專利名稱：具有改進(jìn)的卵污漬洗滌性能的新枯草桿菌酶的制作方法
婕靶改較竄德枯草桿久 subtilase)些薟莞司酶洗蛹錚 洗錚斷詞敝襯荒較竄
婕懊枯草桿久改紡?qiáng)Z、峁瑰、濉⑺橫構(gòu)細(xì)約使帽目薟莞司酶姆遙婕拜草桿久 洗蛹錚夾┟趕從夾低沮橙 鍘
洗蛹業(yè)酶洗浞絞 0稀些酶酶脂酶酶維酶酶塹幕鎩Ｒ滴蓋得
玫酶然詰野偷酶牡墜袒?，DURAZYM_(Novo Nordisk A/S)RELASE_(NovoNordisk A/S)MAXAPEM_(Gist-Brocades N.V.)PURAFECT_(Gene-ncor InternationalInc.)
遙屑舜牡酶澹 EP130756(GENENTECH)(應(yīng)US侔專34606(GENENCOR))EP214435(HENKEL)WO87/04461(AMGEN)WO87/05050(GENEX)EP260105(GENENCOR)ThomasRussellFersht(1985)然(Nature)318375-376ThomasRussellFersht(1987)學(xué)志(J.Mol.Biol)193803-813RusselFersht然328496-500(1987)WO88/08028(Genex)WO88/08033(Amgen)WO95/27049(SOLVAY S.A.)WO95/30011(PROCTER& GAMBLE COMPANY)WO95/30010(PROCTER & GAMBLECOMPANY)WO95/29979(PROCTER & GAMBLE COMPANY)US5.543.302(SOLVAY S.A.)EP251 446(GENENCOR)WO89/06279(NOVO NORDISK A/S)WO91/00345(NOVO NORDISKA/S)EP525 610 Al(SOLVAY)WO94/02618(GIST-BROCADESN.V.)
牽舜玫牡酶偷酶澹 詿墓業(yè)應(yīng)茫要碌母慕牡酶虻鞍酶濉
裕寫詰絲岬懊 硬去盞狻
耍目峁┠僥枯草桿久崗 /硬去鍘
耍詰一媯及一志懈慕洗艿薟莞司酶目薟莞司酶選
a)SEQ ID NO2械1-269位示陌芯88同一緣目薟莞司酶
b)詰細(xì)詠畝目薟莞司酶
i)SEQ ID NO1械1-807位示畝幕
ii)100(i)校
c)煽隆詿蟪Ω司(Escherichia coli)MT173 DSM13306械片系畝目薟莞司酶氬恐蹦枯草桿久 目薟莞司酶88同一緣謀濉
詰詼媯及一址畝幔 氡據(jù)草桿久改盒
詰媯及一直薟莞司酶姆畝幔 選裕
a)SEQ ID NO1械1-807位示暮芯88同一緣畝幔
b)詰細(xì)詠畝目薟莞司酶
i)SEQ ID NO1械1-807位示畝幕
ii)100(i)校
c)隆詿蟪Ω司MT173 DSM13306械片系畝目薟莞司酶氬恐Ｄ盒 8同一緣謀濉
詰姆媯及一職械暮峁瑰，峁股慘恢卉怪篙草桿久桿 詒牡印
詰宸芥，婕耙恢搬構(gòu)?、?轉(zhuǎn)錄頭止藕諾濉
詰媯及一職峁瓜
詰叻媯及一植薟莞司酶姆
(a)誆薟莞司酶細(xì)
(b)湛薟莞司酶
詰詘朔媯及一洗蛹錚∥綽 洗錚枯草桿久
一姆婕澳枯草桿久趕從 械途目薟莞司酶誄去盞途一洗擁姆硬銑去盞姆使硬氡居
卸員群捅挪渭
圖1圖示誑薟莞司酶BPN(a)(BASBPN)捅驢薟莞司酶(b)之卸員取
專械些卸員圓謝斜諾牟鍘
詬細(xì)郾之前榷碌凸鎩
AAla
VVal影
LLeu
IIle
PPro
FPhe
WTrp色
MMet
GGly拾
SSer絲
TThr瞻
CCys裝
YTyr野
NAsn於
QGln勸
DAsp於
EGlu勸
KLys
RArg
HHis榘
XXaa獍
A
G
C
T(DNA)
U(RNA)
凸
詿畝摯薟莞司酶校碌凸誆眨
通幕妝酶薟莞司酶BPN(BASBPN)員慰
GCG9.1GAP卸員齲詼鑰薟司酶斜牛使虜鄭8展鄭8械為塹默值
一址使知目薟莞司酶之畝員齲WO91/00345示卸員取詿蟛糠攏觳⒁
薟莞司酶BPN(BASBPN)捅驢薟莞司酶之員圖1示
耍BASBPN氐缺失筒搿Ｍ 校驢薟莞司酶BASBPN齲3658158162163164位6缺失些缺失圖1嗆(*)示
一碌煞示妝酶械畝危
原始 位 婊荒
耍G195E指195位母拾岜蝗版換
原始謝獍斃宦Ｊ泵 員鋟絞絞疚幻烘換陌幔
位 婊荒
址同源薟莞司酶()
頻兀婊恍穢不要時(shí)
原始 位
原始婊輝扳氨時(shí)只指位茫紓 70
原始/婊荒霸耙幌敵 時(shí)選陌示冢
原始 位{婊荒 1...婊荒 n}
囟謀?，赎P(guān)兌荒鴿，XaaX示魏偉謝
婊
霉勸婊 95位母拾示為
Glyl95GluG195E
獍斃繪換195位母拾示為
Glyl95XaaG195X
Glyl95G195
絲婊 70位獍斃皇疚
Xaal70SerX170S
170Ser170S
址同源薟莞司酶()170Ser指BASBPN械Lysl70Ser魏捅薟莞司酶Argl70Ser(渭圖1)
械原始/婊荒霸耙幌 械陌味裕篩拾?、帷⒔z瞻婊 170位木示為
Argl70{GlyAlaSerThr}R170{GAST}員示
R170GR170AR170SR170T
缺失
195位拾缺失示為
Glyl95*G195*
應(yīng)兀一習(xí)謝缺失195196位拾缺失示為
Glyl95*+Leul96*G195*+L196*
耄
陌謝牟?，G195示為
Glyl95GlyLysG195GK
擼一系陌謝G195LysAla時(shí)植示為
Glyl95GlyLysAlaG195GKA
些攏通小寫母擁氚斃恢澳 謝位煤圓氚斃恍迸∠凳凳┬ 194-196為
194 195 196
BTSAVI A-G-L
194 195 195a 195b 196
A-G-K-A -L
詰陌謝同陌謝時(shí)然諧思蠆ⅰ系實(shí)施校詬拾之拾幔 示為G195GG源
194 195 196
BLSAVI A-G-L
194 195 195a 196
A-G-G-L
194 194a 195 196同一斜浠筆疚狝194AG
些傭員員說遠(yuǎn)準(zhǔn)模冶示G195GG筒應(yīng)示指同募蠆⒈示
畈
詒諾目薟莞司酶BPN薪斜冉希誆盞酶寫缺失時(shí)36位於蒼Ｎ幻茨 示為
*36Asp*36D
危
眉雍歐指蔚謀澹
Argl70Tyr+Glyl95GluR170Y+G195E170195位直野凸勸婊煌甘 巍
耍Tyrl67{GlyAlaSerThr}+Argl70{GlyAlaSerThr}示鹵澹
Tyrl67Gly+Argl70Gly Tyrl67Gly+Argl70Ala
Tyrl67Gly+Argl70Ser Tyr167Gly+Arg170Thr
Tyrl67Ala+Argl70Gly Tyr167Ala+Arg170Ala
Tyrl67Ala+Argl70Ser Tyrl67Ala+Argl70Thr
Tyr167Ser+Arg170Gly Tyr167Ser+Arg170Ala
Tyrl67Ser+Argl70Ser Tyrl67Ser+Argl70Thr
Tyr167Thr+Arg170Gly Tyr167Thr+Arg170Ala
Tyrl67Thr+Argl70Ser Tyrl67Thr+Argl70Thr
婊蝗∪筆ж鍛的 謝(KRH)(DE)牟謝危一小婊灰恍yr167{GlyAlaSerThr}+Arg170{GlyAlaSerThr}謀匕乇亍一細(xì)渭幀
酶
紙獾笆敵得肝 苫)酶(Walsh1979酶應(yīng)(Enzymatic Reaction Mechanisms).W.H.Freeman and CompanySan Francisco3)
位/謝謀
沒指玫陌哦應(yīng)誑薟莞司酶BPN(BASBPN)械陌擰BPN械細(xì)渭圖1Siezen耍使(Protein Engng.)4(1991)719-737
絲岬懊
絲岬懊敢恢催荒妓崗輝參悔 絲謝(WhiteHandlerSmith1973锘г (Principles of Biochemistry)媯琈cGraw-Hill Book司NY271-272頁)
細(xì)絲岬懊改 0,000-45,000俚姆圍凇潛啤水末脂啵 擁酶(也一絲岬懊 啤一為錚 一募緣酶映某些絲岬懊改竝H值為pH9.0-11.0(渭Priest(1977)細(xì)學(xué)(Bacteriological Rev.)41711-753)
薟莞司酶
Siezen絲岬懊敢婚，莩為薟莞司酶(subtilase)使蹋4(1991)719-737Siezen耍學(xué)(Protein Science)6(1997)501-523通170前為薟莞司酶酶絲岬懊改靶酵 苑摹薟莞司酶前通為篩細(xì)絲岬懊崗窼iezen為薟莞司酶一欏Ｑ妓茨枯草桿久稈測(cè)定一些薟莞司酶小源薟莞司酶滸斃的趕肝 Siezen(1997)
薟莞司酶一?，I-S1薟莞司酶準(zhǔn)摹薟莞司酶薟莞司酶168(BSS168)薟莞司酶BPN(BASBPN)薟莞司酶Carlsberg(BLSCAR)(ALCALASE_NOVO NORDISK A/S)涂薟莞司酶DY(BSSDY)
Siezen()識(shí)絲薟莞司酶一?，I-S2強(qiáng)薟莞司酶I-S2酶為強(qiáng)鑰薟莞司酶薟莞司酶PB92(BAALKP)(MAXACAL_Gist-BrocadesNV)薟莞司酶309(BLSAVI)(SAVINASE_NOVO NORDISKA/S)薟莞司酶147(BLS147)(ESPERASE_NOVO NORDISKA/S)圖緣緣酶YaB(BSEYAB)
妝薟莞司酶
鎩白陛草桿久敢恢窼iezen(19911997)目薟莞司酶誚一細(xì)冢渭浴薟莞司酶妝薟莞司酶也譴然源目薟莞司酶校詒摯薟莞司酶緣戮一巍遙妝薟莞司酶通DNA榧計(jì)蹦枯草桿久 J.E.Ness耍然錛 Nature Biotechnology)17893-896(1999)
鎩白陛草桿久肝巴枯草桿久
薟莞司酶
玫鎩拔∥草桿久改謊г 薟莞司酶DNA械糯慰前取目臧貶處婊蝗筆 搿
薟莞司酶
詒校薟莞司酶突目薟莞司酶指殺突目薟莞司酶突源諍原始妝蠆⒉應(yīng)妝酶妝微錚毖吠 圓突突說斜時(shí)突薟莞司酶頻兀突蚧箍DNA榧 妝
同源薟莞司酶
詒校職之同源貌同一浴
為確摯薟莞司酶之同一猿潭齲使同應(yīng)()GCG9.1GAP朔募稅卸員?，之摹同逸x百肥∧
荽說員丶說同源鑰薟莞司酶應(yīng)同源幕圓位強(qiáng)愿荼巍
畝
玫鎩澳奪”指一志氪渴河 糯袒牡什系統(tǒng)式畝?。些姆?源然蟹?，前cDNA突隆約DNA實(shí)蚨ǖ殖(site-directed autogenesis)實(shí)畝帷 姆畝岵煌ㄖ的或，瞪 3未雍止印募員員說遠(yuǎn)準(zhǔn)(紓糄ynanTijan然316774-781985)鎩澳盒∥狣NA小隆暮小頡翱隆DNA小
牡
鎩澳∮凳敝桿笛慈恍
選式校牡駛喜剩乇同源(同源省(渭))
通SDS-PAGE舛凳的慈 0選20選30選峁┩⊿DS-PAGE舛ㄈ 06080選95選99母叨卻式牡省
鎩澳凳∥凳
同源
鎩巴詞≈肝涸疵陛草桿久竿 源細(xì)(吮薟莞司酶之一)
......
囟微源氐鎩 .....謾指枚/薟莞司酶囟源細(xì)囟源細(xì)巡械幕
玫氳懊改地碉“鎩庇 式為一詒薟莞司酶水募幕鎩
玫緣酶酶俜應(yīng)牟泄氐鎩幫”詒應(yīng)為婕懊篙草桿久桿反應(yīng)牟鎩Ｋ反應(yīng)械牡鎩
洗
詒校鎩跋竄∶趕從揮 諧去目系渭實(shí)施2械摹準(zhǔn)洗蛹洗欏
式鎩壩
PR薟莞司酶-R維斯
校P為櫻R薟莞司酶為凇準(zhǔn)洗蛹洗欏泵枯草桿久鋼的 (reflectance)R維斯為凇準(zhǔn)洗蛹洗欏 Savinase_之系姆一渭實(shí)施2械摹準(zhǔn)洗蛹洗欏 圖
圖1示GAP鋅薟莞司酶BPN陌(a)捅驢薟莞司酶陌之卸員取
詒牡一爍趣姆媯改較 目薟莞司酶一址SEQ ID NO2械1-269位示陌(目薟莞司酶)88同一緣目薟莞司酶詒一爍趣實(shí)施校目薟莞司酶SEQ ID NO2械1-269位示陌芯90919293949596979899同一(某為同源薟莞司酶)詒一爍趣實(shí)施校目薟莞司酶SEQ ID NO2械1-269位示陌傘
圓詰屎DNA員鵲全Smith-Waterman員確卸員群同一苑值恪Ｄ記分倔方稡LOSUM50同一躍蠓椒直詰屎DNA卸員取一械牡一謝姆侄詰識(shí)為-12DNA為-16一械謝姆侄緣識(shí)為-2DNA為-4員Fastav20u6姹 W.R.PearsonD.J.Lipman(1988)慕蟹(Improved Tools for Biological sequenceAnalysis)PNAS 852444-2448約W.RPearson(1990)FASTPFASTA目俸斜冉(Rapid and Sensitive sequenceComparison with FASTP and FASTA)酶學(xué)(Methods inEnzymology)18363-98)
通寫卸員齲志SEQ 1D NO2陌械薟莞司酶投知目薟莞司酶陌之同一(園俜時(shí)示)
BLSAVI BLAP BASBPN BLSCAR SEQ ID NO2BLSAVI 100BLAP1) 98.1 100BASBPN 60.4 60.7 100BLSCAR 60.9 61.3 69.5 100SEQ ID NO2 86.6 87.4 59.3 55.8 100
1)BLAP(倩芽吒司(Bacillus lentus)緣酶)US5,352,604
詒一爍趣實(shí)施校目薟莞司酶露?，硕诘细拢选械细?選詬叨細(xì)詠(i)SEQ ID NO1械1-807位示畝幕(ii)俸100(i)(J.SambrookE.F.FritschT.Maniatis1989涌隆實(shí)植幔 Molecular Cloning A Laboratory Manual2dedition)泉郟紐約
SEQ ID NO1械1-807位示畝幕片匣鋅為100?，?00帷 遙應(yīng)帽械水緣目薟莞司酶片稀絲薟莞酶也薔械水緣目薟莞司酶牡位片稀
荼知姆越SEQ ID NO1畝謝片希約SEQ ID NO2陌謝片坪探約涂隆圓同值木摹械水緣薟莞司酶說探愿荼準(zhǔn)Southern印趣值幕cDNA詠約頭應(yīng)幕頡４探囟校應(yīng)為15選25選35岢ひ彩姑較程誡。DNARNA探使謾一探斜約應(yīng)幕(32P3H35S鼗綴)探詒小
耍源票幕DNAcDNA目篩選探詠氡據(jù)草桿久窪NA通脂腔郾烯泳員知爰 幕DNADNA越些目DNADNA轉(zhuǎn)蘋吖潭維鼗說稀為思SEQ ID NO1同源目隆DNASouthern印為目模詠詰細(xì)應(yīng)SEQ ID NO1示暮小浠バ的鼻的禾接絏弒膜些探詠姆印
誄為100某探?，细?荼準(zhǔn)Southern印425SSPE0.3SDS200g/ml瀉捅緣憔獶NA約詰細(xì)25械細(xì)35詬細(xì)50薪預(yù)詠詠
誄為100某探耄 SSC0.2SDS選50(細(xì))選55(械細(xì))一選65(細(xì))細(xì)洗危每15印
誄詿約15約70畝探耄 為荼準(zhǔn)Southern印陸預(yù)詠洗雍詠誆BoltonMcCarthy(1962銥學(xué)院院(Proceedings of the National Academy of Sciences USA)481390)惴═m之5-10媯 .9M NaCl0.09M Tris-HCl pH7.66mM EDTA0.5NP-401Denhardt液1mM疲1mM一疲0.1mM ATP每0.2mg母RNA
誄詿約15約70畝毯幔 0.1SDS6SCC洗15櫻洗一危使6SSC詡Tm之5-10洗危每15印
屑知謂頻陌謝某謝謀取預(yù)誚酶小浠
斜I諧吮匕婊
I
匕婊
()K
H榘
()E勸
D於
Q勸
N於
水 L
I
V影
M
F
W色
Y野
小G拾
A
S絲
T瞻
耍詒一爍趣實(shí)施校SEQ IDNO2陌械目薟莞司酶一謝缺失/搿
乇兀屑知蔚慕峁┠矯 浴
屑舜脅同母慕實(shí)目薟莞司酶?，?目薟莞司酶詒牡摹峒 渭)
耍SEQ ID NO2陌一位么械為(BASBPN)273656768797101104120123167170222224235274
說為目薟莞司酶鹵(BASBPN)K27R*36DS56PN76DS87NG97NR101GS103AV104AV104IV104NV104YH120DN123SY167R170M222SM222AT224SK235LT274A
遙為諢圓位(b)一影謝詿95位103位(BASBPN)一影謝璞灸枯草桿久贛迪竄≡Ｑ∥ 儼一影謝一影謝98位99位之洌 9位100位之洹
遙為SEQ ID NO2陌械目薟莞司酶呋學(xué)同一曰蠆糠呋學(xué)同一緣姆目薟莞司酶選為式也詒姆圍之凇知Ouchterlony雙散通囈娣從︹定呋學(xué)省裕姆通(荻)票呋曰SEQ ID NO2陌械目薟莞司酶謀位系畝隆蹇寡HarboeIngildN.H.AxelsenJ.RollB.Weeks嗉滌局 A Manual of QuantitativeImmunoelectrophoresis)Blackwell Scientific Publication197323攏JohnstoneThorpe實(shí)呋學(xué)(Immunochemistry inPractice)Blackwell Science Publication1982(兀27-31頁)呋學(xué)同一緣目薟莞司酶誆囟呋學(xué)時(shí)同姆式全諢同某態(tài)/同牡泳遷肟寡反應(yīng)目薟莞司酶AxelsenBockKrollN.H.AxelsenJ.RollB.Weeks嗉滌局冊(cè)，BlackwellScientificPublications197310斯呋學(xué)同一緣慕一汀脅呋學(xué)同一緣目薟莞司酶誆囟呋學(xué)時(shí)圓同姆式綺恐弛煌程 蠆糠同牡泳遷肟寡反應(yīng)牡目薟莞司酶N.H.AxelsenJ.RollB.Weeks嗉滌局冊(cè)，BlackwellScientific Publication197311瞬呋學(xué)同一浴
寤骨德″。隆愿碌姆票使茫E.HarlowD.Lane嗉 988?，实?AntibodiesA Laboratory Manual)泉鄢紓Ｅυ
遜SEQ ID NO2示陌械薟莞司酶幕潁氳襟腸桿綧T173莨食專微銼ｆ布斯約2000210戰(zhàn)沒拇蟪Ω司MT173甌Ｚ抵疚保DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH(DSM)Mascheroder Weg 1BD-38124 Braunschweig鹿?jié)。睾为DSM13306
詒爍趣實(shí)施校薟莞司酶煽隆詒睪為DSM13306拇蟪Ω司MT173片系畝目薟莞司酶氬恐蹦枯草桿久 0919293949596979899同一浴
目薟莞司酶殖越洗堋耍為使員-蟹-芄選效暮選詿目牡目薟莞司酶峁┮凰翟ら，殺員猿鄣目薟莞司酶堋
耍牡實(shí)施2欏白枷從枷 欏毖∧枯草桿久改剮т話說誚一摯薟司酶準(zhǔn)洗蛹時(shí)系冉(時(shí)為Savinase_)謾準(zhǔn)洗蛹洗欏臂該枯草桿久溉 準(zhǔn)織系盞椋承狙 薟莞司酶去盞裕原選目薟莞司酶沒斜殖Savinase_愿慕一悴灰 薪一欏
耍詒目目薟莞司酶一摯薟莞司酶凇準(zhǔn)洗蛹洗欏壁奧成 謀準(zhǔn)洗蛹時(shí)同Savinase_缺殖慕洗埽準(zhǔn)洗蛹
6.2 LAS(Nansa 80S)
2 C16-C18脂
4 穎約(Plurafax LF404)
22石P
10.5 Na2CO3
4Na2Si2O5
2 燃諄維(CMC)
6.8 烯液CP5 40
20(式NaBO2.H2O2)
0.2 EDTA
21 Na2SO4
水(平)
通詒牡實(shí)施2卸謂印洗艿母慕卸
詒一淺爍趣實(shí)施校目薟莞司酶凇洗欏筆蔽 為1.5為2選為2.5為3為3.5乇為4為4.5為5
緣兀選目薟莞司酶水平希選械水平希選水平戲準(zhǔn)
目薟莞司酶通準(zhǔn)為夭裕通同薟莞司酶DNA榧 渭WO95/22625J.E.Ness耍然錛 7893-896(1999))
緣兀目薟莞司酶源然源蟹耄 目薟莞司酶細(xì)薟莞司酶細(xì)薟莞酶細(xì)薟莞司酶懈細(xì)薟莞司酶芽吒司模燃芽吒司(Bacillus alkalophilus)芽吒司(Bacillus amyloliquefaciens)芽吒司(Bacillus brevis)狀芽吒司(Bacillus circulans)吒司(Bacillus coagulans)芽吒司(Bacillus lautus)倩芽吒司芽吒司(Bacilluslicheniformis)藪芽吒司(Bacillus megaterium)脂芽吒(Bacillus stearothermophilus)薟芽吒司(Bacillus subtilis)芽吒司(Bacillus thuringiensis)薟莞司酶霉薟莞司酶鉛霉(Streptomyces lividans)霉(Streptomycesmurinus)薟莞司酶細(xì)薟莞司酶蟪Ω俚(俚sp)薟莞司酶
目薟莞司酶也為模選母薟莞司酶絲母(Candida)魯維母(Kluyveromyces)銑母(Pichia)母(Saccharomyces)殖母(Schizosaccharomyces)Yarrowia薟莞司酶吒選絲狀薟莞司酶枝霉(Acremonium)霉(Aspergiilus)癱霉(Aureobasidium)母(Cryptococcus)吆詵劬(Filobasidium)(Fusarium)霉(Humicola)Magnaporthe毛霉(Mucor)絲霉(Myceliophthora)Neocallimastix霉(Neurospora)霉(Paecilomyces)霉(Penicillium)Piromyces(Schizophyllum)捉誥(Talaromyces)揖(Thermoascus)莞霉(Thielavia)Tolypocladium木霉(Trichoderma)薟莞司酶
一爍趣實(shí)施校薟莞司酶為轄母(Saccharomyces carlsbergensis)平母(Saccharomycescerevisiae)腔母(Saccharomyces diastaticus)Saccharomycesdouglasii魯母(Saccharomyces kluyveri)諾亟母(Saccharomyces norbensis)墻母(Saccharomyces oviformis)薟莞司酶
一玫實(shí)施校薟莞司酶為霉(Aspergillusaculeatus)盛霉(Aspergillus awamori)霉(Aspergillusfoetidus)氈霉(Aspergillus japonicus)霉(Aspergillusnidulans)霉(Aspergillus niger)霉(Aspergillus oryzae)狀(Fusarium bactridioides)Fusarium cerealisFusariumcrookwellense(Fusarium culmorum)坦(Fusariumgraminearum)壇(Fusarium graminum)(Fusariumheterosporium)匣木(Fusarium negundi)(Fusariumoxysporum)枝(Fusarium reticulatum)玫色(Fusariumroseum)庸木(Fusarium sambucinum)色(Fusariumsarcochroum)枝(Fusarium sporotrichioides)色(Fusarium sulphureum)Fusarium torulosumFusariumtrichothecioidesFusarium venenatumHumicola insolensHumicolalanuginosa綴毛霉(Mucor miehei)然絲霉(Myceliophthorathermophila)植霉(Neurospora crassa)霉(Penicilliumpurpurogenum)Trichoderma harzianum木霉(Trichodermakoningii)Trichoderma longibrachiatumTrichoderma reesei色木霉(Trichoderma viride)薟莞司酶
?，郑蜏娲?學(xué)同錚，知員識(shí)實(shí)同同一浴
些值木怨說一些銼Ｄ得的 銼 ATCC)志微銼 DSM)直(CBS)農(nóng)業(yè)芯專銼Ｐ (NRRL)
遙圓探影然(戲稀水)微源屑么薟莞司酶詿然微募詒知摹頻篩選一微幕cDNA目苫畝?。一探獾剿W莞司酶畝幔裁痹敝 隆(紓糞ambrook耍1989)
誑隆目薟莞司酶)約(薟莞司酶)畝址屑知模渭凇玫牟慰住
一說圓誑隆沒(/蚨ǖ)謀準(zhǔn)曰帽目薟莞司酶為私一說氈牡實(shí)施()(Sambrook耍(1989)涌隆實(shí)植?，泉实遥泉郏纽约AusubelF.M.(嗉 學(xué)路( Current protocols in Molecular Biology)John WileySons1995HarwoodC.R.CuttingS.M.(嗉 芽吒司姆學(xué)(Molecular Biological Methods forBacillus)John WileySons1990)WO96/34946
遙目薟莞司酶通準(zhǔn)為夭裕通同薟莞司酶DNA榧 渭WO95/22625Stemmer WPC然370389-91(1994))緗 Savinase_幕實(shí)鴨一只植摯薟莞司酶薪DNA?，啥挕妮^竄橫供目薟莞司酶
婕耙恢彪本薟莞司酶姆畝帷
一爍趣實(shí)施校SEQ ID NO1牡1-807位示畝88同一浴選兀碩SEQ ID NO1牡1-807位示畝8990919293949596979899同一浴一爍趣實(shí)施校SEQ ID NO1牡1-807位示畝帷④閨本薟莞司酶牡位片稀然碩SEQ ID NO1牡1-807位示畝傘
SEQ ID NO2陌械畝牡畝?，艆插懍SEQ ID NO1婕 械水緣SEQ ID NO2片系SEQ ID NO1小
SEQ ID NO1為SEQ ID NO1牡1-807位凇去5/3一畝帷
婕半本薟莞司酶姆畝幔 詰細(xì)攏選械細(xì)攏選詬細(xì)攏詠(i)SEQ ID NO1牡1-807位示畝幕(ii)俸100(i)小婕 (i)(ii)幕
詵隆牡畝募屑知模踴DNA蟹?，cDNA票塹稀源些DNA隆畝?，通使知?酶應(yīng)(PCR)目目篩選約泄同峁鼓 隆DNA片稀紓糏nnis耍1990PCR應(yīng)指(PCRA Guide to Methods and Application)Academic Press紐約使暮酶應(yīng)(LCR)蛹轉(zhuǎn)錄(LAT)突諍械(NASBA)
畝通糯玫謀準(zhǔn)隆茫員憬喝晃宦段皇逛復(fù)頻牟同位隆砂諧頭牒草桿久改 片希片喜氳接Ｏ迪 細(xì)懈瞥枚畝隆碩欏DNARNA銑源銑源塹一稀
為目模之同一猿潭舛
氡灸枯草桿久改段囤合趁枯草 酶頻目薟莞司酶潛摹鎩捌的≥草桿 酶指然詰目薟莞司酶式些薟莞司酶某些品式峽然源玫目薟莞司酶同囟幕浴榷浴pH值確詰牟同?。为SEQ ID NO1畝謀氬恐囤的賭幌剮 /通路取洳簧該盒 一摯薟莞司酶陌校應(yīng)酶使茫取剎同陌小取一渭Ford耍1991實(shí)謀(Protein Expression and Purification)295-107
詒員說然取擁墓丶之脅圓鑰薟莞司酶員姆畝畝牡幕要陌謝選洳蝗≡ 知姆綞ㄕ鄙ㄕ 紓 CunninghamWells1989學(xué)(Science)2441081-1085)諍一校詿朔擁每一傻牟謝突?，?擁牡水裕約朔踴緣墓丶謝-酶嗷ッ滴灰餐ㄎ構(gòu)舛ㄎ構(gòu)舜毆峋аё合鼻尖定(紓糳e Vos耍1992學(xué)255306-312Smith耍1992學(xué)志(Journal of Molecular Biology)224899-904Wlodaver耍1992FEBS Letters 30959-64)
峁
婕澳核藉，剎一只芄指說細(xì)詒牡印
遠(yuǎn)址式氡據(jù)草桿久改豐供薟莞司酶謀鎩Ｚ倉霸妒翟和 取詒濉NA味募屑知摹
邪斜銼據(jù)草桿久 幀每一值卸詒薟莞司酶畝說然源摹些邪鵲小嶧∏ 小印藕瀉轉(zhuǎn)錄止印伲邪約轉(zhuǎn)錄頭止藕擰為說峁┴ 位慕頭俳說薟莞司酶畝印
鋅說校為細(xì)識(shí)詒械畝?。泻閷?dǎo)薟莞司酶轉(zhuǎn)錄小涌選細(xì)詒殖轉(zhuǎn)錄緣魏味?，驼d、囟毯雍櫻細(xì)同源源謀細(xì)細(xì)誑薟莞司酶幕謾
指峁棺枷趕缸 說擁實(shí)為碌櫻蟪Ω司遣印色霉(Streptomyces coelicolor)脂腔(dagA薟芽吒司酶(sacB)芽吒司-酶(amyL)脂芽吒司芽塹酶(amyM)芽吒司-酶(amyQ)芽吒司霉酶(penP)薟芽吒司xylAxylB原甩-酶(Villa-Kamaroff耍1978銥學(xué)院院753727-3731)約tac(DeBoer耍1983學(xué)院院8021-25)細(xì)玫(Useful proteins from recombinant bacteria)學(xué)(Scientific American)198024274-94Sambrook耍1989摹
指絲狀細(xì)斜暮峁棺 說擁實(shí)為碌櫻霉TAKA酶Rhiomucor miehei於蛋酶霉馭-酶霉-酶霉盛霉塹酶(glaA)Rhiomucor miehei脂酶霉緣酶霉旃姑該 酶圖鵲酶酶(WO96/00787)約Na2-tpi(院霉馭-酶霉旃姑贛滌 )塹突濉⒇短河河
誚母校擁源幕平母烯酶(ENO-1)平母羌酶(GAL1)平母酶/醛-3-酶(ADH2/GAP)平母3-峒っ改 細(xì)Romanos耍1992母(Yeast 8423-488
謝一說轉(zhuǎn)錄止校為細(xì)識(shí)止轉(zhuǎn)錄小止鋅剎擁薟莞司酶畝3末恕魏選細(xì)蟹庸艿止涌詒
選絲狀細(xì)止擁源幕霉TAKA酶霉塹酶霉酶霉-酶圖鵲酶酶
選慕母細(xì)止擁源幕平母烯酶平母細(xì)色C(CYC1)平母醛-3-酶Romanos耍1992模玫慕母細(xì)止印
謝說鵲校為細(xì)要mRNA欠鵲鋅剎擁牡畝5末恕魏選細(xì)詵庸艿鵲鋅詒
選絲狀細(xì)鵲械源幕霉TAKA酶凸霉旃姑
說慕母細(xì)鵲械源幕平母烯酶(ENO-1)平母3-峒っ鈣僥 雍平母酶/醛-3-酶(ADH2/GAP)
謝薔嶧Ｐ可燦 3末轉(zhuǎn)錄時(shí)細(xì)識(shí)為一紙詹謝轉(zhuǎn)錄mRNA藕諾小魏選細(xì)蟹庸木嶧口
選絲狀細(xì)木嶧翟 霉TAKA酶霉塹酶霉酶鵲酶酶禿霉-酶
母細(xì)玫木嶧疓uoSherman1995細(xì)學(xué)(Molecular Cellular Biology)155983-5990
牡謝藕謀擁薟莞司酶陌末說陌脅指目薟莞司酶細(xì)途謀械5吮園一然誥詰目薟莞司酶謀詼蔚姆系藕謀選兀械5絲園吮械源藕謀謀脅然一藕謀時(shí)要源藕謀選兀然藕謀源藕婊輝促捷草桿久改汾∥何恐改枯 司酶選細(xì)姆途藕謀詒
細(xì)細(xì)效藕謀塹源幕藕謀芽吒司NCIB11837芽塹酶脂芽吒司-酶芽吒司薟莞司酶芽吒司-酶脂芽吒司緣酶(nprTnprSnprM)涂薟芽吒司prsA藕SimonenPalva1993微學(xué)(Microbiological Reviews)57109-137
絲狀細(xì)效藕謀塹祿藕霉TAKA酶霉緣酶霉塹酶Rhizomucor miehei於蛋酶Humicola insolens維酶Humicola lanuginosa脂酶
母細(xì)藕牡源幕平母-平母轉(zhuǎn)酶Romanos耍1992模玫謀
謝潛位誑薟莞司酶末說陌械前謀玫畝知一酶原前(某些為酶原)前一沒謝裕通呋懈呋懈前牡前畝轉(zhuǎn)為幕遠(yuǎn)摹前謀緣源幕潁嚎薟芽吒司緣酶(aprE)薟芽吒司酶(nprT)平母-印Rhizomucor miehei於蛋酶然絲霉酶(WO95/33836)
誑薟莞司酶陌末舜藕暮前時(shí)前薟莞司酶陌末冢藕前陌末凇
細(xì)氐畝牡謀械詰牡也摹些系統(tǒng)實(shí)曰學(xué)碳(詰諢)應(yīng)使沒謀銼回閉迪低吃 統(tǒng)械牡系統(tǒng)lactactrp系統(tǒng)誚母校使ADH2系統(tǒng)GAL1系統(tǒng)絲狀校TAKA-酶印霉塹酶雍霉塹酶涌小械實(shí)為牡小系統(tǒng)校詡裝蝕畝葉嶧乖肛 虻鞍諄些攏牡畝剎印
婕澳橫構(gòu)?、愈Q繭翻止藕諾濉
氡久改橫構(gòu)魏慰煞亟DNA濉
選一取細(xì)耍?，为扰f第靛，母貧染色母疲選兀詰細(xì)?，敝蝗?xì)?，雽⑤犎ト旧黄?br>
選為謀澹ｋ本酶DNA鋅剎DNA轉(zhuǎn)錄慕詼印一說蠆《DNA院叩元鎩吧 印指詼?zhǔn)節(jié)G辭翟つ磕酚管的肥 校轉(zhuǎn)錄通酶DNA卸前小
涌魏選細(xì)詒殖轉(zhuǎn)錄DNA校緣員細(xì)同源源牡時(shí)
細(xì)細(xì)說擁實(shí)祿櫻脂芽吒司芽塹酶頡⒌芽吒司-酶頡⒔芽吒司-酶頡⒖薟芽吒司緣酶小芽司(Bacillus pumilus)木酶刪-PRPL踴司lactrptac印
要氡久窪NA謝鑰剎擁止稀
寤股敗故觀迪 懈頻DNA小
寤乖把∧鼻Ｖ蠶溉 幕潁鑰霉亍霉亍霉亍幕亍壯霉氐鵲目裕亟菁目緣幕
為指酶細(xì)姆途峁┡ 也知為鵲小前原謝前)越藕確擁酶DNA小藕一位詒酶DNA械5恕藕鋅通酶氐幕員一址詰實(shí)幕
詵直穎氡久窪NA小雍選止/藕校通說PCR些瀉徒遣懈蘋息說姆員員說知(渭紓琒ambrook耍)
細(xì)
婕搬構(gòu)細(xì)
氳較感的彪本酶DNA鋅鄣同源源細(xì)同源篩細(xì)然氡久窪NA一剎然寫詰一櫻誑應(yīng)玫一藕瀉/止印同源指然鄣械酶DNA小鎩霸茨≈ 細(xì)斜聿幻窪NA小DNA鋅源一嗆銑傻小
氡綝NA細(xì)魏芄酶細(xì)細(xì)母透叩細(xì)植鎩
通芄酶細(xì)細(xì)實(shí)歉細(xì)芽吒司輳慚窟桿 B.subtilis)芽吒(B.licheniformis)倩芽吒司(B.lentus)芽吒司(B.brevis)脂芽吒司(B.stearothermophilus)燃芽吒司(B.alkalophilus)芽吒司(B.amyloliquefaciens)芽吒司(B.coagulans)狀芽吒司(B.circulans)芽吒司(B.lautus)藪芽吒司(B.megaterium)平芽吒司(B.thuringiensis)乇淺倩芽吒司霉輳γ S.lividans)霉(S.murinus)細(xì)蟪Ω司
細(xì)轉(zhuǎn)通知姆式原轉(zhuǎn)绱┳ 匣使酶態(tài)細(xì)(渭Sambrook耍)
細(xì)蟪Ω司斜酶時(shí)酶一苑芙鑰駐細(xì)(知)細(xì)始隙前一攏細(xì)呀?，裕通?捅約使酶鄣一攏酶通虜眨透壓菘蘋細(xì)使始隙頭牛倩酶
詬細(xì)芽吒司霉斜酶時(shí)酶駐細(xì)校細(xì)小一攏酶砂謝鍘
詒一實(shí)施校細(xì)墻母細(xì)玫摹母醫(yī)母(霉目(Endomycetales))詠母詘知(Fungi Imperfecti)(芽吒(Blastomycetes))慕母誚母鋅芊浠 目模母應(yīng)母學(xué)突(Biology andActivities of Yeast)(SkinnerF.A.PassmoreS.M.DavenportR.R.嗉璖oc.App.Bacteriol.Symposium Series No.91980)慕卸濉
一選實(shí)施校慕母細(xì)羌絲母(Candida)遜母(Hansenula)魯維母(Kluyveromyces)銑母(Pichia)啤平母(母)殖母(Schizosaccharomyces)Yarrowia細(xì)
一選實(shí)施校慕母細(xì)強(qiáng)斯墻母(Saccharomyces carlsbergensis)平母腔母(Saccharomyces diastaticus)Saccharomyces douglasii魯母諾亟母墻母細(xì)
一選實(shí)施校慕母細(xì)魯維母(Kluyveromyces lactis)細(xì)一選實(shí)施校慕母細(xì)Yarrowia lipolytica細(xì)
一選實(shí)施校細(xì)絲狀細(xì)絲狀械(Eumycota)藕丫(Oomycota)諾絲狀式(Hawksworth耍1995模)絲狀園省維亍暇恰薔恰露嗆隙塹木絲為
營通絲映械模碳謝摹嚳茨 平母營通細(xì)芽械模碳謝欠偷摹
一選實(shí)施式校絲狀細(xì)塹預(yù)霉霉霉毛霉絲霉霉霉莞霉Tolypocladium木霉細(xì)
一選實(shí)施式校絲狀細(xì)盛霉霉氈霉霉霉霉細(xì)一選實(shí)施式校絲狀細(xì)歉狀摺FusariumcerealisFusarium crookwellense摺坦摺壇摺摺匣木摺摺枝摺玫色摺庸木摺色摺枝摺色摺Fusarium torulosumFusariumtrichothecioidesFusarium venenatum細(xì)一選實(shí)施式絲狀?yuàn)y細(xì)Fusarium venenatum(Nirenberg sp.nov.)細(xì)
一選實(shí)施式校絲狀細(xì)HumicolainsolensHumicola lanuginosa綴毛霉(Mucor miehei)然絲霉植霉(Neurospora crassa)霉Trichodermaharzianum木霉Trichoderma longibrachiatumTrichodermareesei色木霉(Trichoderma viride)細(xì)
細(xì)通一轉(zhuǎn)目前知姆式緯原濉⒆并細(xì)凇屎轉(zhuǎn)霉細(xì)姆EP238023Yelton耍1984銥學(xué)院院811470-1474轉(zhuǎn)霉姆Malardier耍1989(Gene)78147-156WO96/00787母轉(zhuǎn)姆校BeckerGuarenteAbelsonJ.N.SimonM.I.嗉概囪 學(xué)指(Guide to Yeast Genetics and Molecular Biology)酶學(xué)194恚 82-187頁Academic PressInc.紐約Ito耍1983細(xì)學(xué)志(Journal of Bacteriology)153163Hinnen耍1978銥學(xué)院院751920
目薟莞司酶姆
一婕澳枯草桿久改訪
a)薟莞司酶細(xì)
b)目薟莞司酶
酶DNA械謀轉(zhuǎn)源細(xì)時(shí)使母酶源
紗絲圓同源為牡母叨卻目薟莞司酶鎩
校同源指源帽酶同源細(xì)(酶之)
轉(zhuǎn)細(xì)魏屎鄣細(xì)某目薟莞司酶煞胤詰校然通知姆眨通幕舜蟹細(xì)銨承的抵 脅詠綴筒取
目薟莞司酶途
目薟莞司酶卸止業(yè)途洗蛹業(yè)小吮婕懊趕從鹼，選酶洗祿洗鎩
薟莞司酶洗蛹錚
一說洗蛹詒院實(shí)洗蛹慕一圓渭WO96/34946WO97/07202WO95/30011
舜實(shí)目薟莞司酶殖越洗堋
洗蛹
酶擁洗蛹卸為械幀
紓從計(jì)誠聰聰從 錚と局從計(jì)從 維砘，瞥通揖硬洗蛹錚 瞥洗洗洗灘
一囟姆媯供一職目薟莞司酶洗蛹洗蛹洗蛹園一只酶一值酶脂酶酶酶酶維酶酶露酶酶酶木酶酶酶/酶
一說選酶應(yīng)選洗蛹應(yīng)(pH-耍酶酶志緣)酶應(yīng)效凇
酶實(shí)牡酶遠(yuǎn)鎩⒅詫、微牡酶源微牡酶選摹也學(xué)位虻鞍墜袒突濉Ｄ得桿酷蛋酶酶選微锏懊溉得該岡得甘滴草桿久 乇芽吒司目薟莞司酶紓草桿久窷ovo薟莞司酶Carlsberg薟莞司酶309薟莞司酶147涂薟莞司酶168(WO89/06279)鵲酶酶實(shí)為鵲酶(牛)WO89/06270WO94/25583霉牡酶
玫牡酶實(shí)WO92/19729WO98/20115WO98/20116WO98/34946謀?，貛呕?婊荒卞273657768797101104120123167170194206218222224235274
選目坦牡酶AlcalaseTMSavinaseTMPrimaseTMDuralaseTMEsperaseTMKannaseTM(Novo Nordisk A/S)MaxataseTMMaxacalTMMaxapemTMProperaseTMPurafectTMPurafect OxPTMFN2TMFN3TM(Genencor International Inc.)
脂酶實(shí)脂酶些源細(xì)脂酶也學(xué)位虻鞍墜袒突?。玫脂酶实EP258068EP305216愿霉(Thermomyces之同)H.lanuginosa(T.lanuginosus)脂酶WO96/13580H.Insolens脂酶俚脂酶圓俚(P.Alcaligenes)俚(P.Pseudoalcaligenes)(EP218272)屑俚(P.Cepacia)(EP331376)施霞俚(P.stutzeri)(GB1,372,034)熒俚(P.Fluorescens)俚SD705(WO95/06720WO96/27002)P.wisconsinensis(WO96/12012)芽吒司脂酶鑰薟芽司(Dartois(1993)锘аа Biochemica etBiophysica Acta)1131253-360)脂芽吒司(JP64/744992)小芽吒司(B.Pumilus)(WO91/16422)
實(shí)為WO92/05249WO94/01541EP407225EP260105WO95/35381WO96/00292WO95/30744WO94/25578WO95/14783WO95/22615WO97/04079WO97/07202脂酶濉
選目坦脂酶LipolaseTMLipolase UltraTM(NovoNordisk A/S)
酶實(shí)牡酶(/)些源細(xì)牡酶也學(xué)位虻鞍墜袒突?。脤?芽吒司摩-酶緣芽吒司囟輳 渭GB1,296,839
玫牡酶實(shí)WO94/02597WO94/18314WO96/23873WO97/43424謀?，些?位梅婊荒卞1523105106124128133154156181188190197202208209243264304305391408444
坦牡酶DuramylTMTermamylTMFungamylTMBANTM(Novo Nordisk A/S)RapidaseTMPurastarTM(GenencorInternational Inc.)
維酶實(shí)維酶源細(xì)維酶也渚或蛋白雇誨。實(shí)維酶芽吒俚霉霉莞霉枝霉維酶US4,435,307US5,648,263US5,691178US5,776,757WO89/09259泄Humicola insolens然絲霉卟維酶
乇實(shí)維酶薔色頻募曰維酶維酶實(shí)為EP0495257EP0531372WO96/11262WO96/29397WO98/08940維酶實(shí)為些WO94/07998EP0531315US5,457,046US5,686,593US5,763,254WO95/24471WO98/12307PCT/DK98/00299維酶濉
坦維酶CelluzymeTMCarezymeTM(Novo NordiskA/S)ClazinaseTMPuradax HATM(Genencor International Inc.)KAC-500(B)TM(Kao Corporation)
酶/酶實(shí)墓酶/酶些源植鎩⑾改姑 酶也學(xué)位虻鞍墜袒突濉Ｃ的姑甘礧O93/24618WO95/10602WO98/15257怨傘(Coprinus)(曰腋槍傘(C.cinereus))墓酶
坦墓酶GuardzymeTM(Novo Nordisk A/S)
洗蛹酶通雍一只酶畝蛹通雍些酶蛹洗蛹小洗蛹蛹也嵌蛹蛹瞥狀液濉⒆慈等⊙∠從加際轎 乇欠欠末)液(乇榷液)蚪
欠末US4,106,9914,661,452選知姆狀系實(shí)平為100020000木()(葉PEG)1650櫚ノ簧揮Ｖ 220碳原寫1580櫚ノ恢；脂牡--GB1483591懈屎通應(yīng)玫某膜系實(shí)液酶萍殖傻姆通傭元腔譴榷鼙酶EP238,216姆票
洗蛹魏畏式狀片狀末粘嘔液?。液蠌挠浐水模覅R含嘰70水0-30謝薌蠆緩水摹
洗蛹一一只直約強(qiáng)穎約爰的 雍/雍/穎約謀約一占0.160洗蛹時(shí)通約1約40穎約直-烯(脂)崠 腔脂-烯懟
洗蛹時(shí)通約0.2約40姆約脂苫印鍘諄鎩⒅單掖脂岬ヒ 脂前N-N-()
洗蛹園0-65洗蛹霞石巍巍巍碳巍巍蔚帷 ?、視?huì)、-烯?、?位式(綣篐oechstSKS-6)
洗蛹園一只志酆鎩Ｊ滴甲晃 亍(烯酮)(葉)(烯)(烯-N-)(烯)郾烯/烯峁 圖諄烯鹿/烯峁詫。
洗蛹院鋅馨H2O2源漂系緯曬漂諄罨段 墓位碳巍選兀漂系園前墓帷
洗蛹械酶通碌某榷元腔譴帷 蟣交4-幔 票為WO92/19709WO92/19708鎩
洗蛹園某洗蛹煞維詡菁沫萍蝕儷染稀殺學(xué)準(zhǔn)薌失萍稀
目前為洗蛹魏酶乇潛酶嗟泵肯匆盒 .01-100mg酶剩選每洗液0.05-5mg酶剩乇選每洗液芯0.1-1mg酶實(shí)搿
酶擁WO97/07202洗蛹浞叫 氡疚
通實(shí)員薪細(xì)說些實(shí)為員蟊；圍魏啤
洗蛹校寫煞值攏LAS 直C12TAS 牛XYASClx-ClySS 式2-砘 25EY平為Y摩幕系要為C12-C15
直45EY平為Y摩幕系要為C14-C15
直XYEZS 每摩平Z摩幕系Clx-Cly
櫻 平潭3.8平潭為4.5
C13-C15系/脂
BASF GmbH鄢品為Plurafax LF404CFAAC12-C14N-諄TFAAC16-C18N-諄危 薅凸(SiO2Na2O剩2.0)NaSKS-6 式為-Na2Si2O5木狀碳危 水碳危 MA/AA 14/烯峁詫，平約為80,000郾烯 平為8,000木郾烯錚珺ASF
GmbH郟品為PA30石A 小1-10微追圍詰姆式為
Na12(AlO2SiO2)1227H2O水瞎危水Citric 危水一水漂準(zhǔn)式為
NaBO2H2O2PB4 水水碳危式為2Na2CO33H2O2水碳漂準(zhǔn)TAED葉CMC 燃諄維DETPMP 一(羌諄)Monsanto郟
品為Dequest 2060PVP 烯酮酆EDDS式葉-NN-[SS]旃 菁 25石鄣露50媯 7水石
58石狀菁 12/石18十櫬 0狀危 水HMWPEO 叻芻TAE 25 牛痛(25)
洗蛹實(shí)I
狀維票
直C12 6.515.0石A 26.0蔚5.0酶0.1PVP 0.5TAED 3.0 4.0 18.0踴0.1要 100洗蛹實(shí)II蕓維(芏800g/l)票45AS 8.025E3S2.025E5 3.025E3 3.0TFAA 2.5石A17.0NaSKS-6 12.0 3.0碳 7.0MA/AA5.0CMC 0.4酶 0.1TAED 6.0碳 22.0EDDS 0.3
狀菁3.5
水/要 100
洗蛹實(shí)III
畝洗硬色織乇玫狀織票
LAS 10.7 -
TAS 2.4 -
TFAA - 4.0
45AS 3.1 10.0
45E7 4.0 -
25E3S - 3.0
68E11 1.8 -
25E5 -8.0
15.0 7.0
碳- 10.0
2.5 3.0
石A 32.1 25.0
Na-SKS-6 - 9.0
MA/AA 5.0 5.0
DETPMP0.2 0.8
酶0.10 0.05
2.5 -
5.2 3.0
PVP 0.5 -(4-烯)-N-/烯 - 0.2烯酮墓
1.0 -
踴 0.2 -
水/要100
洗蛹實(shí)IV
木小洗庸械砘”狀織票45AS- 10.0LAS 7.6 -68AS1.3 -45E74.0 -25E3- 5.0椰--諄 1.4 1.0一然5.0 3.0Na-SKS-6- 11.0石A 15.015.0MA/AA 4.0 4.0DETPMP 0.4 0.415.0-碳- 15.0TAED5.0 5.010.010.0HMWPEO - 0.1酶 0.100.05 3.0 5.0碳 10.010.0狀菁 1.0 4.0CMC 0.2 0.1
水/要 100
洗蛹實(shí)V
強(qiáng)液織票式LAS -25.05.0 2.0式25AS 8.0 -式25AE2S 3.0 -25AE78.0 -CFAA 5-DETPMP 1.0 1.0脂 8- -1.0掖 4.0 6.0 2.0 6.0酶 0.10 0.05椰-諄腔一然-3.0 -5.0PVP 2.0 -水/要 100
末遠(yuǎn)洗I
末遠(yuǎn)洗II
末遠(yuǎn)洗III
末遠(yuǎn)洗IV
末遠(yuǎn)洗V
末液木洗穎約系統(tǒng)洗VI
水液態(tài)遠(yuǎn)洗VII
水液態(tài)洗VIII
液遠(yuǎn)洗IX
液遠(yuǎn)洗X
漂卓液遠(yuǎn)洗XI
XIIIIIIIIIVVIX遠(yuǎn)洗錚 曬碳巍
XIIII-VI遠(yuǎn)洗錚還檀呋檀呋紓≮燈的感催 Efficientmanganese catalysts for low-temperature bleaching)然(1994)369637-639幕械一幀
蝦頭
水
詒械水Kilo NOVO酶位(KNPU)示摹幕酶準(zhǔn)品(SAVINASE_)確牟舛ㄇ悔弊悸鄧岡蹲灰 DMC)液械模謀準(zhǔn)為50媯琾H8.3應(yīng)時(shí)9櫻時(shí)3印要時(shí)Novo Nordisk A/SDenmark取AF220/1募校募邪詿為慰
GU歉拾岬ノ晃岡Ｚ弊悸-業(yè)為4015櫻嗟 mmole拾NH2-
也PNA脂學(xué)學(xué)志(Journal ofAmerican Oil Chemists Society)RothgebT.M.GoodlanderB.D.GarrisonP.H.SmithL.A.(1988)牡-----臃應(yīng)舛岡
實(shí)施1-目薟莞司酶墓捅
SEQ ID NO2示械目薟莞司酶位pJRoC112pKH400(WO98/41623)淺啤詒目薟莞司酶同模些械母悖關(guān)吭礳at薟莞司酶轉(zhuǎn)錄始雍Savinase_前/原一碌摹莞司酶蠆糠執(zhí)
一詿蟪Ω司涂薟芽吒司懈啤誑薟芽吒司篩銼灸枯草桿久該得改吠和
PKH400通pJS3(J.Schi_dt耍屎通訊(Protein and Peptide Letters)339-44(1996)暮薟司酶309(Savinase_)暮銑苫拇蟪Ω司-薟芽吒司)牡18413730位BamHI位摹
幕強(qiáng)隆pzero-2(InvitrogenGroningenTheNetherlands)小SEQ ID NO2示械目薟莞司酶全約1240bp PmeI-BamHI片擁pZero-2希酶BamHI-EcoRV踴轉(zhuǎn)態(tài)拇司細(xì)通PCR轉(zhuǎn)證欠誆片危員薟莞司酶一片尾紙脅為pTVB3642000210氈DSMZ睪為DSM13306
薟莞司酶前械模30轉(zhuǎn)臺(tái)(300轉(zhuǎn)/)煤100ml BPX500ml式錐瓶5臁
耍為嘶220錐瓶同時(shí)汀
BPX煞(每)
100g
磨拇 50g
蠖狗 20g
Na2HPO4x12H2O 9g
(Pluronic) 0.1g
業(yè) 10g
械牡忙-酶液模120錄45NaHCO30.1MpH詰9
一婕把窟桿鞠感材蹦枯草 酶2液
1氈誚約1.65000轉(zhuǎn)/35印10拇液使pH值為6.5Seitz Supra S100坦恕
潯窤micon SLY10 UF筒Amicon CH2A UF裝媒順液濃約400ml酶司綴pH7前冉UF濃牟鹿恕薟莞司酶諍0.01M諄幔 .1M0.002M然啤訓(xùn)pH為7幕液械251M然拼癰司洗選
篩司拇玫木械酶緣募趾喜應(yīng)0.01M諄幔 .2M0.002M然頻pH6.5液平750ml Sephadex G25(直5cm)
Sephadex G25玫木械水緣募趾喜應(yīng)煤0.01M諄幔 .2M0.002M然頻pH6.5幕液平150ml CM Sepharose CL 6B詠(直5cm)
酶2同液械0-0.1M荻然洗選
拇校CM Sepharose玫暮械酶募趾喜裝GR81PP膜(Danish Sugar Factories Inc.)Amicon順濃
應(yīng)頭圖約氬借，SEQ ID NO2示械驢薟莞司酶
實(shí)施2-準(zhǔn)洗蛹洗欏
為薟莞司酶詒準(zhǔn)洗蛹械洗埽實(shí)吮準(zhǔn)洗實(shí)椋
洗蛹準(zhǔn)洗蛹
洗蛹 4.0g/l
pH 10.1
洗時(shí)20
露齲 30
水硬齲15dH
酶濃齲10nm(洗蛹液)
系統(tǒng) 10ml氈徒
織/ 5織(_2.5cm)/50ml洗蛹液
圓希 WFK10N()
謀準(zhǔn)洗蛹攏
6.2 LAS(Nansa 80S)
2 C16-C18脂
4 穎約(Plurafax LF404)
22石P
10.5 Na2CO3
4Na2Si2O5
2燃諄維(CMC)
6.8 烯液CP5 40
20 (式NaBO2H2O2)
0.2 EDTA
21 Na2SO4
水 (平)
通系統(tǒng)HC1NaOH洗蛹液pH詰10.1通CaCl2MgC12(Ca2+Mg2+41)水硬鵲詰15dH洗雍織水洗誑懈鎩
戲(R薟莞司酶)牟舛∕acbeth ColorEye 7000photometer(MacbethDivision of Kollmorgen InstrumentsCorporation鹿)460nm陸械摹舛ㄌ的 小
為確瞻值瞬酶洗實(shí)欏 (R瞻)牟舛
然脅慰實(shí)?，袦y(cè)定Savinase_洗堋舛ㄋ Rsavinase)
洗酶式(P)模
P(R薟莞司酶-R瞻)-(Rsavinase-R瞻)
R薟莞司酶-Rsavinase
應(yīng)實(shí)櫸
杉Savinase_齲目薟莞司酶氈殖頻洗堋
實(shí)施3-目薟莞司酶遠(yuǎn)(ADW)械
目薟莞司酶遠(yuǎn)(ADW)械通誑募洗(Somat TurboHenkel Waschmittel GmbH)薪摹玫塹/牛蹋獺一稅食品壓鎩
洗蛹 Somat Turbo
洗蛹4.0g/l
pH10.7(實(shí))
水硬齲 3dH(詠)
露齲55
酶濃齲 20nM40nM莼洗水
櫸 #160; 詬系牡/牛
Cylinda Compact
洗映4預(yù)洗
220ml全脂牛
15械卻小
蹋直18cm
Somat Turbo洗微爐85錄5使械酶失睢
染
220ml全脂牛15Braun UK 20 kitchen machine2櫻撕?，教?jīng)]使染
戲賂夜120錄45使牡時(shí)浴
ADW實(shí)
每一實(shí)校10沒芯Cylinda Compact machine預(yù)洗洗(4)猓 0獺44璞 6椎
遙50g壓嘟
嘟 5.43)小(4.38)(4.32)(4.32)(4.32)(8.76)全脂牛(8.76)(8.76)呀(3.00)飩 2.19)末(4.00)(0.73)水(3mM Ca2++Mg2+)(36.71)
舛ㄍ
墓夥粗 R-值)Minolta Chroma Meter(禿牛CR-300)械同位舛≡干 Rclean)群(Rsoiled)洗(Rafter wash)脅舛
式去牡膜(RPF)
RPF100X(Rafter wash-Rsoiled)/(Rclean-Rsoiled)
櫸 示準(zhǔn)偏)遠(yuǎn)準(zhǔn)酶Savinase_染越堋
系謀
細(xì)薟斯約墓娑ㄚ抵疚保Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH(DSM)Mascheroder Weg 1BD-38124 Braunschweig鹿睪牛
Novo Nordisk A/S票撕轉(zhuǎn)NovozymesA/S
斜<110> 諾酶煞薰司<120> PH004<130> 6137<150> DK PA 2000 00405<151> 2000-03-14<160> 2<170> PatentIn version 3.0<210> 1<211> 807<212> DNA<213> 斯<220><221> CDS<222> (1)..(807)<400> 1gcg caa tcg gta cea tgg gga att agc cgt gtg caa gcc cca gct gcc48Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala1 5 10 15cat aac cgt gga ttg aca ggt tct ggt gta aaa gtt gct gtc ctc gat 96His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30aca ggg ata tcc act cat cca gat cta aat att cgt ggt ggc gca agc 144Thr Gly Ile Ser Thr His Pro Asp Leu Ash Ile Arg Gly Gly Ala Ser
35 40 45ttt gta cca ggg gaa ccg tcg act caa gat ggg aac ggg cac ggg acg 192Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60cac gtt gca gga aca gtg gca gct ctt aat aac tca atc ggt gtg att 240His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser lle Gly Val lle65 70 75 80ggt gtg gcg cea agt gct gat cta tac gct gta aaa gta ctt gga gca 288Gly Val Ala Pro Ser Ala Asp Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95aat ggt aga gga agc gtt agt gga att gct caa ggt eta gag tgg get 336Asn Gly Arg Gly Ser Val Ser Gly Ile Ala Gln Gly Leu Glu Trp Ala
100 105 110gca gcg aat aac atg cat att get aac atg agt ctc ggt agt gat gca 384Ala Ala Asn Asn Met His Ile Ala Asn Met Ser Leu Gly Ser Asp Ala
115 120 125cct agt act aca ctt gag cgt gca gtc aac tac gcg aca agc cag ggt 432Pro Ser Thr Thr Leu Glu Arg Ala Val Asn Tyr Ala Thr Ser Gln Gly
130 135 140gta cta gtt att gca gcg act ggt aac aac ggt tcc ggt tca gta ggc 480Val Leu Val Ile Ala Ala Thr Gly Asn Asn Gly Ser Gly Ser Val Gly145 150 155 160tat cct gct cgt tat gcc aac gca atg gct gta gga gcg act gac caa 528Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln
165 170 175aac aac aga cgt gca aac ttt tct cag tac ggt aca gga att gac atc 576Asn Ash Arg Arg Ala Asn Phe Ser Gln Tyr Gly Thr Gly Ile Asp lle
180 185 190gta gca cct ggc gtt gac att gaa agc acc tac cca ggc agc tct tat624Val Ala Pro Gly Val Asp Ile Glu Ser Thr Tyr Pro Gly Ser Ser Tyr
195 200 205gac agc cta agt ggc aca tca atg gct act cca cat gtc gcc ggc gtc672Asp Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val
210 215 220gcc gca cta gtt aaa caa aag aac cca tct tgg tct aat gta caa att720Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile225 230 235 240cga aat cat cta aag aat acg gca act agt tta gga agc acg aac ttg768Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
245 250 255tat gga agc gga ctt gtt aac gca gaa gcg gca acg cgt807Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
260 265<210> 2<211> 269<212> PRT<213> 斯<400> 2Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala1 5 10 15His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
35 40 45Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val lle65 70 75 80Gly Val Ala Pro Ser Ala Asp Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95Asn Gly Arg Gly Ser Val Ser Gly lle Ala Gln Gly Leu Glu Trp Ala
100 105 110Ala Ala Asn Asn Met His Ile Ala Asn Met Ser Leu Gly Ser Asp Ala
115 120 125Pro Ser Thr Thr Leu Glu Arg Ala Val Asn Tyr Ala Thr Ser Gln Gly
130 135 140Val Leu Val Ile Ala Ala Thr Gly Asn Asn Gly Ser Gly Ser Val Gly145 150 155 160Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln
165 170 175Asn Asn Arg Arg Ala Asn Phe Ser Gln Tyr Gly Thr Gly Ile Asp Ile
180 185 190Val Ala Pro Gly Val Asp Ile Glu Ser Thr Tyr Pro Gly Ser Ser Tyr
195 200 205Asp Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val
210 215 220Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile225 230 235 240Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
245 250 255Tyr Gly Ser Gly Leu Val Ash Ala Glu Ala Ala Thr Arg
260 26權(quán)利要求
1.一摯薟莞司酶選裕
a)SEQ ID NO2牡1-269位示陌芯88同一緣目薟莞司酶
b)詰細(xì)詠畝目薟莞司酶
i)SEQ ID NO1牡1-807位示畝幕
ii)100(i)校
c)煽隆詿蟪Ω司MT173 DSM13306械片系目薟莞司酶氬恐蹦枯草桿久改枯 司酶88同一緣謀濉
2.權(quán)要1目薟莞司酶校詒牡摹準(zhǔn)洗蛹洗欏蹦奧成分的弊枷從 時(shí)
6.2LAS(Nansa 80S)
2C16-C18脂
4穎約(Plurafax LF404)
22石P
10.5Na2CO3
4Na2Si2O5
2燃諄維(CMC)
6.8烯液CP5 40
20(式NaBO2H2O2)
0.2EDTA
21Na2SO4
水(平)絲薟莞司酶同Savinase_缺殖慕洗堋
3.權(quán)要2目薟莞司酶斜為1選為2選為3乇為4為5
4.權(quán)要1-3一目薟莞司酶薟莞司酶械陌SEQ ID NO2牡1-269位示陌芯90919293949596979899同一浴
5.權(quán)要4目薟莞司酶薟莞司酶SEQ IDNO2牡1-269位示陌傘
6.權(quán)要1-3一目薟莞司酶薟莞司酶械陌隆詿蟪Ω司MT173 DSM13306械片系畝目薟莞司酶氬恐蹦枯草桿久 90919293949596979899同一浴
7.權(quán)要6目薟莞司酶薟莞司酶煽隆詿蟪Ω司MT173 DSM13306械片系畝目薟莞司酶氬恐蹦枯草桿久干
8.一權(quán)要目薟莞司酶目薟司酶為SEQ ID NO2牡1-269位示摹一謝婊蝗筆 陌械目薟莞司酶謀濉
9.權(quán)要8目薟莞司酶薟莞司酶一位273656768797101104120123167170222224235274(BASBPN)之一巍
10.權(quán)要9目薟莞司酶選K27R*36DS56PN76DS87NG97NR101GS103AV104AV104IV104NV104YH120DN123SY167XR170XM222SM222AT224SK235LT274A(BASBPN)
11.權(quán)要一目薟莞司酶薟莞酶械細(xì)攏選詬叨細(xì)詠畝耄
i)SEQ ID NO1牡1-807位示畝幕
ii)100(i)片匣小
12.一址畝?，权?-11一目薟莞司酶畝帷
13.一直薟莞司酶姆畝?，?br>
a)SEQ ID NO1牡1-807位示畝88同一緣畝幔
b)詰細(xì)詠畝目薟莞司酶
i)SEQ ID NO1牡1-807位示畝幕
ii)100(i)校
c)隆詿蟪Ω司MT173 DSM13306械系畝目薟莞司酶氬恐Ｄ盒 8同一謀濉
14.權(quán)要13畝?，械?SEQ ID NO1牡1-807位示畝8990919293949596979899同一浴
15.權(quán)要13畝幔的 隆詿蟪Ω司MT173 DSM13306械系畝目薟莞司酶氬恐 990919293949596979899同一浴
16.一趾峁瑰，剎一只芄指薟莞酶說詒牡擁權(quán)要12-15一暮小
17.一澹ㄒ 6暮峁瑰、約轉(zhuǎn)錄頭止藕擰
18.一細(xì)權(quán)要16暮峁瑰。
19.權(quán)要18細(xì)細(xì)為細(xì)選為芽吒司乇淺倩芽吒司
20.權(quán)要18細(xì)細(xì)為母選為絲狀乇霉
21.一權(quán)要1-11一目薟莞司酶姆
(a)薟莞司酶權(quán)要18-20一細(xì)
(b)湛薟莞司酶
22.一洗蛹錚∥綽幌達(dá)，權(quán)要1-11一目薟莞司酶
23.權(quán)要22錚還維酶脂酶酶原酶酶酶塹幕鎩
24.權(quán)要1-11一目薟莞司酶洗蛹械應(yīng)謾
25.權(quán)要1-11一目薟莞司酶誄去途
26.權(quán)要22-23一洗蛹去盞途
27.一洗硬姆使硬權(quán)要22-23喲
28.一殖去硬盞姆使盞硬盞權(quán)要22-23喲
全文摘要
具有改進(jìn)的卵污漬洗滌性能的新枯草桿菌酶。這些枯草桿菌酶在用于諸如清潔或洗滌劑組合物，如洗衣組合物和洗盤組合物，包括自動(dòng)洗盤組合物中時(shí)，表現(xiàn)出優(yōu)良的或改進(jìn)的卵污漬洗滌性能。
文檔編號(hào)C12R1/07GK1418249SQ0180653
公開日2003年5月14日 申請(qǐng)日期2001年3月12日 優(yōu)先權(quán)日2000年3月14日
發(fā)明者F·米克爾森, T·S·法諾, M·N·馬德森, L·B·漢森, P·K·漢森 申請(qǐng)人:諾和酶股份有限公司