬酸鹽5.5碳酸鹽 15.4硅酸鹽 3.0馬來酸丙烯酸共聚物 4.0羧甲基纖維素0.31污垢解脫聚合物 0.30Gly 60Asp+Asn 61Asp+Gly 62Asp+Thr 65Gln 0.2脂酶0.36纖維素酶0.13四水合過硼酸鹽 11.64一水合過硼酸鹽 8.7四乙酰乙二胺5.0二亞乙基三胺五甲基膦酸 0.38硫酸鎂 0.40增白劑 0.19香料，硅氧烷，抑泡劑0.85次要組分至100
b.液體織物清洗組合物本發(fā)明的液體織物清洗組合物包括有效量的一種或多種本發(fā)明的酶變異體，優(yōu)選組合物中活性酶的重量百分比為約0.005％至約5％，更優(yōu)選約0.01％至約1％。通常這種液體織物清洗組合物還包括一種陰離子表面活性劑、一種脂肪酸、一種水溶性助洗劑和水。
本發(fā)明的液體織物清洗組合物的實(shí)例描述于下面的實(shí)施例中。
實(shí)施例48-52液體織物清洗組合物實(shí)施例號(hào)4849505152Val 94Gly+Gly 99Pro+lle106Val0.05 0.03 0.30 0.03 0.10Gly218Pro- - - 0.01 0.20C12-C14烷基硫酸鈉 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00檸檬酸鈉 1.00 1.00 1.00 1.00 1.00C10烷醇乙氧基化物(3)13.00 13.00 13.00 13.00 13.00單乙醇 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1)平衡至100％在實(shí)施例48-50中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Val94Gly+Gly99Pro+Ile106Val，得到基本相似的結(jié)果。
在實(shí)施例51-52中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Val94Gly+Gly99Pro+Ile106Val和Gly218Pro，得到基本相似的結(jié)果。
實(shí)施例53-57液體織物清洗組合物實(shí)施例號(hào)組分 5354555657Gly156Ser 0.05 0.03 0.30 0.03 0.10Ser187Glu - - - 0.01 0.20C12-C14烷基硫酸鈉 20.00 20.00 20.00 20.00 20.002-丁基辛酸5.00 5.00 5.00 5.00 5.00檸檬酸鈉 1.00 1.00 1.00 1.00 1.00C10烷醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00單乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％在實(shí)施例53-55中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gly156Ser，得到基本相似的結(jié)果。
在實(shí)施例56-57中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任意組合代替Gly156Ser和Ser187Glu，得到基本相似的結(jié)果。
實(shí)施例58-59液體織物清洗組合物實(shí)施例號(hào)組分58 59C12-14鏈烯基琥珀酸 3.08.0檸檬酸一水合物 10.0 15.0C12-15烷基硫酸鈉8.08.02倍乙氧基化的C12-15烷醇硫酸鈉 - 3.07倍乙氧基化C12-15烷醇 - 8.05倍乙氧基化C12-15烷醇 8.0-二亞乙基三胺五(亞甲基膦酸) 0.2-油酸1.8-乙醇4.04.0丙二醇 2.02.0Gly101Glu+Ser104Glu+Ser203Asp+Tyr205Ile 0.20.2+Ser210Glu+Thr212Pro聚乙烯吡咯烷酮 1.02.0抑泡劑 0.15 0.15NaOH 至pH7.5過硼酸鹽0.51苯酚磺酸鹽 0.10.2過氧化物酶 0.40.1水及次要組分 至100份在實(shí)施例58和59中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gly101Glu+Ser104Glu+Ser203Asp+Tyr205Ile+Ser210Glu+Thr212Pro，得到基本相似的結(jié)果。
實(shí)施例60-62液體織物清洗組合物實(shí)施例號(hào)組分 60 61 62檸檬酸 7.10 3.00 3.00脂肪酸 2.00 - 2.00乙醇 1.93 3.20 3.20硼酸 2.22 3.50 3.50單乙醇胺 0.71 1.09 1.091，2-丙二醇 7.89 8.00 8.00枯烯磺酸鈉 1.80 3.00 3.00甲酸鈉 0.08 0.08 0.08NaOH 6.70 3.80 3.80硅烷消泡劑 1.16 1.18 1.18Asn 61Glu+Gly 64Ser+Thr 65Gly0.0145 - -Ser129Asp+Gly130Asn - 0.0145 -Leu216Asn- - 0.0145脂酶 0.200 0.200 0.200纖維素酶 - 7.50 7.50污垢解脫聚合物 0.29 0.15 0.15消泡劑 0.06 0.085 0.085增白劑36 0.095 - -增白劑3 - 0.05 0.05C12烷基苯磺酸 9.86 - -C12-15烷基聚乙氧基化(2.5)硫酸鹽 13.80 18.00 18.00C12葡糖酰胺 - 5.00 5.00C12-13烷基聚乙氧基化物(9)2.00 2.00 2.00水、香料和次要組分 平衡至 100％c.條塊狀織物清洗組合物適用于手洗臟衣物的本發(fā)明條塊狀織物清洗組合物包括有效量的一種或多種本發(fā)明的酶變異體，優(yōu)選占組合物的重量百分比為約0.001％至約10％，更優(yōu)選約0.01％至約1％。
本發(fā)明條塊狀織物清洗組合物的實(shí)例描述于下面的實(shí)施例中。
實(shí)施例63-66條塊狀織物清洗組合物實(shí)施例號(hào)組分 6364 65 66Val202Ser+Asn211Asp 0.3 - 0.10.02Gly156Gln+lle164Thr - - 0.40.03C12-C16烷基硫酸鈉 20.0 20.0 20.0 20.00C12-C14N-甲基葡酰胺5.0 5.05.05.00C11-C13烷基苯磺酸鈉10.0 10.0 10.0 10.00碳酸鈉 25.0 25.0 25.0 25.00焦磷酸鈉 7.0 7.07.07.00三聚磷酸鈉 7.0 7.07.07.00沸石A(0.1-10μ) 5.0 5.05.05.00羧甲基纖維素 0.2 0.20.20.20聚丙烯酸鹽(MW1400) 0.2 0.20.20.20椰子油單乙醇酰胺 5.0 5.05.05.00增白劑、香料 0.2 0.20.20.20CaSO41.0 1.01.01.00MgSO41.0 1.01.01.00水 4.0 4.04.04.00填料*平衡至100％*可選自適宜的材料如CaCO3、滑石、粘土、硅酸鹽等等。
在實(shí)施例63-64中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Val202Ser+Asn211Asp，得到基本相似的結(jié)果。
在實(shí)施例65-66中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任何一種組合代替Val202Ser+Asn211Asp和Gly156Gln+Ile164Thr，得到基本相似的結(jié)果。
實(shí)施例67-70條塊狀織物清洗組合物實(shí)施例號(hào)組分 6768 69 70Val204Thr+Pro209Asp+Tyr213Cys 0.3 - 0.10.02Gly126Gln+Thr214Gly+Ser215Glu - 0.30.40.03C12-C16烷基硫酸鈉20.0 20.0 20.0 20.00C12-C14N-甲基葡酰胺 5.0 5.05.05.00C11-C13烷基苯磺酸鈉 10.0 10.0 10.0 10.00碳酸鈉 25.0 25.0 25.0 25.00焦磷酸鈉 7.0 7.07.07.00三聚磷酸鈉 7.0 7.07.07.00沸石A(0.1-10μ)5.0 5.05.05.00羧甲基纖維素 0.2 0.20.20.20聚丙烯酸鹽(MW1400) 0.2 0.20.20.20椰子油單乙醇酰胺 5.0 5.05.05.00增白劑，香料 0.2 0.20.20.20CaSO41.0 1.01.01.00MgSO41.0 1.01.01.00水 4.0 4.04.04.00填料*平衡至100％*可選自適宜的材料如CaCO3、滑石、粘土、硅酸鹽等等。
在實(shí)施例67中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Val204Thr+Pro209Asp+Tyr213Cys，得到基本相似的結(jié)果。
在實(shí)施例68中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gly126Gln+Thr214Gly+Ser215Glu，得到基本相似的結(jié)果。
在實(shí)施例69-70中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體的任何一種組合代替Val204Thr+Pro209AsP+Tyr213Cys和Gly126Gln+Thr214Gly+Ser215Glu，得到基本相似的結(jié)果。
B.其他清洗組合物除上述硬表面清洗組合物、洗碟劑組合物和織物清洗組合物外，本發(fā)明的一種或多種酶變異體還可攙入需要水解不溶性的底物的各種其他清洗組合物中。這類其它組合物包括口腔清潔組合物、假牙清洗組合物和隱性眼鏡清洗組合物。
1.口腔清潔組合物在本發(fā)明另一實(shí)施方案中，在用于去除牙齒或假牙上的蛋白質(zhì)污物的組合物中可含有藥物上可接受量的本發(fā)明的一種或多種酶變異體。這里所用的“口腔清潔組合物”是指牙粉、牙膏、牙膠、牙粉、漱口劑、口腔噴霧劑、口膠、口香糖、錠劑、sachets、片劑、生物膠、預(yù)防膏、牙齒治療液等等。優(yōu)選口腔清潔組合物包括占組合物重量百分比約0.0001％至約20％的一種或多種本發(fā)明的酶變異體，更優(yōu)選約0.001％至約10％，更優(yōu)選約0.01％至約5％，和藥物可接受的載體。這里所用的“藥物可接受”是指藥物、藥劑或惰性成分適于與人或低級(jí)動(dòng)物的組織接觸而不會(huì)有不當(dāng)?shù)亩拘?、不相容性、不穩(wěn)定性、刺激性、過敏反應(yīng)等等，具有相稱的合理的利/弊之比。
通?？谇磺鍧嵔M合物中的口腔清潔成分藥物上可接受的口腔清潔載體成分占該組合物的重量百分比為約50％至99.99％，優(yōu)選約65％至99.99％，更優(yōu)選約65％至99％。
可包含在本發(fā)明口腔清潔組合物中的藥物上可接受的載體成分和可選擇性成分是本領(lǐng)域技術(shù)人員所熟知的。各種組合物類型、用于口腔清潔組合物中的載體成分和可選擇成分描述于Seibel的1992年3月17日公開的美國(guó)專利5096700、Sampathkumar的1991年7月2日公開的美國(guó)專利5028414、Benedict，Bush和Sunbergd的1991年7月2日公開的美國(guó)專利5028415中，這些均在此引作參考文獻(xiàn)。
本發(fā)明口腔清潔組合物的實(shí)例描述于下面的實(shí)施例中。
實(shí)施例71-74牙粉組合物實(shí)施例號(hào)組分 71 72 73 74Thr214Asp2.000 3.500 1.500 2.000山梨醇(70％水溶液) 35.000 35.000 35.000 35.000PEG-6*1.000 1.000 1.000 1.000二氧化硅牙用磨料**20.000 20.000 20.000 20.000氟化鈉 0.243 0.243 0.243 0.243二氧化鈦 0.500 0.500 0.500 0.500糖精鈉 0.286 0.286 0.286 0.286烷基硫酸鈉(27.9％水溶液) 4.000 4.000 4.000 4.000香料 1.040 1.040 1.040 1.040羧乙烯聚合物***0.300 0.300 0.300 0.300角叉膠****0.800 0.800 0.800 0.800水 平衡至100％*PEG-6＝分子量為600的聚乙二醇**由J.M.Huber提供的鑒定為Zeodent119的沉淀硅石***由B.F.Goodrich化學(xué)公司提供的Carbopol****由Hercules化學(xué)公司提供的Lota Carrageenan在實(shí)施例71-74中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Thr214Asp，得到基本相似的結(jié)果。
實(shí)施例75-78漱口劑組合物實(shí)施例號(hào)組分 75 76 77 78Thr132Glu 3.00 7.50 1.00 5.00SDA40烷醇 8.00 8.00 8.00 8.00香料 0.08 0.08 0.08 0.08乳化劑0.08 0.08 0.08 0.08氟化鈉0.05 0.05 0.05 0.05甘油 10.00 10.00 10.00 10.00甜味劑0.02 0.02 0.02 0.02苯甲酸0.05 0.05 0.05 0.05氫氧化鈉 0.20 0.20 0.20 0.20顏料 0.04 0.04 0.04 0.04水 平衡至100％在實(shí)施例75-78中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Thr132Glu，得到基本相似的結(jié)果。
實(shí)施例79-82錠劑組合物實(shí)施例號(hào)組分 79 80 81 82Ser 97Glu+Ile106Leu0.01 0.03 0.10 0.02山梨醇 17.50 17.50 17.50 17.50甘露糖醇 17.50 17.50 17.50 17.50淀粉 13.60 13.60 13.60 13.60甜味劑 1.20 1.20 1.20 1.20香料 11.70 11 70 11.70 11.70色料 0.10 0.10 0.10 0.10玉米糖漿 平衡至100％在實(shí)施例79-82中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Ser97Glu+Ile106Leu，得到基本相似的結(jié)果。
實(shí)施例83-88口香糖組合物實(shí)施例號(hào)組分 83 84 85 86Leu125Ala+Ser129Glu+Gly130Pro 0.03 0.02 0.10 0.05+Ser131Asp山梨醇結(jié)晶 38.44 38.40 38.40 38.40Paloja-T膠基*20.00 20.00 20.00 20.00山梨醇(70％水溶液) 22.00 22.00 22.00 22.00甘露糖醇 10.00 10.00 10.00 10.00甘油 7.56 7.56 7.56 7.56香料 1.00 1.00 1.00 1.00*由L.A.Dreyfus Company提供在實(shí)施例83-86中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Leu125Ala+Ser129Glu+Gly130Pro+Ser131Asp，得到基本相似的結(jié)果。
2.假牙清洗組合物在本發(fā)明另一實(shí)施方案中，在口腔外清洗假牙的假牙清洗組合物包含一種或多種本發(fā)明的酶變異體。這種假牙清洗組合物包括有效量的一種或多種酶變異體，優(yōu)選占組合物重量百分比約0.0001％至約50％的一種或多種酶變異體，更優(yōu)選約0.001％至約35％，更優(yōu)選0.01％至約20％，和假牙清洗載體。各種假牙清洗組合物形式如起泡片等是本領(lǐng)域所熟知的(參見Young的美國(guó)專利5055305，這里引作參考文獻(xiàn))，常常適于摻入一種或多種酶變異體除去假牙上的蛋白質(zhì)污物。
本發(fā)明假牙清洗組合物的實(shí)例描述于下面的實(shí)施例中。
實(shí)施例87-90雙層起泡假牙清洗片實(shí)施例號(hào)組分 87 88 89 90酸層Thr212Pro+Thr219Pro 1.01.50.01 0.05酒石酸24.0 24.0 24.00 24.00碳酸鈉4.04.04.00 4.00氨基磺酸 10.0 10.0 10.00 10.00PEG20,000 4.04.04.00 4.00碳酸氫鈉 24.5 24.5 24.50 24.50過硫酸鉀 15.0 15.0 15.00 15.00酸式焦磷酸鈉 7.07.07.00 7.00焦化的二氧化硅2.02.02.00 2.00TAED*7.07.07.00 7.00蓖麻油基磺基琥珀酸鹽 0.50.50.50 0.50香料 1.01.01.00 1.00堿層一水合過硼酸鈉32.0 32.0 32.00 32.00碳酸氫鈉 19.0 19.0 19.00 19.00EDTA 3.03.03.00 3.00三聚磷酸鈉12.0 12.0 12.00 12.00PEG20,000 2.02.02.00 2.00過硫酸鉀 26.0 26.0 26.00 26.00碳酸鈉2.02.02.00 2.00焦化的二氧化硅2.02.02.00 2.00顏料/香料 2.02.02.00 2.00*四乙酰乙二胺在實(shí)施例87-90中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Thr212Pro+Thr219Pro，得到基本相似的結(jié)果。
3.隱性眼鏡清洗組合物在本發(fā)明另一實(shí)施方案中，隱性眼鏡清洗組合物包含一種或多種本發(fā)明的酶變異體。這種隱性眼鏡清洗組合物包括有效量的一種或多種酶變異體，優(yōu)選占組合物重量百分比約0.01％至約50％的一種或多種酶變異體，更優(yōu)選約0.01％至約20％，更優(yōu)選約1％至約5％，和隱性眼鏡清洗載體。各種隱性眼鏡清洗組合物形式如片劑、液體等是本領(lǐng)域所熟知的(參見Davies，Meaken和Rees的公開于1989年9月5日的美國(guó)專利4863627，Huth，Lam和Kirai的再公開于1988年5月24日的美國(guó)再頒專利32672，Schaferd的公開于1986年9月2日的美國(guó)專利4609493，Ogunbiyi和Smith公開于1987年9月1日的美國(guó)專利4690793，Ogunbiyi，Riedhammer和Smith公開于1986年9月30日的美國(guó)專利4614549，Ogata公開于1981年8月25日的美國(guó)專利4285738，這些在此均引作參考文獻(xiàn))，且常常適于與本發(fā)明一種或多種酶變異體結(jié)合起來除去隱性眼鏡上的蛋白質(zhì)污物。
本發(fā)明的隱性眼鏡清洗組合物的實(shí)例描述于下面的實(shí)施例中。
實(shí)施例91-94隱形眼鏡酶制清洗液實(shí)施例號(hào)組分 9192 93 94Gly156Glu+Thr163Ser+Asn211Ser 0.01 0.50.12.0+Thr212Asn葡萄糖50.00 50.0 50.0 50.0非離子表面活性劑(聚氧乙烯-2.00 2.02.02.0聚氧丙烯共聚物)陰離子表面活性劑(聚氧乙烯-1.00 1.01.01.0烷基苯基醚硫酸酯鈉)氯化鈉1.00 1.01.01.0硼砂 0.30 0.30.30.3水 平衡至100％在實(shí)施例91-94中，用表2-36中列舉的其他枯草桿菌蛋白酶DY變異體代替Gly156Glu+Thr163Ser+Asn211Ser+Thr212Asn，得到基本相似的結(jié)果。
盡管本發(fā)明僅敘述了具體實(shí)施方案，但很顯然本領(lǐng)域的技術(shù)人員可以在不超出本發(fā)明的精神和范圍的情況下對(duì)本發(fā)明作各種改變和修改。本發(fā)明意在后附的權(quán)利要求中包括在本發(fā)明范圍之內(nèi)的所有這些改變。
序列表(1)一般信息(i)申請(qǐng)人BRODE，PHILIP F.
BARNETT，BOBBY L.
RUBINGH，DONN N.(ii)發(fā)明名稱具有降低的吸附作用和增強(qiáng)的水解作用的枯草桿菌蛋白酶DY變異體(iii)序列數(shù)1(iv)通訊地址(A)收件人THE PROCTER&GAMBLE COMPANY(B)街道11810 East Miami River Road(C)城市Ross(D)州OH(E)國(guó)家USA(F)郵政編碼45061(v)計(jì)算機(jī)可讀形式(A)介質(zhì)類型軟盤(B)計(jì)算機(jī)IBM PC兼容機(jī)(C)操作系統(tǒng)PC-DOS/MS-DOS(D)軟件PatentIn Release#1.0，版本1.30(vi)本申請(qǐng)數(shù)據(jù)(A)申請(qǐng)?zhí)?B)申請(qǐng)日(C)分類號(hào)(viii)代理人/代理機(jī)構(gòu)信息(A)姓名HAKE，RICHARD A.
(B)注冊(cè)號(hào)37,343(C)參考/備案號(hào)5606(iX)通訊信息(A)電話(513)627-0087(B)傳真(513)627-0260(2)序列信息SEQ ID NO1(i)序列特征(A)長(zhǎng)度274氨基酸(B)類型氨基酸(C)鏈單鏈(D)拓?fù)浣Y(jié)構(gòu)線性(Xi)序列描述SEQ ID NO1Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val1 5 10 15Gln Ala Gln Gly Tyr Lys Gly Ala Asn Val Lys Val Gly Ile Ile Asp20 25 30Thr Gly Ile Ala Ala Ser His Thr Asp Leu Lys Val Val Gly Gly Ala35 40 45Ser Phe Val Ser Gly Glu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly50 55 60Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val65 70 75 80Leu Gly Val Ala Pro Asn Val Ser Leu Tyr Ala Ile Lys Val Leu Asn85 90 95Ser Ser Gly Ser Gly Thr Tyr Ser Ala Ile Val Ser Gly Ile Glu Trpl00 105 110Ala Thr Gln Asn Gly Leu Asp Val Ile Asn Met Ser Leu Gly Gly Pro115 120 125Ser Gly Ser Thr Ala Leu Lys Gln Ala Val Asp Lys Ala Tyr Ala Ser130 135 140Gly Ile Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Ser145 150 155 160Gln Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val165 170 175Gly Ala Val Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly180 185 190Ala Glu Leu Glu Val Met Ala Pro Gly Val Ser Val Tyr Ser Thr Tyr195 200 205Pro Ser Asn Thr Tyr Thr Ser Leu Asn Gly Thr Ser Met Ala Ser Pro210 215 220His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys Tyr Pro Thr Leu225 230 235 240Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Asn Leu245 250 255Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala260 265 270Ala Gln
權(quán)利要求
1.一種對(duì)野生型枯草桿菌蛋白酶的氨基酸序列進(jìn)行了改良的枯草桿菌蛋白酶DY變異體，該野生型氨基酸序列包括第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)、第五環(huán)區(qū)和第六環(huán)區(qū)；其中改良的氨基酸序列是在一個(gè)或多個(gè)環(huán)區(qū)中一個(gè)或多個(gè)位置上有取代；其中A、當(dāng)取代發(fā)生在第一環(huán)區(qū)時(shí)，在58、59、60、61、62、64或65位的一個(gè)或多個(gè)位置上發(fā)生取代，其中c.取代發(fā)生在第60位時(shí)，取代氨基酸為Asn、Gln、Pro或Ser；f.取代發(fā)生在第64位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；以及g.取代發(fā)生在第65位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Gly、Pro或Ser；B、當(dāng)取代發(fā)生在第二環(huán)區(qū)時(shí)，在94、95、96、97、98、99、100、101、102、103、104、105或106位的一個(gè)或多個(gè)位置上發(fā)生取代，其中a.取代發(fā)生在第94位時(shí)，取代氨基酸為Ala、Cys、Gln、Gly、His、Met、Pro或Ser；b.取代發(fā)生在第95位時(shí)，取代氨基酸為Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Valc.取代發(fā)生在第96位時(shí)，取代氨基酸為Gln或Ser；f.取代發(fā)生在第99位時(shí)，取代氨基酸為Asn、Gln、Pro或Ser；h.取代發(fā)生在第101位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；i.取代發(fā)生在第102位時(shí)，取代氨基酸為Asn、Gln、Gly、Pro或Ser；j.取代發(fā)生在第103位時(shí)，取代氨基酸為Ala、Asn、Cys、Gln、Gly、His、Ile、Leu、Met、Pro或Ser；l.取代發(fā)生在第105位時(shí)，取代氨基酸為Asn、Gln、Gly、His、Pro、Ser或Thr；和m.取代發(fā)生在第106位時(shí)，取代氨基酸為Ala、Cys、Gln、Gly、His、Met、Pro、Ser或Val；C、當(dāng)取代發(fā)生在第三環(huán)區(qū)時(shí)，在125、126、127、128、129、130、131或132位的一個(gè)或多個(gè)位置上發(fā)生取代，其中a.取代發(fā)生在第125位時(shí)，取代氨基酸為Ala、Asn、Cys、Gln、Gly、His、Met、Pro、Ser、Thr或Val；b.取代發(fā)生在第126位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Pro或Serd.取代發(fā)生在第128位時(shí)，取代氨基酸為Asn、Gln或Ser；g.取代發(fā)生在第131位時(shí)，取代氨基酸為Asp或Glu；和h.取代發(fā)生在第132位時(shí)，取代氨基酸為Asn、Gln、Gly、Pro或Ser；D、當(dāng)取代發(fā)生在第四環(huán)區(qū)時(shí)，在153、154、155、156、157、158、159、160、161、162、163、164、165或166位的一個(gè)或多個(gè)位置上發(fā)生取代，其中b.取代發(fā)生在第154位時(shí)，取代氨基酸為Gln或Serd.取代發(fā)生在第156位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；和n.取代發(fā)生在第166位時(shí)，取代氨基酸為Ala、Gly、His、Ile、Leu、Met或Pro；E、當(dāng)取代發(fā)生在第五環(huán)區(qū)時(shí)，在186、187、188、189或190位的一個(gè)或多個(gè)位置上發(fā)生取代，其中c.取代發(fā)生在第188位時(shí)，取代氨基酸為Ala、Asn、Cys、Gln、Gly、His、Ile、Leu、Met、Pro、Ser、Thr、Tyr或ValF、當(dāng)取代發(fā)生在第六環(huán)區(qū)時(shí)，在199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219位的一個(gè)或多個(gè)位置上發(fā)生取代，其中a.取代發(fā)生在第199位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；b.取代發(fā)生在第200位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Gly或Ser；c.取代發(fā)生在第201位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Pro或Ser；d.取代發(fā)生在第202位時(shí)，取代氨基酸為Ala、Cys、Gln、Gly、His、Met、Pro或Ser；f.取代發(fā)生在第204位時(shí)，取代氨基酸為Ala、Cys、Gln、Gly、His、Met、Pro或Ser；g.取代發(fā)生在第205位時(shí)，取代氨基酸為Ala、Asn、Gln、Gly、His、Ile、Leu、Met、Pro或Ser；h.取代發(fā)生在第206位時(shí)，取代氨基酸為Asp或Glu；i.取代發(fā)生在第207位時(shí)，取代氨基酸為Asn、Gln、Gly、Pro或Ser；j.取代發(fā)生在第208位時(shí)，取代氨基酸為Ala、Asn、Cys、Gln、Gly、His、Ile、Met、Pro或Ser；k.取代發(fā)生在第209位時(shí)，取代氨基酸為Asn、Gln、Gly或Ser；、m.取代發(fā)生在第211位時(shí)，取代氨基酸為Gln或Ser；n.取代發(fā)生在第212位時(shí)，取代氨基酸為Asn、Gln、Gly、Pro或Ser；o.取代發(fā)生在第213位時(shí)，取代氨基酸為Ala、Asn、Cys、Gln、Gly、His、Ile、Leu、Met或Pro；p.取代發(fā)生在第214位時(shí)，取代氨基酸為Asn、Gln、Gly、Pro或Ser；r.取代發(fā)生在第216位時(shí)，取代氨基酸為Ala、Asn、Cvs、Gln、Gly、His、Ile、Met、Pro、Ser、Thr或Val；和u.取代發(fā)生在第219位時(shí)，取代氨基酸為Asn、Asp、Gln、Glu、Gly、Pro或Ser；由此與野生型枯草桿菌蛋白酶DY相比，本枯草桿菌蛋白酶DY變異體對(duì)不溶性底物具有降低的吸附作用和增強(qiáng)的水解作用。
2.權(quán)利要求1的枯草桿菌蛋白酶DY變異體，其中在第一環(huán)區(qū)發(fā)生一個(gè)或多個(gè)取代。
3.權(quán)利要求1的枯草桿菌蛋白酶DY變異體，其中在第二環(huán)區(qū)發(fā)生一個(gè)或多個(gè)取代。
4.權(quán)利要求1的枯草桿菌蛋白酶DY變異體，其中在第三環(huán)區(qū)發(fā)生一個(gè)或多個(gè)取代。
5.權(quán)利要求1的枯草桿菌蛋白酶DY變異體，其中在第四環(huán)區(qū)發(fā)生一個(gè)或多個(gè)取代。
6.權(quán)利要求1的枯草桿菌蛋白酶DY變異體，其中在第五環(huán)區(qū)發(fā)生一個(gè)或多個(gè)取代。
7.權(quán)利要求1的枯草桿菌蛋白酶DY變異體，其中在第六環(huán)區(qū)發(fā)生一個(gè)或多個(gè)取代。
8.一種選自硬表面清洗組合物、洗碟劑組合物、口腔清潔組合物、假牙清洗組合物、隱性眼鏡組合物和織物清洗組合物的清洗組合物，其特征在于該清洗組合物包含權(quán)利要求1-7之一的枯草桿菌蛋白酶DY變異體和一種清洗組合物載體；該清洗組合物優(yōu)選是硬表面清洗組合物或織物清洗組合物；優(yōu)選該組合物包含至少約為組合物重量5％的表面活性劑和至少約為組合物重量5％的助洗劑；優(yōu)選該組合物還包含選自溶劑、緩沖劑、酶、污垢解脫劑、粘土類去污垢劑、分散劑、增白劑、抑泡劑、織物柔軟劑、增泡劑、酶穩(wěn)定劑、漂白劑、染料、香料及其混合物的清洗組合物材料。
9.一種編碼權(quán)利要求1-7之任一枯草桿菌蛋白酶DY變異體的DNA序列。
全文摘要
本發(fā)明涉及枯草桿菌蛋白酶DY變異體,其具有對(duì)野生型枯草桿菌蛋白酶DY氨基酸序列進(jìn)行過改良的氨基酸序列,該野生型氨基酸序列包括第一環(huán)區(qū)、第二環(huán)區(qū)、第三環(huán)區(qū)、第四環(huán)區(qū)、第五環(huán)區(qū)和第六環(huán)區(qū);其中改良的氨基酸序列是在一個(gè)或多個(gè)環(huán)區(qū)的特定識(shí)別位置上包括與野生型枯草桿菌蛋白酶DY不同的氨基酸(即取代),由此,枯草桿菌蛋白酶DY變異體與野生型枯草桿菌蛋白酶DY相比對(duì)不溶性底物具有降低的吸附作用和增強(qiáng)的水解作用。本發(fā)明還涉及編碼這種枯草桿菌蛋白酶DY變異體的DNA序列。本發(fā)明也涉及包含該枯草桿菌蛋白酶DY變異體的用于清洗各種表面的組合物。
文檔編號(hào)A61K8/00GK1183810SQ9619379
公開日1998年6月3日 申請(qǐng)日期1996年3月6日 優(yōu)先權(quán)日1995年3月9日
發(fā)明者P·F·布羅德三世, B·L·巴爾內(nèi)特, D·N·魯賓 申請(qǐng)人:普羅格特-甘布爾公司