專利名稱：具有改進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
婕懊枯草桿久改紡奪、峁瑰、濉⑺橫構細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澹珼URAZYM_(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US侔專34606(GENENCOR))EP214435(HENKEL)WO87/04461(AMGEN)WO87/05050(GENEX)EP260105(GENENCOR)ThomasRussellFersht(1985)然(Nature)318375-376ThomasRussellFersht(1987)學志(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 詿墓業應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，婕耙恢搬構濉⒃ 轉錄頭止藕諾濉
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
(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
原始 位
原始婊輝扳氨時只指位茫紓 70
原始/婊荒霸耙幌敵 時選陌示冢
原始 位{婊荒 1...婊荒 n}
囟謀澹關兌荒鴿，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⑨、絲瞻婊 170位木示為
Argl70{GlyAlaSerThr}R170{GAST}員示
R170GR170AR170SR170T
缺失
195位拾缺失示為
Glyl95*G195*
應兀一習謝缺失195196位拾缺失示為
Glyl95*+Leul96*G195*+L196*
耄
陌謝牟耄珿195示為
Glyl95GlyLysG195GK
擼一系陌謝G195LysAla時植示為
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
詰陌謝同陌謝時然諧思蠆ⅰ系實施校詬拾之拾幔 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
些傭員員說遠準模冶示G195GG筒應示指同募蠆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}謀匕乇亍一細渭幀
酶
紙獾笆敵得肝 苫)酶(Walsh1979酶應(Enzymatic Reaction Mechanisms).W.H.Freeman and CompanySan Francisco3)
位/謝謀
沒指玫陌哦應誑薟莞司酶BPN(BASBPN)械陌擰BPN械細渭圖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)細學(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(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絲岬懊崗窼iezen為薟莞司酶一欏Ｑ妓茨枯草桿久稈測定一些薟莞司酶小源薟莞司酶滸斃的趕肝 Siezen(1997)
薟莞司酶一椋琁-S1薟莞司酶準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()識絲薟莞司酶一椋琁-S2強薟莞司酶I-S2酶為強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應妝酶妝微錚毖吠 圓突突說斜時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()GCG9.1GAP朔募稅卸員猓⊥輝百肥∧
荽說員丶說同源鑰薟莞司酶應同源幕圓位強愿荼巍
畝
玫鎩澳奪”指一志氪渴河 糯袒牡什系統式畝帷Ｐ┠酚 源然蟹耄癱DNA突隆約DNA實蚨ǖ殖(site-directed autogenesis)實畝帷 姆畝岵煌ㄖ的或，瞪 3未雍止印募員員說遠準(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(吮薟莞司酶之一)
......
囟微源氐鎩 .....謾指枚/薟莞司酶囟源細囟源細巡械幕
玫氳懊改地碉“鎩庇 式為一詒薟莞司酶水募幕鎩
玫緣酶酶俜應牟泄氐鎩幫”詒應為婕懊篙草桿久桿反應牟鎩Ｋ反應械牡鎩
洗
詒校鎩跋竄∶趕從揮 諧去目系渭實施2械摹準洗蛹洗欏
式鎩壩
PR薟莞司酶-R維斯
校P為櫻R薟莞司酶為凇準洗蛹洗欏泵枯草桿久鋼的 (reflectance)R維斯為凇準洗蛹洗欏 Savinase_之系姆一渭實施2械摹準洗蛹洗欏 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為-12DNA為-16一械謝姆侄緣識為-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(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
詒一爍趣實施校目薟莞司酶露耄囤迪嘎Ｑ⌒迪嘎 選詬叨細詠(i)SEQ ID NO1械1-807位示畝幕(ii)俸100(i)(J.SambrookE.F.FritschT.Maniatis1989涌隆實植幔 Molecular Cloning A Laboratory Manual2dedition)泉郟紐約
SEQ ID NO1械1-807位示畝幕片匣鋅為100幔 00帷 遙應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Southern印趣值幕cDNA詠約頭應幕頡４探囟校應為15選25選35岢ひ彩姑較程誡。DNARNA探使謾一探斜約應幕(32P3H35S鼗綴)探詒小
耍源票幕DNAcDNA目篩選探詠氡據草桿久窪NA通脂腔郾烯泳員知爰 幕DNADNA越些目DNADNA轉蘋吖潭維鼗說稀為思SEQ ID NO1同源目隆DNASouthern印為目模詠詰細應SEQ ID NO1示暮小浠バ的鼻的禾接絏弒膜些探詠姆印
誄為100某探耄肝 荼準Southern印425SSPE0.3SDS200g/ml瀉捅緣憔獶NA約詰細25械細35詬細50薪預詠詠
誄為100某探耄 SSC0.2SDS選50(細)選55(械細)一選65(細)細洗危每15印
誄詿約15約70畝探耄 為荼準Southern印陸預詠洗雍詠誆BoltonMcCarthy(1962銥學院院(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
屑舜脅同母慕實目薟莞司酶澹 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同一曰蠆糠呋學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實呋學(Immunochemistry inPractice)Blackwell Science Publication1982(兀27-31頁)呋學同一緣目薟莞司酶誆囟呋學時同姆式全諢同某態/同牡泳遷肟寡反應目薟莞司酶AxelsenBockKrollN.H.AxelsenJ.RollB.Weeks嗉滌局冊，BlackwellScientificPublications197310斯呋學同一緣慕一汀脅呋學同一緣目薟莞司酶誆囟呋學時圓同姆式綺恐弛煌程 蠆糠同牡泳遷肟寡反應牡目薟莞司酶N.H.AxelsenJ.RollB.Weeks嗉滌局冊，BlackwellScientific Publication197311瞬呋學同一浴
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戰沒拇蟪Ω司MT173甌Ｚ抵疚保DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH(DSM)Mascheroder Weg 1BD-38124 Braunschweig鹿潯Ｘ何狣SM13306
詒爍趣實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洗蛹時系冉(時為Savinase_)謾準洗蛹洗欏臂該枯草桿久溉 準織系盞椋承狙 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洗蛹洗欏壁奧成 謀準洗蛹時同Savinase_缺殖慕洗埽準洗蛹
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)
通詒牡實施2卸謂印洗艿母慕卸
詒一淺爍趣實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
目薟莞司酶通準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 目薟莞司酶細薟莞司酶細薟莞酶細薟莞司酶懈細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
猓Ｍ滄刺 學同錚，知員識實同同一浴
些值木怨說一些銼Ｄ得的 銼 ATCC)志微銼 DSM)直(CBS)農業芯專銼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目苫畝帷Ｒ惶解到薟莞司酶畝幔裁痹敝 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)謀準曰帽目薟莞司酶為私一說氈牡實施()(Sambrook耍(1989)涌隆實植幔狄ＨＥυ糀usubelF.M.(嗉 學路( Current protocols in Molecular Biology)John WileySons1995HarwoodC.R.CuttingS.M.(嗉 芽吒司姆學(Molecular Biological Methods forBacillus)John WileySons1990)WO96/34946
遙目薟莞司酶通準為夭裕通同薟莞司酶DNA榧 渭WO95/22625Stemmer WPC然370389-91(1994))緗 Savinase_幕實鴨一只植摯薟莞司酶薪DNA椋稈∧較竄橫供目薟莞司酶
婕耙恢彪本薟莞司酶姆畝帷
一爍趣實施校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 NO2陌械畝牡畝幔叢鍛琒EQ 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 詰細攏選械細攏選詬細攏詠(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蟹耄琧DNA票塹稀源些DNA隆畝幔ㄊ怪 酶應(PCR)目目篩選約泄同峁鼓 隆DNA片稀紓糏nnis耍1990PCR應指(PCRA Guide to Methods and Application)Academic Press紐約使暮酶應(LCR)蛹轉錄(LAT)突諍械(NASBA)
畝通糯玫謀準隆茫員憬喝晃宦段皇逛復頻牟同位隆砂諧頭牒草桿久改 片希片喜氳接Ｏ迪 細懈瞥枚畝隆碩欏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值確詰牟同濉Ｎ猄EQ 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酶使茫取剎同陌小取一渭Ford耍1991實謀(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(Science)2441081-1085)諍一校詿朔擁每一傻牟謝突洌 擁牡水裕約朔踴緣墓丶謝-酶嗷ッ滴灰餐ㄎ構舛ㄎ構舜毆峋аё合鼻尖定(紓糳e Vos耍1992學255306-312Smith耍1992學志(Journal of Molecular Biology)224899-904Wlodaver耍1992FEBS Letters 30959-64)
峁
婕澳核藉，剎一只芄指說細詒牡印
遠址式氡據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∏ 小印藕瀉轉錄止印伲邪約轉錄頭止藕擰為說峁┴ 位慕頭俳說薟莞司酶畝印
鋅說校為細識詒械畝帷Ｐ洪導薟莞司酶轉錄小涌選細詒殖轉錄緣魏味幔誨、囟毯雍櫻細同源源謀細細誑薟莞司酶幕謾
指峁棺枷趕缸 說擁實為碌櫻蟪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銥學院院753727-3731)約tac(DeBoer耍1983學院院8021-25)細玫(Useful proteins from recombinant bacteria)學(Scientific American)198024274-94Sambrook耍1989摹
指絲狀細斜暮峁棺 說擁實為碌櫻霉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Romanos耍1992母(Yeast 8423-488
謝一說轉錄止校為細識止轉錄小止鋅剎擁薟莞司酶畝3末恕魏選細蟹庸艿止涌詒
選絲狀細止擁源幕霉TAKA酶霉塹酶霉酶霉-酶圖鵲酶酶
選慕母細止擁源幕平母烯酶平母細色C(CYC1)平母醛-3-酶Romanos耍1992模玫慕母細止印
謝說鵲校為細要mRNA欠鵲鋅剎擁牡畝5末恕魏選細詵庸艿鵲鋅詒
選絲狀細鵲械源幕霉TAKA酶凸霉旃姑
說慕母細鵲械源幕平母烯酶(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末轉錄時細識為一紙詹謝轉錄mRNA藕諾小魏選細蟹庸木嶧口
選絲狀細木嶧翟 霉TAKA酶霉塹酶霉酶鵲酶酶禿霉-酶
母細玫木嶧疓uoSherman1995細學(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 司酶選細姆途藕謀詒
細細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(Microbiological Reviews)57109-137
絲狀細效藕謀塹祿藕霉TAKA酶霉緣酶霉塹酶Rhizomucor miehei於蛋酶Humicola insolens維酶Humicola lanuginosa脂酶
母細藕牡源幕平母-平母轉酶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為幕遠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前薟莞司酶陌末冢藕前陌末凇
細氐畝牡謀械詰牡也摹些系統實曰學碳(詰諢)應使沒謀銼回閉迪低吃 統械牡系統lactactrp系統誚母校使ADH2系統GAL1系統絲狀校TAKA-酶印霉塹酶雍霉塹酶涌小械實為牡小系統校詡裝蝕畝葉嶧乖肛 虻鞍諄些攏牡畝剎印
婕澳橫構濉⒂鶴繭翻止藕諾濉
氡久改橫構魏慰煞亟DNA濉
選一取細耍澹舊第靛，母貧染色母疲選兀詰細螅只全細椋將轄去染色一啤
選為謀澹ｋ本酶DNA鋅剎DNA轉錄慕詼印一說蠆《DNA院叩元鎩吧 印指詼使滸辭翟つ磕酚管的肥 校轉錄通酶DNA卸前小
涌魏選細詒殖轉錄DNA校緣員細同源源牡時
細細說擁實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
婕搬構細
氳較感的彪本酶DNA鋅鄣同源源細同源篩細然氡久窪NA一剎然寫詰一櫻誑應玫一藕瀉/止印同源指然鄣械酶DNA小鎩霸茨≈ 細斜聿幻窪NA小DNA鋅源一嗆銑傻小
氡綝NA細魏芄酶細細母透叩細植鎩
通芄酶細細實歉細芽吒司輳慚窟桿 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)細蟪Ω司
細轉通知姆式原轉绱┳ 匣使酶態細(渭Sambrook耍)
細蟪Ω司斜酶時酶一苑芙鑰駐細(知)細始隙前一攏細呀猓Ｍㄏ 捅約使酶鄣一攏酶通虜眨透壓菘蘋細使始隙頭牛倩酶
詬細芽吒司霉斜酶時酶駐細校細小一攏酶砂謝鍘
詒一實施校細墻母細玫摹母醫母(霉目(Endomycetales))詠母詘知(Fungi Imperfecti)(芽吒(Blastomycetes))慕母誚母鋅芊浠 目模母應母學突(Biology andActivities of Yeast)(SkinnerF.A.PassmoreS.M.DavenportR.R.嗉璖oc.App.Bacteriol.Symposium Series No.91980)慕卸濉
一選實施校慕母細羌絲母(Candida)遜母(Hansenula)魯維母(Kluyveromyces)銑母(Pichia)啤平母(母)殖母(Schizosaccharomyces)Yarrowia細
一選實施校慕母細強斯墻母(Saccharomyces carlsbergensis)平母腔母(Saccharomyces diastaticus)Saccharomyces douglasii魯母諾亟母墻母細
一選實施校慕母細魯維母(Kluyveromyces lactis)細一選實施校慕母細Yarrowia lipolytica細
一選實施校細絲狀細絲狀械(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 平母營通細芽械模碳謝欠偷摹
一選實施式校絲狀細塹預霉霉霉毛霉絲霉霉霉莞霉Tolypocladium木霉細
一選實施式校絲狀細盛霉霉氈霉霉霉霉細一選實施式校絲狀細歉狀摺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細一選實施式絲狀妝細Fusarium venenatum(Nirenberg sp.nov.)細
一選實施式校絲狀細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
細通一轉目前知姆式緯原濉⒆并細凇屎轉霉細姆EP238023Yelton耍1984銥學院院811470-1474轉霉姆Malardier耍1989(Gene)78147-156WO96/00787母轉姆校BeckerGuarenteAbelsonJ.N.SimonM.I.嗉概囪 學指(Guide to Yeast Genetics and Molecular Biology)酶學194恚 82-187頁Academic PressInc.紐約Ito耍1983細學志(Journal of Bacteriology)153163Hinnen耍1978銥學院院751920
目薟莞司酶姆
一婕澳枯草桿久改訪
a)薟莞司酶細
b)目薟莞司酶
酶DNA械謀轉源細時使母酶源
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(酶之)
轉細魏屎鄣細某目薟莞司酶煞胤詰校然通知姆眨通幕舜蟹細銨承的抵 脅詠綴筒取
目薟莞司酶途
目薟莞司酶卸止業途洗蛹業小吮婕懊趕從鹼，選酶洗祿洗鎩
薟莞司酶洗蛹錚
一說洗蛹詒院實洗蛹慕一圓渭WO96/34946WO97/07202WO95/30011
舜實目薟莞司酶殖越洗堋
洗蛹
酶擁洗蛹卸為械幀
紓從計誠聰聰從 錚と局從計從 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
一說選酶應選洗蛹應(pH-耍酶酶志緣)酶應效凇
酶實牡酶遠鎩⒅詫、微牡酶源微牡酶選摹也學位虻鞍墜袒突濉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)鵲酶酶實為鵲酶(牛)WO89/06270WO94/25583霉牡酶
玫牡酶實WO92/19729WO98/20115WO98/20116WO98/34946謀澹幣晃 婊荒卞273657768797101104120123167170194206218222224235274
選目坦牡酶AlcalaseTMSavinaseTMPrimaseTMDuralaseTMEsperaseTMKannaseTM(Novo Nordisk A/S)MaxataseTMMaxacalTMMaxapemTMProperaseTMPurafectTMPurafect OxPTMFN2TMFN3TM(Genencor International Inc.)
脂酶實脂酶些源細脂酶也學位虻鞍墜袒突濉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)
酶實牡酶(/)些源細牡酶也學位虻鞍墜袒突濉Ｃ岡 芽吒司摩-酶緣芽吒司囟輳 渭GB1,296,839
玫牡酶實WO94/02597WO94/18314WO96/23873WO97/43424謀澹┮ 位梅婊荒卞1523105106124128133154156181188190197202208209243264304305391408444
坦牡酶DuramylTMTermamylTMFungamylTMBANTM(Novo Nordisk A/S)RapidaseTMPurastarTM(GenencorInternational Inc.)
維酶實維酶源細維酶也渚或蛋白雇誨。實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
乇實維酶薔色頻募曰維酶維酶實為EP0495257EP0531372WO96/11262WO96/29397WO98/08940維酶實為些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/酶實墓酶/酶些源植鎩⑾改姑 酶也學位虻鞍墜袒突濉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懈屎通應玫某膜系實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濉Ｒ合從記核Ｒ匯含嘰70水0-30謝薌蠆緩水摹
洗蛹一一只直約強穎約爰的 雍/雍/穎約謀約一占0.160洗蛹時通約1約40穎約直-烯(脂)崠 腔脂-烯懟
洗蛹時通約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 帷⒁會、-烯帷⒃ 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準薌失萍稀
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酶實搿
酶擁WO97/07202洗蛹浞叫 氡疚
通實員薪細說些實為員蟊；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 危水一水漂準式為
NaBO2H2O2PB4 水水碳危式為2Na2CO33H2O2水碳漂準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V
強液織票式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
末遠洗I
末遠洗II
末遠洗III
末遠洗IV
末遠洗V
末液木洗穎約系統洗VI
水液態遠洗VII
水液態洗VIII
液遠洗IX
液遠洗X
漂卓液遠洗XI
XIIIIIIIIIVVIX遠洗錚 曬碳巍
XIIII-VI遠洗錚還檀呋檀呋紓≮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)示摹幕酶準品(SAVINASE_)確牟舛ㄇ悔弊悸鄧岡蹲灰 DMC)液械模謀準為50媯琾H8.3應時9櫻時3印要時Novo Nordisk A/SDenmark取AF220/1募校募邪詿為慰
GU歉拾岬ノ晃岡Ｚ弊悸-業為4015櫻嗟 mmole拾NH2-
也PNA脂學學志(Journal ofAmerican Oil Chemists Society)RothgebT.M.GoodlanderB.D.GarrisonP.H.SmithL.A.(1988)牡-----臃應舛岡
實施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吭礳at薟莞司酶轉錄始雍Savinase_前/原一碌摹莞司酶蠆糠執
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踴轉態拇司細通PCR轉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轉臺(300轉/)煤100ml BPX500ml式錐瓶5臁
耍為嘶220錐瓶同時汀
BPX煞(每)
100g
磨拇 50g
蠖狗 20g
Na2HPO4x12H2O 9g
(Pluronic) 0.1g
業 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/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
應頭圖約氬借，SEQ ID NO2示械驢薟莞司酶
實施2-準洗蛹洗欏
為薟莞司酶詒準洗蛹械洗埽實吮準洗實椋
洗蛹準洗蛹
洗蛹 4.0g/l
pH 10.1
洗時20
露齲 30
水硬齲15dH
酶濃齲10nm(洗蛹液)
系統 10ml氈徒
織/ 5織(_2.5cm)/50ml洗蛹液
圓希 WFK10N()
謀準洗蛹攏
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
為確瞻值瞬酶洗實欏 (R瞻)牟舛
然脅慰實椋測定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
應實櫸
杉Savinase_齲目薟莞司酶氈殖頻洗堋
實施3-目薟莞司酶遠(ADW)械
目薟莞司酶遠(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)
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櫻撕螅探沒使染
戲賂夜120錄45使牡時浴
ADW實
每一實校10沒芯Cylinda Compact machine預洗洗(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 示準偏)遠準酶Savinase_染越堋
系謀
細薟斯約墓娑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.權要1目薟莞司酶校詒牡摹準洗蛹洗欏蹦奧成分的弊枷從 時
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.權要2目薟莞司酶斜為1選為2選為3乇為4為5
4.權要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.權要4目薟莞司酶薟莞司酶SEQ IDNO2牡1-269位示陌傘
6.權要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.權要8目薟莞司酶薟莞司酶一位273656768797101104120123167170222224235274(BASBPN)之一巍
10.權要9目薟莞司酶選K27R*36DS56PN76DS87NG97NR101GS103AV104AV104IV104NV104YH120DN123SY167XR170XM222SM222AT224SK235LT274A(BASBPN)
11.權要一目薟莞司酶薟莞酶械細攏選詬叨細詠畝耄
i)SEQ ID NO1牡1-807位示畝幕
ii)100(i)片匣小
12.一址畝幔ㄒ -11一目薟莞司酶畝帷
13.一直薟莞司酶姆畝幔
a)SEQ ID NO1牡1-807位示畝88同一緣畝幔
b)詰細詠畝目薟莞司酶
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.權要13畝幔的 SEQ ID NO1牡1-807位示畝8990919293949596979899同一浴
15.權要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要12-15一暮小
17.一澹ㄒ 6暮峁瑰、約轉錄頭止藕擰
18.一細權要16暮峁瑰。
19.權要18細細為細選為芽吒司乇淺倩芽吒司
20.權要18細細為母選為絲狀乇霉
21.一權要1-11一目薟莞司酶姆
(a)薟莞司酶權要18-20一細
(b)湛薟莞司酶
22.一洗蛹錚∥綽幌達，權要1-11一目薟莞司酶
23.權要22錚還維酶脂酶酶原酶酶酶塹幕鎩
24.權要1-11一目薟莞司酶洗蛹械應謾
25.權要1-11一目薟莞司酶誄去途
26.權要22-23一洗蛹去盞途
27.一洗硬姆使硬權要22-23喲
28.一殖去硬盞姆使盞硬盞權要22-23喲
全文摘要
具有改進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，包括自動洗盤組合物中時，表現出優良的或改進的卵污漬洗滌性能。
文檔編號C12R1/07GK1418249SQ0180653
公開日2003年5月14日 申請日期2001年3月12日 優先權日2000年3月14日
發明者F·米克爾森, T·S·法諾, M·N·馬德森, L·B·漢森, P·K·漢森 申請人:諾和酶股份有限公司