CN101128579A - α-淀粉酶变体 - Google Patents
α-淀粉酶变体 Download PDFInfo
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- CN101128579A CN101128579A CNA2005800486042A CN200580048604A CN101128579A CN 101128579 A CN101128579 A CN 101128579A CN A2005800486042 A CNA2005800486042 A CN A2005800486042A CN 200580048604 A CN200580048604 A CN 200580048604A CN 101128579 A CN101128579 A CN 101128579A
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
本发明涉及亲本Termamyl-样α-淀粉酶的变体,该变体具有改变的特性,具体来说是相对于亲本α-淀粉酶增加的淀粉亲和力。
Description
发明领域
本发明涉及,特别是亲本Termamyl-样α-淀粉酶的新变体,尤其是显示改变的特性的变体,特别是改变了淀粉亲和力(相对于亲本),对于所述变体的应用,特别是工业淀粉处理(processing)(如淀粉液化或糖化),这是有利的。
发明背景
α-淀粉酶(α-1,4-葡聚糖-4-葡聚糖水解酶,EC 3.2.1.1)组成催化淀粉和其它直链及支链1,4-葡糖苷寡糖和多糖水解的一组酶。
有大量的专利和科学文献涉及此类在工业上十分重要的酶。许多α-淀粉酶,例如Termamyl-样α-淀粉酶可了解自,如WO 90/11352、WO 95/10603、WO 95/26397、WO 96/23873、WO 96/23874和WO 97/41213。
在最近涉及α-淀粉酶的公开内容中,WO 96/23874提供了称为BA2的Termamyl-样α-淀粉酶的三维X-射线晶体结构数据,BA2由包含本文SEQ IDNO:6中所示氨基酸序列的解淀粉芽孢杆菌(B.amyloliquefaciens)α-淀粉酶的300个N-末端氨基酸残基,以及包含本文SEQ ID NO:4中所示氨基酸序列的地衣芽孢杆菌(B.licheniformis)α-淀粉酶的C-末端的氨基酸301-483(后者可以商品名TermamylTM购得)组成,因此与工业上重要的芽孢杆菌α-淀粉酶(其在本文中包括在术语“Termamyl-样α-淀粉酶”的含义中,尤其包括地衣芽孢杆菌,解淀粉芽孢杆菌,和嗜热脂肪芽孢杆菌(B.stearothermophilus)α-淀粉酶)密切相关。WO 96/23874进一步描述了在对亲本Termamyl-样α-淀粉酶的结构分析的基础上,设计相对于亲本显示改变的特性的亲本Termamyl-样α-淀粉酶变体的方法学。
发明简述
本发明涉及Termamyl-样α-淀粉酶的新的α-淀粉分解变体(突变体),特别是显示出改变的淀粉亲和力(相对于亲本)的变体,所述改变在淀粉的工业处理方面(淀粉液化,糖化等等)是有利的。
本发明人发现与亲本Termamyl-样α-淀粉酶相比,具有改变的特性、特别是改变的淀粉亲和力(affinity)的变体改善了淀粉的转化。
本发明进一步涉及编码本发明变体的DNA构建体,包含本发明变体的组合物,制备本发明变体的方法,以及本发明变体和组合物单独或与其它α-淀粉分解酶(alpha-amylolytic enzymes)联合在各种工业处理中的应用,如淀粉液化,在洗涤剂组合物中,例如洗衣、清洗碗碟(dish washing)和硬表面清洗组合物;乙醇生产,例如燃料,酒类和工业乙醇生产;纺织品(textile)、织物(fabric)或衣物的退浆等。
命名法
在本说明书和权利要求书中,使用了常规的单字母和三字母编码表示氨基酸残基。为便于引用,本发明的α-淀粉酶变体采用下列命名法描述:
原氨基酸:位置:取代的氨基酸
依照该命名法,例如用天冬酰胺取代第30位的丙氨酸表示为:
Ala30Asn或A30N
在相同位置处丙氨酸的缺失表示为:
Ala30*或A30*
以及额外的氨基酸残基例如赖氨酸的插入表示为:
Ala30AlaLys或A30AK
缺失连续的一段氨基酸残基,例如氨基酸残基30-33,表示为(30-33)*或Δ(A30-N33)。
在特定的α-淀粉酶相对于其它α-淀粉酶而言含有“缺失”,并在该位置具有插入,如在第36位插入天冬氨酸的情况下,表示为:
*36Asp或*36D
多个突变由加号隔开,即:
Ala30Asn+Glu34Ser或A30N+E34S
分别表示在第30和34位,天冬酰胺和丝氨酸取代了丙氨酸和谷氨酸。
当在给定位置插入一个或多个可选择的氨基酸残基时,其表示为
A30N,E或
A30N或A30E
此外,当本文鉴定出适于修饰的位置,而没有暗示任何具体的修饰时,应当理解为可用任一氨基酸残基取代该位置存在的氨基酸残基。因此,例如,当提及修饰第30位的丙氨酸,但未具体说明时,应当理解为丙氨酸可缺失,或可用任何其他氨基酸,即下列任一氨基酸来取代:
R、N、D、A、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y、V。
此外,“A30X”表示任何一种下列取代:
A30R、A30N、A30D、A30C、A30Q、A30E、A30G、A30H、A30I、A30L、A30K、A30M、A30F、A30P、A30S、A30T、A30W、A30Y或A30V;或简言之:A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V。
如果使用这种编号方式的亲本酶已具有建议在该位置被取代的所述氨基酸残基,在例如N或V之一存在于野生型中的情况下,使用下列命名法:
“X30N”或“X30N,V”。
因此,其表示其他相应的亲本酶在第30位被“Asn”或“Val”取代。
氨基酸残基特性
带电氨基酸:
Asp、Glu、Arg、Lys、His
带负电荷氨基酸(带有最多负电荷的残基列于最前):
Asp、Glu
带正电荷氨基酸(带有最多正电荷的残基列于最前):
Arg、Lys、His
中性氨基酸:
Gly、Ala、Val、Leu、Ile、Phe、Tyr、Trp、Met、Cys、Asn、Gln、Ser、Thr、Pro
疏水氨基酸残基(具有最大疏水性的残基列于最后):
Gly、Ala、Val、Pro、Met、Leu、Ile、Tyr、Phe、Trp
亲水氨基酸(具有最大亲水性的残基列于最后):
Thr、Ser、Cys、Gln、Asn
发明详述
Termamyl-样α-淀粉酶
众所周知,通过芽孢杆菌(Bacillus spp.)产生的多种α-淀粉酶在氨基酸水平上是高度同源的。例如,已发现包含SEQ ID NO:4中所示的氨基酸序列的地衣芽孢杆菌α-淀粉酶(商品名为TermamylTM)与包含SEQ ID NO:6中所示的氨基酸序列的解淀粉芽孢杆菌α-淀粉酶约89%同源,与包含SEQ IDNO:8中所示的氨基酸序列的嗜热脂肪芽孢杆菌α-淀粉酶约79%同源。其他同源的α-淀粉酶包括来源于芽孢杆菌菌株NCIB 12289、NCIB 12512、NCIB 12513或DSM 9375的α-淀粉酶,皆详述于WO 95/26397,以及Tsukamoto等,Biochemical and Biophysical Research Communications,151(1988),pp.25-31所描述的#707α-淀粉酶。
更多同源的α-淀粉酶包括由EP 0252666中所述的地衣芽孢杆菌菌株(ATCC 27811)所产生的α-淀粉酶,以及WO 91/00353和WO 94/18314中鉴定的α-淀粉酶。其他商用Termamyl-样α-淀粉酶包括在以下列商品名出售的产品中:OptithermTM和TakathermTM(可购自Solvay);MaxamylTM(可购自Gist-brocades/Genencor)、Spezym AATM和Spezyme Delta AATM(可购自Ge-nencor)和KeistaseTM(可购自Daiwa)、PurastarTM ST 5000E、PURASTRATMHPAML(来自Genencor Int.)。
由于发现这些α-淀粉酶之间基本上同源,因此它们被认为属于相同一类的α-淀粉酶,即“Termamyl-样α-淀粉酶”。
因此,在本发明上下文中,术语“Termamyl-样α-淀粉酶”意在指示在氨基酸水平上具有与TermamylTM,即具有本文SEQ ID NO:4中所示的氨基酸序列的地衣芽孢杆菌α-淀粉酶,基本上(substantial)同源的α-淀粉酶。换言之,Termamyl-样α-淀粉酶是具有本文SEQ ID NO:2、4或6中所示的氨基酸序列,和WO 95/26397或Tsukamoto等,1988中的SEQ ID NO:1或2所示的氨基酸序列的α-淀粉酶,或者是:i)显示与至少一种上述氨基酸序列至少60%,优选至少70%,更优选至少75%,甚至更优选至少80%,尤其是至少85%,尤其优选至少90%,甚至尤其更优选至少95%,更优选至少为97%,更优选至少99%同源性的α-淀粉酶,和/或ii)显示与针对至少一种上述α-淀粉酶产生的抗体免疫交叉反应性的α-淀粉酶,和/或iii)由与编码上述特定α-淀粉酶的DNA序列杂交的DNA序列编码的α-淀粉酶,所述编码特定α-淀粉酶的DNA序列分别示于本申请的SEQ ID NO:1、3和5,以及WO 95/26397的SEQ ID NOS:4和5。
同源性(同一性)
同源性可测定为两条序列之间的同一性水平,指示第一序列与第二序列的偏差(derivation)。可通过本领域已知的计算机程序例如GCG程序包中提供的GAP(上述的)适当地测定同源性。因此,可使用具有用于同一性的默认得分矩阵(default scoring matrix)和下列默认参数的Gap GCGv8:用于核酸序列比较,分别为缺口(GAP)产生罚分5.0和缺口延伸罚分0.3,以及用于蛋白序列比较,分别为缺口(GAP)产生罚分3.0和缺口延伸罚分0.1。GAP使用了Needleman和Wunsch,(1970),J.Mol.Biol.48,p.443-453的方法进行比对和计算同一性。
可使用Termamyl和Termamyl-样α-淀粉酶之间的结构对比来鉴定其它Termamyl-样α-淀粉酶中等同的/相应的位置。一种获得所述结构对比的方法是使用GCG程序包中的Pile Up程序,该程序使用默认的缺口罚分值,即缺口产生罚分3.0和缺口延伸罚分0.1。其它结构对比方法包括疏水性聚类分析(Gaboriaud等,(1987),FEBS LETTERS 224,pp.149-155)和反向穿梭法(reverse threading)(Huber,T;Torda,AE,PROTEIN SCIENCE Vol.7,No.1pp.142-149(1998))。α-淀粉酶的特性ii),即可使用针对相关的Termamyl-样α-淀粉酶的至少一个表位产生的、或与之反应的抗体测定免疫交叉反应性。可以是单克隆或多克隆的所述抗体可用本领域已知的方法产生,如Hudson等,Practical Immunology,第3版(1989),Blackwell Scientific Publications中所述的。免疫交叉反应性可使用本领域已知测定法来测定,如蛋白质印迹法或放射免疫扩散,如Hudson等,1989所述。在这方面,在分别具有氨基酸序列SEQ ID NOS:2、4、6或8的α-淀粉酶之间发现了免疫交叉反应性。
杂交
可以在所述α-淀粉酶的完整或部分核苷酸或氨基酸序列的基础上适当地制备寡核苷酸探针,用于鉴定符合上述特性iii)的Termamyl-样α-淀粉酶。
用于检测杂交的合适条件包括在5xSSC中预浸泡,并在20%甲酰胺、5xDenhardt′s溶液、50mM磷酸钠,pH6.8和50mg变性的经超声处理的小牛胸腺DNA溶液中于~40℃预杂交1小时,接着于~40℃,在补充有100mM ATP的相同溶液中杂交18小时,然后于40℃在2xSSC,0.2%SDS中洗涤滤膜三次,每次30分钟(低严谨性),优选于50℃(中等严谨性),更优选于65℃(高严谨性),甚至更优选于~75℃(非常高严谨性)。有关杂交方法的更多细节可见于Sambrook等,Molecular_Cloning:A Laboratory Manual,第2版,ColdSpring Harbor,1989中。
在本发明上下文中,“来源于(derived from)”并不意在只表示由所述生物菌株产生的或可由所述生物菌株产生的α-淀粉酶,还表示由分离自这样的菌株的DNA序列编码的α-淀粉酶以及由所述DNA序列转化的宿主生物产生的α-淀粉酶。最后,该术语还意在表示由合成的和/或cDNA来源的DNA序列编码的,并具有所述α-淀粉酶的鉴定特征的α-淀粉酶。该术语还意在表示亲本α-淀粉酶可以是天然存在的α-淀粉酶的变体,即天然存在的α-淀粉酶的一个或多个氨基酸残基的修饰(插入、取代、缺失)所得到的变体。
亲本杂合α-淀粉酶
亲本α-淀粉酶可以是杂合α-淀粉酶,即包含来源于至少两种α-淀粉酶的部分氨基酸序列组合的α-淀粉酶。
亲本杂合α-淀粉酶可以是在氨基酸同源性和/或免疫交叉反应性和/或DNA杂交(如上所定义的)的基础上可被确定为属于Termamyl-样α-淀粉酶家族的一种杂合α-淀粉酶。在该情况下,杂合α-淀粉酶一般由Termamyl-样α-淀粉酶的至少一个部分和一种或多种其它α-淀粉酶的部分组成,所述其它α-淀粉酶选自微生物(细菌或真菌)和/或哺乳动物来源的Termamyl-样α-淀粉酶或非-Termamyl-样α-淀粉酶。
因此,亲本杂合α-淀粉酶可包含来源于至少两种Termamyl-样α-淀粉酶,或来源于至少一种Termamyl-样和至少一种非-Termamyl-样细菌α-淀粉酶,或来源于至少一种Termamyl-样和至少一种真菌α-淀粉酶的部分氨基酸序列的组合。部分氨基酸序列所来源的Termamyl-样α-淀粉酶可以是例如本文所提及的任何那些特定的Termamyl-样α-淀粉酶。
例如,亲本α-淀粉酶可包含来源于地衣芽孢杆菌菌株的α-淀粉酶的C-末端部分,和来源于解淀粉芽孢杆菌菌株或嗜热脂肪芽孢杆菌菌株的α-淀粉酶的N-末端部分。例如,亲本α-淀粉酶可包含地衣芽孢杆菌α-淀粉酶的C-末端部分的至少430个氨基酸残基,也可包含如a)相应于具有SEQ IDNO:6中所示的氨基酸序列的解淀粉芽孢杆菌α-淀粉酶的37个N端氨基酸残基的氨基酸区段(segment),和相应于具有SEQ ID NO:4中所示的氨基酸序列的地衣芽孢杆菌α-淀粉酶的445个C末端氨基酸残基的氨基酸区段,或b)相应于具有SEQ ID NO:8中所示的氨基酸序列的嗜热脂肪芽孢杆菌α-淀粉酶的68个N-末端氨基酸残基的氨基酸区段,和相应于具有SEQ IDNO:4中所示的氨基酸序列的地衣芽孢杆菌α-淀粉酶的415个C-末端氨基酸残基的氨基酸区段。
在一个优选的实施方案中,亲本Termamyl-样α-淀粉酶是与SEQ ID NO:4中所示的地衣芽孢杆菌α-淀粉酶相同的杂合Termamyl-样α-淀粉酶,不同之处在于:(成熟蛋白的)N-末端的35个氨基酸残基为SEQ ID NO:6中所示的解淀粉芽孢杆菌α-淀粉酶(BAN)的成熟蛋白的N-末端的33个氨基酸残基所取代。上述杂合Termamyl-样α-淀粉酶可进一步具有下列突变:H156Y+A181T+N190F+A209V+Q264S(使用SEQ ID NO:4中的编号),称为LE174。
另一个优选的亲本杂合α-淀粉酶是SEQ ID NO:2中所示的LE429。
非-Termamyl-样α-淀粉酶可以是例如真菌α-淀粉酶、哺乳动物或植物α-淀粉酶或细菌α-淀粉酶(不同于Termamyl-样α-淀粉酶)。这样的α-淀粉酶的具体例子包括米曲霉(Aspergillus oryzae)TAKAα-淀粉酶、黑曲霉(A.niger)酸性α-淀粉酶、枯草芽孢杆菌α-淀粉酶、猪胰腺α-淀粉酶和大麦α-淀粉酶。所有的这些α-淀粉酶都具有已阐明的,与本文所述典型的Termamyl-样α-淀粉酶的结构明显不同的结构。
上述真菌α-淀粉酶,即来源于黑曲霉和米曲霉的α-淀粉酶在氨基酸水平上高度同源,一般认为属于相同的α-淀粉酶家族。来源于米曲霉的真菌α-淀粉酶可以商品名FungamylTM从市场上买到。
此外,当提及Termamyl-样α-淀粉酶的特定变体(本发明的变体)时,所提及的变体是通过常规的方法,修饰(如缺失或取代)特定Termamyl-样α-淀粉酶的氨基酸序列中的特定氨基酸残基而获得的,应理解在等同位置(由各个氨基酸序列之间可能达到最好的氨基酸序列对比所测定的)修饰得到的另一个Termamyl-样α-淀粉酶的变体也包括在本发明的范围内。
本发明变体的一个优选的实施方案是来源于地衣芽孢杆菌α-淀粉酶(作为亲本Termamyl-样α-淀粉酶)的变体,如上述α-淀粉酶中的一个的变体,例如具有SEQ ID NO:4中所示的氨基酸序列的地衣芽孢杆菌α-淀粉酶的变体。
本发明变体的构建
可通过在有助于生产变体的条件下,培养包含编码所述变体的DNA序列的微生物来实现目标变体的构建。所述变体可随后从所得到的培养物中回收。这进一步详述于下。
改变的特性
以下讨论了可在本发明变体中出现的突变与可由其所引起的期望特性改变(相对于亲本Termamyl-样α-淀粉酶的特性)之间的关系。
本发明的第一方面涉及具有α-淀粉酶活性并包含R437W取代的亲本Termamyl-样α-淀粉酶的变体,其中所述位置相应于具有SEQ ID NO:4氨基酸序列的亲本Termamyl-样α-淀粉酶的氨基酸序列的位置。
在淀粉液化过程以及其中涉及α-淀粉酶的其他过程中,增加α-淀粉酶的淀粉亲和力并由此增加如生(raw)淀粉水解(RSH)是有利的。
本发明人已发现通过在仅具有两个色氨酸之一的α-淀粉酶的C-末端区域导入色氨酸残基,由此产生一对色氨酸,形成推定的淀粉结合位点,发现其在吸附至淀粉中起主要作用,因而对于高的淀粉转化率是关键性的。
应当强调的是不仅以下特别提及的Termamyl-样α-淀粉酶可被使用。而且其他商用Termamyl-样α-淀粉酶也可使用。这样的α-淀粉酶的不太详细的列表如下:
EP 0252666(ATCC 27811)中所述的地衣芽孢杆菌菌株产生的α-淀粉酶,和WO 91/00353和WO 94/18314中鉴定的α-淀粉酶。其他商用Termamyl-样地衣芽孢杆菌α-淀粉酶是OptithermTM和TakathermTM(可购自Solvay)、MaxamylTM(可购自Gist-brocades/Genencor)、Spezym AATM Spezyme DeltaAATM(可购自Genencor)和Keistase((可购自Daiwa)。
但是,只有在C-末端不具有两个色氨酸残基的Termamyl-样α-淀粉酶能合适地用作为骨架用于制备本发明变体。
在本发明一个优选的实施方案中,亲本Termamyl-样α-淀粉酶是SEQ IDNO:4或SEQ ID NO:6或它们的变体的α-淀粉酶。
在一个具体的实施方案中,所述变体包含一个或多个下列额外的突变:R176*、G177*、N190F、E469N,更特别是R176*+G177*+N190F,还特别是R176*+G177*+N190F+E469N(使用SEQ ID NO:6中的编号)。
在本发明另一个优选的实施方案中,亲本Termamyl-样α-淀粉酶是SEQID NO:4和SEQ ID NO:6的杂合α-淀粉酶。特别是,所述亲本杂合Termamyl-样α-淀粉酶可以是包含SEQ ID NO:4中所示的地衣芽孢杆菌α-淀粉酶的445个C-末端氨基酸残基和SEQ ID NO:6中所示的来源于解淀粉芽孢杆菌的成熟的α-淀粉酶的37个N-末端氨基酸残基的杂合α-淀粉酶,其可合适地进一步具有下列突变:H156Y+A181T+N190F+A209V+Q264S(使用SEQ ID NO:4中的编号)。该杂合体称为LE174。LE174杂合体可以与更进一步的突变I201F结合以形成具有以下突变H156Y+A181T+N190F+A209V+Q264S+I201F(使用SEQ ID NO:4的编号)的亲本杂合Termamyl-样α-淀粉酶。该杂合变体如SEQ ID NO:2所示,并被用于以下实施例中,称为LE429。
当通过结合LE174与突变I201F(SEQ ID NO:4编号),使用LE429(示于SEQ ID NO:2)作为主链时(即作为亲本Termamyl-样α-淀粉酶),突变/改变,特别是取代、缺失和插入,根据本发明可在一个或多个以下位置上进行:R176*、G177*、E469N(使用SEQ ID NO:6中的编号)。在一个特别的实施方案中,变体包含额外的突变:E469N(使用SEQ ID NO:6中的编号)。在一个更特别的实施方案中,变体包含额外的突变:R176*+G177*+E469N(使用SEQID NO:6中的编号)。
在本发明变体中的常规突变
除以上概述的那些修饰之外,最好让本发明的变体包含一个或多个修饰。
制备α-淀粉酶变体的方法
本领域已知几种将突变导入基因中的方法。在对编码α-淀粉酶的DNA序列克隆的简短讨论之后,将要讨论在编码α-淀粉酶的序列中的特定位点处产生突变的方法。
克隆编码α-淀粉酶的DNA序列
可使用本领域众所周知的多种方法,从产生所述α-淀粉酶的任何细胞或微生物中分离出编码亲本α-淀粉酶的DNA序列。首先,应使用源自产生待研究之α-淀粉酶的生物的染色体DNA或信使RNA构建基因组DNA和/或cDNA文库。然后,如果α-淀粉酶的氨基酸序列是已知的,可合成同源的经标记的寡核苷酸探针,并使用该探针从制备自所述生物的基因组文库中鉴定编码α-淀粉酶的克隆。或者,可将含有与已知α-淀粉酶基因同源之序列的经标记的寡核苷酸探针用作探针,使用较低严谨性的杂交和洗涤条件鉴定编码α-淀粉酶的克隆。
另一种鉴定编码α-淀粉酶的克隆的方法包括:将基因组DNA片段插入表达载体,例如质粒,用所得到的基因组DNA文库转化α-淀粉酶-阴性细菌,然后将经转化的细菌铺于含有α-淀粉酶底物的琼脂上,由此使得表达α-淀粉酶的克隆得以鉴定。
或者,可通过已建立的标准方法合成制备编码酶的DNA序列,所述方法例如S.L.Beaucage和M.H.Camthers(1981)所述的膦酰胺(phosphoroamidite)法或Matthes等(1984)所述的方法。在膦酰胺法中,可在例如自动化的DNA合成仪中合成寡核苷酸,对其进行纯化、退火、连接并克隆至适当的载体中。
最终,根据标准技术,DNA序列可以是通过连接合成的,基因组的或cDNA来源的片段(适当时是对应于完整DNA序列的多个部分的片段)而制备的混合的基因组和合成来源,混合的合成和cDNA来源或混合的基因组和cDNA来源的DNA序列。也可以使用特定的引物,例如US4,683,202或R.K.Saiki等(1988)所述的引物通过聚合酶链反应(PCR)制备DNA序列。
位点定向诱变
一旦分离出编码α-淀粉酶的DNA序列,并鉴定出期望的突变位点,可使用合成的寡核苷酸导入突变。这些寡核苷酸含有侧接于期望的突变位点的核苷酸序列;在寡核苷酸合成期间插入突变的核苷酸。在一种特定的方法中,在携有α-淀粉酶基因的载体中产生桥连α-淀粉酶-编码序列的DNA单链缺口。然后,使含有期望的突变的合成核苷酸与该单链DNA的同源部分退火。然后用DNA聚合酶I(Klenow片段)补平其余缺口,并使用T4连接酶连接构建体。该方法的一个特定的例子描述于Morinaga等(1984)。US4,760,025公开了通过对盒进行微小的改变而导入编码多个突变的寡核苷酸。但是,由于可导入不同长度的多个寡核苷酸,因此通过Morinaga法可在任何一次导入甚至更多个的突变。
Nelson和Long(1989)描述了另一种将突变导入编码α-淀粉酶的DNA序列的方法。所述方法包括以3个步骤产生含有期望的突变的PCR片段,所述突变是通过使用化学合成的DNA链作为PCR反应的一个引物而导入的。通过用限制性内切核酸酶裂解,可从PCR-产生的片段中分离出携有突变的DNA片段,并将该片段重新插入表达质粒中。
随机诱变
可在翻译为本文所示氨基酸序列的基因的至少3个部分中,或在整个基因内适当地进行随机诱变,所述诱变可以是定域的(localised)或区域-特异性的随机诱变。
利用本领域已知的任何方法,可以方便地对编码亲本α-淀粉酶的DNA序列进行随机诱变。
相对于上文,本发明的另一方面涉及一种产生亲本α-淀粉酶的变体的方法,例如,其中相对于亲本,变体显示改变的淀粉亲和力,所述方法包括:
(a)对编码亲本α-淀粉酶的DNA序列进行随机诱变,
(b)在宿主细胞中表达步骤(a)中获得的突变的DNA序列,和
(c)筛选表达相对于亲本α-淀粉酶具有改变的淀粉亲和力的α-淀粉酶变体的宿主细胞。
优选使用掺杂的引物进行本发明上述方法中的步骤(a)。例如,可使用适当的物理或化学诱变剂,通过使用适当的寡核苷酸,或通过对DNA序列进行PCR以产生诱变来进行随机诱变。此外,可使用这些诱变剂的任何组合来进行随机诱变。诱变剂可以是例如诱导转换、颠换(transversion)、倒位、颠倒(scrambling)、缺失和/或插入的试剂。适用于本发明目的的物理或化学诱变剂的例子包括紫外线(UV)照射、羟胺、N-甲基-N′-硝基-N-亚硝基胍(MNNG)、O-甲基羟胺、亚硝酸、甲磺酸乙酯(EMS)、亚硫酸氢钠(sodiumbisulphite)、甲酸和核苷酸类似物。当使用这样的诱变剂时,一般通过在适于发生诱变的条件下,在选定诱变剂的存在下温育要被诱变的编码亲本酶的DNA序列来进行诱变,然后筛选具有期望的特性的突变DNA。当利用寡核苷酸进行诱变时,在合成要被改变位置处的寡核苷酸的过程中,寡核苷酸可以掺杂或掺入有3种非亲本核苷酸。掺杂或掺入的目的是避免不需要的氨基酸的密码子。可通过任何公开的技术如使用PCR、LCR或在适当时使用任何DNA聚合酶和连接酶,将掺杂(doped)或掺入(spiked)的寡核苷酸掺入编码α-淀粉酶的DNA。优选地,使用“不变的随机掺杂”进行掺杂,其中在每个位置中的野生型和突变的百分比是预先确定的。此外,掺杂可以导致优先导入某些核苷酸,从而优先导入一个或多个特定的氨基酸残基。例如,进行掺杂可使得在每个位置中导入90%野生型和10%突变。选择掺杂方案的其它考虑是基于遗传学以及蛋白质-结构的限制。可使用DOPE程序制订掺杂方案,所述程序可特别地确保避免导入终止密码子。当使用PCR-产生的诱变时,可在增加核苷酸错误掺入的条件下,对经化学处理或未经处理的编码亲本α-淀粉酶的基因进行PCR(Deshler 1992;Leung等,Technique,Vol.1,1989,pp.11-15)。可使用大肠杆菌(Fowler等,Molec.Gen.Genet.,133,1974,pp.179-191),酿酒酵母或任何其它微生物的增变株对编码α-淀粉酶的DNA进行随机诱变,诱变方法包括例如:将含有亲本糖基化酶的质粒转化到增变株中,培养含有质粒的增变株,从增变株中分离突变的质粒。随后,将突变的质粒转化至表达生物中。要被诱变的DNA序列可方便地存在于基因组或cDNA文库中,所述文库制备自表达亲本α-淀粉酶的生物。或者,DNA序列可以存在于适当的载体例如质粒或噬菌体上,可与诱变剂一起温育或要不然暴露于诱变剂中。要被诱变的DNA也可存在于宿主细胞中,或者整合于所述细胞的基因组中,或者存在于细胞所携带的载体上。最后,要被诱变的DNA也可以是分离的形式。很清楚要接受随机诱变的DNA序列优选是cDNA或基因组DNA序列。在某些情况下可在实施表达步骤b)或筛选步骤c)之前方便地扩增突变的DNA序列。可根据本领域已知的方法进行这样的扩增,本发明优选的方法是:使用根据亲本酶的DNA或氨基酸序列制备的寡核苷酸引物进行PCR扩增。与诱变剂温育或暴露于诱变剂之后,通过在允许发生表达的条件下培养携有突变DNA序列的适当宿主细胞来表达突变的DNA。用于此目的的宿主细胞可以是已用突变的DNA序列(任选地存在于载体上)转化的宿主细胞,或者是在诱变处理过程中携有编码亲本酶的DNA序列的宿主细胞。适当宿主细胞的例子如下:革兰氏阳性细菌,例如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌(Bacilluslentus)、短芽孢杆菌(Bacillus brevis)、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌(Bacillus alkalophilus)、解淀粉芽孢杆菌、凝结芽孢杆菌(Bacillus coagulans)、环状芽孢杆菌(Bacillus circulans)、灿烂芽孢杆菌(Bacillus lautus)、巨大芽孢杆菌(Bacillus megaterium)、苏云金芽孢杆菌(Bacillus thuringiensis)、浅青紫链霉菌(Streptomyces lividans)或鼠灰链霉菌(Streptomyces murinus);和革兰氏阴性细菌,例如大肠杆菌(E.coli)。突变的DNA序列可进一步包含能使突变的DNA序列表达的DNA序列。
定域随机诱变
随机诱变可有利地定域于所述亲本α-淀粉酶的一部分。例如,当酶的某些区域已被鉴定为对酶的给定特性特别重要时,以及当预期修饰能导致具有改善特性的变体时,定域随机诱变可能是有利的。当亲本酶的三级结构已被阐明,且所述结构与酶的功能相关时,一般可鉴定出这样的区域。
通过使用上述的PCR产生的诱变技术或本领域已知的任何其它适当的技术,可以方便地进行定域的或区域-特异性的随机诱变。或者,通过例如插入适当的载体来分离编码要被修饰的DNA序列部分的DNA序列,随后可使用上述任何诱变方法对所述部分进行诱变。
提供α-淀粉酶变体的备选方法
提供α-淀粉酶变体的备选方法包括本领域已知的基因改组方法,所述方法包括例如WO 95/22625(来自Affymax Technologies N.V.)和WO96/00343(来自Novo NordiskA/S)中所述的方法。
表达α-淀粉酶变体
根据本发明,可使用表达载体,以酶的形式表达通过上述方法,或通过本领域已知的任何其它方法产生的编码变体的DNA序列,所述表达载体通常包括编码启动子、操纵子、核糖体结合位点、翻译起始信号的控制序列,并任选包括阻遏基因或多种激活基因。
携有编码本发明α-淀粉酶变体的DNA序列的重组表达载体可以是能对其方便地进行重组DNA操作的任何载体,载体的选择常取决于其要被导入的宿主细胞。因此,载体可以是自主复制的载体,即作为染色体外实体存在的载体,所述载体的复制不依赖于染色体的复制,如质粒、噬菌体或染色体外元件、微型染色体或人工染色体。或者,载体可以是当导入宿主细胞时,被整合入宿主细胞基因组,并与整合了该载体的染色体一起复制的载体。
在载体中,DNA序列应与适当的启动子序列可操作地连接。启动子可以是在选定宿主细胞中显示出转录活性的任何DNA序列,并可来源于编码与宿主细胞同源或异源之蛋白质的基因。指导编码本发明α-淀粉酶变体之DNA序列转录,尤其是在细菌宿主中的转录的适当启动子的例子是:大肠杆菌lac操纵子的启动子、天蓝色链霉菌(Streptomyces coelicolor)琼脂酶基因dagA启动子、地衣芽孢杆菌α-淀粉酶基因(amyL)启动子、嗜热脂肪芽孢杆菌产麦芽糖淀粉酶基因(amyM)启动子,解淀粉芽孢杆菌α-淀粉酶(amyQ)启动子、枯草芽孢杆菌xylA和xylB基因的启动子等。对于在真菌宿主中转录,有用的启动子的例子是来源于编码下列酶的基因的启动子:米曲霉TAKA淀粉酶,米赫根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定性α-淀粉酶、黑曲霉葡糖淀粉酶、米赫根毛霉脂肪酶、米曲霉碱性蛋白酶、米曲霉丙糖磷酸异构酶或构巢曲霉(A.nidulans)乙酰胺酶。
本发明的表达载体还可含有适当的转录终止子,以及在真核生物中,还可含有与编码本发明α-淀粉酶变体的DNA序列可操作地连接的聚腺苷酸化序列。终止和聚腺苷酸化序列可适当地来源于与启动子相同的来源。
载体可进一步包含能使载体在所述宿主细胞中复制的DNA序列。这样的序列的例子是质粒pUC19、pACYC177、pUB110、pE194、pAMB1和pIJ702的复制起点。
载体还可含有选择标记,例如其产物可以弥补宿主细胞缺陷的基因,如枯草芽孢杆菌或地衣芽孢杆菌的dal基因,或能赋予抗生素抗性的基因,所述抗生素抗性例如氨苄青霉素、卡那霉素、氯霉素或四环素抗性。此外,载体可含有曲霉属选择标记,例如amdS、argB、niaD和sC,产生潮霉素抗性的标记,或者可通过如WO 91/17243中所述的共转化来完成的选择。
尽管细胞内表达在某些方面(如当使用某些细菌作为宿主细胞时)有利,但一般优选在细胞外表达。一般而言,本文所述的芽孢杆菌属α-淀粉酶包含允许表达的蛋白酶分泌至培养基的前区。必要时,该前区可为不同的前区或信号序列所取代,通过取代编码各个前区的DNA序列可以方便地实现此目的。
用于分别连接编码α-淀粉酶变体的本发明DNA构建体、启动子、终止子和其它元件,并将它们插入含有复制所需信息的适当载体的方法是本领域技术人员众所周知的(参见例如,Sambrook等,Molecular Cloning:ALaboratory Manual,第2版,Cold Spring Harbor,1989)。
在重组生产本发明的α-淀粉酶变体中,包含上文所定义的本发明DNA构建体或表达载体的本发明细胞可以有利地用作为宿主细胞。可以方便地通过将编码变体的本发明DNA构建体(以一个或多个拷贝)整合至宿主染色体中,用所述DNA构建体转化细胞。一般认为整合是有利的,因为整合后DNA序列可以在细胞中更加稳定地维持。根据常规方法,例如通过同源或异源重组可以将DNA构建体整合至宿主染色体中。或者,可用与不同类型的宿主细胞有关的上述表达载体转化细胞。
本发明的细胞可以是高等生物的细胞,例如哺乳动物或昆虫的细胞,但优选其为微生物细胞,例如细菌或真菌(包括酵母)的细胞。
适合的细菌为革兰氏阳性菌,例如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌、短芽孢杆菌、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌、环状芽孢杆菌、灿烂芽孢杆菌、巨大芽孢杆菌、苏云金芽孢杆菌、浅青紫链霉菌或鼠灰链霉菌,或革兰氏阴性菌例如大肠杆菌。细菌的转化可受例如原生质体转化或以感受态细胞自身已知的方式使用该细胞而影响。
酵母生物可优选糖酵母属或裂殖酵母属(Schizosaccharomyces),例如酿酒酵母(Saccharomyces cerevisiae)。丝状真菌优选属于曲霉属,例如,米曲霉或黑曲霉。真菌细胞可通过一种方法转化,该方法涉及原生质体形成、原生质体转化、然后细胞壁以其自身已知的方式再生。适合用于曲霉属宿主细胞转化的方法如EP 238 023所述。
在另一方面,本发明涉及一种生产本发明α-淀粉酶变体的方法,该方法包含在有利于变体生产的条件下培养如上所述的宿主细胞以及从细胞和/或培养基中回收变体。
用于培养细胞的培养基可以是适于培养所述宿主细胞以及获得本发明α-淀粉酶变体表达的任何常规培养基。适当的培养基可从供应商处购得,或者可根据公开的配方(例如美国典型培养物保藏中心目录中所述的)制备。
通过众所周知的方法,包括通过离心或过滤分离培养基与细胞,利用诸如硫酸铵等盐沉淀培养基中的蛋白质成分,接着通过利用层析法(chromatography),例如离子交换层析,亲和层析等,可以从培养基中方便地回收宿主细胞分泌的α-淀粉酶变体。
工业应用
本发明的α-淀粉酶变体具有能进行多种工业应用的有价值的特点。具体来说,本发明酶变体适于作为洗衣、碗碟清洗、硬表面清洗的洗涤剂组合物的成分来应用。
具有改变的特性的本发明的变体可用于淀粉处理,具体是淀粉转化,尤其是淀粉液化(参见例如US3,912,590,EP专利申请号252730和63909,WO 99/19467,以及WO 96/28567,所有文献在此并入作为参考)。还涉及的是用于淀粉转化目的的组合物,其除本发明变体之外,还可包含葡糖淀粉酶、支链淀粉酶和其他α-淀粉酶。
此外,本发明的变体还特别用于由淀粉或整谷物/谷粒(whole grain)生产增甜剂和乙醇(参见例如US专利号5,231,017,在此并入作为参考),例如燃料、酒类和工业乙醇。
本发明的变体还可用于纺织品(textile)、织物(fabric)或衣物的退浆(参见例如WO 95/21247,US专利4,643,736,EP 119,920,在此并入作为参考)、啤酒生产或酿造、纸浆和纸生产以及饲料和食品生产。
淀粉转化
常规的淀粉转化处理例如液化核糖化处理描述于如US专利号3,912,590和EP专利申请号252,730和63,909中,在此并入作为参考。
在一个实施方案中,将淀粉降解为低分子量的碳水化合物成分例如糖或脂肪代用品的淀粉转化处理包括脱支步骤。
淀粉至糖的转化
在将淀粉转化为糖的情况下,淀粉被解聚。这样的解聚处理由预处理步骤和两或三步的连续处理步骤(即液化处理、糖化处理和取决于所需最终产物任选的异构化处理)组成。
天然淀粉的预处理
天然淀粉由显微镜下可见的颗粒组成,所述颗粒在室温下不溶于水。当加热淀粉浆水溶液时,颗粒膨胀并最终爆裂,将淀粉分子分散在溶液中。在该“凝胶化”处理过程中,粘度急剧增加了。由于在工业过程中一般固体含量为30-40%,因此淀粉必须变稀或“液化”,以使得其能易于处理。现今,降低粘度主要通过酶促降解进行。
液化
在液化步骤过程中,使用α-淀粉酶长链淀粉降解为支链和线性的短单元(麦芽糖糊精)。液化处理在105-110℃下进行5-10分钟,然后在95℃下进行1-2小时。pH在5.5-6.2之间。为了在这些条件下确保最佳的酶稳定性,添加1mM钙(40ppm游离钙离子)。在该处理后,液化的淀粉应具有10-15的“糖化率”(DE)。
糖化
在液化处理后,通过添加葡糖淀粉酶(如AMG)和诸如异淀粉酶(US专利号4,335,208)或支链淀粉酶(如PromozymeTM)(US专利号4,560,651)的脱支酶将麦芽糖糊精转化为葡萄糖。在该步骤之前,将pH降低到低于4.5,保持高温(高于95℃)以失活液化的α-淀粉酶,以减少称为“潘糖前体”的短寡糖形成,所述“潘糖前体”无法为脱支酶所完全水解。
将温度降低到60℃,并添加葡糖淀粉酶和脱支酶。糖化处理持续进行24-72小时。
正常地,当在液化步骤后变性α-淀粉酶时,约0.2-0.5%的糖化产物是无法为支链淀粉酶所降解的支链三糖62-α-葡糖基麦芽糖(潘糖)。如果在糖化过程中,来自液化步骤的活性淀粉酶存在(即不变性),则该水平可高达1-2%,其是极其不期望的,因为其明显降低了糖化率。
异构化
当期望的最终糖产物是例如高果糖糖浆时,可将葡萄糖糖浆转化为果糖。在糖化处理后,将pH增加到6-8,优选pH7.5,并通过离子交换除去钙。然后使用例如固定化的葡糖异构酶(例如SweetzymeTM IT)将葡萄糖糖浆转化为高果糖糖浆。
乙醇生产
一般而言,由整谷物/谷粒生产乙醇(酒精)可分成4个主要的步骤
-研磨
-液化
-糖化
-发酵
研磨
研磨谷粒以便打开其结构并供进一步处理。所用到的两种处理是湿磨或干磨。在干磨中,完整的谷粒(kernel)被研磨并用于处理的剩余步骤中。湿磨提供了十分良好的胚和粗粉(meal)(淀粉粒和蛋白质)的分离,有些例外的是可用于糖浆的并行生产。
液化
在液化处理中,通过水解为麦芽糖糊精使淀粉粒溶解,大部分的DP高于4。可通过酸处理或α-淀粉酶酶催化进行水解。酸水解在受限的基础上使用。原料可以是磨碎的整谷物/谷粒后来自淀粉处理的侧线馏分(side stream)。
酶促液化通常以三步热淤浆(hot slurry)法来进行。将淤浆加热至60-95℃,优选80-85℃,并添加酶。然后在95-140℃喷射(jet-cooked)加热淤浆,优选105-125℃,冷却至60-95℃,并添加更多的酶以获得最终的水解。液化处理在pH4.5-6.5下进行,通常在pH5-6下进行。磨碎的和液化的谷粒也称为醪液(mash)。
糖化
为产生酵母能代谢的低分子量糖类DP1-3,来自液化的麦芽糖糊精应进一步水解。一般通过葡糖淀粉酶酶催化进行水解,或者可以使用α-葡糖苷酶或酸性α-淀粉酶。完全的糖化步骤可延续72小时,但是,所共有的预糖化通常只进行40-90分钟,然后在发酵过程(SSF)中完成糖化。糖化一般在30-65℃的温度和pH4.5下进行,通常在60℃左右进行。
发酵
将通常来自糖酵母属菌种的酵母添加到醪液中,并发酵24-96小时,例如通常发酵35-60小时,温度在26-34℃,通常在约32℃,以及pH为pH3-6,优选pH4-5左右。
注意到广泛使用的处理是同时进行糖化和发酵(SSF)处理,其中对于糖化来说没有保温(holding)阶段,意味着酵母和酶要同时添加。当在高于50℃的温度下引入预糖化步骤是进行SSF所共有的之时,只要在发酵之前同时添加酵母和酶即可。
蒸馏
发酵后蒸馏醪液以提取乙醇。
根据本发明的方法获得的乙醇可用作为例如燃料乙醇;饮料乙醇即饮料酒精;或工业乙醇。
副产品
发酵中留下的是谷物/谷粒,其通常以液体形式或干燥形式用作为动物饲料。
本领域技术人员众所周知关于如何进行液化、糖化、发酵、蒸馏和乙醇回收的更多细节。
根据本发明的方法,糖化和发酵可同时或独立进行。
纸浆和纸生产
本发明的碱性α-淀粉酶还可用于由淀粉加固(starch reinforced)的废纸和纸板生产木质纤维(lignocellulosic)材料,例如纸浆、纸和纸板,特别是其中在高于7的pH下进行再制浆,并且淀粉酶通过降解加固淀粉而促进废料的分解。本发明的α-淀粉酶能特别用于由涂有浆糊的印刷纸生产造纸纸浆的处理。该处理可如WO 95/14807中所述的进行,包括以下步骤:
a)分解(disintegrating)纸以产生纸浆,
b)在步骤a)之前、过程中或之后,用淀粉降解酶处理,和
c)在步骤a)和b)之后,将油墨(ink)颗粒与纸浆分离。
本发明的α-淀粉酶还可特别用于改性淀粉,其中酶促改性的淀粉与诸如碳酸钙、高岭土(kaolin)和粘土的碱性填料一起用于造纸。使用本发明的碱性α-淀粉酶,有可能在填料存在下改性淀粉,从而可供更简单的综合工艺使用。
纺织品、织物或衣物的退浆
在纺织品、织物或衣物退浆中,本发明的α-淀粉酶还可以是十分有用的。在纺织加工工业中,在退浆处理中α-淀粉酶通常用作为助剂以促进在织布过程中在纬纱(weft yarns)上起保护涂层作用的含淀粉胶料的除去。对于确保要在随后织物被洗炼(scoured)、漂白和染色的处理中获得最好的结果,重要的是在织布后要完全除去胶料涂层。优选的是酶促淀粉分解,因为其对纤维材料不产生任何有害影响。为了降低加工成本并增加工厂生产量,退浆处理往往与洗炼和漂白步骤结合进行。在这样的情况下,非酶助剂例如碱化剂或氧化剂通常用于分解淀粉,因为传统的α-淀粉酶与高pH水平及漂白剂并不非常相容。由于使用了具有相当侵蚀性的化学品,淀粉胶料的非酶分解导致了一些纤维损伤。因此,期望使用本发明的α-淀粉酶,因为它们在碱性溶液中具有改善的特性。当退浆含有纤维素的织物或纺织品时,α-淀粉酶可单独或与纤维素酶结合使用。
退浆和漂白处理是本领域众所周知的。例如,这样的处理描述于WO95/21247、US专利4,643,736、EP 119,920中,在此并入作为参考。
用于退浆的市售产品包括来自Novozymes A/S的AQUAZYME和AQUAZYMEULTRA。
啤酒生产
在啤酒生产过程中,本发明的α-淀粉酶还是十分有用的;α-淀粉酶一般在淀粉糖化过程中添加。
洗涤剂组合物
可添加本发明的α-淀粉酶,并因此成为洗涤剂组合物的成分。
本发明的洗涤剂组合物可例如配制为手洗或机洗衣服的洗涤剂组合物,包括适于染色织物预处理的洗衣添加剂组合物和漂洗添加的织物柔顺剂组合物,或可配制为用于一般家庭硬表面清洁操作的洗涤剂组合物,或可配制用于手洗或机洗碗碟操作。
在一个特定的方面,本发明提供了包含本发明的酶的洗涤添加剂。所述洗涤添加剂以及洗涤剂组合物可包含一种或多种其他的酶,例如蛋白酶,脂肪酶,过氧化物酶,不同的(another)淀粉分解酶,如不同的(another)α-淀粉酶、葡糖淀粉酶、产麦芽糖淀粉酶、CGT酶(CGTase)和/或纤维素酶、甘露聚糖酶(例如来自Novozymes,Denmark的MANNAWAYTM)、果胶酶、果胶裂解酶、角质酶(cutinase)和/或漆酶。
一般而言,所选择的酶的特性应与所选择的洗涤剂相容(即在最适pH下与其他酶或非酶成分相容等),并且所述酶应以有效量存在。
蛋白酶:适合的蛋白酶包括那些动物、植物或微生物来源的蛋白酶。微生物来源的蛋白酶是优选的。包括化学改性或蛋白质工程化的突变体。蛋白酶可以是丝氨酸蛋白酶或金属蛋白酶,优选碱性微生物蛋白酶或胰蛋白酶样蛋白酶。碱性蛋白酶的例子是枯草杆菌蛋白酶,特别是那些来源于芽孢杆菌的枯草杆菌蛋白酶,如枯草杆菌蛋白酶Novo、枯草杆菌蛋白酶Carlsberg、枯草杆菌蛋白酶309、枯草杆菌蛋白酶147和枯草杆菌蛋白酶168(描述于WO 89/06279)。胰蛋白酶样蛋白酶的例子是胰蛋白酶(如猪或牛来源的)以及WO 89/06270和WO 94/25583中描述的镰孢属(Fusarium)蛋白酶。
有用的蛋白酶的例子是WO 92/19729、WO 98/20115、WO 98/20116和WO 98/34946中描述的变体,特别是在一个或多个以下位置具有取代的变体:27、36、57、76、87、97、101、104、120、123、167、170、194、206、218、222、224、235和274。
优选的市售蛋白酶包括ALCALASE、SAVINASE、PRIMASE、DURALASE、ESPERASE和KANNASE(来自Novozymes A/S),MAXATASE、MAXACAL、MAXAPEM、PROPERASE、PURAFECT、PURAFECT OXP、FN2、FN3、FN4(Genencor International Inc.)。
脂肪酶:适合的脂肪酶包括那些细菌或真菌来源的脂肪酶。包括化学改性或蛋白质工程化的突变体。有用的脂肪酶的例子包括来自腐质霉(Humicola)(同义词Thermomyces)的脂肪酶,如来自EP 258 068和EP 305 216中所述的H.lanuginosa(T.lanuginosus)的脂肪酶,或来自WO 96/13580中所述的H.insolens的脂肪酶,假单胞菌属(Pseudomonas)脂肪酶,如来自产碱假单胞菌(P.alcaligenes)或类产碱假单胞菌(P.pseudoalcaligenes)(EP 218272)、洋葱假单胞菌(P.cepacia)(EP 331 376)、施氏假单胞菌(P.stutzeri)(GB1,372,034)、荧光假单胞菌(P.fluorescens)、假单胞菌菌株SD 705(WO95/06720和WO 96/27002)、P.wisconsinensis(WO 96/12012)的脂肪酶,芽孢杆菌属脂肪酶,如来自枯草芽孢杆菌(Dartois等(1993),Biochemica etBiophysica Acta,1131,253-360)、嗜热脂肪芽孢杆菌(JP 64/744992)或短小芽孢杆菌(B.puumilus)(WO 91/16422)的脂肪酶。
其他的例子是脂肪酶变体,例如那些描述于WO 92/05249、WO94/01541、EP 407 225、EP 260 105、WO 95/35381、WO 96/00292、WO95/30744、WO 94/25578、WO 95/14783、WO 95/22615、WO 97/04079和WO 97/07202中的。
优选的市售脂肪酶包括LIPOLASETM和LIPOLASEULTRATM(Novozymes A/S)。
淀粉酶:适合的淀粉酶(α和/或β)包括那些细菌或真菌来源的淀粉酶。包括化学改性或蛋白质工程化的突变体。淀粉酶包括,例如获得自芽孢杆菌,如详述于GB 1,296,839中的地衣芽孢杆菌的特定菌株的α-淀粉酶。有用的α-淀粉酶的例子是WO 94/02597、WO 94/18314、WO 96/23873和WO97/43424中描述的变体,特别是在一个或多个以下位置具有取代的变体:15、23、105、106、124、128、133、154、156、181、188、190、197、202、208、209、243、264、304、305、391、408和444。
市售α-淀粉酶是DURAMYLTM、LIQUEZYMETM、TERMAMYLTM、NATALASETM、SUPRAMYLTM、STAINZYMETM、FUNGAMyLTM和BANTM(Novozymes A/S),RAPIDASETM、PURASTARTM和PURASTAROXAMTM(来自Genencor International Inc.)。
纤维素酶:适合的纤维素酶包括细菌或真菌来源的那些纤维素酶。包括化学改性或蛋白质工程化的突变体。适合的纤维素酶包括来自芽孢杆菌属、假单胞菌属、腐质霉属(Humicola)、镰孢属(Fusarium)、梭孢壳属(Thielavia)、枝顶孢属(Acremonium)的纤维素酶,例如US4,435,307、US5,648,263、US5,691,178、US5,776,757和WO 89/09259中所披露的Humicolainsolens、嗜热毁丝霉(Myceliophthora thermophila)和尖孢镰孢(Fusariumoxysporum)产生的真菌纤维素酶。
特别适合的纤维素酶是具有颜色护理效果的碱性或中性纤维素酶。这样的纤维素酶的例子是描述于EP 0 495 257、EP 0 531 372、WO 96/11262、WO 96/29397、WO 98/08940中的纤维素酶。其他例子是诸如WO 94/07998、EP 0 531 315、US5,457,046、US5,686,593、US5,763,254、WO 95/24471、WO 98/12307和PCT/DK98/00299中描述的那些的纤维素酶变体。
市售纤维素酶包括CELLUZYME和CAREZYME(Novozymes A/S),CLAZINASE和PURADAX HA(Genencor International Inc.)以及KAC-500(B)(Kao Corporation)。
过氧化物酶/氧化酶:适合的过氧化物酶/氧化酶包括那些植物、细菌或真菌来源的过氧化物酶/氧化酶。包括化学改性或蛋白质工程化的突变体。有用的过氧化物酶的例子包括如WO 93/24618、WO 95/10602和WO98/15257中所述的来自鬼伞(Coprinus),如来自灰盖鬼伞(C.cinereus)的过氧化物酶及其变体。
市售过氧化物酶包括GUARDZYME(Novozymes A/S)。
通过添加含有一种或多种酶的分离的添加剂或通过添加包含所有这些酶的组合的添加剂,可将洗涤剂酶包括在洗涤剂组合物中。本发明的洗涤添加剂即分离的添加剂或组合的添加剂可配制成例如颗粒、液体、淤浆等。优选的洗涤添加剂配方是颗粒状的,特别是无粉尘(non-dusting)颗粒,液体的,特别是稳定液体或淤浆的。
可例如US4,106,991和4,661,452中所披露的生产无粉尘颗粒,并可任选地通过本领域已知的方法进行包被。蜡涂层材料的例子是具有1000-20000平均分子量的聚(环氧乙烷)(ethylene oxide)制品(聚乙二醇,PEG);既有16-50个环氧乙烷单元的乙氧基化壬酚;其中醇含有12-20个碳原子以及存在15-80个环氧乙烷单元的乙氧基化脂肪族醇;脂肪族醇;脂肪酸和脂肪酸甘油单酯、二酯和三酯。在GB 1483591中给出了适用于流化床技术的成膜涂层材料的例子。根据所确定的方法,液体酶制剂可以例如通过添加诸如丙二醇的多元醇、糖或糖醇、乳酸或硼酸来稳定。可根据EP 238,216中所披露的方法制备保护的酶。
本发明的洗涤剂组合物可以任何方便的形式存在,如条、片、粉末、颗粒、糊或液体。液体洗涤剂可以是含水的,一般包含高达70%水和0-30%有机溶剂,或是不含水的。
洗涤剂组合物包含可以是非离子型表面活性剂的一种或多种表面活性剂,包括半极性和/或阴离子和/或阳离子和/或两性离子型表面活性剂。按重量计算表面活性剂通常以0.1%-60%的水平存在。
当包括在其中时,洗涤剂应通常含有约1%至约40%的阴离子型表面活性剂,例如直链烷基苯磺酸盐/酯(alkylbenzenesulfonate)、α-烯属磺酸盐/酯、烷基硫酸盐/酯(alkyl sulfate)(脂肪醇硫酸盐/酯(fatty alcohol sulfate))、脂肪醇乙氧基硫酸盐/酯(alcohol ethoxysulfate)、仲烷基磺酸盐/酯(secondaryalkanesulfonate)、α-磺基脂肪酸甲酯(sulfo fatty acid methyl ester)、烷基或烯基琥珀酸或脂肪酸盐(soap)。
当包括在其中时,洗涤剂(detergent)应通常约0.2%至约40%的非离子型表面活性剂,例如脂肪醇乙氧基化物、壬酚乙氧基化物、烷基多糖苷、烷基二甲胺化氧化物(alkyldimethylamine-oxide)、乙氧基化脂肪酸单乙醇酰胺(ethoxylated fatty acid monoethanol-amide)、脂肪酸单乙醇酰胺、多羟基烷基脂肪酸酰胺或葡萄糖胺(“葡糖胺”)的N-酰基N-烷基衍生物。
洗涤剂可含有0-65%的洗涤增效助剂(builder)或络合剂,例如沸石、二磷酸盐/酯、三磷酸盐/酯、膦酸盐/酯(phosphonate)、碳酸盐/酯、柠檬酸盐/酯、次氮基三乙酸(nitrilotriacetic acid)、乙二胺四乙酸、二亚乙基三胺五醋酸、烷基或烯基琥珀酸、可溶性硅酸盐/酯或分层(layered)硅酸盐/酯(如来自Hoechst的SKS-6)。
洗涤剂可包含一种或多种聚合物。例子是羧甲基纤维素、聚(乙烯吡咯烷酮)、聚(乙二醇)、聚(乙烯醇)、聚(乙烯基吡啶-N-氧化物)、聚(乙烯咪唑)、诸如的聚丙烯酸酯的聚羧化物(polycarboxylates)、马来酸/丙烯酸共聚物以及甲基丙烯酸月桂酯/丙烯酸共聚物。
洗涤剂可含有漂白体系,所述漂白体系可包含诸如过硼酸盐/酯或过碳酸盐/酯的H2O2源,所述H2O2源可与诸如四乙酰基乙二胺或壬酰基羟苯磺酸盐/酯(nonanoyloxyben-zenesul-fonate)的过酸形成的漂白活化剂结合。或者,漂白体系可包含例如酰胺、二酰亚胺或砜型过氧酸。
可使用常规稳定剂稳定本发明洗涤剂组合物的酶,所述常规稳定剂例如诸如丙二醇或甘油的多元醇、糖或糖醇、乳酸、硼酸、或硼酸衍生物如芳香族硼酸酯、或苯基硼酸衍生物例如4-甲酰苯基硼酸以及可配制的组合物,如WO 92/19709和WO 92/19708中所述的。
洗涤剂还可含有其他常见的洗涤剂成分,例如包括粘土的织物调节剂、发泡剂(foam boosters)、抑泡剂、防腐剂、污垢悬浮剂、防污垢再沉积剂、染料、杀菌剂、荧光增白剂、水溶助长剂、晦暗抑制剂或香料。
预计任何酶都能存在于洗涤剂组合物中,特别是本发明的酶,可以相应于每升洗涤液0.001-100mg酶类蛋白质的量添加,优选以每升洗涤液0.005-5mg酶类蛋白质,更优选以每升洗涤液0.01-1mg酶类蛋白质,以及特别是以每升洗涤液0.1-1mg酶类蛋白质的量添加。
本发明的酶可额外地掺入到WO 97/07202中披露的洗涤剂配方中,其在此并入作为参考。
碗碟清洗洗涤剂组合物
本发明的酶还可用于碗碟清洗洗涤剂组合物中,包括以下:
1)粉末自动碗碟清洗组合物
非离子型表面活性剂 | 0.4-2.5% |
偏硅酸钠 | 0-20% |
二硅酸钠 | 3-20% |
三磷酸钠 | 20-40% |
碳酸钠 | 0-20% |
高硼酸钠 | 2-9% |
四乙酰基乙二胺(TAED) | 1-4% |
硫酸钠 | 5-33% |
酶类 | 0.0001-0.1% |
2)粉末自动碗碟清洗组合物
非离子型表面活性剂(如脂肪醇乙氧基化物) | 1-2% |
二硅酸钠 | 2-30% |
碳酸钠 | 10-50% |
膦酸钠 | 0-5% |
柠檬酸三钠脱水物 | 9-30% |
次氮基三乙酸钠(NTA) | 0-20% |
高硼酸钠一水合物 | 5-10% |
四乙酰基乙二胺(TAED) | 1-2% |
聚丙烯酸酯聚合物(如马来酸/丙烯酸共聚物) | 6-25% |
酶类 | 0.0001-0.1% |
香料 | 0.1-0.5% |
水 | 5-10 |
3)粉末自动碗碟清洗组合物
非离子型表面活性剂 | 0.5-2.0% |
二硅酸钠 | 25-40% |
柠檬酸钠 | 30-55% |
碳酸钠 | 0-29% |
碳酸氢钠 | 0-20% |
高硼酸钠一水合物 | 0-15% |
四乙酰基乙二胺(TAED) | 0-6% |
马来酸/丙烯酸共聚物 | 0-5% |
粘土 | 1-3% |
聚氨基酸 | 0-20% |
聚丙烯酸钠 | 0-8% |
酶类 | 0.0001-0.1% |
4)粉末自动碗碟清洗组合物
非离子型表面活性剂 | 1-2% |
沸石MAP | 15-42% |
二硅酸钠 | 30-34% |
柠檬酸钠 | 0-12% |
碳酸钠 | 0-20% |
高硼酸钠一水合物 | 7-15% |
四乙酰基乙二胺(TAED) | 0-3% |
聚合物 | 0-4% |
马来酸/丙烯酸共聚物 | 0-5% |
有机膦酸盐 | 0-4% |
粘土 | 1-2% |
酶类 | 0.0001-0.1% |
硫酸钠 | 余量 |
5)粉末自动碗碟清洗组合物
非离子型表面活性剂 | 1-7% |
二硅酸钠 | 18-30% |
柠檬酸三钠 | 10-24% |
碳酸钠 | 12-20% |
单过硫酸盐(2KHSO5.KHSO4.K2SO4) | 15-21% |
漂白稳定剂 | 0.1-2% |
马来酸/丙烯酸共聚物 | 0-6% |
二亚乙基三胺五醋酸盐,五钠盐 | 0-2.5% |
酶类 | 0.0001-0.1% |
硫酸钠,水 | 余量 |
6)带有清洁表面活性剂体系的粉末和液体碗碟清洗组合物
非离子型表面活性剂 | 0-1.5% |
十八烷基二甲胺N-氧化物脱水物 | 0-5% |
80∶20wt.C18/C16混合的十八烷基二甲胺N-氧化物二水合物和十六烷基二甲胺N-氧化物脱水物 | 0-4% |
70∶30wt.C18/C16混合的无水的十八烷基二(羟乙基)胺N-氧化物和无水的十六烷基二(羟乙基)胺N-氧化物 | 0-5% |
具有3的平均乙氧基化度的C13-C15烷基乙氧基硫酸盐/酯 | 0-10% |
具有3的平均乙氧基化度的C12-C15烷基乙氧基硫酸盐/酯 | 0-5% |
具有12的平均乙氧基化度的C13-C15乙氧基化醇 | 0-5% |
具有9的平均乙氧基化度的C12-C15乙氧基化醇混合物 | 0-6.5% |
具有30的平均乙氧基化度的C13-C15乙氧基化醇混合物 | 0-4% |
二硅酸钠 | 0-33% |
三磷酸钠 | 0-46% |
柠檬酸钠 | 0-28% |
柠檬酸 | 0-29% |
碳酸钠 | 0-20% |
高硼酸钠一水合物 | 0-11.5% |
四乙酰基乙二胺(TAED) | 0-4% |
马来酸/丙烯酸共聚物 | 0-7.5% |
硫酸钠 | 0-12.5% |
酶类 | 0.0001-0.1% |
7)非水液体自动碗碟清洗组合物
液体非离子型表面活性剂(如脂肪醇乙氧基化物) | 2.0-10.0% |
碱金属硅酸盐 | 3.0-15.0% |
碱金属磷酸盐 | 20.0-40.0% |
选自高级乙二醇、聚乙二醇、多氧化物、乙二醇醚的液体载体 | 25.0-45.0% |
稳定剂(如磷酸偏酯和C16-C18链烷醇) | 0.5-7.0% |
抑泡剂(如硅氧烷) | 0-1.5% |
酶类 | 0.0001-0.1% |
8)非水液体碗碟清洗组合物
液体非离子型表面活性剂(如脂肪醇乙氧基化物) | 2.0-10.0% |
硅酸钠 | 3.0-15.0% |
碱金属碳酸盐 | 7.0-20.0% |
柠檬酸钠 | 0.0-1.5% |
稳定体系(如细碎的硅氧烷(silicone)和低分子量二烃基聚乙二醇醚的混合物) | 0.5-7.0% |
低分子量聚丙烯酸酯聚合物 | 5.0-15.0% |
粘土胶体增稠剂(如膨润土(bentonite)) | 0.0-10.0% |
羟丙基纤维素聚合物 | 0.0-0.6% |
酶类 | 0.0001-0.1% |
选自高级乙二醇、聚乙二醇、多氧化物和乙二醇醚的液体载体 | 余量(Balance) |
9)触变性液体自动碗碟清洗组合物
C12-C14脂肪酸 | 0-0.5% |
嵌段共聚物表面活性剂 | 1.5-15.0% |
柠檬酸钠 | 0-12% |
三聚磷酸钠(sodium tripolyphosphate) | 0-15% |
碳酸钠 | 0-8% |
三硬脂酸铝 | 0-0.1% |
异丙基苯磺酸钠 | 0-1.7% |
聚丙烯酸酯增稠剂 | 1.32-2.5% |
聚丙烯酸钠 | 2.4-6.0% |
硼酸 | 0-4.0% |
甲酸钠 | 0-0.45% |
甲酸钙 | 0-0.2% |
正癸基二苯醚二磺酸钠(Sodium n-decydiphenyloxide disulphonate) | 0-4.0% |
单乙醇胺(MEA) | 0-1.86% |
氢氧化钠(50%) | 1.9-9.3% |
1,2-丙二醇 | 0-9.4% |
酶 | 0.0001-0.1% |
抑泡剂,染料,香料,水 | 余量 |
10)液体自动碗碟清洗组合物
脂肪醇乙氧基化合物 | 0-20% |
脂肪酸磺酸酯 | 0-30% |
十二烷基硫酸钠 | 0-20% |
烷基聚糖苷 | 0-21% |
油酸 | 0-10% |
二硅酸钠一水合物 | 18-33% |
柠檬酸钠脱水物 | 18-33% |
硬脂酸钠 | 0-2.5% |
高硼酸钠一水合物 | 0-13% |
四乙酰基乙二胺(TAED) | 0-8% |
马来酸/丙烯酸共聚物 | 4-8% |
酶类 | 0.0001-0.1% |
11)含有保护的漂白粒子的液体自动碗碟清洗组合物
硅酸钠 | 5-10% |
焦磷酸四钾 | 15-25% |
三磷酸钠 | 0-2% |
碳酸钾 | 4-8% |
保护的漂白粒子,如氯 | 5-10% |
聚合增稠剂 | 0.7-1.5% |
氢氧化钾 | 0-2% |
酶类 | 0.0001-0.1% |
水 | 余量 |
12)如1)、2)、3)、4)、6)和10)中所述的自动碗碟清洗组合物,其中过硼酸盐/酯为过碳酸盐/酯所取代。
13)如1)-6)中所述的自动碗碟清洗组合物,其还含有锰催化剂。锰催化剂可例如是″Efficient manganese catalysts for low-temperature bleaching″,Nature 369,1994,pp.637-639.中描述的化合物的一种。
原料和方法
酶:
LE174:杂合α-淀粉酶:
LE174是一种杂合Termamyl-样α-淀粉酶,除N-末端35个氨基酸残基(成熟蛋白的)已为BAN(成熟蛋白)(即SEQ ID NO:6所示的解淀粉芽孢杆菌α-淀粉酶)的N-末端33个残基所取代之外,其与Termamyl序列(即SEQ IDNO:4所示的地衣芽孢杆菌α-淀粉酶)一致,其进一步具有以下突变:
H156Y+A181T+N190F+A209V+Q264S(SEQ ID NO:4).
LE429杂合α-淀粉酶变体:
LE429是一种杂合Termamyl-样α-淀粉酶,除N-末端35个氨基酸残基(成熟蛋白的)已为BAN(成熟蛋白)(即SEQ ID NO:6所示的解淀粉芽孢杆菌α-淀粉酶)的N-末端33个残基所取代之外,其与Termamyl序列(即SEQ IDNO:4所示的地衣芽孢杆菌α-淀粉酶)一致,其进一步具有以下突变:
H156Y+A181T+N190F+A209V+Q264S+I201F(SEQ ID NO:4)。LE429示于SEQ ID NO:2,并通过SOE-PCR(Higuchi等1988,Nucleic Acids Research16:7351)构建。
来源于黑曲霉的葡糖淀粉酶具有示于WO 00/04136中的氨基酸序列,如SEQ ID No:2或所披露的变体之一。
来源于黑曲霉的酸性真菌α-淀粉酶。
底物:
获得自Sigma-Aldrich的小麦淀粉(S-5127)。
发酵和α-淀粉酶变体的纯化
在来自-80℃储存的具有10μg/ml卡那霉素的LB-琼脂平板上划线接种携有相关的表达质粒的枯草芽孢杆菌菌株,并在37℃下培养过夜。
将克隆转移入在500ml摇瓶中的补充有10μg/ml卡那霉素的100mlBPX培养基中。
BPX培养基成分:
马铃薯淀粉 100g/l
大麦粉 50g/l
BAN 5000 SKB 0.1g/l
酪蛋白酸钠 10g/l
大豆粉 20g/l
Na2HPO4,12H2O 9g/l
PluronicTM 0.1g/l
培养物在37℃下以270rpm振荡培养5天。
通过以4500rpm离心20-25分钟,从发酵培养液中除去细胞和细胞碎片。之后过滤上清液以获得完全清澈的溶液。在UF-过滤器(10000截留分子量膜)上浓缩并洗涤滤液,并将缓冲液改变为20mM醋酸,pH5.5。将UF-滤液加在S-sepharose F.F.上,通过使用在相同缓冲液中的0.2M NaCl一步洗脱来进行洗脱。对10mM Tris,pH9.0透析洗脱物并加在Q-sepharose F.F.上,用超过6倍柱体积的0-0.3M NaCl的线性梯度进行洗脱。汇集合有活性(通过Phadebas测定法测定的)的级分,调节pH至pH7.5并在5分钟内通过用0.5%W/V活性炭处理除去残留的颜色。
活性测定(KNU)
可使用马铃薯淀粉作为底物测定淀粉分解活性。该方法是基于用酶分解改性马铃薯淀粉,并接着通过将淀粉/酶溶液的样品与碘溶液混合进行反应。最初,与有色玻璃标准相比,出现稍黑的蓝色,但在淀粉分解过程中,蓝色变浅并逐渐地变为红棕色。
1000个Novoα淀粉酶单位(KNU)规定为在标准条件(即在37℃+/-0.05;0.0003M Ca2+;和pH5.6)下,糊精化5.26g淀粉干物质——Merck可溶性淀粉(Amylum solubile)的酶的量。
文件夹AF 9/6更详细地描述了该分析方法,函索Novozymes A/S,Denmark即可获得,该文件夹在此并入作为参考。
葡糖淀粉酶活性(AGU)
1个Novo葡糖淀粉酶单位(AGU)规定为在37℃和pH4.3下,每分钟水解1微摩尔麦芽糖的酶的量。
通过在使用来自Boehringer Mannheim,124036的葡萄糖GOD-Perid试剂盒之后改良的方法(AEL-SM-0131,函索Novozymes即可获得)将该活性确定为AGU/ml。标准:AMG-标准,批号7-1195,195 AGU/ml。 375μL底物(在50mM醋酸钠,pH4.3中的1%麦芽糖)在37℃下温育5分钟。添加在醋酸钠中稀释的25μL酶。在10分钟后通过添加100μL 0.25M NaOH停止反应。将20μL转移到96孔微量滴定板中,并添加200μL GOD-Perid溶液(124036,Boehringer Mannheim)。在室温下30分钟后,在650nm处测定吸光度,并根据AMG-标准以AGU/ml计算活性。更详细地描述该分析方法的文件夹(AEL-SM-0131)函索Novozymes A/S,Denmark即可获得,该文件夹在此并入作为参考。
酸性α-淀粉酶活性(AFAU)
酸性α-淀粉酶活性可以以AFAU(酸性真菌α-淀粉酶单位)测定,其中是相对于酶标准进行测定的。
所使用的标准是AMG 300L(来自Novozymes A/S,葡糖淀粉酶野生型黑曲霉G1,也披露于Boel等(1984),EMBO J.3(5),p.1097-1102和WO 92/00381中)。在该AMG中的中性α-淀粉酶在室温下贮藏3周后从大约1FAU/mL降低到低于0.05FAU/mL。
根据以下描述测定该AMG标准中的酸性α-淀粉酶活性。在该方法中,1 AFAU规定为在标准条件下,每小时降解5.26mg淀粉干物质固形物(drysolids)的酶的量。
碘与淀粉形成蓝色络合物,但不与其降解产物形成蓝色络合物。因此,颜色强度与淀粉浓度成正比。使用在指定分析条件下作为淀粉浓度减少的逆比色法测定淀粉酶活性。
α-淀粉酶
淀粉+碘 ? 糊精+寡糖
40℃,pH 2.5
蓝色/紫色 t=23秒. 脱色
标准条件/反应条件:(每分钟)
底物: 淀粉,大约0.17g/L
缓冲液: 柠檬酸盐,大约0.03M
碘(I2): 0.03 g/L
CaCl2: 1.85mM
pH: 2.50-0.05
温育温度: 40℃
反应时间: 23秒
波长: λ=590nm
酶浓度: 0.025 AFAU/mL
酶工作范围:0.01-0.04 AFAU/mL
更多细节优选这些能见于函索Novozymes A/S即可获得的EB-SM-0259.02/01中的,在此并入作为参考。
糖分布和溶解的干固形物的测定
通过HPLC测定淀粉水解产物的糖成分,随后计算葡萄糖产率为DX.°BRIX,并通过折光率(refractive index)测定法测定淀粉水解产物的溶解(可溶)干固形物。
α-淀粉酶活性的测定
通过使用Phadebas药片作为底物的方法测定α-淀粉酶活性。Phadebas药片(Phadebas淀粉酶检验标准,提供自Pharmacia Diagnostic)含有交联的不溶性蓝色淀粉聚合物,其已与牛血清白蛋白和缓冲物质混合并压片。
对于每一次测定,将一片悬浮于含有5ml 50mM Britton-Robinson缓冲液(50mM醋酸,50mM磷酸,50mM硼酸,0.1mM CaCl2,用NaOH调节pH至所需值)的试管中。于所需温度下在水浴中进行该测试。在x ml的50mMBritton-Robinson缓冲液中稀释要被测试的α-淀粉酶。将1ml该α-淀粉酶溶液添加到5ml 50mM Britton-Robinson缓冲液中。通过α-淀粉酶释放出可溶的蓝色片段水解淀粉。在620nm处用分光光度计测定所得到的蓝色溶液的吸光度,其是α-淀粉酶活性的函数。
重要的是在10-15分钟的温育(测试时间)后测定的620nm吸光度要在0.2-2.0的620nm处吸光度单位范围内。在该吸光度范围内,在活性和吸光度之间存在线性(朗伯-比尔(Lambert-Beer)定律)。因此应调节酶的稀释以适合该标准。在给定的一组条件(温度、pH、反应时间、缓冲液条件)下,1mg给定的α-淀粉酶应水解一定量的底物并会产生蓝色。在620nm处测定颜色强度。在给定组的条件下,所测定的吸光度与所述α-淀粉酶的比活性(纯的α-淀粉酶蛋白的每毫克活性)成正比。
测定比活性
使用Phadebas测定法(Pharmacia)测定比活性为活性/毫克酶。
测定pH活性分布(pH稳定性)
将变体贮藏在20mM TRIS pH7.5,0.1mM,CaCl2中,并在30℃下,50mM Britton-Robinson,0.1mM CaCl2中测试。使用上述Phadebas测定法,在pH4.0、4.5、5.0、5.5、6.0、7.0、8.0、9.0(9.5)、9.5、10和10.5下测定pH活性。
实施例
实施例1
构建Termamyl变体LE429
在枯草芽孢杆菌中由称为pDN1528的质粒表达Termamyl(地衣芽孢杆菌α-淀粉酶SEQ ID NO:4)。该质粒含有编码Termamyl的完整基因,其表达受其自己的启动子amyL所指导。此外,该质粒含有来自于质粒pUB110的复制起点ori,和赋予对氯霉素抗性的来自于质粒pC194的cat基因。pDN1528示于WO 96/23 874的图9中。制备一种含有SEQ ID NO:3编码区主要部分的特定的诱变载体。称为pJeEN1的该载体的主要特征包括来自于pUC质粒的复制起点,赋予对氯霉素抗性的cat基因以及bla基因的含有移码的变型,其野生型通常赋予对氨苄青霉素的抗性(ampR表型)。该突变型产生ampS表型。质粒pJeEN1示于WO 96/23874的图10中,大肠杆菌复制起点ori、bla、cat、Termamyl淀粉酶基因的5′-截短变型以及所选择的限制酶切位点指示在质粒上。
除具有掺入的“选择引物”(引物#6616,参见以下)的质粒是基于含有具有修复的bla基因的质粒转化的大肠杆菌细胞ampR表型进行选择,而不使用Deng和Nickoloff所概述的限制性内切酶消化选择之外,通过Deng和Nickoloff(1992,Anal.Biochem.200,pp.81-88)所述的方法将突变导入amyL。用于该诱变的化学品和酶获得自Stratagene的Chameleon诱变试剂盒(目录号200509)。
在检验变体质粒中的DNA序列之后,将含有期望的改变的截短的基因作为PstI-EcoRI片段亚克隆入pDN1528,并转化入蛋白酶-和淀粉酶-缺失的枯草芽孢杆菌菌株SHA273(描述于WO 92/11357和WO 95/10603)中,以便表达变体酶。
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体V54W:
PG GTC GTA GGC ACC GTA GCC CCAATC CGC TTG(SEQ ID NO:9)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体A52W+V54W:
PG GTC GTA GGC ACC GTA GCC CCAATC CCA TTG GCT CG(SEQ IDNO:10)
引物#6616(5′到3′从左到右书写;P表示5′磷酸):
P CTG TGA CTG GTG AGTACT CAA CCAAGT C(SEQ ID NO:11)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体V54E:
PGG TCG TAG GCA CCG TAG CCC TCA TCC GCT TG(SEQ ID NO:12)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体V54M:
PGG TCG TAG GCA CCG TAG CCC ATA TCC GCT TG(SEQ ID NO:13)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体V54I:
PGG TCG TAG GCA CCG TAG CCAATA TCC GCT TG(SEQ ID NO:14)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体Y290E和Y290K:
PGC AGC ATG GAA CTG CTY ATG AAG AGG CAC GTC AAA C(SEQID NO:15)
Y表示C和T的等量混合。通过DNA测序验证编码第290位的谷氨酸或赖氨酸的密码子的存在。
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体N190F:
PCA TAG TTG CCG AAT TCA TTG GAA ACT TCC C(SEQ ID NO:16)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体N188P+N190F:
PCA TAG TTG CCG AAT TCA GGG GAA ACT TCC CAA TC(SEQ IDNO:17)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体H140K+H142D:
PCC GCG CCC CGG GAA ATC AAA TTT TGT CCA GGC TTT AAT TAG(SEQ ID NO:18)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体H156Y:
PCA AAA TGG TAC CAA TAC CAC TTA AAA TCG CTG(SEQ ID NO:19)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体A181T:
PCT TCC CAA TCC CAA GTC TTC CCT TGA AAC(SEQ ID NO:20)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体A209V:
PCTT AAT TTC TGC TAC GAC GTC AGG ATG GTC ATA ATC(SEQ IDNO:21)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体Q264S:
PCG CCC AAG TCA TTC GAC CAG TAC TCA GCT ACC GTA AAC(SEQ ID NO:22)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体S187D:
PGC CGT TTT CAT TGT CGA CTT CCC AAT CCC(SEQ ID NO:23)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体DELTA(K370-G371-D372)(即缺失氨基酸残基370、371和372):
PGG AAT TTC GCG CTG ACT AGT CCC GTA CAT ATC CCC(SEQ IDNO:24)
通过使用以下诱变引物(5′到3′从左到右书写)构建Termamyl变体DELTA(D372-S373-Q374):
PGG CAG GAA TTT CGC GAC CTT TCG TCC CGT ACA TAT C(SEQID NO:25)
通过用切割pDN1528-样质粒两次产生1116bp片段和3850bp载体部分(即含有质粒复制起点)的限制性内切酶ClaI消化含有A181T的pDN1528-样质粒(即在amyL中含有引起A181T改变的突变的pDN1528)和含有A209V的pDN1528-样质粒(即在amyL中含有引起A209V改变的突变的pDN1528),将Termamyl变体A181T和A209V结合为A181T+A209V。在琼脂糖凝胶上分离后,通过QIAquick凝胶抽提试剂盒(购自QIAGEN)纯化含有A209V突变的片段和含有A181T突变的载体部分。连接片段和载体并转化入上述提及的蛋白酶和淀粉酶缺失的枯草芽孢杆菌菌株。对来自amy+(在含有淀粉的琼脂平板上亮区)和氯霉素抗性的转化体的质粒分析质粒上两种突变的存在。
以类似于上述的方法,使用限制性内切酶Acc65I和EcoRI结合H156Y和A209V,给出H156Y+A209V。
通过使用限制性内切酶Acc65I和HindIII将H156Y+A209V和A181T+A209V结合为H156Y+A181T+A209V。
利用SOE-PCR方法(Higuchi等1988,Nucleic Acids Research16:7351),Termamyl变体H156Y+A181T+A209V成熟部分的35个N-末端残基被解淀粉芽孢杆菌α-淀粉酶(SEQ ID NO:4)(在本发明上下文中称为BAN)的33个N-末端残基所取代,如下:
引物19364(序列5’-3’):CCT CAT TCT GCA GCA GCA GCC GTA AATGGC ACG CTG(SEQ ID NO:26)
引物19362:CCA GAC GGC AGT AAT ACC GAT ATC CGA TAA ATGTTC CG(SEQ ID NO:27)
引物19363:CGG ATATCG GTA TTA CTG CCG TCT GGA TTC(SEQ IDNO:28)
引物1C:CTC GTC CCA ATC GGT TCC GTC(SEQ ID NO:29)
根据厂商的说明书,使用来自Boehringer Mannheim的Pwo耐热聚合酶进行标准PCR(聚合酶链反应),温度循环:94℃ 5分钟,25个循环(94℃ 30秒、50℃ 45秒、72℃ 1分钟),72℃ 10分钟。
在称为PCR1的第一次PCR中,使用引物19364和19362,对含有编码解淀粉芽孢杆菌α-淀粉酶的基因的DNA片段扩增出大约130bp片段。
在称为PCR2的另一次PCR中,使用引物19363和1C,对模板pDN1528扩增出大约400bp片段。
PCR1和PCR2纯化自琼脂糖凝胶并在使用引物19364和1C的PCR3中被用作为模板,得到大约520bp的片段。因此,该片段含有融合至编码从第35个氨基酸起的Termamy的DNA的一部分的编码来自BAN的N-末端的DNA的一部分。
通过用限制性内切酶PstI和SacII消化、连接并如先前所述的转化枯草芽孢杆菌菌株,将520bp片段亚克隆入pDN1528-样质粒(含有编码Termamyl变体H156Y+A181T+A209V的基因)中。通过在提取的来自amy+和氯霉素抗性转化体的质粒中进行DNA测序验证在限制酶切位点PstI和SacII之间的DNA序列。
最终的构建体含有来自BAN的正确N-末端和H156Y+A181T+A209V,称为BAN(1-35)+H156Y+A181T+A209V。
除pJeEN1中的amyL序列为编码Termamyl变体BAN(1-35)+H156Y+A181T+A209V的DNA序列所取代之外,通过如上所述进行诱变,将N190F与BAN(1-35)+H156Y+A181T+A209V结合,给出BAN(1-35)+H156Y+A181T+N190F+A209V。
除pJeEN中的amyL序列为编码Termamyl变体BAN(1-35)+H156Y+A181T+A209V的DNA序列所取代之外,通过如上所述进行诱变,将Q264S与BAN(1-35)+H156Y+A181T+A209V结合,给出BAN(1-35)+H156Y+A181T+A209V+Q264S。
利用限制性内切酶BsaHI(在A209V突变附近导入BsaHI位点)和PstI,将BAN(1-35)+H156Y+A181T+A209V+Q264S和BAN(1-35)+H156Y+A181T+N190F+A209V结合入BAN(1-35)+H156Y+A181T+N190F+A209V+Q264S。
通过如上所述进行诱变,将I201F与BAN(1-35)+H156Y+A181T+N190F+A209V+Q264S结合,给出BAN(1-35)+H156Y+A181T+N190F+A209V+Q264S+I201F(SEQ ID NO:2)。使用诱变引物AM100,导入I201F取代并同时除去ClaI限制酶切位点,其利于容易鉴别突变。
引物AM100:
5’GATGTATGCCGACTTCGATTATGACC 3’(SEQ ID NO:30)
实施例2
构建具有改变的淀粉亲和力的Termamyl-样α-淀粉酶变体
构建LE1153 (LE429+R437W):
通过PCR,使用结合淀粉酶基因下游的载体引物CAAX37和诱变引物CAAX447扩增来自pDN1528-样质粒(在编码来自SEQ ID NO:4的淀粉酶的基因中具有BAN(1-35)+H156Y+A181T+N190F+I201F+A209V+Q264S突变)的大约450bp DNA片段。
450bp片段纯化自琼脂糖凝胶并用作为大(mega)引物与引物1B一起对相同的模板进行在第二PCR。
用限制性内切酶Pst I和Avr II消化所得到的大约1800bp片段,纯化所得到的大约1600bp DNA片段并与用相同的酶消化的pDN1528-样质粒连接。用连接和氯霉素抗性的转化体转化感受态枯草芽孢杆菌SHA273(低淀粉酶和蛋白酶)细胞,并通过DNA测序检查以验证质粒上正确突变的存在。
引物CAAX37:
5’CTCATGTTTGACAGCTTATCATCGATAAGC 3’(SEQ ID NO:31)
引物1B:
5’CCGATTGCTGACGCTGTTATTTGC 3’(SEQ ID NO:32)
引物CAAX447:
5’CCCGGTGGGGCAAAGTGGATGTATGTCGGCCGG 3’(SEQ ID NO:33)
构建LE1154:
除使用两种诱变引物CAAX447和CAAX448之外,以类似的方法构建BAN/Termamyl杂合+H156Y+A181T+N190F+A209V+Q264S+[R437W+E469N]。
引物CAAX448:
5’CGGAAGGCTGGGGAAATTTTCACGTAAACGGC 3’(SEQ ID NO:34)
实施例3
构建具有改变的淀粉亲和力的BAN-样α-淀粉酶变体:(R176*+G177*)
BAN(解淀粉芽孢杆菌α-淀粉酶SEQ ID NO:6)由类似于描述于实施例1中的pDN1528的质粒在枯草芽孢杆菌中表达。该称为pCA330-BAN的BAN质粒含有取代限定为SEQ ID NO:4中的氨基酸1-483的编码地衣芽孢杆菌α-淀粉酶成熟部分的基因的限定为SEQ ID NO:6中的氨基酸1-483的编码BAN成熟部分的基因。
解淀粉芽孢杆菌α-淀粉酶的变体示于SEQ ID NO:2,包含两个氨基酸缺失R176和G177以及N190F取代(使用SEQ ID NO:6中的编号),与野生型解淀粉芽孢杆菌α-淀粉酶相比,具有改善的稳定性。该变体在下文中称为BAN-var003。
为改善所述α-淀粉酶变体的淀粉亲和力和水解能力,使用Sarkar和Sommer,1990(BioTechniques 8:404-407)所述的大引物方法进行位点定向诱变:
构建BE1001:BAN-var003+R437W:
通过PCR,使用结合淀粉酶基因下游的载体引物CAAX37和诱变引物CABX437扩增来自pCA330-BAN质粒(在编码来自SEQ ID NO:6的淀粉酶的基因中含有BAN-var003突变)的大约450bp DNA片段。
450bp片段纯化自琼脂糖凝胶并用作为大引物与引物1B一起对相同的模板进行第二PCR。
用限制性内切酶Pst I和Avr II消化所得到的大约1800bp片段,纯化所得到的大约1600bp DNA片段并与用相同的酶消化的pCA330-样质粒连接。用连接和氯霉素抗性的转化体转化感受态枯草芽孢杆菌SHA273(低淀粉酶和蛋白酶)细胞,并通过DNA测序检查以验证质粒上正确突变的存在。
引物CABX437:
5’GGTGGGGCAAAGTGGATGTATGTCGGC 3’(SEQ ID NO:35)
构建BE1004:
除使用两种诱变引物CABX437和CABX438之外,以类似方法构建BAN-var003淀粉酶+[R437W+E469N]。
CABX438:
5’GGAAGGCTGGGGAAACTTTCACGTAAACG3’(SEQ ID NO:36)
实施例4
Termamyl LC对LE1153和LE1154
该实施例说明了与Termamyl LC相比,使用根据本发明的细菌α-淀粉酶(LE1153和LE1154)将粒状小麦淀粉转化为葡萄糖。
通过在搅拌下将247.5g小麦淀粉添加到502.5ml水中,制备具有33%干固形物(DS)粒状淀粉的淤浆。用HCl将pH调节到4.5。将粒状淀粉浆分配在100ml锥形烧瓶中,每瓶75g。在60℃水浴中温育烧瓶,带有磁力搅拌。在0小时处,将表1中所给出的酶活性剂量给予烧瓶。在24、48和73以及94小时后提取样品。
表1.使用的酶活性水平
α-淀粉酶+/-取代KNU/kg DS | 葡糖淀粉酶AGU/kg DS | 酸性真菌α-淀粉酶AFAU/kgDS |
100.0 | 200 | 50 |
使用以下方法测定总的干固形物淀粉。通过添加过量的α-淀粉酶(300KNU/kg干固形物)并将样品置于95℃油浴中45分钟完全水解淀粉。随后将样品冷却至60℃,并添加过量的葡糖淀粉酶(600AGU/kg DS),接着在60℃下温育2小时。
在通过0.22微米过滤器过滤后,通过折光率测定法对样品测定淀粉水解产物中的可溶性干固形物。通过HPLC测定糖分布。葡萄糖的量计算为DX。结果示于表2和3中。
表2.在100KNU/kg DSα-淀粉酶用量下,可溶性干固形物占总的干物质的百分数
酶 | 24小时 | 48小时 | 73小时 | 94小时 |
Termamyl LCLE1153LE1154 | 83.788.386.7 | 8791.290.3 | 89.793.291.9 | 90.394.693.0 |
表3.在100KNU/kg DS α-淀粉酶用量下,可溶性水解产物的DX
酶 | 24小时 | 48小时 | 73小时 | 94小时 |
Termamyl LCLE1153LE1154 | 72.077.174.0 | 82.087.186.6 | 83.888.487.8 | 83.888.587.8 |
实施例5
BAN对R437W变体
该实施例说明了与BAN WT相比,使用根据本发明的BAN R437W变体的细菌α-淀粉酶将粒状小麦淀粉转化为葡萄糖。
通过在搅拌下将247.5g小麦淀粉添加到502.5ml水中,制备具有33%干固形物(DS)粒状淀粉的淤浆。用HCl将pH调节到4.5。将粒状淀粉浆分配在100ml锥形烧瓶中,每瓶75g。在60℃水浴中温育烧瓶,带有磁力搅拌。在0小时处,将表1中所给出的酶活性给予烧瓶。在24、48和73以及94小时后提取样品。
表1.使用的酶活性水平
α-淀粉酶+/-取代KNU/kg DS | 葡糖淀粉酶AGU/kg DS | 酸性真菌α-淀粉酶AFAU/kgDS |
100.0 | 200 | 50 |
使用以下方法测定总的干固形物淀粉。通过添加过量的α-淀粉酶(300KNU/kg干固形物)并将样品置于95℃油浴中45分钟完全水解淀粉。随后将样品冷却至60℃,并添加过量的葡糖淀粉酶(600AGU/kg DS),接着在60℃下温育2小时。
在通过0.22微米过滤器过滤后,通过折光率测定法对样品测定淀粉水解产物中的可溶性干固形物。通过HPLC测定糖分布。葡萄糖的量计算为DX。结果示于表4和5中。
表4.在100KNU/kg DSα-淀粉酶用量下,可溶性干固形物占总的干物质的百分数
酶 | 96小时 |
BAN WT变体R437W | 95.695.8 |
表5.在100KNU/kg DSα-淀粉酶用量下,可溶性水解产物的DX
酶 | 96小时 |
BAN WT变体R437W | 92.3892.52 |
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序列表
<110>诺维信公司(Novozymes A/S)
<120>α-淀粉酶变体
<130>
<160>40
<170>PatentIn Ver.2.1
<210>1
<211>1443
<212>DNA
<213>解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<220>
<221>CDS
<222>(1)..(1143)
<400>1
gta aat ggc acg ctg atg cag tat ttt gaa tgg tat acg ccg aac gac 48
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
ggc cag cat tgg aaa cga ttg cag aat gat gcg gaa cat tta tcg gat 96
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
atc ggt att act gcc gtc tgg att ccc ccg gca tat aag gga acg agc 144
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser
35 40 45
caa gcg gat gtg ggc tac ggt gct tac gac ctt tat gat tta ggg gag 192
Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc aca aaa gga gag 240
Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu
65 70 75 80
ctg caa tct gcg atc aaa agt ctt cat tcc cgc gac att aac gtt tac 288
Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr
85 90 95
ggg gat gtg gtc atc aac cac aaa ggc ggc gct gat gcg acc gaa gat 336
Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp
100 105 110
gta acc gcg gtt gaa gtc gat ccc gct gac cgc aac cgc gta att tca 384
Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser
115 120 125
gga gaa cac cta att aaa gcc tgg aca cat ttt cat ttt ccg ggg cgc 432
Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg
130 135 140
ggc agc aca tac agc gat ttt aag tgg tat tgg tac cat ttt gac gga 480
Gly Ser Thr Tyr Ser Asp Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly
145 150 155 160
acc gat tgg gac gag tcc cga aag ctg aac cgc atc tat aag ttt caa 528
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln
165 170 175
ggg aag act tgg gat tgg gaa gtt tcc aat gaa ttc ggc aac tat gat 576
Gly Lys Thr Trp Asp Trp Glu Val Ser Asn Glu Phe Gly Asn Tyr Asp
180 185 190
tat ttg atg tat gcc gac ttt gat tat gac cat cct gat gtc gta gca 624
Tyr Leu Met Tyr Ala Asp Phe Asp Tyr Asp His Pro Asp Val Val Ala
195 200 205
gag att aag aga tgg ggc act tgg tat gcc aat gaa ctg caa ttg gac 672
Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp
210 215 220
ggt ttc cgt ctt gat gct gtc aaa cac att aaa ttt tct ttt ttg cgg 720
Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg
225 230 235 240
gat tgg gtt aat cat gtc agg gaa aaa acg ggg aag gaa atg ttt acg 768
Asp Trp yal Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr
245 250 255
gta gct gag tac tgg tcg aat gac ttg ggc gcg ctg gaa aac tat ttg 816
Val Ala Glu Tyr Trp Ser Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu
260 265 270
aac aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt cat tat 864
Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr
275 280 285
cag ttc cat gct gca tcg aca cag gga ggc ggc tat gat atg agg aaa 912
Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys
290 295 300
ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg gtt aca 960
Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr
305 310 315 320
ttt gtc gat aac cat gat aca cag ccg ggg caa tcg ctt gag tcg act 1008
Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr
325 330 335
gtc caa aca tgg ttt aag ccg ctt gct tac gct ttt att ctc aca agg 1056
Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg
340 345 350
gaa tct gga tac cct cag gtt ttc tac ggg gat atg tac ggg acg aaa 1104
Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys
355 360 365
gga gac tcc cag cgc gaa att cct gcc ttg aaa cac aaa att gaa ccg 1152
Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro
370 375 380
atc tta aaa gcg aga aaa cag tat gcg tac gga gca cag cat gat tat 1200
Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr
385 390 395 400
ttc gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac agc tcg 1248
Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser
405 410 415
gtt gca aat tca ggt ttg gcg gca tta ata aca gac gga ccc ggt ggg 1296
Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly
420 425 430
gca aag cga atg tat gtc ggc cgg caa aac gcc ggt gag aca tgg cat 1344
Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His
435 440 445
gac att acc gga aac cgt tcg gag ccg gtt gtc atc aat tcg gaa ggc 1392
Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly
450 455 460
tgg gga gag ttt cac gta aac ggc ggg tcg gtt tca att tat gtt caa 1440
Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln
465 470 475 480
aga 1443
Arg
<210>2
<211>481
<212>PRT
<213>解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400>2
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser
35 40 45
Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu
65 70 75 80
Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr
85 90 95
Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp
100 105 110
Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser
115 120 125
Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg
130 135 140
Gly Ser Thr Tyr Ser Asp Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly
145 150 155 160
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln
165 170 175
Gly Lys Thr Trp Asp Trp Glu Val Ser Asn Glu Phe Gly Asn Tyr Asp
180 185 190
Tyr Leu Met Tyr Ala Asp Phe Asp Tyr Asp His Pro Asp Val Val Ala
195 200 205
Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp
210 215 220
Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg
225 230 235 240
Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr
245 250 255
Val Ala Glu Tyr Trp Ser Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu
260 265 270
Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr
275 280 285
Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys
290 295 300
Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr
305 310 315 320
Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr
325 330 335
Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg
340 345 350
Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys
355 360 365
Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro
370 375 380
Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr
385 390 395 400
Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser
405 410 415
Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly
420 425 430
Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His
435 440 445
Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly
450 455 460
Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln
465 470 475 480
Arg
<210>3
<211>1920
<212>DNA
<213>地衣芽孢杆菌(Bacillus licheniformis)
<220>
<221>CDS
<222>(421)..(1872)
<400>3
cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60
gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120
agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag 180
aagtgaagaa gcagagaggc tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240
ttttggaaga aaatataggg aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300
tcataggttt cacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360
cgattgctga cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420
gca aat ctt aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc 468
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
aat gac ggc caa cat tgg agg cgt ttg caa aac gac tcg gca tat ttg 516
Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
gct gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga 564
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
acg agc caa gcg gat gtg ggc tac ggt gct tac gac ctt tat gat tta 612
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
ggg gag ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc aca aaa 660
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
gga gag ctg caa tct gcg atc aaa agt ctt cat tcc cgc gac att aac 708
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
gtt tac ggg gat gtg gtc atc aac cac aaa ggc ggc gct gat gcg acc 756
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
gaa gat gta acc gcg gtt gaa gtc gat ccc gct gac cgc aac cgc gta 804
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
att tca gga gaa cac cta att aaa gcc tgg aca cat ttt cat ttt ccg 852
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
ggg cgc ggc agc aca tac agc gat ttt aaa tgg cat tgg tac cat ttt 900
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
gac gga acc gat tgg gac gag tcc cga aag ctg aac cgc atc tat aag 948
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
ttt caa gga aag gct tgg gat tgg gaa gtt tcc aat gaa aac ggc aac 996
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
tat gat tat ttg atg tat gcc gac atc gat tat gac cat cct gat gtc 1044
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
gca gca gaa att aag aga tgg ggc act tgg tat gcc aat gaa ctg caa 1092
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
ttg gac ggt ttc cgt ctt gat gct gtc aaa cac att aaa ttt tct ttt 1140
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
ttg cgg gat tgg gtt aat cat gtc agg gaa aaa acg ggg aag gaa atg 1188
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
ttt acg gta gct gaa tat tgg cag aat gac ttg ggc gcg ctg gaa aac 1236
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
tat ttg aac aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt 1284
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
cat tat cag ttc cat gct gca tcg aca cag gga ggc ggc tat gat atg 1332
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
agg aaa ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg 1380
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
gtt aca ttt gtc gat aac cat gat aca cag ccg ggg caa tcg ctt gag 1428
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
tcg act gtc caa aca tgg ttt aag ccg ctt gct tac gct ttt att ctc 1476
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
aca agg gaa tct gga tac cct cag gtt ttc tac ggg gat atg tac ggg 1524
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
acg aaa gga gac tcc cag cgc gaa att cct gcc ttg aaa cac aaa att 1572
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
gaa ccg atc tta aaa gcg aga aaa cag tat gcg tac gga gca cag cat 1620
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
gat tat ttc gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
agc tcg gtt gca aat tca ggt ttg gcg gca tta ata aca gac gga ccc 1716
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc ggt gag aca 1764
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
tgg cat gac att acc gga aac cgt tcg gag ccg gtt gtc atc aat tcg 1812
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460
gaa ggc tgg gga gag ttt cac gta aac ggc ggg tcg gtt tca att tat 1860
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
gtt caa aga tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912
Val Gln Arg
tttatttt 1920
<210>4
<211>483
<212>PRT
<213>地衣芽孢杆菌(Bacillus licheniformis)
<400>4
Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15
Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30
Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro
130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys
165 170 175
Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205
Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His
385 390 395 400
Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445
Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Arg
<210>5
<211>2604
<212>DNA
<213>解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<220>
<221>-10_signal
<222>(707)..(712)
<220>
<221>-35_signal
<222>(729)..(734)
<220>
<221>RBS
<222>(759)..(762)
<220>
<221>sig_peptide
<222>(770)..(862)
<220>
<221>mat_peptide
<222>(863)..(2314)
<220>
<221>终止子
<222>(2321)..(2376)
<220>
<221>CDS
<222>(863)..(2314)
<400>5
aagcttcaag cggtcaatcg gaatgtgcat ctcgcttcat acttaggttt tcacccgcat 60
attaagcagg cgtttttgaa ccgtgtgaca gaagctgttc gaaaccccgg cgggcggttt 120
gattttaagg ggggacagta tgctgcctct tcacattaat ctcagcggaa aaagaatcat 180
cattgctggc gggggcaatg ttgcattaag aaggctgaaa cggtgtttcc ggaaggcgct 240
gatattaccg tgatcagtct gagcctgcct gaaattaaaa agctggcgga tgaaggacgc 300
atccgctgga ttccccggag aattgaaatg aaagatctca agcccgcttt tttcattatt 360
gccgcgacaa atgaccgagg cgtgaatcag gagatagccg caaacgcttc tgaaacgcag 420
ctggtcaact gtgtaagcaa ggctgaacaa ggcagcgtat atatgccgaa gatcatccgc 480
aaagggcgca ttcaagtatc agtatcaaca agcggggcaa gccccgcaca tacgaaaaga 540
ctggctgaaa acattgagcc tttgatgact gatgatttgg ctgaagaagt ggatcgattg 600
tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc atcagacagg gtatttttta 660
tgctgtccag actgtccgct gtgtaaaaaa taggaataaa ggggggttgt tattatttta 720
ctgatatgta aaatataatt tgtataagaa aatgagaggg agaggaaaca tgattcaaaa 780
acgaaagcgg acagtttcgt tcagacttgt gcttatgtgc acgctgttat ttgtcagttt 840
gccgat taca aaaacatcag cc gta aat ggc acg ctg atg cag tat ttt gaa 892
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu
1 5 10
tgg tat acg ccg aac gac ggc cag cat tgg aaa cga ttg cag aat gat 940
Trp Tyr Thr Pro Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp
15 20 25
gcg gaa cat tta tcg gat atc gga atc act gcc gtc tgg att cct ccc 988
Ala Glu His Leu Ser Asp Ile Gly Ile Thr Ala Val Trp Ile Pro Pro
30 35 40
gca tac aaa gga ttg agc caa tcc gat aac gga tac gga cct tat gat 1036
Ala Tyr Lys Gly Leu Ser Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp
45 50 55
ttg tat gat tta gga gaa ttc cag caa aaa ggg acg gtc aga acg aaa 1084
Leu Tyr Asp Leu Gly Glu Phe Gln Gln Lys Gly Thr Val Arg Thr Lys
60 65 70
tac ggc aca aaa tca gag ctt caa gat gcg atc ggc tca ctg cat tcc 1132
Tyr Gly Thr Lys Ser Glu Leu Gln Asp Ala Ile Gly Ser Leu His Ser
75 80 85 90
cgg aac gtc caa gta tac gga gat gtg gtt ttg aat cat aag gct ggt 1180
Arg Asn Val Gln Val Tyr Gly Asp Val Val Leu Asn His Lys Ala Gly
95 100 105
gct gat gca aca gaa gat gta act gcc gtc gaa gtc aat ccg gcc aat 1228
Ala Asp Ala Thr Glu Asp Val Thr Ala Val Glu Val Asn Pro Ala Asn
110 115 120
aga aat cag gaa act tcg gag gaa tat caa atc aaa gcg tgg acg gat 1276
Arg Asn Gln Glu Thr Ser Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp
125 130 135
ttt cgt ttt ccg ggc cgt gga aac acg tac agt gat ttt aaa tgg cat 1324
Phe Arg Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp His
140 145 150
tgg tat cat ttc gac gga gcg gac tgg gat gaa tcc cgg aag atc agc 1372
Trp Tyr His Phe Asp Gly Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser
155 160 165 170
cgc atc ttt aag ttt cgt ggg gaa gga aaa gcg tgg gat tgg gaa gta 1420
Arg Ile Phe Lys Phe Arg Gly Glu Gly Lys Ala Trp Asp Trp Glu Val
175 180 185
tca agt gaa aac ggc aac tat gac tat tta atg tat gct gat gtt gac 1468
Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp
190 195 200
tac gac cac cct gat gtc gtg gca gag aca aaa aaa tgg ggt atc tgg 1516
Tyr Asp His Pro Asp Val Val Ala Glu Thr Lys Lys Trp Gly Ile Trp
205 210 215
tat gcg aat gaa ctg tca tta gac ggc ttc cgt att gat gcc gcc aaa 1564
Tyr Ala Asn Glu Leu Ser Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys
220 225 230
cat att aaa ttt tca ttt ctg cgt gat tgg gtt cag gcg gtc aga cag 1612
His Ile Lys Phe Ser Phe Leu Arg Asp Trp Val Gln Ala Val Arg Gln
235 240 245 250
gcg acg gga aaa gaa atg ttt acg gtt gcg gag tat tgg cag aat aat 1660
Ala Thr Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn
255 260 265
gcc ggg aaa ctc gaa aac tac ttg aat aaa aca agc ttt aat caa tcc 1708
Ala Gly Lys Leu Glu Asn Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser
270 275 280
gtg ttt gat gtt ccg ctt cat ttc aat tta cag gcg gct tcc tca caa 1756
Val Phe Asp Val Pro Leu His Phe Asn Leu Gln Ala Ala Ser Ser Gln
285 290 295
gga ggc gga tat gat atg agg cgt ttg ctg gac ggt acc gtt gtg tcc 1804
Gly Gly Gly Tyr Asp Met Arg Arg Leu Leu Asp Gly Thr Val Val Ser
300 305 310
agg cat ccg gaa aag gcg gtt aca ttt gtt gaa aat cat gac aca cag 1852
Arg His Pro Glu Lys Ala Val Thr Phe Val Glu Asn His Asp Thr Gln
315 320 325 330
ccg gga cag tca ttg gaa tcg aca gtc caa act tgg ttt aaa ccg ctt 1900
Pro Gly Gln Ser Leu Glu Ser Thr Val Gln Thr Trp Phe Lys Pro Leu
335 340 345
gca tac gcc ttt att ttg aca aga gaa tcc ggt tat cct cag gtg ttc 1948
Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe
350 355 360
tat ggg gat atg tac ggg aca aaa ggg aca tcg cca aag gaa att ccc 1996
Tyr Gly Asp Met Tyr Gly Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro
365 370 375
tca ctg aaa gat aat ata gag ccg att tta aaa gcg cgt aag gag tac 2044
Ser Leu Lys Asp Asn Ile Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr
380 385 390
gca tac ggg ccc cag cac gat tat att gac cac ccg gat gtg atc gga 2092
Ala Tyr Gly Pro Gln His Asp Tyr Ile Asp His Pro Asp Val Ile Gly
395 400 405 410
tgg acg agg gaa ggt gac agc tcc gcc gcc aaa tca ggt ttg gcc gct 2140
Trp Thr Arg Glu Gly Asp Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala
415 420 425
tta atc acg gac gga ccc ggc gga tca aag cgg atg tat gcc ggc ctg 2188
Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu
430 435 440
aaa aat gcc ggc gag aca tgg tat gac ata acg ggc aac cgt tca gat 2236
Lys Asn Ala Gly Glu Thr Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp
445 450 455
act gta aaa atc gga tct gac ggc tgg gga gag ttt cat gta aac gat 2284
Thr Val Lys Ile Gly Ser Asp Gly Trp Gly Glu Phe His Val Asn Asp
460 465 470
ggg tcc gtc tcc att tat gtt cag aaa taa ggtaataaaa aaacacctcc 2334
Gly Ser Val Ser Ile Tyr Val Gln Lys
475 480
aagctgagtg cgggtatcag cttggaggtg cgtttatttt ttcagccgta tgacaaggtc 2394
ggcatcaggt gtgacaaata cggtatgctg gctgtcatag gtgacaaatc cgggttttgc 2454
gccgtttggc tttttcacat gtctgatttt tgtataatca acaggcacgg agccggaatc 2514
tttcgccttg gaaaaataag cggcgatcgt agctgcttcc aatatggatt gttcatcggg 2574
atcgctgctt ttaatcacaa cgtgggatcc 2604
<210>6
<211>483
<212>PRT
<213>解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400>6
Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp
1 5 10 15
Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp
20 25 30
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser
35 40 45
Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu
50 55 60
Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu
65 70 75 80
Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr
85 90 95
Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp
100 105 110
Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser
115 120 125
Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg
130 135 140
Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly
115 150 155 160
Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg
165 170 175
Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val
195 200 205
Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser
210 215 220
Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe
225 230 235 240
Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn
260 265 270
Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu
275 280 285
His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met
290 295 300
Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala
305 310 315 320
Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile
370 375 380
Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His
385 390 395 400
Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr
435 440 445
Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser
450 455 460
Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr
465 470 475 480
Val Gln Lys
<210>7
<211>1548
<212>DNA
<213>嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)
<220>
<221>CDS
<222>(1)..(1548)
<400>7
gcc gca ccg ttt aac ggc acc atg atg cag tat ttt gaa tgg tac ttg 48
Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
1 5 10 15
ccg gat gat ggc acg tta tgg acc aaa gtg gcc aat gaa gcc aac aac 96
Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
20 25 30
tta tcc agc ctt ggc atc acc gct ctt tgg ctg ccg ccc gct tac aaa 144
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45
gga aca agc cgc agc gac gta ggg tac gga gta tac gac ttg tat gac 192
Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60
ctc ggc gaa ttc aat caa aaa ggg acc gtc cgc aca aaa tac gga aca 240
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
65 70 75 80
aaa gct caa tat ctt caa gcc att caa gcc gcc cac gcc gct gga atg 288
Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
85 90 95
caa gtg tac gcc gat gtc gtg ttc gac cat aaa ggc ggc gct gac ggc 336
Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110
acg gaa tgg gtg gac gcc gtc gaa gtc aat ccg tcc gac cgc aac caa 384
Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
115 120 125
gaa atc tcg ggc acc tat caa atc caa gca tgg acg aaa ttt gat ttt 432
Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe
130 135 140
ccc ggg cgg ggc aac acc tac tcc agc ttt aag tgg cgc tgg tac cat 480
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His
145 150 155 160
ttt gac ggc gtt gat tgg gac gaa agc cga aaa ttg agc cgc att tac 528
Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
165 170 175
aaa ttc cgc ggc atc ggc aaa gcg tgg gat tgg gaa gta gac acg gaa 576
Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
180 185 190
aac gga aac tat gac tac tta atg tat gcc gac ctt gat atg gat cat 624
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205
ccc gaa gtc gtg acc gag ctg aaa aac tgg ggg aaa tgg tat gtc aac 672
Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn
210 215 220
aca acg aac att gat ggg ttc cgg ctt gat gcc gtc aag cat att aag 720
Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240
ttc agt ttt ttt cct gat tgg ttg tcg tat gtg cgt tct cag act ggc 768
Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly
245 250 255
aag ccg cta ttt acc gtc ggg gaa tat tgg agc tat gac atc aac aag 816
Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys
260 265 270
ttg cac aat tac att acg aaa aca gac gga acg atg tct ttg ttt gat 864
Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp
275 280 285
gcc ccg tta cac aac aaa ttt tat acc gct tcc aaa tca ggg ggc gca 912
Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala
290 295 300
ttt gat atg cgc acg tta atg acc aat act ctc atg aaa gat caa ccg 960
Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro
305 310 315 320
aca ttg gcc gtc acc ttc gtt gat aat cat gac acc gaa ccc ggc caa 1008
Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln
325 330 335
gcg ctg cag tca tgg gtc gac cca tgg ttc aaa ccg ttg gct tac gcc 1056
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala
340 345 350
ttt att cta act cgg cag gaa gga tac ccg tgc gtc ttt tat ggt gac 1104
Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365
tat tat ggc att cca caa tat aac att cct tcg ctg aaa agc aaa atc 1152
Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile
370 375 380
gat ccg ctc ctc atc gcg cgc agg gat tat gct tac gga acg caa cat 1200
Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
gat tat ctt gat cac tcc gac atc atc ggg tgg aca agg gaa ggg ggc 1248
Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly
405 410 415
act gaa aaa cca gga tcc gga ctg gcc gca ctg atc acc gat ggg ccg 1296
Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
gga gga agc aaa tgg atg tac gtt ggc aaa caa cac gct gga aaa gtg 1344
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445
ttc tat gac ctt acc ggc aac cgg agt gac acc gtc acc atc aac agt 1392
Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser
450 455 460
gat gga tgg ggg gaa ttc aaa gtc aat ggc ggt tcg gtt tcg gtt tgg 1440
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp
465 470 475 480
gtt cct aga aaa acg acc gtt tct acc atc gct cgg ccg atc aca acc 1488
Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr
485 490 495
cga ccg tgg act ggt gaa ttc gtc cgt tgg acc gaa cca cgg ttg gtg 1536
Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val
500 505 510
gca tgg cct tga 1548
Ala Trp Pro
515
<210>8
<211>515
<212>PRT
<213>嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)
<400>8
Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
1 5 10 15
Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
20 25 30
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45
Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
65 70 75 80
Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
85 90 95
Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
100 105 110
Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
115 120 125
Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe
130 135 140
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His
145 150 155 160
Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
165 170 175
Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu
180 185 190
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205
Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn
210 215 220
Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
225 230 235 240
Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly
245 250 255
Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys
260 265 270
Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp
275 280 285
Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala
290 295 300
Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro
305 310 315 320
Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln
325 330 335
Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala
340 345 350
Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365
Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile
370 375 380
Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly
405 410 415
Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445
Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser
450 455 460
Asp Gly Trp Gly Glu Phe Lys Val Asn Gly GIy Ser Val Ser Val Trp
465 470 475 480
Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr
485 490 495
Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val
500 505 510
Ala Trp Pro
515
<210>9
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>9
ggtcgtaggc accgtagccc caatccgctt g 31
<210>10
<211>36
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>10
ggtcgtaggc accgtagccc caatcccatt ggctcg 36
<210>11
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>11
ctgtgact gg tgagtactca accaagtc 28
<210>12
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>12
ggtcgtaggc accgtagccc tcatccgctt g 31
<210>13
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>13
ggtcgtaggc accgtagccc atatccgctt g 31
<210>14
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>14
ggtcgtaggc accgtagcca atatccgctt g 31
<210>15
<211>36
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>15
gcagcatgga actgctyatg aagaggcacg tcaaac 36
<210>16
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>16
catagttgcc gaattcattg gaaacttccc 30
<210>17
<211>34
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>17
catagttgcc gaattcaggg gaaacttccc aatc 34
<210>18
<211>41
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>18
ccgcgccccg ggaaatcaaa ttttgtccag gctttaatta g 41
<210>19
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>19
caaaatggta ccaataccac ttaaaatcgc tg 32
<210>20
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>20
cttcccaatc ccaagtcttc ccttgaaac 29
<210>21
<211>36
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>21
cttaatttct gctacgacgt caggatggtc ataatc 36
<210>22
<211>38
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>22
cgcccaagtc attcgaccag tactcagcta ccgtaaac 38
<210>23
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>23
gccgttttca ttgtcgactt cccaatccc 29
<210>24
<211>35
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>24
ggaatttcgc gctgactagt cccgtacata tcccc 35
<210>25
<211>36
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>25
ggcaggaatt tcgcgacctt tcgtcccgta catatc 36
<210>26
<211>36
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>26
cctcattctg cagcagcagc cgtaaatggc acgctg 36
<210>27
<211>38
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>27
ccagacggca gtaataccga tatccgataa atgttccg 38
<210>28
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>28
cggatatcgg tattactgcc gtctggattc 30
<210>29
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>29
ctcgtcccaa tcggttccgt c 21
<210>30
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>30
gatgtatgcc gacttcgatt atgacc 26
<210>31
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>31
ctcatgtttg acagcttatc atcgataagc 30
<210>32
<211>24
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>32
ccgattgctg acgctgttat ttgc 24
<210>33
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>33
cccggtgggg caaagtggat gtatgtcggc cgg 33
<210>34
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>34
cggaaggctg gggaaatttt cacgtaaacg gc 32
<210>35
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>35
ggtggggcaa agtggatgta tgtcggc 27
<210>36
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>36
ggaaggctgg ggaaactttc acgtaaacg 29
Claims (20)
1.具有α-淀粉酶活性并包含取代R437W的亲本Termamyl-样α-淀粉酶的变体,其中所述位置相应于具有SEQ ID NO:4之氨基酸序列的亲本Termamyl-样α-淀粉酶的氨基酸序列的位置。
2.权利要求1的变体,其中亲本α-淀粉酶是SEQ ID NO:4和SEQ ID NO:6的杂合α-淀粉酶。
3.权利要求1-2任一项的变体,其中亲本杂合α-淀粉酶是包含如SEQ IDNO:4所示的地衣芽孢杆菌α-淀粉酶的445个C-末端氨基酸残基和如SEQID NO:6所示的来源于解淀粉芽孢杆菌的α-淀粉酶的37个N-末端氨基酸残基的杂合α-淀粉酶。
4.权利要求1-3任一项的变体,其中亲本杂合Termamyl-样α-淀粉酶进一步具有以下突变:H156Y+A181T+N190F+A209V+Q264S(使用SEQ ID NO:4中的编号)或LE174。
5.权利要求1-3任一项的变体,其中亲本杂合Termamyl-样α-淀粉酶进一步具有以下突变:H156Y+A181T+N190F+A209V+Q264S+I201F(使用SEQID NO:4的编号)或LE429。
6.权利要求1-5任一项的变体,其中变体包含一个或多个以下额外的突变:R176*、G177*、E469N(使用SEQ ID NO:6中的编号)。
7.权利要求1-6任一项的变体,其中变体包含额外的突变:E469N(使用SEQ ID NO:6中的编号)。
8.权利要求1-6任一项的变体,其中变体包含额外的突变:R176*+G177*+E469N(使用SEQ ID NO:6中的编号)。
9.权利要求1的变体,其中亲本α-淀粉酶是SEQ ID NO:4或SEQ IDNO:6的α-淀粉酶。
10.权利要求9的变体,其中变体包含一个或多个以下额外的突变:R176*、G177*、N190F、E469N(使用SEQ ID NO:6中的编号)。
11.权利要求10的变体,其中变体包含额外的突变:R176*+G177*+N190F(使用SEQ ID NO:6中的编号)。
12.权利要求11的变体,其中变体包含额外的突变:E469N(使用SEQ IDNO:6中的编号)。
13.权利要求1-12任一项的变体,其中亲本Termamyl-样α-淀粉酶具有与SEQ ID NO:4具有至少60%、优选70%、更优选至少80%、甚至更优选至少约90%、甚至更优选至少95%、甚至更优选至少97%以及甚至更优选至少约99%同一性水平的氨基酸序列。
14.一种包含编码根据权利要求1-13任一项的α-淀粉酶变体的DNA序列的DNA构建体。
15.一种携有根据权利要求14的DNA构建体的重组表达载体。
16.一种用根据权利要求14的DNA构建体或根据权利要求15的载体转化的细胞。
17.权利要求16的细胞,其是微生物,特别是细菌或真菌,例如革兰氏阳性细菌,如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌、短芽孢杆菌、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌、环状芽孢杆菌、灿烂芽孢杆菌或苏云金芽孢杆菌。
18.一种包含权利要求1-13的α-淀粉酶变体的组合物。
19.一种生产根据权利要求1-13任一项的变体的方法,其中在有利于产生变体的条件下培养根据权利要求16-17任一项的细胞,并随后从培养物中回收变体。
20.权利要求1-13任一项的α-淀粉酶变体或权利要求18的组合物在淀粉液化;在洗涤剂组合物,例如洗衣、碗碟清洗和硬表面清洁组合物;乙醇生产,例如燃料、饮用和工业乙醇生产;纺织品、织物或衣物的退浆中的应用。
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PCT/DK2005/000817 WO2006066594A2 (en) | 2004-12-23 | 2005-12-22 | Alpha-amylase variants |
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EP (1) | EP1831360A2 (zh) |
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CN (1) | CN101128579B (zh) |
AU (1) | AU2005318696B2 (zh) |
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CN102341495A (zh) * | 2009-03-10 | 2012-02-01 | 丹尼斯科美国公司 | 巨大芽孢杆菌菌株DSM90相关的α-淀粉酶及其使用方法 |
CN105483099A (zh) * | 2008-06-06 | 2016-04-13 | 丹尼斯科美国公司 | 具有改良特性的嗜热脂肪土芽孢杆菌α-淀粉酶(AMYS)变体 |
CN106047844A (zh) * | 2016-08-01 | 2016-10-26 | 安徽工程大学 | 一种具有高麦芽糖生成率的真菌α‑淀粉酶变体及其制备方法 |
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KR100511499B1 (ko) * | 1995-02-03 | 2005-12-21 | 노보자임스 에이/에스 | 소정 특성을 가지는 알파-아밀라제 돌연변이체를 디자인하는 방법 |
US6187576B1 (en) * | 1997-10-13 | 2001-02-13 | Novo Nordisk A/S | α-amylase mutants |
US6197565B1 (en) * | 1998-11-16 | 2001-03-06 | Novo-Nordisk A/S | α-Amylase variants |
JP4745503B2 (ja) * | 1999-03-31 | 2011-08-10 | ノボザイムス アクティーゼルスカブ | アルカリα−アミラーゼ活性を有するポリペプチド及びそれらをコードする核酸 |
EP3000881A3 (en) * | 2001-05-15 | 2016-07-20 | Novozymes A/S | Alpha-amylase variant with altered properties |
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2005
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- 2005-12-22 EP EP05823004A patent/EP1831360A2/en not_active Withdrawn
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- 2005-12-22 JP JP2007547183A patent/JP5166880B2/ja not_active Expired - Fee Related
- 2005-12-22 CN CN2005800486042A patent/CN101128579B/zh not_active Expired - Fee Related
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105483099A (zh) * | 2008-06-06 | 2016-04-13 | 丹尼斯科美国公司 | 具有改良特性的嗜热脂肪土芽孢杆菌α-淀粉酶(AMYS)变体 |
CN105483099B (zh) * | 2008-06-06 | 2020-06-23 | 丹尼斯科美国公司 | 具有改良特性的嗜热脂肪土芽孢杆菌α-淀粉酶(AMYS)变体 |
CN102341495A (zh) * | 2009-03-10 | 2012-02-01 | 丹尼斯科美国公司 | 巨大芽孢杆菌菌株DSM90相关的α-淀粉酶及其使用方法 |
CN106047844A (zh) * | 2016-08-01 | 2016-10-26 | 安徽工程大学 | 一种具有高麦芽糖生成率的真菌α‑淀粉酶变体及其制备方法 |
CN106047844B (zh) * | 2016-08-01 | 2020-05-22 | 安徽工程大学 | 一种具有高麦芽糖生成率的真菌α-淀粉酶变体及其制备方法 |
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CN101128579B (zh) | 2013-10-02 |
MX2007007494A (es) | 2007-08-15 |
WO2006066594A2 (en) | 2006-06-29 |
CA2593920A1 (en) | 2006-06-29 |
AU2005318696A1 (en) | 2006-06-29 |
EP1831360A2 (en) | 2007-09-12 |
US20090275078A1 (en) | 2009-11-05 |
WO2006066594A3 (en) | 2006-08-31 |
AU2005318696B2 (en) | 2010-12-16 |
JP5166880B2 (ja) | 2013-03-21 |
JP2008524995A (ja) | 2008-07-17 |
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