專利名稱：葡萄糖異構(gòu)酶突變體的應(yīng)用的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及分子生物技術(shù)領(lǐng)域，具體地說，涉及利用基因突變技術(shù)制備高活力、或高活力并耐熱的葡萄糖異構(gòu)酶突變體的方法、所獲得的突變體及其應(yīng)用。
背景技術(shù)：
葡萄糖異構(gòu)酶(Xylose isomerase，E.C.5.3.1.5，簡稱XI)、或稱木糖異構(gòu)酶(Glucose isomerase)是戊糖發(fā)酵途徑的關(guān)鍵酶。該酶是最重要的酶制劑之一(Kaneko，et al.，Biosci Biotechnol Biochem，64940-947，2000)。在生物能源工業(yè)中，人們正在試圖使用該酶并連同其它纖維素與半纖維素降解途徑中的關(guān)鍵酶商業(yè)化地生產(chǎn)生物乙醇。例如，從木聚糖至5-磷酸木酮糖的酶解途徑如下木聚糖在內(nèi)-β-1，4-木聚糖酶的作用下生成低聚木聚，后者再在β-木糖苷酶的作用下生成木糖，該木糖又可被葡萄糖異構(gòu)酶(即木糖異構(gòu)酶)催化生成木酮糖，最后該木酮糖在β-木酮糖激酶的作用下，生成5-磷酸木酮糖。
迫于環(huán)境污染和石油緊缺的壓力，世界上許多國家，如巴西、美國、加拿大，早已以甘蔗或玉米為原料工業(yè)化生產(chǎn)燃料乙醇。中國也已利用玉米或其他糧食生產(chǎn)生物乙醇。由于自然界中生物質(zhì)資源十分豐富，世界上多個國家的科學(xué)家正在探索以飽含纖維素與半纖維素的殘余農(nóng)作物秸稈等為原料，工業(yè)化地生產(chǎn)生物乙醇。葡萄糖異構(gòu)酶是生物降解半纖維素的關(guān)鍵酶之一。高活力、或高活力并耐熱的葡萄糖異構(gòu)酶有利于降低生物乙醇的生產(chǎn)成本，進(jìn)而實現(xiàn)工業(yè)化生產(chǎn)生物乙醇。
世界各地的科學(xué)家對從耐熱菌中篩選并通過蛋白質(zhì)工程的方法培育耐熱、高催化活性的葡萄糖異構(gòu)酶進(jìn)行了許多研究。如J.G.Zeikus及其合作者從Thermoanaerobacterium saccharolyticum、Thermotoga neapolitana等數(shù)種耐高溫菌中分離并研究了耐高溫葡萄糖異構(gòu)酶(見Lee et al.，Journal of General Microbiology，1391227-1234，1993；Vieille et al.，Methods Enzymology，330215-24，2001；Lee et al.，Journal of General Microbiology，1391241-1243，1993；Scriprapundh et al.，Protein Engineering，13259-265，2000；Scriprapundh et al.，Protein Engineering，16683-690，2003；Zeikuset al.，US Patent No 5,656,497)。這些或其它來源的耐高溫葡萄糖異構(gòu)酶之活力較低，尚未見用于工業(yè)化生產(chǎn)生物乙醇。因此，目前仍存在高活力、或高活力并耐熱的葡萄糖異構(gòu)酶的需求。

發(fā)明內(nèi)容
本發(fā)明人對利用遺傳工程和蛋白質(zhì)工程技術(shù)對來源于T.saccharolyticum的葡萄糖異構(gòu)酶進(jìn)行改良所獲得的一系列突變體的酶活性進(jìn)行了研究，從而提供了該突變體在較高溫度下使用并能夠高活性地催化D-木糖轉(zhuǎn)變?yōu)槟就堑膽?yīng)用。
本發(fā)明人通過對T.saccharolyticum葡萄糖異構(gòu)酶基因進(jìn)行定點突變，隨后在MacConkey培養(yǎng)基上篩選，從而取得一系列高催化活性、或具高催化活性并耐熱的葡萄糖異構(gòu)酶突變體。具體而言，利用分子生物學(xué)技術(shù)，構(gòu)建含有親本葡萄糖異構(gòu)酶基因的載體質(zhì)粒，然后設(shè)定定點突變的位點以及突變后的氨基酸種類，再合成適當(dāng)?shù)囊?，以所述的含親本葡萄糖異構(gòu)酶基因的載體質(zhì)?；蚱溲苌|(zhì)粒為模板，PCR擴增DNA片段、裝配所擴增的DNA片段以及PCR擴增全長突變基因。通過將該全長突變基因克隆到適當(dāng)?shù)妮d體上并轉(zhuǎn)化適當(dāng)?shù)乃拗骷?xì)胞，經(jīng)培養(yǎng)篩選出具有葡萄糖異構(gòu)酶活性的陽性克隆。從陽性克隆中提取質(zhì)粒DNA，進(jìn)行DNA序列測定分析，以確定引入的突變。最后，以D-木糖為底物測定具有所設(shè)定位點突變的葡萄糖異構(gòu)酶的酶活力，將該酶活力與親本的酶活力相比較，選取具有比親本的活力高的催化活性的葡萄糖異構(gòu)酶突變體，將之用于將D-木糖轉(zhuǎn)變?yōu)槟就堑墓に?、降解纖維素的工藝以及生產(chǎn)生物乙醇的工藝。
在上述篩選突變體的過程中，所述載體可為任何適當(dāng)?shù)妮d體。例如，適用的載體包括但不限于原核表達(dá)載體pGEMT-Easy，pRSET和pET21；包括但不限于真核表達(dá)載體pYD1和pYES2/GS；包括但不限于克隆載體pUC18/19和pBluscript-SK。
所獲得的葡萄糖異構(gòu)酶突變體基因可以在原核細(xì)胞或真核細(xì)胞胞內(nèi)表達(dá)，也可采用本領(lǐng)域已知的任何其它適當(dāng)方法實現(xiàn)在原核細(xì)胞或真核細(xì)胞胞外表達(dá)。
所述載體的微生物宿主細(xì)胞為原核細(xì)胞或真核細(xì)胞。所述原核微生物包括但不限于大腸桿菌、結(jié)芽孢桿菌、枯草芽胞桿菌、巨大芽胞桿菌(如巨大芽胞桿菌BP931)、T.saccharolyticum和鏈霉菌(如Streptomyces diastaticus M1033)。所述真核微生物包括但不限于釀酒酵母和畢赤巴斯德酵母(如畢赤巴斯德酵母GS115/9891)。
在根據(jù)本發(fā)明的應(yīng)用中，以序列表中的序列2為參考序列，所述葡萄糖異構(gòu)酶突變體具有至少一個、至少兩個、至少三個、至少四個、至少五個、至少六個、至少七個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的至少一個突變，并且在以D-木糖為底物生成木酮糖的反應(yīng)中所述突變體具有比親本高出至少50％的酶催化活性。所述的葡萄糖異構(gòu)酶突變體還可用于半纖維素的降解過程，并適宜用于生產(chǎn)生物乙醇。
以序列表中的序列2為參考序列，優(yōu)選的是，所述第87位的苯丙氨酸(Phe)突變?yōu)榧琢虬彼?Met)、或亮氨酸(Leu)；第139位的色氨酸突變?yōu)榻z氨酸(Ser)、或賴氨酸(Lys)、或半胱氨酸(Cys)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或天冬酰胺(Asn)、或天門冬氨酸(Asp)；第182位的精氨酸突變?yōu)楸彼?Ala)、或脯氨酸(Pro)、或絲氨酸(Ser)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或纈氨酸(Val)；第187位的纈氨酸突變?yōu)楦拾彼?Gly)、或丙氨酸(Ala)、或脯氨酸(Pro)；第217位的纈氨酸(Val)突變?yōu)榫彼?Arg)、或甘氨酸(Gly)；第260位的天門冬氨酸(Asp)突變?yōu)楣劝彼?Glu)、或丙氨酸(Ala)；第276位的苯丙氨酸(Phe)突變?yōu)楦拾彼?Gly)、或蘇氨酸(Thr)；和/或第299位的蘇氨酸突變?yōu)楫惲涟彼?Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或甲硫氨酸(Met)、或谷氨酸(Glu)。
更為優(yōu)選的是，在本發(fā)明的應(yīng)用中，所述的突變體具有序列表中序列5至序列40之一(即SEQ ID NO.5至SEQ ID NO.40中之一)所示的氨基酸序列。
最優(yōu)選的是，在本發(fā)明的應(yīng)用中，所述葡萄糖異構(gòu)酶突變體包含下文表2中所列的那些突變的氨基酸序列。因為表2所列的突變體具有高的催化活性并耐熱。例如，一個具有七個點突變的突變體MGI4-34的比活性較親本高305.9％，且在80℃反應(yīng)19.2小時后仍保持50％或以上的活力。另一個具有七個點突變的突變體MGI4-35的比活性較親本高296.3％，且在80℃反應(yīng)大于24小時后仍保持50％或以上的活力。
在本發(fā)明的應(yīng)用中，所述葡萄糖異構(gòu)酶突變體可以未經(jīng)純化粗酶形式使用，也可以是經(jīng)部分純化的或完全純化的形式。如果需要，還可利用本領(lǐng)域已知的固化技術(shù)將所述葡萄糖異構(gòu)酶突變體制成固相酶或固相細(xì)胞?？梢詫⒈磉_(dá)葡萄糖異構(gòu)酶突變體的表達(dá)載體轉(zhuǎn)入細(xì)菌(如產(chǎn)酸克雷伯菌和運動發(fā)酵單胞菌)、酒精酵母菌或其它宿主(如畢赤巴斯德酵母)中以加速木糖的代謝。具體地講，所述葡萄糖異構(gòu)酶突變體的表達(dá)菌，包括天然或工程的生物乙醇發(fā)酵用的細(xì)菌，如產(chǎn)酸克雷伯菌(Klebsiella oxytoca)和運動發(fā)酵單胞菌(Zymomonas mobilis)；天然或工程的生物乙醇發(fā)酵用的釀酒酵母真菌；以及天然或工程的生物乙醇發(fā)酵用的畢赤巴斯德酵母真菌等。
定義本申請文本中所用的術(shù)語“親本”系指來自Thermoanaerobacterium saccharolyticum ATCC 49915的葡萄糖異構(gòu)酶，其核苷酸序列如序列1所示，氨基酸序列如序列2所示。本發(fā)明中親本基因的核苷酸序列與公布的T.saccharolyticum葡萄糖異構(gòu)酶的基因序列(Lee et al.，Journal of General Microbiology，1391227-1234，1993；GenBank L09699)相比有兩個核苷酸的差異，即與基因庫(GenBank L09699)的核苷酸序列相比，本發(fā)明中親本基因第241-242位為GC，相應(yīng)的第81位氨基酸序列為丙氨酸(Ala)，GenBankL09699對應(yīng)處、第241-242位核苷酸序列為CG，相應(yīng)的第81位氨基酸序列為精氨酸(Arg)。
本申請文本中所用的術(shù)語“參考序列”，當(dāng)其為核苷酸序列時，系指序列表中的序列1，當(dāng)其為氨基酸序列時，是指序列表中的序列2。在將參考序列和突變的葡萄糖異構(gòu)酶序列進(jìn)行排序比較時，可以手工進(jìn)行，也可以用計算機進(jìn)行(目前有許多可供利用的計算機軟件，例如CLUSTALW，AMAS，DIALIGN程序等)。
本申請文本中所用的術(shù)語“位點”或“第x位”是指當(dāng)本發(fā)明的葡萄糖異構(gòu)酶突變體的序列與參考序列的排序?qū)Ρ冗_(dá)最大同源性時，被比較的葡萄糖異構(gòu)酶突變體的各核苷酸或氨基酸所對應(yīng)的參考序列中的核苷酸或氨基酸位置。
本申請文本所用的術(shù)語“葡萄糖異構(gòu)酶突變體”是指一種以序列表中序列2所示氨基酸序列為參考序列，在第87位、第139位、第182位、第187位、第217位、第260位、第276位、第299位具有至少一個氨基酸的差異，并且以D-木糖為底物生成木酮糖時其具有比親本高出至少50％的葡萄糖異構(gòu)酶催化活性的酶。因此，在本專利申請中，所述葡萄糖異構(gòu)酶突變體包括具有序列4所示氨基酸序列的突變體、序列4的保守取代形式、增加或缺失一個或幾個氨基酸的形式、氨基端截斷的形式、羧基端截斷的形式、以及序列4的部分或全部串聯(lián)重復(fù)形式。
在本申請文本所用的氨基酸三字母或單字母表達(dá)方式，采用IUPAC規(guī)定的氨基酸代碼(Eur.J.Biochem.，1389-37，1984)。


圖1親本葡萄糖異構(gòu)酶與葡萄糖異構(gòu)酶突變體在80℃的熱穩(wěn)定。MGI-4、MGI4-34和MGI4-35為葡萄糖異構(gòu)酶突變體，具體請參見實施例8和15。
具體實施例方式
下列實施例僅用于說明本發(fā)明而不應(yīng)視為限定本發(fā)明的范圍。
實施例中未注明具體條件者，按常規(guī)條件或制造商建議的條件進(jìn)行。
實施例1親本基因的擴增及pGEMT-TS的構(gòu)建根據(jù)基因庫(GenBank L09699)基因序列設(shè)計引物T1和T2(見表1)。利用引物對T1和T2從T.saccharolyticum ATCC 49915(購自ATCC，USA)中擴增葡萄糖異構(gòu)酶親本基因。
擴增條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1，400nM引物T2，1.5U Taq DNA聚合酶(Promega，USA)，用接種環(huán)挑取少許T.saccharolyticum菌體，再用無菌水調(diào)反應(yīng)體積至50μl。
PCR擴增反應(yīng)程序為95℃3分鐘，40圈循環(huán)95℃50秒、50℃30秒和72℃1分鐘，最后72℃10分鐘。將擴增的產(chǎn)物(長約1.5kb)連接至載體pGEMT-Easy，得質(zhì)粒pGEMT-TS。經(jīng)DNA測序確定親本葡萄糖異構(gòu)酶的核苷酸序列為序列1，相應(yīng)的氨基酸序列為序列2。與基因庫(GenBank L09699)的核苷酸序列相比，本發(fā)明中親本基因第241-242位為GC，相應(yīng)的第81位氨基酸序列為丙氨酸(Ala)。GenBankL09699對應(yīng)處、第241-242位核苷酸序列為CG，相應(yīng)的第81位氨基酸序列為精氨酸(Arg)。
實施例2葡萄糖異構(gòu)酶位點139之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對139FF和139FR(見表1)，將親本氨基酸序列中第139位點的Trp(W)突變?yōu)镻he(F)，獲得突變體MGI-W139F。引物對T1和T2見實施例1。
利用引物對T1和139FR，擴增T1FR片段，引物對139FF和T2，擴增FFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物139FR或400nM引物139FF和400nM引物T2，1.5UPfu DNA聚合酶(Promega，USA)，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1FR片段和FFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μMdCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段與20ng FFT2片段，用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-W139F。將MGI-W139F與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-W139F。將質(zhì)粒pGEMT-MGI-W139F轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-W139F DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-W139F氨基酸序列見序列表序列5，比活力見表2。
實施例3葡萄糖異構(gòu)酶位點182之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對182AF和182AR(見表1)，將親本氨基酸序列中第182位點的Arg(R)突變?yōu)锳la(A)，獲得突變體MGI-R182A。引物T1和T2見實施例1。
利用引物對T1和182AR，擴增T1AR片段，引物對182AF和T2，擴增AFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物182AR或400nM引物182AF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1AR片段和AFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1AR片段與20ng AFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到全長突變基因MGI-R182A。將MGI-R182A與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-R182A。將質(zhì)粒pGEMT-MGI-R182A轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-R182ADNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-R182A的氨基酸序列見序列表序列6。
實施例4葡萄糖異構(gòu)酶位點187之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對187SF和187SR(見表1)，將親本氨基酸序列中第187位點的Phe(F)突變?yōu)镾er(S)，獲得突變體MGI-F187S。引物T1和T2見實施例1。
利用引物對T1和187SR，擴增T1SR片段，引物對187SF和T2，擴增SFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物187SR或400nM引物T2和400nM引物187SF，1.5UPfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1SR片段和SFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1SR片段與20ngSFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick ExtractionGel Kit回收，得到全長突變基因MGI-F187S。將MGI-F187S與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-F187S。將質(zhì)粒pGEMT-MGI-F187S轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-F187SDNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-F187S的氨基酸序列見序列表序列7。
實施例5葡萄糖異構(gòu)酶位點299之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對299QF和299QR(見表1)，將親本氨基酸序列中第299位點的Thr(T)突變?yōu)镚ln(Q)，獲得突變體MGI-T299Q。引物T1和T2見實施例1。
利用引物對T1和299QR，擴增T1QR片段，引物對299QF和T2，擴增QFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物299QR或400nM引物299QF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1QR片段和QFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1QR片段與20ng QFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到全長突變基因MGI-T299Q。將MGI-T299Q與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-T299Q。將質(zhì)粒pGEMT-MGI-T299Q轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-T299QDNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-T299Q的氨基酸序列見序列表序列8，比活力見表2。
實施例6葡萄糖異構(gòu)酶雙突變組合MGI-2之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按實施例2擴增和回收T1FR片段，按實施例3擴增和回收AFT2片段。用引物對139FF(如實施例2)和182AR(如實施例3)擴增和回收FFAR片段。FFAR片段擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 139FF和400nM 182AR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到FFAR片段。然后擴增全長基因。擴增反應(yīng)條件為20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ngT1FR片段，20ng FFAR片段和20ng AFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick DNA回收試劑盒，得到全長突變基因MGI-2。將MGI-2與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-2。將質(zhì)粒pGEMT-MGI-2轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-2 DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-2序列含有W139F和R182A的二個突變。MGI-2的氨基酸序列見序列表序列9，比活力見表2。
實施例7葡萄糖異構(gòu)酶三突變組合MGI-3之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按實施例2擴增和回收T1FR片段，按實施例5擴增和回收QFT2片段，按實施例6擴增和回收FFAR片段。用引物對182AF(如實施例3)和299QR(如實施例5)擴增和回收AFQR片段。AFQR片段擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM 182AF和400nM 299QR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到AFQR片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段，20ng FFAR片段，20ng AFQR片段和20ng QFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-3。將MGI-3與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-3。將質(zhì)粒pGEMT-MGI-3轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-3DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-3序列含有W139F、R182A和T299Q的三突變。MGI-3的氨基酸序列見序列表序列10，比活力見表2。
實施例8葡萄糖異構(gòu)酶四突變組合MGI-4之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按實施例2擴增和回收T1FR片段，按實施例5擴增和回收QFT2片段，按實施例6擴增和回收FFAR片段。用引物對182AF(如實施例3)和187SR(如實施例4)擴增AFSR片段。AFSR片段擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μMdCTP，50μM dGTP，400nM 182AF和400nM 187SR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到AFSR片段。用引物對187SF(如實施例4)和299QR(如實施例5)擴增SFQR片段。SFQR片段擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM 187SF和400nM 299QR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到SFQR片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段，20ng FFAR片段，20ng AFSR片段，20ng SFQR片段和20ng QFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-4。將MGI-4與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-4。將質(zhì)粒pGEMT-MGI-4轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coliHB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-4DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-4序列含有W139F、R182A、F187S和T299Q的四突變。MGI-4的氨基酸序列見序列表序列11，比活力見表2。
實施例9葡萄糖異構(gòu)酶四突變組合MGI-4之139位點的飽和突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-MGI-4(見實施例8)為模板，設(shè)計引物對139SF和139SR(見表1)，將親本氨基酸序列中第139位點的Phe(F)突變?yōu)镾er(S)，獲得突變體MGI4-F139S。引物對T1和T2見實施例1。
利用引物對T1和139SR，擴增T1SR片段，引物對139SF和T2，擴增SFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物139SR或400nM引物139SF和400nM引物T2，1.5UPfu DNA聚合酶(Promega，USA)，20ng pGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1SR片段和SFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5UPfu DNA聚合酶，20ng T1SR片段與20ng SFT2片段，用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI4-F139S。將MGI4-F139S與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI4-F139S。將質(zhì)粒pGEMT-MGI4-F139S轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI4-F139S DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI4-F139S序列含有F139S、R182A、F187S和T299Q的四突變。MGI4-F139S氨基酸序列見序列表序列12，比活力見表2。
按照類似步構(gòu)建突變體MGI4-F139K、MGI4-F139C、MGI4-F139I、MGI4-F139T、MGI4-F139N和MGI4-F139D，所用引物見表1。所得諸突變體的氨基酸序列見序列表序列13-18，比活力見表2。
實施例10葡萄糖異構(gòu)酶四突變組合MGI-4之182位點的飽和突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-MGI-4(見實施例8)為模板，設(shè)計引物對182PF和182PR(見表1)，將親本氨基酸序列中第182位點的Ala(A)突變?yōu)镻ro(P)，獲得突變體MGI4-A182P。引物T1和T2見實施例1。
利用引物對T1和182PR，擴增T1PR片段，引物對182PF和T2，擴增PFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物182PR或400nM引物182PF和400nM引物T2，1.5UPfu DNA聚合酶，20ng pGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1PR片段和PFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1PR片段與20ng PFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到全長突變基因MGI4-A182P。將MGI4-A182P與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI4-A182P。將質(zhì)粒pGEMT-MGI4-A182P轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI4-A182P DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI4-A182P序列含有W139F、A182P、F187S和T299Q的四突變。MGI4-A182P的氨基酸序列見序列表序列19，比活力見表2。
按照類似步構(gòu)建突變體MGI4-A182S、MGI4-A182I、MGI4-A182T和MGI4-A182V，所用引物見表1。所得諸突變體的氨基酸序列見序列表序列20-23，比活力見表2。
實施例11葡萄糖異構(gòu)酶四突變組合MGI-4之187位點的飽和突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-MGI-4(見實施例8)為模板，設(shè)計引物對187GF和187GR(見表1)，將親本氨基酸序列中第187位點的Ser(S)突變?yōu)镚ly(G)，獲得突變體MGI4-S187G。引物T1和T2見實施例1。
利用引物對T1和187GR，擴增T1GR片段，引物對187GF和T2，擴增GFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％ Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物187GR或400nM引物T2和400nM引物187GF，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1GR片段和GFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1GR片段與20ng GFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到全長突變基因MGI4-S187G。將MGI4-S187G與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI4-S187G。將質(zhì)粒pGEMT-MGI4-S187G轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI4-S187G DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI4-S187G序列含有W139F、R182A、S187G和T299Q的四突變。MGI4-S187G的氨基酸序列見序列表序列24，比活力見表2。
按照類似步構(gòu)建突變體MGI4-S187A和MGI4-S187P，所用引物見表1。所得諸突變體的氨基酸序列見序列表序列25-26，比活力見表2。
實施例12葡萄糖異構(gòu)酶四突變組合MGI-4之299位點的飽和突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以質(zhì)粒pGEMT-MGI-4(見實施例8)為模板，設(shè)計引物對299IF和299IR(見表1)，將親本氨基酸序列中第299位點的Gln(Q)突變?yōu)镮le(I)，獲得突變體MGI4-Q299I。引物T1和T2見實施例1。
利用引物對T1和299IR，擴增T1IR片段，引物對299IF和T2，擴增IFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物299IR或400nM引物299IF和400nM引物T2，1.5U PfuDNA聚合酶，20ng pGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1IR片段和IFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1IR片段與20ngIFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick ExtractionGel Kit回收，得到全長突變基因MGI4-Q299I。將MGI4-Q299I與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI4-Q299I。將質(zhì)粒pGEMT-MGI4-Q299I轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI4-Q299IDNA，經(jīng)DNA測序確定引入的點突變無誤。MGI4-Q299I序列含有W139F、R182A、F187S和Q299I的四突變。MGI4-Q299I的氨基酸序列見序列表序列27，比活力見表2。
按照類似步構(gòu)建突變體MGI4-Q299Y、MGI4-Q299C、MGI4-Q299M和MGI4-Q299E，所用引物見表1。所得諸突變體的氨基酸序列見序列表序列28-31，比活力見表2。
實施例13葡萄糖異構(gòu)酶五突變組合MGI4-F87L、MGI4-F87M、MGI4-V217R、MGI4-D260E和MGI4-F276G之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.Humana Press，1993)之描述。
以質(zhì)粒pGEMT-MGI-4(見實施例8)為模板，設(shè)計引物對87LF和87LR(見表1)，將MGI-4氨基酸序列中第87位點的Phe(F)突變?yōu)長eu(L)，獲得突變體MGI4-F87L。引物對T1和T2見表1。
用引物對T1和87LR，擴增T1LR片段，用引物對87LF和T2，擴增LFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物87LR或400nM引物87LF和400nM引物T2，1.5U PfuDNA聚合酶(Promega，USA)，20ng pGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1LR片段和LFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μMdCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段與20ng LFT2片段，用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI4-F87L。將MGI4-F87L與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI4-F87L。將質(zhì)粒pGEMT-MGI4-F87L轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI4-F87L DNA，經(jīng)DNA測序確定引入的點突變無誤。所得突變體序列含有F87L、W139F、R182A、F187S和T299Q五個突變。MGI4-F87L氨基酸序列見序列表序列32，比活力見表2。
按照類似步構(gòu)建突變體MGI4-F87M，所用引物見表1。MGI4-F87M突變體序列含有F87M、W139F、R182A、F187S和T299Q五個突變。MGI4-F87M的氨基酸序列見序列表序列33，比活力見表2。按照類似步構(gòu)建突變體MGI4-V217R引物見表1。所得突變體序列含有W139F、R182A、F187S、V217R和T299Q五個突變。MGI4--V217R的氨基酸序列見序列表序列34，比活力見表2。按照類似步構(gòu)建突變體MGI4-D260E所用引物見表1。所得突變體序列含有W139F、R182A、F187S、D260E和T299Q五個突變。MGI4-D260E的氨基酸序列見序列表序列35，比活力見表2。按照類似步構(gòu)建突變體MGI4-F276G所用引物見表1。所得突變體序列含有W139F、R182A、F187S、F276G和T299Q五個突變。MGI4-F276G的氨基酸序列見序列表序列36，比活力見表2。
實施例14葡萄糖異構(gòu)酶六突變組合MGI4-24和MGI4-25之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按實施例13擴增和回收T1LR片段。用引物對87LF和260AR(見表1)擴增和回收LFAR片段。LFAR片段擴增反應(yīng)條件為20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM 87LF和400nM 260AR，1.5U Pfu DNA聚合酶，20ngpGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到LFAR片段。用引物對260AF和T2(見表1)擴增和回收AFT2片段。AFT2片段擴增反應(yīng)條件為20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM 260AF和400nM T2，1.5U Pfu DNA聚合酶，20ngpGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到AFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段，20ng LFAR片段和20ng AFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick DNA回收試劑盒，得到全長突變基因MGI4-24。將MGI4-24與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI4-24。將質(zhì)粒pGEMT-MGI4-24轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI4-24DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI4-24的氨基酸序列見序列表序列37。所得突變體序列含有F87L、W139F、R182A、F187S、T299Q和D260A六個突變，比活力見表2。
按照類似步構(gòu)建突變體MGI4-25。突變體MGI4-25所用引物對為T1和87LR、87LF和276TR、276TF和T2(均見表1)，所得突變體序列含有F87L、W139F、R182A、F187S、T299Q和F276T六個突變。所得突變體的氨基酸序列見序列表序列38，比活力見表2。
實施例15葡萄糖異構(gòu)酶七突變組合MGI4-34和MGI4-35之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按實施例13擴增和回收T1LR片段。用引物對87LF和217GR(見表1)擴增和回收LFGR片段。LFGR片段擴增反應(yīng)條件為20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM 87LF和400nM 217GR，1.5U Pfu DNA聚合酶，20ngpGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到LFGR片段。用引物對217GF和276TR(見表1)擴增和回收GFTR片段。GFTR片段擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM (NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 217GF和400nM 276TR，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到GFTR片段。用引物對276TF和T2(見表1)擴增和回收TFT2片段。TFT2片段擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 276TF和400nM T2，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到TFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段，20ng LFGR片段，20ng GFTR片段和20ng TFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃3分鐘，35圈循環(huán)95℃50秒、52℃30秒和72℃3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI4-34。將MGI4-34與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI4-34。將質(zhì)粒pGEMT-MGI4-34轉(zhuǎn)入感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI4-34DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI4-34序列含有F87L、W139F、R182A、F187S、T299Q、V217G和F276T七個突變。MGI4-34的氨基酸序列見序列表序列39，比活力見表2。
按照類似步驟構(gòu)建七突變體MGI4-35。突變體MGI4-35所用引物對為T1和87LR、87LF和217GR、217GF和260AR、260AF和T2(均見表1)，所得突變體序列含有F87L、W139F、R182A、F187S、T299Q、V217G和D260A七突變。所得突變體的氨基酸序列見序列表序列40，比活力見表2。
擴增親本葡萄糖異構(gòu)酶(親本)與實施例1-12所葡萄糖異構(gòu)酶突變體所用的引物如下表1所列。
表1



實施例16親本葡萄糖異構(gòu)酶的提取與純化葡萄糖異構(gòu)酶的提取與純化主要參考Lee et al.，Journal ofGeneral Microbiology，1391227-1234(1993)。
將含親本葡萄糖異構(gòu)酶基因的質(zhì)粒pGEMT-TS轉(zhuǎn)化感受態(tài)細(xì)菌細(xì)胞E.coli HB101，在MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上、37℃培養(yǎng)36小時。接種單個克隆在5ml LB液體培養(yǎng)基(含50mg/L氨芐青霉素)中培養(yǎng)16小時。離心收集菌體，并懸浮于1ml 20mM磷酸鈉緩沖液(pH 6.5)中，加CoCl2和MgCl2至終濃度分別為250μM和5mM。然后用超聲波裂解細(xì)菌細(xì)胞。離心(10℃，17，800g，15分鐘)并收集上清液為粗提蛋白。粗提蛋白經(jīng)80℃熱處理10分鐘后，離心(10℃，17，800g，15分鐘)去沉淀，即為部分純化的葡萄糖異構(gòu)酶，可用于酶活性的測定。
實施例17葡萄糖異構(gòu)酶突變體的提取與純化葡萄糖異構(gòu)酶突變體MGI4-35的提取與純化與實施例16同，惟所用質(zhì)粒為pGEMT-MGI4-35。其它葡萄糖異構(gòu)酶突變體也照實施例16所述提取與純化。
實施例18親本葡萄糖異構(gòu)酶活性的測定配底物溶液A含1.0M的D-木糖、20mM磷酸鈉緩沖溶液(pH6.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至6.5。取底物溶液A 90μl，然后加入10μl按實施例16制備的葡萄糖異構(gòu)酶。反應(yīng)于80℃進(jìn)行10分鐘。將反應(yīng)物置冰上以終止反應(yīng)。反應(yīng)產(chǎn)物D-木酮糖通過半胱氨酸-咔唑法測定。測定方法參見Dische et al.，J.Biol.Chem，192583-587，1951)以及Nakamura，Agr.Biol.Chem.32701-706，1968)。用CoomassiePlusProtein Assay Reagent Kit(PIERCE，USA)并結(jié)合SDS聚丙烯酰胺膠電泳測定酶蛋白濃度。一單位酶比活性定義為在上述條件下每分鐘轉(zhuǎn)化一微摩爾D-木糖為木酮糖所需之酶量。下列表2顯示親本葡萄糖異構(gòu)酶之比活性。
實施例19葡萄糖異構(gòu)酶突變體活性的測定葡萄糖異構(gòu)酶突變體MGI4-35活性測定與實施例18同。其它葡萄糖異構(gòu)酶突變體的活性也照實施例18所述測定。下表2顯示諸葡萄糖異構(gòu)酶突變體與親本葡萄糖異構(gòu)酶(親本)比活性之差異。
表2親本葡萄糖異構(gòu)酶與葡萄糖異構(gòu)酶突變體比活性之差異


實施例20親本葡萄糖異構(gòu)酶熱穩(wěn)定性測定置按實施例16獲得的部分純化的親本葡萄糖異構(gòu)酶于7管1.5ml離心管中。每管離心管分別加入200μl酶液，并加入200μl礦物油。將離心管置于80℃水浴中，0小時、4小時、12小時、24小時后分別取出一管酶液，離心(10℃，17，800g，20分鐘)后，取上清液，按實施例18所述、測定葡萄糖異構(gòu)酶殘余蛋白和殘余比活性。圖1顯示親本葡萄糖異構(gòu)酶在80℃的熱穩(wěn)定。
實施例21葡萄糖異構(gòu)酶突變體熱穩(wěn)定性測定葡萄糖異構(gòu)酶突變體MGI-4、MGI4-34、或MGI4-35熱穩(wěn)定性測定與實施例20同。圖1顯示葡萄糖異構(gòu)酶突變體MGI-4、MGI4-34、或MGI4-35在80℃的熱穩(wěn)定。如圖所示，親本葡萄糖異構(gòu)酶的活性在80℃的半衰期為13.4小時；MGI-4的活性在80℃的半衰期為21.4小時；MGI4-34的活性在80℃的半衰期為19.2小時；MGI4-35的活性在80℃的半衰期大于24小時。
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序列表<110>百瑞全球有限公司(BioRight Worldwide Company Ltd.)<120>葡萄糖異構(gòu)酶突變體的應(yīng)用(Use of Glucose Isomerase Mutants)<130>FI-050438-59<160>40<170>PatentIn version 3.2<210>1<211>1320<212>DNA<213>Thermoanaerobacterium saccharolyticum<400>1atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat60ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag 120catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa 240gcaagggtag aagcagcatt tgagtttttt gataagataa atgcaccttt cttctgcttc 300catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat 360acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgtggggt 420accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct 480gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt 540ggccgcgaaa actacgtatt ttggggtgga agagaagggt acgagacgct tctcaataca 600gatatggagt tagagcttga taactttgca agatttttgc acatggctgt tgactatgca 660aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa 720catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttgac 780aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcattcca cgacttccaa 840catgagctaa gatacgccag aataaacggt gtattaggat caattgacgc aaatacaggc 900gacatgcttt tgggatggga tacggaccag ttccctacag atatacgcat gacaacgctt 960gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca 1020aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg 1080gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac 1140aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc 1200ggtaaagctg acttcaagag ccttgaaaag tatgcattag agcacagcca gattgtaaac 1260aaatcaggca gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa 1320<210>2<211>439
<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>2Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435
<210>3<211>1320<212>DNA<213>Thermoanaerobacterium saccharolyticum<220>
<221>misc_feature<222>(259)..(261)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for phenylalanine.
<220>
<221>misc_feature<222>(415)..(417)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for typtophan.
<220>
<221>misc_feature<222>(544)..(546)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid Arginine.
<220>
<221>misc_feature<222>(559)..(561)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for phenylalanine.
<220>
<221>misc_feature<222>(649)..(651)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid valine.
<220>
<221>misc_feature<222>(778)..(780)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for aspartic acid.
<220>
<221>misc_feature
<222>(826)..(828)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for phenylalanine.
<220>
<221>misc_feature<222>(895)..(897)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for threonine.
<400>3atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat60ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag 120catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa 240gcaagggtag aagcagcann ngagtttttt gataagataa atgcaccttt cttctgcttc 300catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat 360acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgnnnggt 420accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct 480gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt 540ggcnnngaaa actacgtann ntggggtgga agagaagggt acgagacgct tctcaataca 600gatatggagt tagagcttga taactttgca agatttttgc acatggctnn ngactatgca 660aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa 720catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttnnn 780aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcannnca cgacttccaa 840catgagctaa gatacgccag aataaacggt gtattaggat caattgacgc aaatnnnggc 900gacatgcttt tgggatggga tacggaccag ttccctacag atatacgcat gacaacgctt 960gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca 1020aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg 1080gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac 1140aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc 1200ggtaaagctg acttcaagag ccttgaaaag tatgcattag agcacagcca gattgtaaac 1260aaatcaggca gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa 1320<210>4<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<220>
<221>MISC_FEATURE<222>(87)..(87)<223>Xaa represents any amino acid residue other than phenylalanine.
<220>
<221>MISC_FEATURE<222>(139)..(139)<223>Xaa represents any amino acid residue other than tryptophan.
<220>
<221>MISC_FEATURE<222>(182)..(182)<223>Xaa represents any amino acid residue other than Arginine.
<220>
<221>MISC_FEATURE<222>(187)..(187)<223>Xaa represents any amino acid residue other than phenylalanine.
<220>
<221>MISC_FEATURE<222>(217)..(217)<223>Xaa represents any amino acid residue other than valine.
<220>
<221>MISC_FEATURE<222>(260)..(260)<223>Xaa represents any amino acid residue other than aspartic acid.
<220>
<221>MISC_FEATURE<222>(276)..(276)<223>Xaa represents any other amino acid residue other than phenylalanine.
<220>
<221>MISC_FEATURE<222>(299)..(299)<223>Xaa represents any other amino acid residue other than threonine.
<400>4Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Xaa Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Xaa Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Xaa Glu Asn Tyr Val Xaa Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Xaa Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Xaa Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Xaa His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Xaa Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>5<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>5Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Ghy Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>6<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>6
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>7<211>439<212>PRT
<213>Thermoanaerobacterium saccharolyticum<400>7Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435
<210>8<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>8Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430
Asn Gln Tyr Leu Phe Ala Glu435<210>9<211>439<212>PRT<213>Thermoanaerobacter Iactoethylicum<400>9Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>10<211>439<212>PRT<213>Thermoanaerobacter Iactoethylicum<400>10Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>11<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>11Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu
130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>12<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>12Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ser Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>13<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>13Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Lys Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>14<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>14Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Cys Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>15<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>15Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ile Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>16<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>16Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Thr Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>17<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>17Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Asn Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>18<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum
<400>18Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Asp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>19
<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>19Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Pro Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu
435<210>20<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>20Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ser Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>21<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>21Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175Thr Lys Glu Leu Gly Ile Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>22<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>22Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Thr Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>23<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>23Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Val Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>24<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>24Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Gly Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Va Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>25<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>25Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ala Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>26<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>26Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Pro Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>27<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>27Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Ile Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>28<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>28Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Tyr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>29<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>29Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Cys Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>30<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>30
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Met Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>31<211>439
<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>31Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Glu Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435
<210>32<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>32Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430
Asn Gln Tyr Leu Phe Ala Glu435<210>33<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>33Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Met Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>34<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>34Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Arg Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>35<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>35Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Glu Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>36<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>36Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Gly His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>37<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>37Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Ala Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>38<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>38Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Thr His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>39<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>39Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Gly Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Thr His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>40<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>40Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Gly Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Ala Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu43權(quán)利要求
1.一種葡萄糖異構(gòu)酶突變體的應(yīng)用，其特征在于以序列表中的序列2為參考序列所述的突變體具有至少一個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的至少一個突變，并且在以D-木糖為底物生成木酮糖的反應(yīng)中所述突變體具有比親本高出至少50％的酶催化活性。
2.權(quán)利要求1所述的應(yīng)用，其中第87位的苯丙氨酸(Phe)突變?yōu)榧琢虬彼?Met)、或亮氨酸(Leu)。
3.權(quán)利要求1所述的應(yīng)用，其中第139位的色氨酸突變?yōu)楸奖彼?Phe)、或絲氨酸(Ser)、或賴氨酸(Lys)、或半胱氨酸(Cys)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或天冬酰胺(Asn)、或天門冬氨酸(Asp)。
4.權(quán)利要求3所述的應(yīng)用，其中所述的突變體具有序列表中SEQID NO.5所示的氨基酸序列。
5.權(quán)利要求1所述的應(yīng)用，其中第182位的精氨酸突變?yōu)楸彼?Ala)、或脯氨酸(Pro)、或絲氨酸(Ser)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或纈氨酸(Val)。
6.權(quán)利要求5所述的應(yīng)用，其中所述的突變體具有序列表中SEQID NO.6所示的氨基酸序列。
7.權(quán)利要求1所述的應(yīng)用，其中第187位的苯丙氨酸突變?yōu)楦拾彼?Gly)、或丙氨酸(Ala)、或絲氨酸(Ser)、或脯氨酸(Pro)。
8.權(quán)利要求7所述的應(yīng)用，其中所述的突變體具有序列表中SEQID NO.7所示的氨基酸序列。
9.權(quán)利要求1所述的應(yīng)用，其中第217位的纈氨酸(Val)突變?yōu)榫彼?Arg)、或甘氨酸(Gly)。
10.權(quán)利要求1所述的應(yīng)用，其中第260位的天門冬氨酸(Asp)突變?yōu)楣劝彼?Glu)、或丙氨酸(Ala)。
11.權(quán)利要求1所述的應(yīng)用，其中第276位的苯丙氨酸(Phe)突變?yōu)楦拾彼?Gly)、或蘇氨酸(Thr)。
12.權(quán)利要求1所述的應(yīng)用，其中第299位的蘇氨酸突變?yōu)楣劝滨０?Gln)、或異亮氨酸(Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或甲硫氨酸(Met)、或谷氨酸(Glu)。
13.權(quán)利要求12所述的應(yīng)用，其中所述的突變體具有序列表中SEQ ID NO.8所示的氨基酸序列。
14.權(quán)利要求1所述的應(yīng)用，其中所述的突變體具有至少兩個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突變。
15.權(quán)利要求14所述的應(yīng)用，其中所述的突變體具有序列表中SEQ ID NO.9所示的氨基酸序列。
16.權(quán)利要求14所述的應(yīng)用，其中所述的突變體具有至少三個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突變。
17.權(quán)利要求16所述的應(yīng)用，其中所述的突變體具有序列表中SEQ ID NO.10所示的氨基酸序列。
18.權(quán)利要求16所述的應(yīng)用，其中所述的突變體具有至少四個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突變。
19.權(quán)利要求18所述的應(yīng)用，其中所述的突變體具有序列表中SEQ ID NO.11至SEQ ID NO.31之一所示的氨基酸序列。
20.權(quán)利要求18所述的應(yīng)用，其中所述的突變體具有至少五個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突變。
21.權(quán)利要求20所述的應(yīng)用，其中所述的突變體具有序列表中SEQ ID NO.32至SEQ ID NO.36之一所示的氨基酸序列。
22.權(quán)利要求20所述的應(yīng)用，其中所述的突變體具有至少六個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突變。
23.權(quán)利要求22所述的應(yīng)用，其中所述的突變體具有序列表中SEQ ID NO.37或SEQ ID NO.38所示的氨基酸序列。
24.權(quán)利要求23所述的應(yīng)用，其中所述的突變體具有至少七個選自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突變。
25.權(quán)利要求24所述的應(yīng)用，其中所述的突變體具有序列表中SEQ ID NO.39或SEQ ID NO.40所示的氨基酸序列。
26.權(quán)利要求1所述的應(yīng)用，其中所述的突變體用于從半纖維素轉(zhuǎn)化成乙醇。
全文摘要
本發(fā)明披露應(yīng)用基因突變技術(shù)改良的一系列高催化活性Thermoanaerobacterium saccharolyticum葡萄糖異構(gòu)酶突變體的應(yīng)用。這些葡萄糖異構(gòu)酶突變體可以木糖為底物高效率地生成木酮糖。
文檔編號C12N9/92GK1966702SQ200510123630
公開日2007年5月23日 申請日期2005年11月18日 優(yōu)先權(quán)日2005年11月18日
發(fā)明者王駿, 傅榮昭, 張?zhí)熳?申請人:百瑞全球有限公司