专利名称：具有改进的卵污渍洗涤性能的新枯草杆菌酶的制作方法
婕靶改较窜德枯草杆久 subtilase)些莶莞司酶洗蛹铮 洗铮断词敝衬荒较窜
婕懊枯草杆久改纺夺、峁瑰、濉⑺横构细约使帽目莶莞司酶姆遥婕拜草杆久 洗蛹铮夹┟赶从夹低沮橙 铡
洗蛹业酶洗浞绞 0稀些酶酶脂酶酶维酶酶堑幕铩Ｒ滴盖得
玫酶然诘野偷酶牡坠袒澹珼URAZYM_(Novo Nordisk A/S)RELASE_(NovoNordisk A/S)MAXAPEM_(Gist-Brocades N.V.)PURAFECT_(Gene-ncor InternationalInc.)
遥屑舜牡酶澹 EP130756(GENENTECH)(应US侔专34606(GENENCOR))EP214435(HENKEL)WO87/04461(AMGEN)WO87/05050(GENEX)EP260105(GENENCOR)ThomasRussellFersht(1985)然(Nature)318375-376ThomasRussellFersht(1987)学志(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.)
牵舜玫牡酶偷酶澹 诖墓业应茫要碌母慕牡酶虻鞍酶濉
裕写诘丝岬懊 硬去盏狻
耍目峁┠侥枯草杆久岗 /硬去铡
耍诘一妫及一志懈慕洗艿莶莞司酶目莶莞司酶选
a)SEQ ID NO2械1-269位示陌芯88同一缘目莶莞司酶
b)诘细咏亩目莶莞司酶
i)SEQ ID NO1械1-807位示亩幕
ii)100(i)校
c)煽隆诖蟪Ω司(Escherichia coli)MT173 DSM13306械片系亩目莶莞司酶氩恐蹦枯草杆久 目莶莞司酶88同一缘谋濉
诘诙妫及一址亩幔 氡据草杆久改盒
诘妫及一直莶莞司酶姆亩幔 选裕
a)SEQ ID NO1械1-807位示暮芯88同一缘亩幔
b)诘细咏亩目莶莞司酶
i)SEQ ID NO1械1-807位示亩幕
ii)100(i)校
c)隆诖蟪Ω司MT173 DSM13306械片系亩目莶莞司酶氩恐Ｄ盒 8同一缘谋濉
诘姆妫及一职械暮峁瑰，峁股惨恢卉怪篙草杆久杆 诒牡印
诘宸芥，婕耙恢搬构濉⒃ 转录头止藕诺濉
诘妫及一职峁瓜
诘叻妫及一植莶莞司酶姆
(a)诓莶莞司酶细
(b)湛莶莞司酶
诘诎朔妫及一洗蛹铮∥绰 洗铮枯草杆久
一姆婕澳枯草杆久赶从 械途目莶莞司酶诔去盏途一洗拥姆硬铣去盏姆使硬氡居
卸员群捅挪渭
图1图示诳莶莞司酶BPN(a)(BASBPN)捅驴莶莞司酶(b)之卸员取
专械些卸员圆谢斜诺牟铡
诟细郾之前榷碌凸铩
AAla
VVal影
LLeu
IIle
PPro
FPhe
WTrp色
MMet
GGly拾
SSer丝
TThr瞻
CCys装
YTyr野
NAsn於
QGln劝
DAsp於
EGlu劝
KLys
RArg
HHis榘
XXaa獍
A
G
C
T(DNA)
U(RNA)
凸
诖亩挚莶莞司酶校碌凸诓眨
通幕妆酶莶莞司酶BPN(BASBPN)员慰
GCG9.1GAP卸员龋诙钥莶司酶斜牛使虏郑8展郑8械为堑默值
一址使知目莶莞司酶之亩员龋WO91/00345示卸员取诖蟛糠拢觳⒁
莶莞司酶BPN(BASBPN)捅驴莶莞司酶之员图1示
耍BASBPN氐缺失筒搿Ｍ 校驴莶莞司酶BASBPN龋3658158162163164位6缺失些缺失图1呛(*)示
一碌煞示妆酶械亩危
原始 位 婊荒
耍G195E指195位母拾岜蝗版换
原始谢獍毙宦Ｊ泵 员锓绞绞疚幻烘换陌幔
位 婊荒
址同源莶莞司酶()
频兀婊恍秽不要时
原始 位
原始婊辉扳氨时只指位茫纾 70
原始/婊荒霸耙幌敌 时选陌示冢
原始 位{婊荒 1...婊荒 n}
囟谋澹关兑荒鸽，XaaX示魏伟谢
婊
霉劝婊 95位母拾示为
Glyl95GluG195E
獍毙绘换195位母拾示为
Glyl95XaaG195X
Glyl95G195
丝婊 70位獍毙皇疚
Xaal70SerX170S
170Ser170S
址同源莶莞司酶()170Ser指BASBPN械Lysl70Ser魏捅莶莞司酶Argl70Ser(渭图1)
械原始/婊荒霸耙幌 械陌味裕筛拾帷⑨、丝瞻婊 170位木示为
Argl70{GlyAlaSerThr}R170{GAST}员示
R170GR170AR170SR170T
缺失
195位拾缺失示为
Glyl95*G195*
应兀一习谢缺失195196位拾缺失示为
Glyl95*+Leul96*G195*+L196*
耄
陌谢牟耄珿195示为
Glyl95GlyLysG195GK
撸一系陌谢G195LysAla时植示为
Glyl95GlyLysAlaG195GKA
些拢通小写母拥氚毙恢澳 谢位煤圆氚毙恍迸∠凳凳┬ 194-196为
194 195 196
BTSAVI A-G-L
194 195 195a 195b 196
A-G-K-A -L
诘陌谢同陌谢时然谐思虿ⅰ系实施校诟拾之拾幔 示为G195GG源
194 195 196
BLSAVI A-G-L
194 195 195a 196
A-G-G-L
194 194a 195 196同一斜浠笔疚狝194AG
些佣员员说远准模冶示G195GG筒应示指同募虿⒈示
畈
诒诺目莶莞司酶BPN薪斜冉希诓盏酶写缺失时36位於苍Ｎ幻茨 示为
*36Asp*36D
危
眉雍欧指蔚谋澹
Argl70Tyr+Glyl95GluR170Y+G195E170195位直野凸劝婊煌甘 巍
耍Tyrl67{GlyAlaSerThr}+Argl70{GlyAlaSerThr}示卤澹
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∪笔ж锻的 谢(KRH)(DE)牟谢危一小婊灰恍yr167{GlyAlaSerThr}+Arg170{GlyAlaSerThr}谋匕乇亍一细渭帧
酶
纸獾笆敌得肝 苫)酶(Walsh1979酶应(Enzymatic Reaction Mechanisms).W.H.Freeman and CompanySan Francisco3)
位/谢谋
没指玫陌哦应诳莶莞司酶BPN(BASBPN)械陌拧BPN械细渭图1Siezen耍使(Protein Engng.)4(1991)719-737
丝岬懊
丝岬懊敢恢催荒妓岗辉参悔 丝谢(WhiteHandlerSmith1973锘г (Principles of Biochemistry)妫琈cGraw-Hill Book司NY271-272页)
细丝岬懊改 0,000-45,000俚姆围凇潜啤水末脂啵 拥酶(也一丝岬懊 啤一为铮 一募缘酶映某些丝岬懊改竝H值为pH9.0-11.0(渭Priest(1977)细学(Bacteriological Rev.)41711-753)
莶莞司酶
Siezen丝岬懊敢婚，莩为莶莞司酶(subtilase)使蹋4(1991)719-737Siezen耍学(Protein Science)6(1997)501-523通170前为莶莞司酶酶丝岬懊改靶酵 苑摹莶莞司酶前通为筛细丝岬懊岗窼iezen为莶莞司酶一椤Ｑ妓茨枯草杆久秆测定一些莶莞司酶小源莶莞司酶浒毙的赶肝 Siezen(1997)
莶莞司酶一椋琁-S1莶莞司酶准摹莶莞司酶莶莞司酶168(BSS168)莶莞司酶BPN(BASBPN)莶莞司酶Carlsberg(BLSCAR)(ALCALASE_NOVO NORDISK A/S)涂莶莞司酶DY(BSSDY)
Siezen()识丝莶莞司酶一椋琁-S2强莶莞司酶I-S2酶为强钥莶莞司酶莶莞司酶PB92(BAALKP)(MAXACAL_Gist-BrocadesNV)莶莞司酶309(BLSAVI)(SAVINASE_NOVO NORDISKA/S)莶莞司酶147(BLS147)(ESPERASE_NOVO NORDISKA/S)图缘缘酶YaB(BSEYAB)
妆莶莞司酶
铩白陛草杆久敢恢窼iezen(19911997)目莶莞司酶诮一细冢渭浴莶莞司酶妆莶莞司酶也谴然源目莶莞司酶校诒挚莶莞司酶缘戮一巍遥妆莶莞司酶通DNA榧计蹦枯草杆久 J.E.Ness耍然锛 Nature Biotechnology)17893-896(1999)
铩白陛草杆久肝巴枯草杆久
莶莞司酶
玫铩拔∥草杆久改谎г 莶莞司酶DNA械糯慰前取目臧贬处婊蝗笔 搿
莶莞司酶
诒校莶莞司酶突目莶莞司酶指杀突目莶莞司酶突源诤原始妆虿⒉应妆酶妆微铮毖吠 圆突突说斜时突莶莞司酶频兀突蚧箍DNA榧 妆
同源莶莞司酶
诒校职之同源貌同一浴
为确挚莶莞司酶之同一猿潭龋使同应()GCG9.1GAP朔募税卸员猓⊥辉百肥∧
荽说员丶说同源钥莶莞司酶应同源幕圆位强愿荼巍
亩
玫铩澳夺”指一志氪渴河 糯袒牡什系统式亩帷Ｐ┠酚 源然蟹耄癱DNA突隆约DNA实蚨ǖ殖(site-directed autogenesis)实亩帷 姆亩岵煌ㄖ的或，瞪 3未雍止印募员员说远准(纾糄ynanTijan然316774-781985)铩澳盒∥狣NA小隆暮小颉翱隆DNA小
牡
铩澳∮凳敝杆笛慈恍
选式校牡驶喜剩乇同源(同源省(渭))
通SDS-PAGE舛凳的慈 0选20选30选峁┩⊿DS-PAGE舛ㄈ 06080选95选99母叨却式牡省
铩澳凳∥凳
同源
铩巴词≈肝涸疵陛草杆久竿 源细(吮莶莞司酶之一)
......
囟微源氐铩 .....谩指枚/莶莞司酶囟源细囟源细巡械幕
玫氲懊改地碉“铩庇 式为一诒莶莞司酶水募幕铩
玫缘酶酶俜应牟泄氐铩帮”诒应为婕懊篙草杆久杆反应牟铩Ｋ反应械牡铩
洗
诒校铩跋窜∶赶从挥 谐去目系渭实施2械摹准洗蛹洗椤
式铩坝
PR莶莞司酶-R维斯
校P为樱R莶莞司酶为凇准洗蛹洗椤泵枯草杆久钢的 (reflectance)R维斯为凇准洗蛹洗椤 Savinase_之系姆一渭实施2械摹准洗蛹洗椤 图
图1示GAP锌莶莞司酶BPN陌(a)捅驴莶莞司酶陌之卸员取
诒牡一烁趣姆妫改较 目莶莞司酶一址SEQ ID NO2械1-269位示陌(目莶莞司酶)88同一缘目莶莞司酶诒一烁趣实施校目莶莞司酶SEQ ID NO2械1-269位示陌芯90919293949596979899同一(某为同源莶莞司酶)诒一烁趣实施校目莶莞司酶SEQ ID NO2械1-269位示陌伞
圆诘屎DNA员鹊全Smith-Waterman员确卸员群同一苑值恪Ｄ记分倔方稡LOSUM50同一跃蠓椒直诘屎DNA卸员取一械牡一谢姆侄诘识为-12DNA为-16一械谢姆侄缘识为-2DNA为-4员Fastav20u6姹 W.R.PearsonD.J.Lipman(1988)慕蟹(Improved Tools for Biological sequenceAnalysis)PNAS 852444-2448约W.RPearson(1990)FASTPFASTA目俸斜冉(Rapid and Sensitive sequenceComparison with FASTP and FASTA)酶学(Methods inEnzymology)18363-98)
通写卸员龋志SEQ 1D NO2陌械莶莞司酶投知目莶莞司酶陌之同一(园俜时示)
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)缘酶)US5,352,604
诒一烁趣实施校目莶莞司酶露耄囤迪嘎Ｑ⌒迪嘎 选诟叨细咏(i)SEQ ID NO1械1-807位示亩幕(ii)俸100(i)(J.SambrookE.F.FritschT.Maniatis1989涌隆实植幔 Molecular Cloning A Laboratory Manual2dedition)泉郏纽约
SEQ ID NO1械1-807位示亩幕片匣锌为100幔 00帷 遥应帽械水缘目莶莞司酶片稀丝莶莞酶也蔷械水缘目莶莞司酶牡位片稀
荼知姆越SEQ ID NO1亩谢片希约SEQ ID NO2陌谢片坪探约涂隆圆同值木摹械水缘莶莞司酶说探愿荼准Southern印趣值幕cDNA咏约头应幕颉４探囟校应为15选25选35岢ひ彩姑较程诫。DNARNA探使谩一探斜约应幕(32P3H35S鼗缀)探诒小
耍源票幕DNAcDNA目筛选探咏氡据草杆久窪NA通脂腔郾烯泳员知爰 幕DNADNA越些目DNADNA转苹吖潭维鼗说稀为思SEQ ID NO1同源目隆DNASouthern印为目模咏诘细应SEQ ID NO1示暮小浠バ的鼻的禾接絏弑膜些探咏姆印
诔为100某探耄肝 荼准Southern印425SSPE0.3SDS200g/ml泻捅缘憔獶NA约诘细25械细35诟细50薪预咏咏
诔为100某探耄 SSC0.2SDS选50(细)选55(械细)一选65(细)细洗危每15印
诔诖约15约70亩探耄 为荼准Southern印陆预咏洗雍咏诓BoltonMcCarthy(1962铱学院院(Proceedings of the National Academy of Sciences USA)481390)惴═m之5-10妫 .9M NaCl0.09M Tris-HCl pH7.66mM EDTA0.5NP-401Denhardt液1mM疲1mM一疲0.1mM ATP每0.2mg母RNA
诔诖约15约70亩毯幔 0.1SDS6SCC洗15樱洗一危使6SSC诩Tm之5-10洗危每15印
屑知谓频陌谢某谢谋取预诮酶小浠
斜I谐吮匕婊
I
匕婊
()K
H榘
()E劝
D於
Q劝
N於
水 L
I
V影
M
F
W色
Y野
小G拾
A
S丝
T瞻
耍诒一烁趣实施校SEQ IDNO2陌械目莶莞司酶一谢缺失/搿
乇兀屑知蔚慕峁┠矫 浴
屑舜胁同母慕实目莶莞司酶澹 目莶莞司酶诒牡摹峒 渭)
耍SEQ ID NO2陌一位么械为(BASBPN)273656768797101104120123167170222224235274
说为目莶莞司酶卤(BASBPN)K27R*36DS56PN76DS87NG97NR101GS103AV104AV104IV104NV104YH120DN123SY167R170M222SM222AT224SK235LT274A
遥为诨圆位(b)一影谢诖95位103位(BASBPN)一影谢璞灸枯草杆久赣迪窜≡Ｑ∥ 俨一影谢一影谢98位99位之洌 9位100位之洹
遥为SEQ ID NO2陌械目莶莞司酶呋学同一曰虿糠呋学同一缘姆目莶莞司酶选为式也诒姆围之凇知Ouchterlony双散通呓娣从︹定呋学省裕姆通(荻)票呋曰SEQ ID NO2陌械目莶莞司酶谋位系亩隆蹇寡HarboeIngildN.H.AxelsenJ.RollB.Weeks嗉涤局 A Manual of QuantitativeImmunoelectrophoresis)Blackwell Scientific Publication197323拢JohnstoneThorpe实呋学(Immunochemistry inPractice)Blackwell Science Publication1982(兀27-31页)呋学同一缘目莶莞司酶诓囟呋学时同姆式全诨同某态/同牡泳迁肟寡反应目莶莞司酶AxelsenBockKrollN.H.AxelsenJ.RollB.Weeks嗉涤局册，BlackwellScientificPublications197310斯呋学同一缘慕一汀胁呋学同一缘目莶莞司酶诓囟呋学时圆同姆式绮恐弛煌程 虿糠同牡泳迁肟寡反应牡目莶莞司酶N.H.AxelsenJ.RollB.Weeks嗉涤局册，BlackwellScientific Publication197311瞬呋学同一浴
寤骨德″。隆愿碌姆票使茫E.HarlowD.Lane嗉 988澹抵 AntibodiesA Laboratory Manual)泉鄢纾Ｅυ
逊SEQ ID NO2示陌械莶莞司酶幕颍氲襟肠杆綧T173莨食专微锉ｆ布斯约2000210战没拇蟪Ω司MT173瓯Ｚ抵疚保DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH(DSM)Mascheroder Weg 1BD-38124 Braunschweig鹿浔Ｘ何狣SM13306
诒烁趣实施校莶莞司酶煽隆诒睾为DSM13306拇蟪Ω司MT173片系亩目莶莞司酶氩恐蹦枯草杆久 0919293949596979899同一浴
目莶莞司酶殖越洗堋耍为使员-蟹-芄选效暮选诖目牡目莶莞司酶峁┮凰翟ら，杀员猿鄣目莶莞司酶堋
耍牡实施2椤白枷从枷 椤毖∧枯草杆久改剐т话说诮一挚莶司酶准洗蛹时系冉(时为Savinase_)谩准洗蛹洗椤臂该枯草杆久溉 准织系盏椋承狙 莶莞司酶去盏裕原选目莶莞司酶没斜殖Savinase_愿慕一悴灰 薪一椤
耍诒目目莶莞司酶一挚莶莞司酶凇准洗蛹洗椤壁奥成 谋准洗蛹时同Savinase_缺殖慕洗埽准洗蛹
6.2 LAS(Nansa 80S)
2 C16-C18脂
4 颖约(Plurafax LF404)
22石P
10.5 Na2CO3
4Na2Si2O5
2 燃谆维(CMC)
6.8 烯液CP5 40
20(式NaBO2.H2O2)
0.2 EDTA
21 Na2SO4
水(平)
通诒牡实施2卸谓印洗艿母慕卸
诒一浅烁趣实施校目莶莞司酶凇洗椤笔蔽 为1.5为2选为2.5为3为3.5乇为4为4.5为5
缘兀选目莶莞司酶水平希选械水平希选水平戏准
目莶莞司酶通准为夭裕通同莶莞司酶DNA榧 渭WO95/22625J.E.Ness耍然锛 7893-896(1999))
缘兀目莶莞司酶源然源蟹耄 目莶莞司酶细莶莞司酶细莶莞酶细莶莞司酶懈细莶莞司酶芽吒司模燃芽吒司(Bacillus alkalophilus)芽吒司(Bacillus amyloliquefaciens)芽吒司(Bacillus brevis)状芽吒司(Bacillus circulans)吒司(Bacillus coagulans)芽吒司(Bacillus lautus)倩芽吒司芽吒司(Bacilluslicheniformis)薮芽吒司(Bacillus megaterium)脂芽吒(Bacillus stearothermophilus)莶芽吒司(Bacillus subtilis)芽吒司(Bacillus thuringiensis)莶莞司酶霉莶莞司酶铅霉(Streptomyces lividans)霉(Streptomycesmurinus)莶莞司酶细莶莞司酶蟪Ω俚(俚sp)莶莞司酶
目莶莞司酶也为模选母莶莞司酶丝母(Candida)鲁维母(Kluyveromyces)铣母(Pichia)母(Saccharomyces)殖母(Schizosaccharomyces)Yarrowia莶莞司酶吒选丝状莶莞司酶枝霉(Acremonium)霉(Aspergiilus)瘫霉(Aureobasidium)母(Cryptococcus)吆诜劬(Filobasidium)(Fusarium)霉(Humicola)Magnaporthe毛霉(Mucor)丝霉(Myceliophthora)Neocallimastix霉(Neurospora)霉(Paecilomyces)霉(Penicillium)Piromyces(Schizophyllum)捉诰(Talaromyces)揖(Thermoascus)莞霉(Thielavia)Tolypocladium木霉(Trichoderma)莶莞司酶
一烁趣实施校莶莞司酶为辖母(Saccharomyces carlsbergensis)平母(Saccharomycescerevisiae)腔母(Saccharomyces diastaticus)Saccharomycesdouglasii鲁母(Saccharomyces kluyveri)诺亟母(Saccharomyces norbensis)墙母(Saccharomyces oviformis)莶莞司酶
一玫实施校莶莞司酶为霉(Aspergillusaculeatus)盛霉(Aspergillus awamori)霉(Aspergillusfoetidus)毡霉(Aspergillus japonicus)霉(Aspergillusnidulans)霉(Aspergillus niger)霉(Aspergillus oryzae)状(Fusarium bactridioides)Fusarium cerealisFusariumcrookwellense(Fusarium culmorum)坦(Fusariumgraminearum)坛(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缀毛霉(Mucor miehei)然丝霉(Myceliophthorathermophila)植霉(Neurospora crassa)霉(Penicilliumpurpurogenum)Trichoderma harzianum木霉(Trichodermakoningii)Trichoderma longibrachiatumTrichoderma reesei色木霉(Trichoderma viride)莶莞司酶
猓Ｍ沧刺 学同铮，知员识实同同一浴
些值木怨说一些锉Ｄ得的 锉 ATCC)志微锉 DSM)直(CBS)农业芯专锉Ｐ (NRRL)
遥圆探影然(戏稀水)微源屑么莶莞司酶诖然微募诒知摹频筛选一微幕cDNA目苫亩帷Ｒ惶解到莶莞司酶亩幔裁痹敝 隆(纾糞ambrook耍1989)
诳隆目莶莞司酶)约(莶莞司酶)亩址屑知模渭凇玫牟慰住
一说圆诳隆没(/蚨ǖ)谋准曰帽目莶莞司酶为私一说毡牡实施()(Sambrook耍(1989)涌隆实植幔狄ＨＥυ糀usubelF.M.(嗉 学路( Current protocols in Molecular Biology)John WileySons1995HarwoodC.R.CuttingS.M.(嗉 芽吒司姆学(Molecular Biological Methods forBacillus)John WileySons1990)WO96/34946
遥目莶莞司酶通准为夭裕通同莶莞司酶DNA榧 渭WO95/22625Stemmer WPC然370389-91(1994))缃 Savinase_幕实鸭一只植挚莶莞司酶薪DNA椋秆∧较窜横供目莶莞司酶
婕耙恢彪本莶莞司酶姆亩帷
一烁趣实施校SEQ ID NO1牡1-807位示亩88同一浴选兀硕SEQ ID NO1牡1-807位示亩8990919293949596979899同一浴一烁趣实施校SEQ ID NO1牡1-807位示亩帷④闺本莶莞司酶牡位片稀然硕SEQ ID NO1牡1-807位示亩伞
SEQ ID NO2陌械亩牡亩幔丛锻琒EQ ID NO1婕 械水缘SEQ ID NO2片系SEQ ID NO1小
SEQ ID NO1为SEQ ID NO1牡1-807位凇去5/3一亩帷
婕半本莶莞司酶姆亩幔 诘细拢选械细拢选诟细拢咏(i)SEQ ID NO1牡1-807位示亩幕(ii)俸100(i)小婕 (i)(ii)幕
诜隆牡亩募屑知模踊DNA蟹耄琧DNA票堑稀源些DNA隆亩幔ㄊ怪 酶应(PCR)目目筛选约泄同峁鼓 隆DNA片稀纾糏nnis耍1990PCR应指(PCRA Guide to Methods and Application)Academic Press纽约使暮酶应(LCR)蛹转录(LAT)突诤械(NASBA)
亩通糯玫谋准隆茫员憬喝晃宦段皇逛复频牟同位隆砂谐头牒草杆久改 片希片喜氲接Ｏ迪 细懈瞥枚亩隆硕椤DNARNA铣源铣源堑一稀
为目模之同一猿潭舛
氡灸枯草杆久改段囤合趁枯草 酶频目莶莞司酶潜摹铩捌的≥草杆 酶指然诘目莶莞司酶式些莶莞司酶某些品式峡然源玫目莶莞司酶同囟幕浴榷浴pH值确诘牟同濉Ｎ猄EQ ID NO1亩谋氩恐囤的赌幌剐 /通路取洳簧该盒 一挚莶莞司酶陌校应酶使茫取刹同陌小取一渭Ford耍1991实谋(Protein Expression and Purification)295-107
诒员说然取拥墓丶之胁圆钥莶莞司酶员姆亩亩牡幕要陌谢选洳蝗≡ 知姆缍ㄕ鄙ㄕ 纾 CunninghamWells1989学(Science)2441081-1085)诤一校诖朔拥每一傻牟谢突洌 拥牡水裕约朔踊缘墓丶谢-酶嗷ッ滴灰餐ㄎ构舛ㄎ构舜殴峋аё合鼻尖定(纾糳e Vos耍1992学255306-312Smith耍1992学志(Journal of Molecular Biology)224899-904Wlodaver耍1992FEBS Letters 30959-64)
峁
婕澳核藉，刹一只芄指说细诒牡印
远址式氡据草杆久改丰供莶莞司酶谋铩Ｚ仓霸妒翟和 取诒濉NA味募屑知摹
邪斜锉据草杆久 帧每一值卸诒莶莞司酶亩说然源摹些邪鹊小峄∏ 小印藕泻转录止印伲邪约转录头止藕拧为说峁┴ 位慕头俳说莶莞司酶亩印
锌说校为细识诒械亩帷Ｐ洪导莶莞司酶转录小涌选细诒殖转录缘魏味幔诲、囟毯雍樱细同源源谋细细诳莶莞司酶幕谩
指峁棺枷赶缸 说拥实为碌樱蟪Ω司遣印色霉(Streptomyces coelicolor)脂腔(dagA莶芽吒司酶(sacB)芽吒司-酶(amyL)脂芽吒司芽堑酶(amyM)芽吒司-酶(amyQ)芽吒司霉酶(penP)莶芽吒司xylAxylB原甩-酶(Villa-Kamaroff耍1978铱学院院753727-3731)约tac(DeBoer耍1983学院院8021-25)细玫(Useful proteins from recombinant bacteria)学(Scientific American)198024274-94Sambrook耍1989摹
指丝状细斜暮峁棺 说拥实为碌樱霉TAKA酶Rhiomucor miehei於蛋酶霉驭-酶霉-酶霉盛霉堑酶(glaA)Rhiomucor miehei脂酶霉缘酶霉旃姑该 酶图鹊酶酶(WO96/00787)约Na2-tpi(院霉驭-酶霉旃姑赣涤 )堑突濉⒇短河河
诮母校拥源幕平母烯酶(ENO-1)平母羌酶(GAL1)平母酶/醛-3-酶(ADH2/GAP)平母3-峒っ改 细Romanos耍1992母(Yeast 8423-488
谢一说转录止校为细识止转录小止锌刹拥莶莞司酶亩3末恕魏选细蟹庸艿止涌诒
选丝状细止拥源幕霉TAKA酶霉堑酶霉酶霉-酶图鹊酶酶
选慕母细止拥源幕平母烯酶平母细色C(CYC1)平母醛-3-酶Romanos耍1992模玫慕母细止印
谢说鹊校为细要mRNA欠鹊锌刹拥牡亩5末恕魏选细诜庸艿鹊锌诒
选丝状细鹊械源幕霉TAKA酶凸霉旃姑
说慕母细鹊械源幕平母烯酶(ENO-1)平母3-峒っ钙侥 雍平母酶/醛-3-酶(ADH2/GAP)
谢蔷峄Ｐ可灿 3末转录时细识为一纸詹谢转录mRNA藕诺小魏选细蟹庸木峄口
选丝状细木峄翟 霉TAKA酶霉堑酶霉酶鹊酶酶秃霉-酶
母细玫木峄疓uoSherman1995细学(Molecular Cellular Biology)155983-5990
牡谢藕谋拥莶莞司酶陌末说陌胁指目莶莞司酶细途谋械5吮园一然诰诘目莶莞司酶谋诙蔚姆系藕谋选兀械5丝园吮械源藕谋谋胁然一藕谋时要源藕谋选兀然藕谋源藕婊辉促捷草杆久改汾∥何恐改枯 司酶选细姆途藕谋诒
细细效藕谋堑源幕藕谋芽吒司NCIB11837芽堑酶脂芽吒司-酶芽吒司莶莞司酶芽吒司-酶脂芽吒司缘酶(nprTnprSnprM)涂莶芽吒司prsA藕SimonenPalva1993微学(Microbiological Reviews)57109-137
丝状细效藕谋堑禄藕霉TAKA酶霉缘酶霉堑酶Rhizomucor miehei於蛋酶Humicola insolens维酶Humicola lanuginosa脂酶
母细藕牡源幕平母-平母转酶Romanos耍1992模玫谋
谢潜位诳莶莞司酶末说陌械前谋玫亩知一酶原前(某些为酶原)前一没谢裕通呋懈呋懈前牡前亩转为幕远摹前谋缘源幕颍嚎莶芽吒司缘酶(aprE)莶芽吒司酶(nprT)平母-印Rhizomucor miehei於蛋酶然丝霉酶(WO95/33836)
诳莶莞司酶陌末舜藕暮前时前莶莞司酶陌末冢藕前陌末凇
细氐亩牡谋械诘牡也摹些系统实曰学碳(诘诨)应使没谋锉回闭迪低吃 统械牡系统lactactrp系统诮母校使ADH2系统GAL1系统丝状校TAKA-酶印霉堑酶雍霉堑酶涌小械实为牡小系统校诩装蚀亩叶峄乖肛 虻鞍谆些拢牡亩刹印
婕澳横构濉⒂鹤茧翻止藕诺濉
氡久改横构魏慰煞亟DNA濉
选一取细耍澹旧第靛，母贫染色母疲选兀诘细螅只全细椋将辖去染色一啤
选为谋澹ｋ本酶DNA锌刹DNA转录慕诙印一说虿《DNA院叩元铩吧 印指诙使浒辞翟つ磕酚管的肥 校转录通酶DNA卸前小
涌魏选细诒殖转录DNA校缘员细同源源牡时
细细说拥实禄樱脂芽吒司芽堑酶颉⒌芽吒司-酶颉⒔芽吒司-酶颉⒖莶芽吒司缘酶小芽司(Bacillus pumilus)木酶删-PRPL踊司lactrptac印
要氡久窪NA谢钥刹拥止稀
寤股败故观迪 懈频DNA小
寤乖把∧鼻Ｖ蚕溉 幕颍钥霉亍霉亍霉亍幕亍壮霉氐鹊目裕亟菁目缘幕
为指酶细姆途峁┡ 也知为鹊小前原谢前)越藕确拥酶DNA小藕一位诒酶DNA械5恕藕锌通酶氐幕员一址诘实幕
诜直颖氡久窪NA小雍选止/藕校通说PCR些泻徒遣懈苹息说姆员员说知(渭纾琒ambrook耍)
细
婕搬构细
氲较感的彪本酶DNA锌鄣同源源细同源筛细然氡久窪NA一刹然写诘一樱诳应玫一藕泻/止印同源指然鄣械酶DNA小铩霸茨≈ 细斜聿幻窪NA小DNA锌源一呛铣傻小
氡綝NA细魏芄酶细细母透叩细植铩
通芄酶细细实歉细芽吒司辏惭窟杆 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)状芽吒司(B.circulans)芽吒司(B.lautus)薮芽吒司(B.megaterium)平芽吒司(B.thuringiensis)乇浅倩芽吒司霉辏γ S.lividans)霉(S.murinus)细蟪Ω司
细转通知姆式原转绱┳ 匣使酶态细(渭Sambrook耍)
细蟪Ω司斜酶时酶一苑芙钥驻细(知)细始隙前一拢细呀猓Ｍㄏ 捅约使酶鄣一拢酶通虏眨透压菘苹细使始隙头牛倩酶
诟细芽吒司霉斜酶时酶驻细校细小一拢酶砂谢铡
诒一实施校细墙母细玫摹母医母(霉目(Endomycetales))咏母诎知(Fungi Imperfecti)(芽吒(Blastomycetes))慕母诮母锌芊浠 目模母应母学突(Biology andActivities of Yeast)(SkinnerF.A.PassmoreS.M.DavenportR.R.嗉璖oc.App.Bacteriol.Symposium Series No.91980)慕卸濉
一选实施校慕母细羌丝母(Candida)逊母(Hansenula)鲁维母(Kluyveromyces)铣母(Pichia)啤平母(母)殖母(Schizosaccharomyces)Yarrowia细
一选实施校慕母细强斯墙母(Saccharomyces carlsbergensis)平母腔母(Saccharomyces diastaticus)Saccharomyces douglasii鲁母诺亟母墙母细
一选实施校慕母细鲁维母(Kluyveromyces lactis)细一选实施校慕母细Yarrowia lipolytica细
一选实施校细丝状细丝状械(Eumycota)藕丫(Oomycota)诺丝状式(Hawksworth耍1995模)丝状园省维亍暇恰蔷恰露呛隙堑木丝为
营通丝映械模碳谢摹喾茨 平母营通细芽械模碳谢欠偷摹
一选实施式校丝状细堑预霉霉霉毛霉丝霉霉霉莞霉Tolypocladium木霉细
一选实施式校丝状细盛霉霉毡霉霉霉霉细一选实施式校丝状细歉状摺FusariumcerealisFusarium crookwellense摺坦摺坛摺摺匣木摺摺枝摺玫色摺庸木摺色摺枝摺色摺Fusarium torulosumFusariumtrichothecioidesFusarium venenatum细一选实施式丝状妆细Fusarium venenatum(Nirenberg sp.nov.)细
一选实施式校丝状细HumicolainsolensHumicola lanuginosa缀毛霉(Mucor miehei)然丝霉植霉(Neurospora crassa)霉Trichodermaharzianum木霉Trichoderma longibrachiatumTrichodermareesei色木霉(Trichoderma viride)细
细通一转目前知姆式纬原濉⒆并细凇屎转霉细姆EP238023Yelton耍1984铱学院院811470-1474转霉姆Malardier耍1989(Gene)78147-156WO96/00787母转姆校BeckerGuarenteAbelsonJ.N.SimonM.I.嗉概囱 学指(Guide to Yeast Genetics and Molecular Biology)酶学194恚 82-187页Academic PressInc.纽约Ito耍1983细学志(Journal of Bacteriology)153163Hinnen耍1978铱学院院751920
目莶莞司酶姆
一婕澳枯草杆久改访
a)莶莞司酶细
b)目莶莞司酶
酶DNA械谋转源细时使母酶源
纱丝圆同源为牡母叨却目莶莞司酶铩
校同源指源帽酶同源细(酶之)
转细魏屎鄣细某目莶莞司酶煞胤诘校然通知姆眨通幕舜蟹细铵承的抵 胁咏缀筒取
目莶莞司酶途
目莶莞司酶卸止业途洗蛹业小吮婕懊赶从硷，选酶洗禄洗铩
莶莞司酶洗蛹铮
一说洗蛹诒院实洗蛹慕一圆渭WO96/34946WO97/07202WO95/30011
舜实目莶莞司酶殖越洗堋
洗蛹
酶拥洗蛹卸为械帧
纾从计诚聪聪从 铮と局从计从 维砘，瞥通揖硬洗蛹铮 瞥洗洗洗滩
一囟姆妫供一职目莶莞司酶洗蛹洗蛹洗蛹园一只酶一值酶脂酶酶酶酶维酶酶露酶酶酶木酶酶酶/酶
一说选酶应选洗蛹应(pH-耍酶酶志缘)酶应效凇
酶实牡酶远铩⒅诧、微牡酶源微牡酶选摹也学位虻鞍坠袒突濉Ｄ得杆酷蛋酶酶选微锏懊溉得该冈得甘滴草杆久 乇芽吒司目莶莞司酶纾草杆久窷ovo莶莞司酶Carlsberg莶莞司酶309莶莞司酶147涂莶莞司酶168(WO89/06279)鹊酶酶实为鹊酶(牛)WO89/06270WO94/25583霉牡酶
玫牡酶实WO92/19729WO98/20115WO98/20116WO98/34946谋澹币晃 婊荒卞273657768797101104120123167170194206218222224235274
选目坦牡酶AlcalaseTMSavinaseTMPrimaseTMDuralaseTMEsperaseTMKannaseTM(Novo Nordisk A/S)MaxataseTMMaxacalTMMaxapemTMProperaseTMPurafectTMPurafect OxPTMFN2TMFN3TM(Genencor International Inc.)
脂酶实脂酶些源细脂酶也学位虻鞍坠袒突濉Ｃ抵甘礒P258068EP305216愿霉(Thermomyces之同)H.lanuginosa(T.lanuginosus)脂酶WO96/13580H.Insolens脂酶俚脂酶圆俚(P.Alcaligenes)俚(P.Pseudoalcaligenes)(EP218272)屑俚(P.Cepacia)(EP331376)施霞俚(P.stutzeri)(GB1,372,034)荧俚(P.Fluorescens)俚SD705(WO95/06720WO96/27002)P.wisconsinensis(WO96/12012)芽吒司脂酶钥莶芽司(Dartois(1993)锘аа Biochemica etBiophysica Acta)1131253-360)脂芽吒司(JP64/744992)小芽吒司(B.Pumilus)(WO91/16422)
实为WO92/05249WO94/01541EP407225EP260105WO95/35381WO96/00292WO95/30744WO94/25578WO95/14783WO95/22615WO97/04079WO97/07202脂酶濉
选目坦脂酶LipolaseTMLipolase UltraTM(NovoNordisk A/S)
酶实牡酶(/)些源细牡酶也学位虻鞍坠袒突濉Ｃ冈 芽吒司摩-酶缘芽吒司囟辏 渭GB1,296,839
玫牡酶实WO94/02597WO94/18314WO96/23873WO97/43424谋澹┮ 位梅婊荒卞1523105106124128133154156181188190197202208209243264304305391408444
坦牡酶DuramylTMTermamylTMFungamylTMBANTM(Novo Nordisk A/S)RapidaseTMPurastarTM(GenencorInternational Inc.)
维酶实维酶源细维酶也渚或蛋白雇诲。实维酶芽吒俚霉霉莞霉枝霉维酶US4,435,307US5,648,263US5,691178US5,776,757WO89/09259泄Humicola insolens然丝霉卟维酶
乇实维酶蔷色频募曰维酶维酶实为EP0495257EP0531372WO96/11262WO96/29397WO98/08940维酶实为些WO94/07998EP0531315US5,457,046US5,686,593US5,763,254WO95/24471WO98/12307PCT/DK98/00299维酶濉
坦维酶CelluzymeTMCarezymeTM(Novo NordiskA/S)ClazinaseTMPuradax HATM(Genencor International Inc.)KAC-500(B)TM(Kao Corporation)
酶/酶实墓酶/酶些源植铩⑾改姑 酶也学位虻鞍坠袒突濉Ｃ的姑甘礧O93/24618WO95/10602WO98/15257怨伞(Coprinus)(曰腋枪伞(C.cinereus))墓酶
坦墓酶GuardzymeTM(Novo Nordisk A/S)
洗蛹酶通雍一只酶亩蛹通雍些酶蛹洗蛹小洗蛹蛹也嵌蛹蛹瞥状液濉⒆慈等⊙∠从加际轿 乇欠欠末)液(乇榷液)蚪
欠末US4,106,9914,661,452选知姆状系实平为100020000木()(叶PEG)1650榈ノ簧挥Ｖ 220碳原写1580榈ノ恢；脂牡--GB1483591懈屎通应玫某膜系实液酶萍殖傻姆通佣元腔谴榷鼙酶EP238,216姆票
洗蛹魏畏式状片状末粘呕液濉Ｒ合从记核Ｒ汇含叽70水0-30谢芗虿缓水摹
洗蛹一一只直约强颖约爰的 雍/雍/颖约谋约一占0.160洗蛹时通约1约40颖约直-烯(脂)岽 腔脂-烯怼
洗蛹时通约0.2约40姆约脂苫印铡谆铩⒅单掖脂岬ヒ 脂前N-N-()
洗蛹园0-65洗蛹霞石巍巍巍碳巍巍蔚帷 帷⒁会、-烯帷⒃ 位式(绻篐oechstSKS-6)
洗蛹园一只志酆铩Ｊ滴甲晃 亍(烯酮)(叶)(烯)(烯-N-)(烯)郾烯/烯峁 图谆烯鹿/烯峁诧。
洗蛹院锌馨H2O2源漂系纬晒漂谆罨段 墓位碳巍选兀漂系园前墓帷
洗蛹械酶通碌某榷元腔谴帷 虮交4-幔 票为WO92/19709WO92/19708铩
洗蛹园某洗蛹煞维诩菁沫萍蚀俪染稀杀学准芗失萍稀
目前为洗蛹魏酶乇潜酶嗟泵肯匆盒 .01-100mg酶剩选每洗液0.05-5mg酶剩乇选每洗液芯0.1-1mg酶实搿
酶拥WO97/07202洗蛹浞叫 氡疚
通实员薪细说些实为员蟊；围魏啤
洗蛹校写煞值拢LAS 直C12TAS 牛XYASClx-ClySS 式2-砘 25EY平为Y摩幕系要为C12-C15
直45EY平为Y摩幕系要为C14-C15
直XYEZS 每摩平Z摩幕系Clx-Cly
樱 平潭3.8平潭为4.5
C13-C15系/脂
BASF GmbH鄢品为Plurafax LF404CFAAC12-C14N-谆TFAAC16-C18N-谆危 薅凸(SiO2Na2O剩2.0)NaSKS-6 式为-Na2Si2O5木状碳危 水碳危 MA/AA 14/烯峁诧，平约为80,000郾烯 平为8,000木郾烯铮珺ASF
GmbH郏品为PA30石A 小1-10微追围诘姆式为
Na12(AlO2SiO2)1227H2O水瞎危水Citric 危水一水漂准式为
NaBO2H2O2PB4 水水碳危式为2Na2CO33H2O2水碳漂准TAED叶CMC 燃谆维DETPMP 一(羌谆)Monsanto郏
品为Dequest 2060PVP 烯酮酆EDDS式叶-NN-[SS]旃 菁 25石鄣露50妫 7水石
58石状菁 12/石18十榇 0状危 水HMWPEO 叻刍TAE 25 牛痛(25)
洗蛹实I
状维票
直C12 6.515.0石A 26.0蔚5.0酶0.1PVP 0.5TAED 3.0 4.0 18.0踊0.1要 100洗蛹实II芸维(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
状菁3.5
水/要 100
洗蛹实III
亩洗硬色织乇玫状织票
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 -
踊 0.2 -
水/要100
洗蛹实IV
木小洗庸械砘”状织票45AS- 10.0LAS 7.6 -68AS1.3 -45E74.0 -25E3- 5.0椰--谆 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状菁 1.0 4.0CMC 0.2 0.1
水/要 100
洗蛹实V
强液织票式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-谆腔一然-3.0 -5.0PVP 2.0 -水/要 100
末远洗I
末远洗II
末远洗III
末远洗IV
末远洗V
末液木洗颖约系统洗VI
水液态远洗VII
水液态洗VIII
液远洗IX
液远洗X
漂卓液远洗XI
XIIIIIIIIIVVIX远洗铮 晒碳巍
XIIII-VI远洗铮还檀呋檀呋纾≮灯的感催 Efficientmanganese catalysts for low-temperature bleaching)然(1994)369637-639幕械一帧
虾头
水
诒械水Kilo NOVO酶位(KNPU)示摹幕酶准品(SAVINASE_)确牟舛ㄇ悔弊悸邓冈蹲灰 DMC)液械模谋准为50妫琾H8.3应时9樱时3印要时Novo Nordisk A/SDenmark取AF220/1募校募邪诖为慰
GU歉拾岬ノ晃冈Ｚ弊悸-业为4015樱嗟 mmole拾NH2-
也PNA脂学学志(Journal ofAmerican Oil Chemists Society)RothgebT.M.GoodlanderB.D.GarrisonP.H.SmithL.A.(1988)牡-----臃应舛冈
实施1-目莶莞司酶墓捅
SEQ ID NO2示械目莶莞司酶位pJRoC112pKH400(WO98/41623)浅啤诒目莶莞司酶同模些械母悖关吭礳at莶莞司酶转录始雍Savinase_前/原一碌摹莞司酶虿糠执
一诖蟪Ω司涂莶芽吒司懈啤诳莶芽吒司筛锉灸枯草杆久该得改吠和
PKH400通pJS3(J.Schi_dt耍屎通讯(Protein and Peptide Letters)339-44(1996)暮莶司酶309(Savinase_)暮铣苫拇蟪Ω司-莶芽吒司)牡18413730位BamHI位摹
幕强隆pzero-2(InvitrogenGroningenTheNetherlands)小SEQ ID NO2示械目莶莞司酶全约1240bp PmeI-BamHI片拥pZero-2希酶BamHI-EcoRV踊转态拇司细通PCR转证欠诓片危员莶莞司酶一片尾纸胁为pTVB3642000210毡DSMZ睾为DSM13306
莶莞司酶前械模30转台(300转/)煤100ml BPX500ml式锥瓶5臁
耍为嘶220锥瓶同时汀
BPX煞(每)
100g
磨拇 50g
蠖狗 20g
Na2HPO4x12H2O 9g
(Pluronic) 0.1g
业 10g
械牡忙-酶液模120录45NaHCO30.1MpH诘9
一婕把窟杆鞠感材蹦枯草 酶2液
1毡诮约1.65000转/35印10拇液使pH值为6.5Seitz Supra S100坦恕
浔窤micon SLY10 UF筒Amicon CH2A UF装媒顺液浓约400ml酶司缀pH7前冉UF浓牟鹿恕莶莞司酶诤0.01M谆幔 .1M0.002M然啤训pH为7幕液械251M然拼痈司洗选
筛司拇玫木械酶缘募趾喜应0.01M谆幔 .2M0.002M然频pH6.5液平750ml Sephadex G25(直5cm)
Sephadex G25玫木械水缘募趾喜应煤0.01M谆幔 .2M0.002M然频pH6.5幕液平150ml CM Sepharose CL 6B咏(直5cm)
酶2同液械0-0.1M荻然洗选
拇校CM Sepharose玫暮械酶募趾喜装GR81PP膜(Danish Sugar Factories Inc.)Amicon顺浓
应头图约氩借，SEQ ID NO2示械驴莶莞司酶
实施2-准洗蛹洗椤
为莶莞司酶诒准洗蛹械洗埽实吮准洗实椋
洗蛹准洗蛹
洗蛹 4.0g/l
pH 10.1
洗时20
露龋 30
水硬龋15dH
酶浓龋10nm(洗蛹液)
系统 10ml毡徒
织/ 5织(_2.5cm)/50ml洗蛹液
圆希 WFK10N()
谋准洗蛹拢
6.2 LAS(Nansa 80S)
2 C16-C18脂
4 颖约(Plurafax LF404)
22石P
10.5 Na2CO3
4Na2Si2O5
2燃谆维(CMC)
6.8 烯液CP5 40
20 (式NaBO2H2O2)
0.2 EDTA
21 Na2SO4
水 (平)
通系统HC1NaOH洗蛹液pH诘10.1通CaCl2MgC12(Ca2+Mg2+41)水硬鹊诘15dH洗雍织水洗诳懈铩
戏(R莶莞司酶)牟舛∕acbeth ColorEye 7000photometer(MacbethDivision of Kollmorgen InstrumentsCorporation鹿)460nm陆械摹舛ㄌ的 小
为确瞻值瞬酶洗实椤 (R瞻)牟舛
然胁慰实椋测定Savinase_洗堋舛ㄋ Rsavinase)
洗酶式(P)模
P(R莶莞司酶-R瞻)-(Rsavinase-R瞻)
R莶莞司酶-Rsavinase
应实榉
杉Savinase_龋目莶莞司酶毡殖频洗堋
实施3-目莶莞司酶远(ADW)械
目莶莞司酶远(ADW)械通诳募洗(Somat TurboHenkel Waschmittel GmbH)薪摹玫堑/牛蹋獭一税食品压铩
洗蛹 Somat Turbo
洗蛹4.0g/l
pH10.7(实)
水硬龋 3dH(咏)
露龋55
酶浓龋 20nM40nM莼洗水
榉 #160; 诟系牡/牛
Cylinda Compact
洗映4预洗
220ml全脂牛
15械却小
蹋直18cm
Somat Turbo洗微炉85录5使械酶失睢
染
220ml全脂牛15Braun UK 20 kitchen machine2樱撕螅探没使染
戏赂夜120录45使牡时浴
ADW实
每一实校10没芯Cylinda Compact machine预洗洗(4)猓 0獭44璞 6椎
遥50g压嘟
嘟 5.43)小(4.38)(4.32)(4.32)(4.32)(8.76)全脂牛(8.76)(8.76)呀(3.00)饨 2.19)末(4.00)(0.73)水(3mM Ca2++Mg2+)(36.71)
舛ㄍ
墓夥粗 R-值)Minolta Chroma Meter(秃牛CR-300)械同位舛≡干 Rclean)群(Rsoiled)洗(Rafter wash)胁舛
式去牡膜(RPF)
RPF100X(Rafter wash-Rsoiled)/(Rclean-Rsoiled)
榉 示准偏)远准酶Savinase_染越堋
系谋
细莶斯约墓娑ㄚ抵疚保Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH(DSM)Mascheroder Weg 1BD-38124 Braunschweig鹿睾牛
Novo Nordisk A/S票撕转NovozymesA/S
斜<110> 诺酶煞薰司<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权利要求
1.一挚莶莞司酶选裕
a)SEQ ID NO2牡1-269位示陌芯88同一缘目莶莞司酶
b)诘细咏亩目莶莞司酶
i)SEQ ID NO1牡1-807位示亩幕
ii)100(i)校
c)煽隆诖蟪Ω司MT173 DSM13306械片系目莶莞司酶氩恐蹦枯草杆久改枯 司酶88同一缘谋濉
2.权要1目莶莞司酶校诒牡摹准洗蛹洗椤蹦奥成分的弊枷从 时
6.2LAS(Nansa 80S)
2C16-C18脂
4颖约(Plurafax LF404)
22石P
10.5Na2CO3
4Na2Si2O5
2燃谆维(CMC)
6.8烯液CP5 40
20(式NaBO2H2O2)
0.2EDTA
21Na2SO4
水(平)丝莶莞司酶同Savinase_缺殖慕洗堋
3.权要2目莶莞司酶斜为1选为2选为3乇为4为5
4.权要1-3一目莶莞司酶莶莞司酶械陌SEQ ID NO2牡1-269位示陌芯90919293949596979899同一浴
5.权要4目莶莞司酶莶莞司酶SEQ IDNO2牡1-269位示陌伞
6.权要1-3一目莶莞司酶莶莞司酶械陌隆诖蟪Ω司MT173 DSM13306械片系亩目莶莞司酶氩恐蹦枯草杆久 90919293949596979899同一浴
7.权要6目莶莞司酶莶莞司酶煽隆诖蟪Ω司MT173 DSM13306械片系亩目莶莞司酶氩恐蹦枯草杆久干
8.一权要目莶莞司酶目莶司酶为SEQ ID NO2牡1-269位示摹一谢婊蝗笔 陌械目莶莞司酶谋濉
9.权要8目莶莞司酶莶莞司酶一位273656768797101104120123167170222224235274(BASBPN)之一巍
10.权要9目莶莞司酶选K27R*36DS56PN76DS87NG97NR101GS103AV104AV104IV104NV104YH120DN123SY167XR170XM222SM222AT224SK235LT274A(BASBPN)
11.权要一目莶莞司酶莶莞酶械细拢选诟叨细咏亩耄
i)SEQ ID NO1牡1-807位示亩幕
ii)100(i)片匣小
12.一址亩幔ㄒ -11一目莶莞司酶亩帷
13.一直莶莞司酶姆亩幔
a)SEQ ID NO1牡1-807位示亩88同一缘亩幔
b)诘细咏亩目莶莞司酶
i)SEQ ID NO1牡1-807位示亩幕
ii)100(i)校
c)隆诖蟪Ω司MT173 DSM13306械系亩目莶莞司酶氩恐Ｄ盒 8同一谋濉
14.权要13亩幔的 SEQ ID NO1牡1-807位示亩8990919293949596979899同一浴
15.权要13亩幔的 隆诖蟪Ω司MT173 DSM13306械系亩目莶莞司酶氩恐 990919293949596979899同一浴
16.一趾峁瑰，刹一只芄指莶莞酶说诒牡拥权要12-15一暮小
17.一澹ㄒ 6暮峁瑰、约转录头止藕拧
18.一细权要16暮峁瑰。
19.权要18细细为细选为芽吒司乇浅倩芽吒司
20.权要18细细为母选为丝状乇霉
21.一权要1-11一目莶莞司酶姆
(a)莶莞司酶权要18-20一细
(b)湛莶莞司酶
22.一洗蛹铮∥绰幌达，权要1-11一目莶莞司酶
23.权要22铮还维酶脂酶酶原酶酶酶堑幕铩
24.权要1-11一目莶莞司酶洗蛹械应谩
25.权要1-11一目莶莞司酶诔去途
26.权要22-23一洗蛹去盏途
27.一洗硬姆使硬权要22-23哟
28.一殖去硬盏姆使盏硬盏权要22-23哟
全文摘要
具有改进的卵污渍洗涤性能的新枯草杆菌酶。这些枯草杆菌酶在用于诸如清洁或洗涤剂组合物，如洗衣组合物和洗盘组合物，包括自动洗盘组合物中时，表现出优良的或改进的卵污渍洗涤性能。
文档编号C12R1/07GK1418249SQ0180653
公开日2003年5月14日 申请日期2001年3月12日 优先权日2000年3月14日
发明者F·米克尔森, T·S·法诺, M·N·马德森, L·B·汉森, P·K·汉森 申请人:诺和酶股份有限公司