CN116323910A - α-淀粉酶突变体 - Google Patents
α-淀粉酶突变体 Download PDFInfo
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- CN116323910A CN116323910A CN202180067664.8A CN202180067664A CN116323910A CN 116323910 A CN116323910 A CN 116323910A CN 202180067664 A CN202180067664 A CN 202180067664A CN 116323910 A CN116323910 A CN 116323910A
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- amino acid
- gly
- mutant
- asn
- val
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Abstract
本发明提供能够在低温下发挥功能、同时维持或提高了稳定性和/或清洗性能的α-淀粉酶。该α-淀粉酶突变体为包含与序列编号2所示的氨基酸序列的G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的1处或多处氨基酸残基的改变的亲本α-淀粉酶的突变体,上述亲本α-淀粉酶或α-淀粉酶突变体相对于序列编号4所示的氨基酸序列具有至少90%的序列同一性。
Description
技术领域
本发明涉及α-淀粉酶的突变体。
背景技术
α-淀粉酶除了在淀粉工业、酿造工业、纤维工业、医药品工业和食品工业等广泛的产业领域中利用以外,还已知在清洗剂中的配合适应性,作为去除淀粉质的污渍的成分而在自动餐具清洗机用的餐具清洗剂或衣物用洗涤剂等中配合。
作为在清洗剂用途中有用的α-淀粉酶,已知有来自芽孢杆菌(Bacillus sp.)KSM-1378(FERM BP-3048)株的α-淀粉酶AP1378(专利文献1)、作为来自地衣芽孢杆菌的α-淀粉酶的Termamyl或Duramyl(注册商标),除此以外还有来自芽孢杆菌(Bacillus sp.)DSM12649株的α-淀粉酶AA560(专利文献2)、来自芽孢杆菌(Bacillus sp.)SP722株的α-淀粉酶SP722(专利文献3的序列编号4)、来自噬纤维菌属的α-淀粉酶CspAmy2(专利文献4)等。另外,关于这些α-淀粉酶,报道有为了面向特定用途改善功能而进行了改变、例如提高了在洗涤剂中的稳定性的突变体等(专利文献5)。
近年来,从环境保护和减少清洗成本的观点出发,降低餐具清洗或洗涤清洗、特别是在洗衣房的洗涤清洗时的温度被认为很重要,另外还希望缩短清洗时间。然而,包括淀粉酶的大部分酶的最适温度比在低温清洗时通常设定的温度高,因此,大量淀粉质污渍难以完全去除。
因此,寻找在低温下也可以保持清洗性能、淀粉分解活性且污渍除去效果高的α-淀粉酶是重要的。
专利文献1:国际公开第94/26881号
专利文献2:国际公开第00/60060号
专利文献3:国际公开第06/002643号
专利文献4:国际公开第2014/164777号
专利文献5:国际公开第98/044126号
发明内容
本发明涉及以下方案。
1)一种α-淀粉酶突变体,其为包含与序列编号2所示的氨基酸序列的G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的1处或多处氨基酸残基的改变的亲本α-淀粉酶的突变体,上述亲本α-淀粉酶或α-淀粉酶突变体相对于序列编号4所示的氨基酸序列具有至少90%的序列同一性。
2)编码1)的突变体的多核苷酸。
3)包含2)的多核苷酸的载体或DNA片段。
4)含有3)的载体或DNA片段的转化细胞。
5)包含1)的突变体的清洗剂组合物。
附图说明
图1是2个氨基酸缺失突变体的稳定性评价。
具体实施方式
本发明涉及提供能够在低温下发挥功能、同时维持或提高了稳定性和/或清洗性能的α-淀粉酶。
本发明的发明人以在低温下发挥功能的α-淀粉酶作为亲本,成功得到了具有比该亲本α-淀粉酶高的清洗性能和/或稳定性的α-淀粉酶突变体。
根据本发明,能够提供具有比亲本α-淀粉酶高的清洗性能和/或稳定性的α-淀粉酶突变体。通过使用这样的α-淀粉酶突变体,即使在低温也能够进行发挥优异的淀粉污渍除去效果的清洗。
在本说明书中,“淀粉酶”(EC3.2.1.1;α-D-(1→4)-葡聚糖水解酶)是指催化淀粉以及其它直链或支链1,4-糖苷寡糖或多糖类的水解的酶群。α-淀粉酶活性能够通过测定由淀粉的酶分解得到的还原末端的生成量来确定。另外,不限定于此,例如也能够通过测定Phadebas这样的色素交联淀粉由酶分解得到的色素的游离来确定(Soininen,K.,M.Ceska,and H.Adlercreutz."Comparison between a new chromogenicα-amylase test(Phadebas)and the Wohlgemuth amyloclastic method in urine."Scandinavianjournal of clinical and laboratory investigation 30.3(1972):291-297.)。
在本说明书中,氨基酸序列或核苷酸序列的同一性可以通过Lipman-Pearson法(Science,1985,227:1435-1441)计算。具体而言,使用遗传信息处理软件GENETYX Ver.12的同源性解析(Search homology)程序,将Unit size to compare(ktup)作为2进行解析而计算。
在本说明书中,“氨基酸残基”是指构成蛋白质的20种氨基酸残基、丙氨酸(Ala或A)、精氨酸(Arg或R)、天冬酰胺(Asn或N)、天冬氨酸(Asp或D)、半胱氨酸(Cys或C)、谷氨酰胺(Gln或Q)、谷氨酸(Glu或E)、甘氨酸(Gly或G)、组氨酸(His或H)、异亮氨酸(Ile或I)、亮氨酸(Leu或L)、赖氨酸(Lys或K)、甲硫氨酸(Met或M)、苯丙氨酸(Phe或F)、脯氨酸(Pro或P)、丝氨酸(Ser或S)、苏氨酸(Thr或T)、色氨酸(Trp或W)、酪氨酸(Tyr或Y)和缬氨酸(Val或V)。
在本说明书中,氨基酸的位置和突变体的记载使用公认的IUPAC的单字母的氨基酸符号表示如下。
规定位置的氨基酸用[氨基酸、位置]表示。例如位置226的苏氨酸表示为“T226”。
关于氨基酸的“取代”,用[原始的氨基酸、位置、取代后的氨基酸]表示。例如位置226的苏氨酸被取代为丙氨酸用“T226A”表示。
关于氨基酸的“缺失”,用[原始的氨基酸、位置、Δ]表示。例如,位置181处的丝氨酸的缺失用“S181Δ”表示。
关于氨基酸的“插入”,用[原始的氨基酸、位置、原始的氨基酸、插入后的氨基酸]表示。例如在位置195的甘氨酸后插入赖氨酸表示为“G195GK”。多个氨基酸的插入用[原始的氨基酸、位置、原始的氨基酸、插入后的氨基酸1号、插入后的氨基酸2号等]表示。例如在位置195的甘氨酸后插入赖氨酸和丙氨酸表示为“G195GKA”。
包含多个改变的突变体通过加法记号(“+”)表示。例如,“R170Y+G195E”分别表示位置170的精氨酸被取代为酪氨酸且位置195的甘氨酸被取代为谷氨酸。
在1个位置能够导入不同改变的情况下,不同的改变用斜线(“/”)分开,例如“R170Y/E”表示位置170的精氨酸被取代为酪氨酸或谷氨酸。
在本说明书中,启动子等的控制区域与基因的“可发挥作用地连结”是指基因与控制区域以该基因在该控制区域的控制下能够表达的方式连结。基因与控制区域的“可发挥作用地连结”的步骤是本领域技术人员所公知的。
在本说明书中,关于基因的“上游”和“下游”是指该基因的转录方向的上游和下游。例如“配置在启动子的下游的基因”是指该基因存在于DNA正义链中在启动子的3'侧的意思,基因的上游是指DNA正义链中该基因的5'侧的区域。
在本说明书中,对于细胞的功能或性状、特性使用的术语“本来”是为了表示该功能性质、形状在该细胞从最初就存在而使用的。对照而言,术语“外来”是为了表示并不是在该细胞中从最初就存在,而是从外部导入的功能性质、形状而使用的。例如“外来”基因或多核苷酸是指从外部导入细胞的基因或多核苷酸。外来基因或多核苷酸可以来自与其所导入的细胞同种的生物,也可以来自不同种的生物(即异源基因或多核苷酸)。
<突变体>
本发明的突变体为亲本α-淀粉酶的突变体,其包含与序列编号2所示的氨基酸序列的G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的1个或多个氨基酸残基的改变。
即,“突变体”是指在构成亲本α-淀粉酶的氨基酸中,1处或多处规定位置的氨基酸残基被改变的具有α-淀粉酶活性的多肽。这样的规定位置的氨基酸残基改变是用于提高清洗性能和/或在洗涤剂中的稳定性的改变,因此,该突变体与亲本α-淀粉酶相比,具有更高的清洗性能和/或稳定性。
在本发明的突变体中,氨基酸残基的改变部位(突变位置)是与序列编号2所示的氨基酸序列的G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置。
其中,序列编号2所示的氨基酸序列是构成α-淀粉酶YR288的氨基酸序列,本发明的突变体中的突变位置根据该氨基酸序列的氨基酸编号进行编号。
YR288是在NCBI蛋白质序列数据库中作为WP_100346362.1登录的蛋白质,根据本申请人,是被确定为低温下的淀粉分解活性和清洗性能高的α-淀粉酶的蛋白质(日本特愿2020-121626)。
氨基酸序列上的“与……的位置相当”能够通过将目标序列和参照序列(在本发明中为序列编号2所示的氨基酸序列)以获得最大同源性的方式进行比较(比对)来确定。氨基酸序列的比对能够使用公知的算法实行,其步骤是本领域技术人员公知的。例如,比对能够通过以缺省设定使用Clustal W多重比对程序(Thompson,J.D.et al,1994,Nucleic AcidsRes.22:4673-4680)来进行。或者也可以使用Clustal W的修订版Clustal W2或Clustalomega。Clustal W、Clustal W2和Clustal omega例如能够在欧洲生物信息学研究所(European Bioinformatics Institute:EBI[www.ebi.ac.uk/index.html])或日本国立遗传学研究所运营的日本DNA数据库(DDBJ[www.ddbj.nig.ac.jp/searches-j.html])的网站上利用。通过上述比对,与参照序列的任意的位置匹配的目标序列的位置被视为与该任意的位置相当的位置。
只要是本领域技术人员,能够为了优化而将上述得到的氨基酸序列的比对进一步进行微调整。这样的最适比对优选考虑氨基酸序列的类似性、所插入的空格的频度等来确定。这里氨基酸序列的类似性是指在比对2个氨基酸序列时,在这两个序列中存在同一或类似的氨基酸残基的位置的数量相对于全长氨基酸残基数的比例(%)。类似的氨基酸残基是指构成蛋白质的20种氨基酸中,极性或电荷的方面彼此具有类似的性质、产生所谓的保守取代这样的氨基酸残基。包含这样的类似氨基酸残基的组是本领域技术人员熟知的,例如可以分别列举精氨酸与赖氨酸或谷氨酰胺;谷氨酸与天冬氨酸或谷氨酰胺;丝氨酸与苏氨酸或丙氨酸;谷氨酰胺与天冬酰胺或精氨酸;亮氨酸与异亮氨酸等,但不限定于这些。
在本发明中,“亲本α-淀粉酶”是指为了得到本发明的突变体而要进行改变的基准α-淀粉酶。亲本可以是天然(野生型)多肽或其突变体。
在本发明中,亲本α-淀粉酶或α-淀粉酶突变体在氨基酸序列中与序列编号4所示的氨基酸序列具有至少90%、优选至少95%、更优选至少96%、更优选至少97%、更优选至少98%、更优选至少99%的同一性。
其中,由序列编号4所示的氨基酸序列构成的α-淀粉酶是在由序列编号2所示的氨基酸序列构成的α-淀粉酶(YR288)中缺失了与R178和T180对应的氨基酸残基的(R178Δ+T180Δ)α-淀粉酶突变体。如后述的实施例所示,包含与序列编号2所示的氨基酸序列的R178、G179、T180和G181的各位置相当的位置的2个氨基酸残基的缺失的以YR288为亲本α-淀粉酶的突变体在洗涤剂中的稳定性比YR288飞跃性地提高。因此,在序列编号2所示的氨基酸序列或与其具有至少90%、优选至少95%、更优选至少96%、更优选至少97%、更优选至少98%、更优选至少99%的同一性的氨基酸序列中,缺失了与序列编号2所示的氨基酸序列的R178、G179、T180和G181的各位置相当的位置的2处以上的氨基酸残基的α-淀粉酶突变体包含由序列编号4所示的氨基酸序列构成的α-淀粉酶,均可以为本发明的突变体的亲本α-淀粉酶。
其中,作为缺失2处以上的氨基酸残基,优选列举R178Δ+T180Δ、G179Δ+T180Δ、R178Δ+G179Δ、R178Δ+G181Δ、G179Δ+G181Δ等,更优选R178Δ+T180Δ。
作为由与序列编号4所示的氨基酸序列具有至少90%的同一性的氨基酸序列构成的α-淀粉酶,除此以外还可以列举来自弯曲芽孢杆菌(Bacillus flexus)的α-淀粉酶DE0178、来自芽孢杆菌属(Bacillus sp.)的α-淀粉酶RU2C等(日本特愿2020-121626)。
在上述规定位置的1或多处进行的氨基酸残基的改变可以列举氨基酸残基的取代、插入和/或缺失。其中,“取代”是指将处于某个位置的氨基酸被不同的氨基酸取代的意思,“缺失”是指处于某个位置的氨基酸的除去,“插入”是指以与处于某个位置的氨基酸相邻且直接连续的方式添加氨基酸的意思。
在本发明中,突变部位可以是1处或多处,优选为2处以上,可以列举更优选2~15处、更优选3~15处、更优选5~10处。
其中,从提高清洗性能或稳定性的观点出发,该突变体优选为相对于序列编号4所示的氨基酸序列具有至少90%的同一性的α-淀粉。另外,突变体只要保持作为上述突变体的性质即可,可以包含任意数量的保守氨基酸取代。
与序列编号2所示的氨基酸序列的G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置所示的突变部位中,从提高稳定性的观点出发,优选列举与N126、E187、N192、F205、R211、H240、S241、Y242的各位置相当的位置。
其中,作为优选的2处以上的改变,例如可以列举以下的a)~f)的改变的组合。
a)选自与E187、F205和H240的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变、与选自与N126、N192、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变的组合。
b)选自与E187和H240的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变、与选自与N126、N192、F205、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变的组合。
c)选自与F205和H240的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变、与选自与N126、E187、N192、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变的组合。
d)与F205的位置相当的氨基酸残基的改变、与选自与N126、E187、N192、R211、H240、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变的组合。
e)与H240的位置相当的氨基酸残基的改变、与选自与N126、E187、N192、F205、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变的组合。
f)与R211的位置相当的氨基酸残基的改变、与选自与N126、E187、N192、F205、H240、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变的组合。
与G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的氨基酸残基的改变的优选方式表示如下。
即,G5优选被取代为E、D、P、R或K(G5E/D/P/R/K);
S38优选被取代为N(S38N);
T49优选被取代为Q(T49Q);
Q96优选被取代为R或K(Q96R/K);
N126优选被取代为Y(N126Y);
T129优选被取代为I(T129I);
G140优选被取代为Y、F或W(G140Y/F/W);
F153优选被取代为W(F153W);
Q167优选被取代为E(Q167E);
G179优选被取代为D或H(G179D/H);
W186优选被取代为L(W186L);
E187优选被取代为P(E187P);
N192优选被取代为F(N192F);
M199优选被取代为L、T、A、N、Q、S、V或I(M199L/T/A/N/Q/S/V/I);
Y200优选被取代为G(Y200G);
L203优选被取代为Y、M或F(L203Y/M/F);
Y205优选被取代为F(Y205F);
D206优选被取代为R、E、N、T或G(D206R/E/N/T/G);
R211优选被取代为L、V或I(R211L/V/I);
K215优选被取代为F(K215F);
H240优选被取代为F(H240F);
S241优选被取代为A、Q、D、L、Y、P、H(S241A/Q/D/L/Y/P/H);
Y242优选被取代为F(Y242F);
G244优选被取代为K、W、L或R(G244K/W/L/R);
E257优选被取代为T(E257T);
F259优选被取代为W(F259W);
K278优选被取代为L、D、W、I、H、S、T、N、Q、V、A、Y或F(K278L/D/W/I/H/S/T/N/Q/V/A/Y/F);
H283优选被取代为Q(H283Q);
S284优选被取代为W(S284W);
A288优选被取代为F(A288F);
H295优选被取代为Y(H295Y);
Y296优选被取代为A(Y296A);
N303优选被取代为R、E、S、G、V、D、T或A(N303R/E/S/G/V/D/T/A);
T320优选被取代为D或E(T320D/E);
S331优选被取代为T(S331T);
L348优选被取代为I(L348I);
Y360优选被取代为C、M、L或V(Y360C/M/L/V);
W408位优选被取代为P(W408P);
L429优选被取代为V(L429V);
V430优选被取代为M(V430M);
G433优选在G后插入S(G433GS);
A434优选被取代为V(A434V);
W439优选被取代为R(W439R);
N471优选被取代为T(N471T);
G476优选被取代为A、P、E、S、F、R或K(G476A/P/E/S/F/R/K);
G477优选被取代为E(G477E)。
接着,将有助于提高清洗性能的优选突变的组合表示于下述表1-1~1-4。因此,至少具有该组合突变的突变体是特别有助于提高清洗性能的突变体。
[表1-1]
G5R+W186L+E257T |
G5R+E257T+G433GS |
G5R+Y360L+G433GS |
G5R+Q96K+E257T |
G5R+Y360L+W408P |
G5R+F259W+S284W |
G5R+W186L+F259W |
G5R+S284W+T320E |
Q96K+F259W+G433GS |
Q96K+W186L+W439R |
Q96K+W186L+E257T |
Q96K+W408P+N471T |
Qg6K+E257T+W408P |
Q96K+W408P+G433GS |
Q96K+F259W+N471T |
Q96K+Y360L+A434V |
Q96K+F259W+W408P |
W186L+E257T+Y360L |
W186L+W408P+N471T |
W186L+E257T+W408P |
W186L+E257T+N471T |
E257T+A434V+N471T |
E257T+W408P+A434V |
F259W+S284W+W439R |
F259W+S284W+Y360L |
F259W+T320E+G433GS |
S284W+T320E+Y360L |
S284W+W408P+G433GS |
S284W+T320E+A434V |
T320E+Y360L+G433GS |
[表1-2]
T320E+W439R+N471T |
T320E+G433GS+N471T |
T320E+Y360L+W439R |
Y360L+A434V+N471T |
G5R+S284W+W439R |
Q96K+E257T+T320E+W408P |
Q96K+E257T+W408P+A434V |
Q96K+E257T+W408P+N471T |
Q96K+E257T+T320E+W408P+A434V |
Q96K+E257T+T320E+W408P+N471T |
Q96K+T320E+Y360L+W408P+A434V |
Q96K+T320E+Y360L+A434V+N471T |
E257T+T320E+W408P+A434V+N471T |
G5R+Q96K+W186L+E257T+T320E+W439R |
G5R+Q96K+W186L+E257T+S284W+G433GS |
G5R+F259W+S284W+T320E+W439R+N471T |
G5R+Q96K+W186L+Y360L+W408P+G433GS |
G5R+Q96K+W186L+E257T+W408P+W439R |
G5R+Q96K+F259W+Y360L+W408P+W439R |
G5R+E257T+F259W+S284W+Y360L+N471T |
G5R+Q96K+W186L+F259W+Y360L+G433GS |
G5R+F259W+S284W+T320E+Y360L+A434V |
Q96K+E257T+F259W+T320E+G433GS+N471T |
Q96K+W186L+E257T+S284W+W439R+N471T |
Q96K+S284W+T320E+W408P+W439R+N471T |
Q96K+W186L+E257T+W408P+A434V+N471T |
Q96K+W186L+F259W+Y360L+W439R+N471T |
G5R+Q96K+Y360L+W408P+G433GS |
G5R+Q96K+F259W+Y360L+G433GS |
Q96K+F259W+Y360L+W439R+N471T |
[表1-3]
Q96K+E257T+W408P+A434V+N471T |
E257T+Y360L |
E257T+Y360L+W408P |
E257T+Y360L+G476K |
E257T+Y360L+W408P+G476K |
G5R+S38N |
Q96K+S38N |
S38N+E257T |
S38N+F259W |
S38N+S284W |
S38N+T320D |
S38N+T320E |
S38N+Y360L |
S38N+W408P |
S38N+N471T |
S38N+G476A |
S38N+G476K |
S38N+G476E |
S38N+N471T+G476K |
S38N+N471T+G476K+G477E |
E257T+Y360C |
E257T+Y360M |
E257T+Y360V |
E257T+G476K+Y360C |
E257T+G476K+Y360M |
E257T+G476K+Y360V |
G5R+W186L |
G5R+E257T |
G5R+Y360L |
S284W+T320E |
Q96K+F259W |
[表1-4]
Q96K+W186L |
Q96K+W408P |
G5R+S284W |
G5R+W439R |
S38N+S284W |
S38N+T320D |
接着,将有助于提高稳定性的优选突变的组合表示于下述表2-1~2-4。因此,至少具有该组合突变的突变体是特别有助于提高稳定性的突变体。另外,组合有表1-1~1-4的组合突变与表2-1~2-4的组合突变的突变从实现清洗性能和稳定性的提高的方面来看也是优选的。
[表2-1]
F205Y+H240F+Y242F |
N192F+F205Y+H240F+Y242F |
E187P+N192F |
H240F+Y242F |
H240F+Y242F+S331T |
N126Y+T129I+L203Y+F205Y |
N126Y+T129I+H240F+Y242F+G244W |
N126Y+T129I+H283Q+A288F |
H240F+Y242F+G244W |
H240F+G244W |
Y242F+G244W |
L203Y+F205Y |
N126Y+T129I |
H283Q+A288F |
S241Q+Y242F |
S241F+G244W |
S241Q+Y242F+G244W |
H240F+S241Q+G244W |
H240F+S241Q+Y242F |
T129I+E187P+G244W+S331T |
S241Q+H283Q+A288F+S331T |
N126Y+T129I+G140W+E187P |
N126Y+G179D+N192F+L203Y |
N126Y+F205Y+Y242F+A288F |
L203Y+R211I+Y242F+H283Q |
G179D+E187P+R2111+H240F |
G140W+L203Y+Y242F+G244W |
H240F+S241Q+Y242F+G244W |
F205Y+H240F+S241Q |
F205Y+H240F+S241Q+Y242F |
[表2-2]
F205Y+S241Q |
E187P+H240F+S241Q |
E187P+H240F+S241Q+Y242F |
E187P+S241Q+Y242F |
E187P+H240F+Y242F |
N126Y+E187P |
T129I+E187P |
G140W+E187P |
E187P+L203Y |
E187P+F205Y |
E187P+S241Q |
E187P+Y242F |
E187P+G244W |
E187P+H283Q |
E187P+S331T |
N126Y+T129I+E187P |
E187P+N192F+F205Y+H240F+Y242F |
M199L+F205Y+R211I+H240F+S241Q+Y242F |
H240F+S241Q |
E187P+R211I |
E187P+N192F+M199L |
E187P+F205Y+H240F+Y242F |
E187P+N192F+Y242F |
E187P+N192F+R211I |
E187P+N192F+M199L+R211I |
E187P+N192F+F205Y+Y242F |
E187P+N192F+F205Y+H240F+Y242F |
[表2-3]
N192F+S241Q |
N192F+H240F+S241Q |
N192F+F205Y+H240F+S241Q |
N192F+F205Y+H240F+S241Q+Y242F |
R211I+H240F+S241Q |
R211I+H240F+S241Q+Y242F |
F205Y+R211I+H240F+S241Q |
F205Y+R211I+H240F+S241Q+Y242F |
G179H+M199L+F205Y+R211I+H240F+S241Q+Y242F |
G1了9H+F205Y+R211I+H240F+S241Q+Y242F |
G179H+F205Y+H240F+S241Q+Y242F |
G179H+E187P+N192F |
E187P+M199L |
M199L+H240F+S241Q |
F205Y+H240F+S241A |
F205Y+H240F+S241D |
H240F+S241A |
H240F+S241D |
N126Y+R211I |
N126Y+H240F |
E187P+H240F |
N192F+F205Y |
N192F+R211I |
N192F+H240F |
F205Y+R211I |
F205Y+H240F |
R211I+H240F |
R211I+S241Q |
R211I+Y242F |
[表2-4]
N192F+M199L+F205Y+R211I+H240F+S241Q+Y242F |
G179H+N192F+M199L+F205Y+R211I+H240F+S241Q+Y242F |
N192F+F205Y+R211I+H240F+S241Q+Y242F |
G179H+N192F+F205Y+R211I+H240F+S241Q+Y242F |
N192F+R211I+H240F+S241Q+Y242F |
N192F+F205Y+R211I+H240F+S241Q |
E187P+K278L |
E187P+K278D |
E187P+K278W |
E187P+K278I |
E187P+K278H |
E187P+K278S |
E187P+K278T |
E187P+K278N |
E187P+K278Q |
E187P+K278V |
E187P+K278A |
E187P+K278Y |
E187P+K278F |
E187P+S241L+K278W |
E187P+S241Y+K278I |
E187P+S241P+K278W |
E187P+S241L+K278I |
E187P+S241P+K278I |
E187P+S241Y+K278W |
E187P+S241F+K278W |
E187P+S241Y+K278Y |
E187P+S241Y+K278F |
E187P+S241Y+K278H |
E187P+S241Y+K278L |
E187P+S241H+K278W |
接着,将有助于提高清洗性能和稳定性的优选突变的组合表示于下述表3。因此,至少具有该组合突变的突变体是特别有助于提高清洗性能和稳定性的突变体。
[表3]
G5R+E187P+N192F+S284W+W439R |
G5R+E187P+N192F+Y360L+G433GS |
Q96K+E187P+N192F+F259W+G433GS |
F205Y+H240F+S241Q+Y242F+E257T+Y360L |
F205Y+H240F+S241Q+Y242F+E257T+Y360L+W408P |
F205Y+H240F+S241Q+Y242F+E257T+Y360L+G476K |
F205Y+R211I+H240F+S241Q+Y242F+E257T+Y360L |
F205Y+H240F+S241Q+Y242F+E257T+Y360L+W408P+G476K |
M199L+F205Y+H240F+S241Q+Y242F+E257T+Y360L+W408P+G476K |
M199L+F205Y+H240F+Y242F+E257T+Y360L+S241Q+G476K |
M199L+F205Y+R211I+H240F+S241Q+Y242F+E257T+Y360L+W408P+G476K |
M199L+F205Y+H240F+S241Q+Y242F+E257T+S331T+Y360L+W408P+G476K |
N126Y+M199L+F205Y+H240F+S241Q+Y242F+E257T+Y360L+W408P+G476K |
E187P+M199L+F205Y+H240F+S241Q+Y242F+E257T+Y360L+G476K+W408P |
G5R+Q96K+E187P+N192F+M199L+Y242F+F259W+S331T+Y360L+W439R |
G5R+Q96K+E187P+N192F+M199L+R211I+Y242F+F259W+Y360L+W439R |
G179H+N192F+M199L+F205Y+R2111+H240F+S241Q+Y242F+E257T+Y360L+W408P+G476K |
S38N+G179H+N192F+M199L+F205Y+R2111+H240F+S241Q+Y242F+N471T+G476K+G477E |
Q96K+E187P+N192F+R211I+F259W+G433GS |
S38N+M199L+F205Y+R211I+H240F+S241Q+Y242F+N471T+G476K+G477E |
Q96K+G179H+N192F+M199L+F205Y+R211I+H240F+S241Q+Y242F+F259W+G433GS |
E187P+N192F+R211I+E257T+Y360L+W408P+G476K |
Q96K+M199L+F205Y+R211I+H240F+S241Q+Y242F+F259W+G433GS |
G5R+M199L+F205Y+R2111+H240F+S241Q+Y242F+S284W+W439R |
G5R+G179H+N192F+M199L+F205Y+R211I+H240F+S241Q+Y242F+S284W+W439R |
<编码本发明的突变体的多核苷酸>
本发明的突变体能够使用本技术领域中公知的各种突变导入技术来制造。例如,能够通过使编码其基准氨基酸序列的亲本α-淀粉酶基因(基准α-淀粉酶基因)内的编码改变对象的氨基酸残基的多核苷酸突变成编码改变后的氨基酸残基的多核苷酸,进而从该突变基因表达突变体来制造。
编码本发明的突变体的多核苷酸可以为单链或双链DNA、RNA、或人工核酸的形态,或者可以为cDNA、或不含内含子的化学合成DNA。
在本发明中,作为使亲本α-淀粉酶的氨基酸残基突变的手段,能够使用本技术领域中公知的各种突变导入技术。例如能够通过在编码亲本α-淀粉酶的氨基酸序列的多核苷酸(以下也称为亲本基因)中,使编码要突变的氨基酸残基的核苷酸序列突变成编码突变后的氨基酸残基的核苷酸序列,得到编码本发明的突变体的多核苷酸。
在亲本基因中导入目标突变基本上能够使用本领域技术人员公知的各种定点突变导入法进行。定点突变导入法例如能够通过反向PCR法或退火法等任意的方法进行。也能够使用市售的定点突变导入用试剂盒(例如,Stratagene公司的QuickChange II Site-Directed Mutagenesis Kit、QuickChange Multi Site-Directed Mutagenesis Kit等)。
在亲本基因中的定点突变导入最通常地能够使用包含要导入的核苷酸突变的突变用引物来进行。该突变用引物以与亲本基因中包含编码要突变的氨基酸残基的核苷酸序列的区域退火、且包含代替编码该要突变的氨基酸残基的核苷酸序列(密码子)而具有编码突变后的氨基酸残基的核苷酸序列(密码子)的核苷酸序列的方式设计即可。编码突变前和突变后的氨基酸残基的核苷酸序列(密码子)只要是本领域技术人员就可以根据通常的教科书等适当识别并选择。或者,定点突变导入也可以使用如下方法:分别使用包含要导入的核苷酸突变的互补的2个引物,将突变部位的上游侧和下游侧分别扩增得到的DNA片段利用SOE(splicing by overlap extension,重叠延伸拼接)-PCR(Gene,1989,77(1):p61-68)连结成1个的方法。
包含亲本基因的模板DNA能够从上述产生α-淀粉酶的微生物通过常规方法提取基因组DNA,或者提取RNA通过逆转录来合成cDNA而制备。或者也可以根据亲本α-淀粉酶的氨基酸序列,化学合成对应的核苷酸序列,作为模板DNA使用。将包含编码由序列编号4所示的氨基酸序列构成的α-淀粉酶的碱基序列的DNA序列表示为序列编号3,将包含编码由序列编号2所示的氨基酸序列构成的α-淀粉酶(YR288)的碱基序列的DNA序列表示为序列编号1。
突变用引物能够通过亚磷酰胺法(Nucleic Acids R4esearch,1989,17:7059-7071)等公知的寡聚核苷酸合成法制作。这样的引物合成例如可以使用市售的寡聚核苷酸合成装置(ABI公司制等)实施。通过使用包含该突变用引物的引物组,以亲本基因为模板DNA,进行上述的定点突变导入,能够得到编码具有目标突变的本发明的突变体的多核苷酸。
编码该本发明的突变体的多核苷酸可以是单链或双链的DNA、cDNA、RNA或其它人工核酸。该DNA、cDNA和RNA也可以化学合成。另外,该多核苷酸在开放阅读框(ORF)的基础上,也可以包含非翻译区域(UTR)的核苷酸序列。另外,该多核苷酸可以根据本发明的突变多肽产生用的转化体的种类,进行密码子优化。各种生物所使用的密码子的信息可以从Codon Usage Database([www.kazusa.or.jp/codon/])获得。
<载体或DNA片段>
所得到的编码本发明的突变体的多核苷酸可以被整合入载体。作为含有该多核苷酸的载体的种类,没有特别限定,可以是质粒、噬菌体、噬菌粒、粘粒、病毒、YAC载体、穿梭载体等任意的载体。另外该载体没有限定,优选为能够在细菌内、优选在杆菌属细菌(例如枯草杆菌或其突变株)内扩增的载体,更优选是能够在杆菌属细菌内诱导所导入基因的表达的表达载体。其中,能够在杆菌属细菌和其它生物中都能够复制的载体即穿梭载体由于能够重组生产本发明的突变体,因此能够优选使用。作为优选的载体的例子,没有限定,可以列举:pHA3040SP64、pHSP64R或pASP64(日本专利第3492935号)、pHY300PLK(能够转化大肠杆菌和枯草杆菌这两者的表达载体;Jpn J Genet,1985,60:235-243)、pAC3(NucleicAcids Res,1988,16:8732)等的穿梭载体;pUB110(J Bacteriol,1978,134:318-329)、pTA10607(Plasmid,1987,18:8-15)等的能够用于杆菌属细菌的转化的质粒载体等。另外也可以使用来自大肠杆菌的质粒载体(例如pET22b(+)、pBR322、pBR325、pUC57、pUC118、pUC119、pUC18、pUC19、pBluescript等)。
上述载体可以包括包含DNA的复制起始区域或复制起点的DNA区域。或者,在上述载体中,也可以在编码本发明的突变体的多核苷酸(即突变体基因)的上游可发挥作用地连结有用于开始该基因的转录的启动子区域、终止子区域、或用于使所表达的蛋白质分泌到细胞外的分泌信号区域等的控制序列。此外,基因与控制序列“可发挥作用地连结”是指基因和控制区域以该基因能够在通过该控制区域的控制下表达的方式配置。
上述启动子区域、终止子、分泌信号区域等的控制序列的种类没有特别限定,可以根据要导入的宿主,适当选择通常使用的启动子和分泌信号序列来使用。例如作为能够整合入载体的控制序列的优选例,可以列举Bacllus sp.KSM-S237株的纤维素酶基因的启动子、分泌信号序列等。
或者,也可以在上述本发明的载体中进一步整合用于选择被适当导入了该载体的宿主的标记物基因(例如,氨苄青霉素、新霉素、卡那霉素、氯霉素等药剂的耐性基因)。或者,在宿主使用营养缺陷型株的情况下,可以将编码所要求的营养的合成酶的基因作为标记物基因整合入载体。另外或者使用为了生育所需的特定代谢的选择培养基的情况下,也可以将该代谢的相关基因作为标记物基因整合入载体。作为这样的代谢相关基因的例子,可以列举用于利用乙酰胺作为氮源的乙酰胺酶基因。
编码上述本发明的突变体的多核苷酸与控制序列和标记物基因的连结能够用SOE(splicing by overlap extension,重叠延伸拼接)-PCR法(Gene,1989,77:61-68)等该领域公知的方法进行。连结的片段向载体的导入步骤在该领域中为公知的。
<转化细胞>
通过将包含编码本发明的突变体的多核苷酸的载体导入宿主,或者将包含编码本发明的突变体的多核苷酸的DNA片段导入宿主的基因组,能够得到本发明的转化细胞。
作为宿主细胞,可以列举细菌、丝状菌等的微生物。作为细菌的例子,可以列举大肠杆菌(Escherichia coli)、属于葡萄球菌属(Staphylococcus)、肠球菌属(Enterococcus)、李斯特菌属(Listeria)、杆菌属(Bacillus)的细菌等。其中,优选大肠杆菌和杆菌属细菌(例如枯草杆菌Bacillus subtilis Marburg No.168(枯草杆菌168株)或其突变株)。作为枯草杆菌突变株的例子,可以列举J.Biosci.Bioeng.,2007,104(2):135-143中记载的蛋白酶9重缺陷株KA8AX、以及Biotechnol.Lett.,2011,33(9):1847-1852中记载的蛋白酶8重缺陷株中提高了蛋白质的折叠效率的D8PA株。作为丝状菌的例子,可以列举木霉属(Trichoderma)、曲霉属(Aspergillus)、根霉属(Rizhopus)等。
作为向宿主导入载体的方法,可以使用原生质体法、电穿孔法等该领域中通常使用的方法。通过对适当进行了导入的菌株选择标记物基因的表达、营养缺陷型等作为指标,能够得到导入了载体的目标转化体。
或者,也可以将连结有编码本发明的突变体的多核苷酸、控制序列和标记物基因的片段直接导入宿主的基因组。例如通过SOE-PCR法等,构建在上述连结片段的两端添加了与宿主的基因组互补的序列的DNA片段,将其导入宿主,在宿主基因组与该DNA片段之间发生同源重组,而将编码本发明的突变体的多核苷酸导入宿主的基因组。
对这样操作得到的导入了编码本发明的突变体的多核苷酸或包含其的载体的转化体用适当的培养基进行培养,则该载体上的编码蛋白质的基因表达,产生本发明的突变体。本领域技术人员能够根据该转化体的微生物的种类,适当选择该转化体的培养中使用的培养基。
或者,本发明的突变体也可以使用无细胞翻译体系从编码本发明的突变体的多核苷酸或其转录产物表达。“无细胞翻译体系”是指在将成为宿主的细胞机械性地破坏而得到的悬浮液中加入蛋白质的翻译所必须的氨基酸等试剂,构成in vitro(体外)转录翻译体系或in vitro翻译体系。
上述培养物或由无细胞翻译体系所生成的本发明的突变体能够通过将蛋白质纯化中使用的通常的方法、例如离心分离、硫酸铵沉淀、凝胶色谱、离子交换色谱、亲和色谱等单独使用或适当组合使用来进行分离或纯化。此时,在转化体内的载体上编码本发明的α-淀粉酶突变体的基因与分泌信号序列可发挥作用地连结的情况下,所生成的蛋白质被分泌到细胞外,因此能够更容易地从培养物回收。从培养物回收的蛋白质可以用公知的手段进一步纯化。
这样操作得到的本发明的突变体具有比亲本α-淀粉酶提高的清洗性能和/或稳定性。
这里,“提高的清洗性能”是指比亲本α-淀粉酶提高的清洗效果,例如在洗涤或清洗工序中引起污渍的除去的能力。
清洗性能可以使用该技术领域中公知的方法评价。例如,裁切为规定大小的污染布插入96孔测试板的孔中,添加洗涤剂溶液和酶溶液在规定条件下进行清洗处理。对清洗结束后的清洗液测定488nm的吸光度,求出与空白的差量ΔA488作为清洗力。通过将突变体的ΔA488除以亲本α-淀粉酶的ΔA488,能够求出相对清洗力。
另外,“提高的稳定性”是指比亲本α-淀粉酶提高了的在清洗剂存在下的α-淀粉酶活性的维持能力。
稳定性可以使用该技术领域中公知的方法评价。例如在洗涤剂中添加酶溶液,处理规定时间后测定α-淀粉酶活性,算出经由该处理的每单位时间(h)的失活速度,计算半衰期(h)。通过将突变体的半衰期(h)除以亲本α-淀粉酶的半衰期(h),能够求出相对稳定性。
本发明的突变体作为各种清洗剂组合物配合用酶有用,特别是作为适于低温清洗的清洗剂组合物配合用酶有用。
这里,作为“低温”,可以列举40℃以下、35℃以下、30℃以下、25℃以下,还可以列举5℃以上、10℃以上、15℃以上。另外,可以列举5~40℃、10~35℃、15~30℃、15~25℃。
清洗剂组合物中的本发明的突变体的配合量只要是该蛋白质显示活性的量即可,没有特别限制,例如,相对于清洗剂组合物每1kg,优选为1mg以上,更优选10mg以上,更优选50mg以上,且优选为5000mg以下,更优选1000mg以下,更优选500mg以下。另外优选为1~5000mg,更优选为10~1000mg,更优选为50~500mg。
清洗剂组合物除了本发明的突变体以外还可以并用各种酶。例如水解酶、氧化酶、还原酶、转移酶、裂解酶、异构酶、连接酶、合成酶等。其中,优选与本发明的蛋白质不同的淀粉酶、蛋白酶、纤维素酶、角蛋白酶、酯酶、角质酶、脂肪酶、支链淀粉酶、果胶酶、甘露聚糖酶、葡萄糖苷酶、葡聚糖酶、胆固醇氧化酶、过氧化物酶、漆酶等,特别优选蛋白酶、纤维素酶、淀粉酶、脂肪酶。
作为蛋白酶,可以列举市售的Alcalase、Esperase、Everlase、Savinase、Kannase、Progress Uno(注册商标;诺维信公司)、PREFERENZ、EFFECTENZ、EXCELLENZ(注册商标;杜邦公司)、Lavergy(注册商标;BASF公司)、以及KAP(花王)等。
作为纤维素酶,可以列举Celluclean、Carezyme(注册商标;诺维信公司)、以及KAC、日本特开平10-313859号公报记载的杆菌属KSM-S237株所生产的碱性纤维素酶、日本特开2003-313592的号公报记载的突变碱性纤维素酶(以上,花王)等。
作为淀粉酶,可以列举Termamyl、Duramyl、Stainzyme、Stainzyme Plus、AmplifyPrime(注册商标;诺维信公司)、PREFERENZ、EFFECTENZ(注册商标;杜邦公司)、以及KAM(花王)等。
作为脂肪酶,可以列举Lipolase、Lipex(注册商标;诺维信公司)等。
清洗剂组合物中可以配合公知的清洗剂成分,作为该公知的清洗剂成分,可以列举例如如下物质。
(1)表面活性剂
表面活性剂优选在清洗剂组合物中配合0.5~60质量%,特别优选在粉末状清洗剂组合物中配合10~45质量%、在液体清洗剂组合物中配合20~90质量%。另外在本发明的清洗剂组合物为洗衣房用衣物清洗剂、自动餐具清洗机用清洗剂的情况下,表面活性剂通常配合1~10质量%,优选配合1~5质量%。
作为清洗剂组合物所用的表面活性剂,能够列举阴离子性表面活性剂、非离子性表面活性剂、两性表面活性剂、阳离子性表面活性剂的1种或组合,优选为阴离子性表面活性剂、非离子性表面活性剂。
作为阴离子性表面活性剂,优选碳原子数10~18的醇的硫酸酯盐、碳原子数8~20的醇的烷氧化物的硫酸酯盐、烷基苯磺酸盐、石蜡烃磺酸盐、α-烯烃磺酸盐、内部烯烃磺酸盐、α-磺基脂肪酸盐、α-磺基脂肪酸烷基酯盐或脂肪酸盐。在本发明中特别优选选自烷基链的碳原子数为10~14的、更优选12~14的直链烷基苯磺酸盐和亚烷基链的碳原子数为12~20的、更优选16~18的内部烯烃砜中的一种以上的阴离子性表面活性剂,作为平衡离子,优选碱金属盐、胺类,特别优选钠和/或钾、单乙醇胺、二乙醇胺。内部烯烃磺酸例如可以参照WO2017/098637。
作为非离子性表面活性剂,优选聚氧亚烷基烷基(碳原子数8~20)醚、烷基多聚糖苷、聚氧亚烷基烷基(碳原子数8~20)苯基醚、聚氧亚烷基脱水山梨糖醇脂肪酸(碳原子数8~22)酯、聚氧亚烷基二醇脂肪酸(碳原子数8~22)酯、聚氧乙烯聚氧丙烯嵌段聚合物。特别是作为非离子性表面活性剂,优选在碳原子数10~18的醇加成了4~20摩尔环氧乙烷或环氧丙烷等的环氧烷烃而成的〔HLB值(用Griffin法算出)为10.5~15.0、优选为11.0~14.5这样的〕聚氧亚烷基烷基醚。
(2)二价金属离子捕集剂
二价金属离子捕集剂可以配合0.01~50质量%、优选配合5~40质量%。作为本发明清洗剂组合物中使用的二价金属离子捕集剂,可以列举三聚磷酸盐、焦磷酸盐、正磷酸盐等的缩合磷酸盐、沸石等的铝硅酸盐、合成层状结晶性硅酸盐、次氮基三乙酸盐、乙二胺四乙酸盐、柠檬酸盐、异柠檬酸盐、聚缩醛羧酸盐等。其中特别优选结晶性铝硅酸盐(合成沸石),A型、X型、P型沸石中,特别优选A型。合成沸石适合使用平均一次粒径0.1~10μm、特别是0.1~5μm的沸石。
(3)碱剂
碱剂可以配合0.01~80质量%、优选配合1~40质量%。粉末洗涤剂的情况下,可以列举被统称为重灰或轻灰的碳酸钠等的碱金属碳酸盐、以及JIS1号、2号、3号等的非晶质的碱金属硅酸盐。这些无机性的碱剂在洗涤剂干燥时在颗粒的骨架形成中有效,能够得到比较硬、流动性优异的洗涤剂。作为这些以外的碱,可以列举倍半碳酸钠、碳酸氢钠等,另外三聚磷酸盐等的磷酸盐也具有作为碱剂的作用。另外,作为液体洗涤剂中使用的碱剂,除了上述碱剂以外,还可以使用氢氧化钠、以及单乙醇胺、二乙醇胺或三乙醇胺,也可以作为活性剂的平衡离子使用。
(4)再污染防止剂
再污染防止剂可以配合0.001~10质量%、优选配合1~5质量%。作为本发明清洗剂组合物中使用的再污染防止剂,可以列举聚乙二醇、羧酸系聚合物、聚乙烯醇、聚乙烯吡咯烷酮等。其中,羧酸系聚合物除了防止再污染能力以外,还具有捕集金属离子的功能、使固体颗粒污渍从衣物分散到洗涤浴中的作用。羧酸系聚合物为丙烯酸、甲基丙烯酸、衣康酸等的均聚物或共聚物,作为共聚物,上述单体与马来酸共聚得到的共聚物是合适的,优选分子量为数千~10万。除了上述羧酸系聚合物以外,聚缩水甘油酸盐等的聚合物、羧甲基纤维素等的纤维素衍生物、以及聚天冬氨酸等的氨基羧酸系的聚合物也具有金属离子捕集剂、分散剂和再污染防止能力,故而优选。
(5)漂白剂
例如过氧化氢、过碳酸盐等的漂白剂优选配合1~10质量%。在使用漂白剂时,可以配合四乙酰乙二胺(TAED)或日本特开平6-316700号公报记载等的漂白活化剂(激活剂)0.01~10质量%。
(6)荧光剂
作为清洗剂组合物中使用的荧光剂,可以列举联苯型荧光剂(例如Tinopal CBS-X等)或二苯乙烯型荧光剂(例如DM型荧光染料等)。荧光剂优选配合0.001~2质量%。
(7)其它成分
清洗剂组合物中可以含有衣物用洗涤剂的领域中公知的助洗剂(builder)、柔顺剂、还原剂(亚硫酸盐等)、抑泡剂(有机硅等)、香料、防菌防霉剂(Proxel[商品名]、苯甲酸等)、其它添加剂。
清洗剂组合物可以将由上述方法得到的本发明的蛋白质和上述公知的清洗成分组合,按照常规方法制造。洗涤剂的形态可以根据用途进行选择,可以制成例如液体、粉体、颗粒、膏、固形等。
这样操作得到的清洗剂组合物可以作为衣物清洗剂、餐具清洗剂、漂白剂、硬质表面清洗用清洗剂、排水管清洗剂、假牙清洗剂、医疗器具用的杀菌清洗剂等使用,优选列举衣物清洗剂、餐具清洗剂,更优选列举洗衣房用衣物清洗剂(洗衣房用洗涤洗剂)、通过手洗的餐具清洗剂、自动餐具清洗机用清洗剂。
另外,该清洗剂组合物适于在40℃以下、35℃以下、30℃以下、25℃以下且5℃以上、10℃以上、15℃以上使用。另外,适于在5~40℃、10~35℃、15~30℃、15~25℃使用。
关于上述实施方式,在本发明中还公开了以下的方案。
<1>一种α―淀粉酶突变体,其为包含与序列编号2所示的氨基酸序列的G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的1处或多处氨基酸残基的改变的亲本α-淀粉酶的突变体,上述亲本α-淀粉酶或α-淀粉酶突变体相对于序列编号4所示的氨基酸序列具有至少90%的序列同一性。
<2>如<1>所述的突变体,其中,上述与G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的氨基酸残基的改变分别为G5E/D/P/R/K、S38N、T49Q、Q96R/K、N126Y、T129I、G140Y/F/W、F153W、Q167E、G179D/H、W186L、E187P、N192F、M199L/T/A/N/Q/S/V/I、Y200G、L203Y/M/F、Y205F、D206R/E/N/T/G、R211V/L/I、K215F、H240F、S241A/Q/D/L/Y/P/H、Y242F、G244K/W/L/R、E257T、F259W、K278L/D/W/I/H/S/T/N/Q/V/A/Y/F、H283Q、S284W、A288F、H295Y、Y296A、N303R/E/S/G/V/D/T/A、T320D/E、S331T、L348I、Y360C/M/L/V、W408P、L429V、V430M、G433GS、A434V、W439R、N471T、G476A/P/E/S/F/R/K和G477E。
<3>如<1>或<2>所述的突变体,其中,氨基酸残基的改变为2处以上的改变。
<4>如<1>所述的突变体,其中,上述氨基酸残基的改变为与N126、E187、N192、F205、R211、H240、S241和Y242的各位置相当的位置的氨基酸残基的改变。
<5>如<4>所述的突变体,其中,上述与N126、E187、N192、F205、R211、H240、S241和Y242的各位置相当的位置的氨基酸残基的改变分别为N126Y、E187P、N192F、F205Y、R211I、H240F、S241Q和Y242F。
<6>如<4>或<5>所述的突变体,其中,氨基酸残基的改变为2处以上的改变。
<7>如<1>或<2>所述的突变体,其包含选自与E187、F205和H240的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变、和选自与N126、N192、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变。
<8>如<1>或<2>所述的突变体,其包含选自与E187和H240的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变、和选自与N126、N192、F205、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变。
<9>如<1>或<2>所述的突变体,其包含选自与F205和H240的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变、和选自与N126、E187、N192、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变。
<10>如<1>或<2>所述的突变体,其包含与F205的位置相当的氨基酸残基的改变、和选自与N126、E187、N192、R211、H240、S241和Y242的各位置相当的氨基酸残基的至少1个以上的氨基酸残基的改变。
<11>如<1>或<2>所述的突变体,其包含与H240的位置相当的氨基酸残基的改变、和选自与N126、E187、N192、F205、R211、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变。
<12>如<1>或<2>所述的突变体,其包含与R211的位置相当的氨基酸残基的改变、和选自与N126、E187、N192、F205、H240、S241和Y242的各位置相当的氨基酸残基中的至少1个以上的氨基酸残基的改变。
<13>如<1>或<2>所述的突变体,其中,亲本α-淀粉酶为序列编号2所示的氨基酸序列中缺失了R178和G179的氨基酸残基的α-淀粉酶突变体。
<14>如<1>、<2>或<13>所述的突变体,其中,突变体至少包含选自上述表1-1~1-4所示的突变的组合中的突变。
<15>如<1>、<2>或<13>所述的突变体,其中,突变体至少包含选自上述表2-1~2-4所示的突变的组合中的突变。
<16>如<1>、<2>或<13>所述的突变体,其中,突变体包含选自上述表1-1~1-4所示的突变的组合中的突变和选自上述表2-1~2-4所示的突变的组合中的突变。
<17>如<1>、<2>或<13>所述的突变体,其中,突变体至少包含选自上述表3所示的突变的组合中的突变。
<18>编码<1>~<17>中任一项所述的突变体的多核苷酸。
<19>包含<18>的多核苷酸的载体或DNA片段。
<20>含有<19>的载体或DNA片段的转化细胞。
<21>如<20>所述的转化细胞,其为微生物。
<22>包含<1>~<17>的突变体的清洗剂组合物。
<23>如<22>所述的清洗剂组合物,其为衣物清洗剂或餐具清洗剂。
<24>如<23>所述的清洗剂组合物,其为粉末或液体。
<25>如<22>~<24>中任一项所述的清洗剂组合物,其在低温下使用。
<26>如<25>所述的清洗剂组合物,其在5~40℃的温度下使用。
实施例
(1)YR288突变体表达质粒的构建
记载在下述的实施例所记载的YR288突变体的构建方法。使用在5'末端具有15个碱基与反向引物的互补序列且包含突变序列的正向引物、和以紧挨着突变序列之前的碱基为5'末端的反向引物作为突变导入用引物对。以日本特愿2020-121626的实施例中记载的YR288表达质粒pHY-YR288或本实施例中制成的YR288突变体表达质粒为模板,使用突变导入用引物对,进行PCR。在将多个片段连结的情况下,使用各个PCR产物,按照In-Fusion,HDCloning kit(Clontech)的操作说明,进行In-Fusion反应。通过原生质体法将PCR产物或In-Fusion反应液对枯草杆菌进行转化,获得保持目标YR288突变体表达质粒的转化体。
(2)酶生产培养
在分注有添加了15ppm四环素的LB培养基300μL的96孔深孔板接种(1)中得到的重组枯草杆菌菌落,然后在30℃以210rpm培养过夜。第二天,将培养液6μL接种到分注有2×L-麦芽糖培养基(2%胰蛋白胨、1%酵母提取物、1%NaCl、7.5%麦芽糖、7.5ppm硫酸锰五水合物、0.04%氯化钙二水合物、15ppm四环素;%为(w/v)%)100μL的96孔深孔板中,在30℃以210rpm培养2天后,通过离心分离回收由菌体产生的包含酶的培养上清,作为酶溶液。
(3)培养上清的蛋白质浓度测定
培养上清的蛋白质浓度测定中使用蛋白质测定快速试剂盒Wako II(ProteinAssay Rapid Kit Wako II)(富士胶片和光纯药株式会社)。通过将不具有淀粉酶表达盒的pHY300PLK(Takara Bio)导入株的培养上清的蛋白质浓度作为空白,计算出培养上清中的淀粉酶的浓度。
(4)活性测定
使用保护了非还原末端的亚乙基对硝基苯基-α-D-麦芽七糖苷(Et-G7-pNP)作为底物。使α-葡萄糖苷酶作用于通过α-淀粉酶对Et-G7-pNP的作用生成的低聚麦芽糖-pNP,使pNP游离,测定伴随pNP生成的吸光度的增加速度,由此能够求出α-淀粉酶活性。使用Et-G7-pNP与作为包含α-葡萄糖苷酶的α-淀粉酶活性测定试剂的AMY-EL(Serotec)的RI液和RII液以2∶1混合得到的溶液作为底物溶液。将底物溶液100μL和适当稀释的酶样品10μL在96孔测试板的各孔中混合,在30℃测定405nm的吸光度变化(OD/min)。将与空白(不添加酶的样品)的差量ΔOD/min作为活性值。
(5)第178-181位的2个氨基酸缺失突变体的稳定性评价
通过实施例(1)中记载的方法,将YR288(序列编号2)作为亲本多肽构建图1所记载的突变体。在用离子交换水稀释成10%(v/v)的市售的液体衣物用洗涤剂(花王株式会社、Attack 3X)中添加酶溶液,在50℃保温15分钟后进行活性测定。将50℃处理后的样品的活性值除以50℃处理前的样品的活性值,再乘以100,由此算出残存活性(%)。通过使R178、G179、T180、G181中任意的2个残基缺失,稳定性大幅提高(图1)。
(6)清洗力评价
从CFT公司获得裁切成直径5.5mm的圆形的CS-26污染布来使用。在96孔测试板的各孔各插入2片CS-26圆形污染布,各加入200μL用自来水稀释到3000倍的市售的液体衣物用洗涤剂(花王株式会社、Attack 0)。各加入10μL用自来水稀释成3ppm的酶溶液,进行密封在20℃用灵敏型混合器(cute mixer)以1200rpm振荡15分钟。在清洗结束后,将100μL的清洗液移到新的96孔测试板,测定488nm的吸光度。将代替酶溶液而加入了自来水的样品作为空白,求出与空白的差量ΔA488作为清洗力。将各突变体的ΔA488除以亲本多肽的ΔA488,由此求出相对清洗力。
(7)稳定性评价
在用离子交换水稀释成10%(v/v)的市售的液体衣物用洗涤剂(花王株式会社、Attack 3X或花王株式会社、Attack 0)中添加酶溶液,在50℃保温30分钟~18小时后进行活性测定。将50℃处理前的样品的活性值作为初始活性,计算经由50℃处理每单位时间(h)的失活速度,由此计算半衰期(h)。将各突变体的半衰期(h)除以亲本多肽的半衰期(h),从而求出相对稳定性。
(8)突变筛选
通过实施例(1)中记载的方法,将YR288 R178Δ+T180Δ(序列编号4)作为亲本多肽构建各种突变体。通过实施例(6)和(7)中记载的方法评价突变体的性能。相对清洗力和/或相对稳定性为1.1以上的情况视为性能得到提高。以下的突变体的性能得到提高。
G5E、G5D、G5P、G5R、G5K、S38N、T49Q、Q96R、Q96K、N126Y、T129I、G140Y、G140F、G140W、F153W、Q167E、G179D、G179H、W186L、E187P、N192F、M199L、M199T、M199A、M199N、M199Q、M199S、M199V、M199I、Y200G、L203Y、L203M、L203F、Y205F、D206R、D206E、D206N、D206T、D206G、R211V、R211L、R211I、K215F、H240F、S241A、S241Q、S241D、Y242F、G244K、G244W、G244L、G244R、E257T、F259W、H283Q、S284W、A288F、H295Y、Y296A、N303R、N303E、N303S、N303G、N303V、N303D、N303T、N303A、T320D、T320E、S331T、L348I、Y360C、Y360M、Y360L、Y360V、W408P、L429V、V430M、G433GS、A434V、W439R、N471T、G476A、G476P、G476E、G476S、G476F、G476R、G476K、G477E
(9)多重突变体的清洗力评价
通过实施例(1)中记载的方法,将YR288 R178Δ+T180Δ(序列编号4)作为亲本多肽,构建包含2个以上实施例(8)中得到的突变的各种突变体。通过实施例(6)中记载的方法评价突变体的清洗力。在以下表示其结果。
[表4-1]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对清洗力 |
G5R+W186L+E257T | 2.7 |
G5R+E257T+G433GS | 2.6 |
G5R+Y360L+G433GS | 3 |
G5R+Q96K+E257T | 2.5 |
G5R+Y360L+W408P | 2.6 |
G5R+F259W+S284W | 1.8 |
G5R+W186L+F259W | 1.2 |
G5R+S284W+T320E | 2.5 |
Q96K+F259W+G433GS | 3 |
Q96K+W186L+W439R | 3.2 |
Q96K+W186L+E257T | 1.2 |
Q96K+W408P+N471T | 2.6 |
Q96K+E257T+W408P | 2.1 |
Q96K+W408P+G433GS | 3 |
Q96K+F259W+N471T | 2.8 |
Q96K+Y360L+A434V | 2.6 |
Q96K+F259W+W408P | 2.9 |
W186L+E257T+Y360L | 2.8 |
W186L+W408P+N471T | 1.7 |
W186L+E257T+W408P | 1.3 |
W186L+E257T+N471T | 2.5 |
E257T+A434V+N471T | 1.1 |
E257T+W408P+A434V | 2.1 |
F259W+S284W+W439R | 1.7 |
F259W+S284W+Y360L | 2.2 |
F259W+T320E+G433GS | 2.3 |
S284W+T320E+Y360L | 1.9 |
S284W+W408P+G433GS | 2.6 |
S284W+T320E+A434V | 2.2 |
T320E+Y360L+G433GS | 1.8 |
[表4-2]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对清洗力 |
T320E+W439R+N471T | 1.9 |
T320E+G433GS+N471T | 2.1 |
T320E+Y360L+W439R | 2.6 |
Y360L+A434V+N471T | 2.2 |
G5R+S284W+W439R | 3.1 |
Q96K+E257T+T320E+W408P | 2.6 |
Q96K+E257T+W408P+A434V | 2.1 |
Q96K+E257T+W408P+N471T | 2.5 |
Q96K+E257T+T320E+W408P+A434V | 2.9 |
Q96K+E257T+T320E+W408P+N471T | 2.9 |
Q96K+T320E+Y360L+W408P+A434V | 2.3 |
Q96K+T320E+Y360L+A434V+N471T | 2.1 |
E257T+T320E+W408P+A434V+N471T | 2.8 |
G5R+Q96K+W186L+E257T+T320E+W439R | 2.3 |
G5R+Q96K+W186L+E257T+S284W+G433GS | 1.4 |
G5R+F259W+S284W+T320E+W439R+N471T | 2.5 |
G5R+Q96K+W186L+Y360L+W408P+G433GS | 3.3 |
G5R+Q96K+W186L+E257T+W408P+W439R | 1.9 |
G5R+Q96K+F259W+Y360L+W408P+W439R | 3 |
G5R+E257T+F259W+S284W+Y360L+N471T | 2.8 |
G5R+Q96K+W186L+F259W+Y360L+G433GS | 3.2 |
G5R+F259W+S284W+T320E+Y360L+A434V | 2.2 |
Q96K+E257T+F259W+T320E+G433GS+N471T | 2.8 |
Q96K+W186L+E257T+S284W+W439R+N471T | 3.1 |
Q96K+S284W+T320E+W408P+W439R+N471T | 3 |
Q96K+W186L+E257T+W408P+A434V+N471T | 3.3 |
Q96K+W186L+F259W+Y360L+W439R+N471T | 3.4 |
G5R+Q96K+Y360L+W408P+G433GS | 3.1 |
G5R+Q96K+F259W+Y360L+G433GS | 3.1 |
Q96K+F259W+Y360L+W439R+N471T | 3 |
[表4-3]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对清洗力 |
Q96K+E257T+W408P+A434V+N471T | 2.6 |
E257T+Y360L | 2.6 |
E257T+Y360L+W408P | 2.8 |
E257T+Y360L+G476K | 3.1 |
E257T+Y360L+W408P+G476K | 3.3 |
G5R+S38N | 2.2 |
Q96K+S38N | 2.1 |
S38N+E257T | 2.2 |
S38N+F259W | 2.2 |
S38N+S284W | 2.6 |
S38N+T320D | 2.4 |
S38N+T320E | 2 |
S38N+Y360L | 2.3 |
S38N+W408P | 2 |
S38N+N471T | 2 |
S38N+G476A | 1.9 |
S38N+G476K | 2.2 |
S38N+G476E | 1.9 |
S38N+N471T+G476K | 2.5 |
S38N+N471T+G476K+G477E | 2.6 |
E257T+Y360C | 1.9 |
E257T+Y360M | 2.5 |
E257T+Y360V | 2.3 |
E257T+G476K+Y360C | 2.5 |
E257T+G476K+Y360M | 3.1 |
E257T+G476K+Y360V | 2.7 |
G5R+W186L | 3.1 |
G5R+E257T | 1.8 |
G5R+Y360L | 2 |
S284W+T320E | 2 |
Q96K+F259W | 2.2 |
[表4-4]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对清洗力 |
Q96K+W186L | 2 |
Q96K+W408P | 2.1 |
G5R+S284W | 2.4 |
G5R+W439R | 3 |
S38N+S284W | 2.6 |
S38N+T320D | 2.4 |
(10)多重突变体的稳定性评价
通过实施例(1)中记载的方法,将YR288 R178Δ+T180Δ(序列编号4)作为亲本多肽,构建包含2个以上实施例(8)中得到的突变的各种突变体。通过实施例(7)中记载的方法评价突变体的稳定性。在以下表示其结果。
[表5-1]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对稳定性 |
F205Y+H240F+Y242F | 17.1 |
N192F+F205Y+H240F+Y242F | 34.6 |
E187P+N192F | 66.3 |
H240F+Y242F | 9.7 |
H240F+Y242F+S331T | 15.7 |
N126Y+T129I+L203Y+F205Y | 4.3 |
N126Y+T129I+H240F+Y242F+G244W | 27.2 |
N126Y+T129I+H283Q+A288F | 7 |
H240F+Y242F+G244W | 9.6 |
H240F+G244W | 7.8 |
Y242F+G244W | 1.8 |
L203Y+F205Y | 1.7 |
N126Y+T129I | 2.5 |
H283Q+A288F | 2.8 |
S241Q+Y242F | 7.5 |
S241F+G244W | 8.2 |
S241Q+Y242F+G244W | 7.7 |
H240F+S241Q+G244W | 40 |
H240F+S241Q+Y242F | 39.7 |
T129I+E187P+G244W+S331T | 40.7 |
S241Q+H283Q+A288F+S331T | 10.4 |
N126Y+T129I+G140W+E187P | 61.6 |
N126Y+G179D+N192F+L203Y | 3.1 |
N126Y+F205Y+Y242F+A288F | 5.7 |
L203Y+R211I+Y242F+H283Q | 3.3 |
G179D+E187P+R211I+H240F | 44.6 |
G140W+L203Y+Y242F+G244W | 2.7 |
H240F+S241Q+Y242F+G244W | 38.7 |
F205Y+H240F+S241Q | 47.3 |
F205Y+H240F+S241Q+Y242F | 57.9 |
[表5-2]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对稳定性 |
F205Y+S241Q | 7.8 |
E187P+H240F+S241Q | 38.9 |
E187P+H240F+S241Q+Y242F | 43 |
E187P+S241Q+Y242F | 18 |
E187P+H240F+Y242F | 73.5 |
N126Y+E187P | 62.1 |
T129I+E187P | 36.7 |
G140W+E187P | 36.7 |
E187P+L203Y | 42.6 |
E187P+F205Y | 35.3 |
E187P+S241Q | 34.6 |
E187P+Y242F | 36.9 |
E187P+G244W | 36.6 |
E187P+H283Q | 34.5 |
E187P+S331T | 44.6 |
N126Y+T129I+E187P | 64.7 |
E187P+N192F+F205Y+H240F+Y242F | 99.2 |
M199L+F205Y+R211I+H240F+S241Q+Y242F | 112.9 |
H240F+S241Q | 33 |
E187P+R211I | 61.1 |
E187P+N192F+M199L | 88.1 |
E187P+F205Y+H240F+Y242F | 81.7 |
E187P+N192F+Y242F | 61.1 |
E187P+N192F+R211I | 119.6 |
E187P+N192F+M199L+R211I | 88.4 |
E187P+N192F+F205Y+Y242F | 70.6 |
E187P+N192F+F205Y+H240F+Y242F | 77.9 |
[表5-3]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对稳定性 |
N192F+S241Q | 7.9 |
N192F+H240F+S241Q | 60.7 |
N192F+F205Y+H240F+S241Q | 71.3 |
N192F+F205Y+H240F+S241Q+Y242F | 69.9 |
R211I+H240F+S241Q | 76.3 |
R211I+H240F+S241Q+Y242F | 77.2 |
F205Y+R211I+H240F+S241Q | 83 |
F205Y+R211I+H240F+S241Q+Y242F | 94.8 |
G179H+M199L+F205Y+R211I+H240F+S241Q+Y242F | 99.8 |
G179H+F205Y+R211I+H240F+S241Q+Y242F | 76.2 |
G179H+F205Y+H240F+S241Q+Y242F | 56.7 |
G179H+E187P+N192F | 65.7 |
E187P+M199L | 45.3 |
M199L+H240F+S241Q | 40.6 |
F205Y+H240F+S241A | 17.1 |
F205Y+H240F+S241D | 87.4 |
H240F+S241A | 15.1 |
H240F+S241D | 77 |
N126Y+R211I | 5.2 |
N126Y+H240F | 13.1 |
E187P+H240F | 76.9 |
N192F+F205Y | 3.6 |
N192F+R211I | 9.2 |
N192F+H240F | 20 |
F205Y+R211I | 6.4 |
F205Y+H240F | 12.1 |
R211I+H240F | 23 |
R211I+S241Q | 18 |
R211I+Y242F | 5.8 |
[表5-4]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对稳定性 |
N192F+M199L+F205Y+R211I+H240F+S241Q+Y242F | 84.8 |
G179H+N192F+M199L+F205Y+R211I+H240F+S241Q+Y242F | 147.8 |
N192F+F205Y+R211I+H240F+S241Q+Y242F | 116.7 |
G179H+N192F+F205Y+R211I+H240F+S241Q+Y242F | 98.7 |
N192F+R211I+H240F+S241Q+Y242F | 107.8 |
N192F+F205Y+R211I+H240F+S241Q | 102.6 |
通过实施例(1)中记载的方法,将YR288 R178Δ+T180Δ(序列编号4)作为亲本多肽,构建各种突变体。通过实施例(7)中记载的方法,评价突变体的稳定性。在以下表示其结果。
[表5-5]
酶(亲本:R178Δ+T180Δ(序列编号4)) | 相对稳定性 |
E187P+K278L | 81.8 |
E187P+K278D | 56.5 |
E187P+K278W | 106 |
E187P+K278I | 82.2 |
E187P+K278H | 80.3 |
E187P+K278S | 54.1 |
E187P+K278T | 63.4 |
E187P+K278N | 47.5 |
E187P+K278Q | 62.1 |
E187P+K278V | 63 |
E187P+K278A | 50.1 |
E187P+K278Y | 114.9 |
E187P+K278F | 102.4 |
E187P+S241L+K278W | 131.2 |
E187P+S241Y+K278I | 75.1 |
E187P+S241P+K278W | 40.7 |
E187P+S241L+K278I | 37.9 |
E187P+S241P+K278I | 102.6 |
E187P+S241Y+K278W | 47.6 |
E187P+S241F+K278W | 78.6 |
E187P+S241Y+K278Y | 109.7 |
E187P+S241Y+K278F | 105.8 |
E187P+S241Y+K278H | 47.9 |
E187P+S241Y+K278L | 49.8 |
E187P+S241H+K278W | 96.6 |
(11)多重突变体的清洗力和稳定性的评价
通过实施例(1)中记载的方法,将YR288 R178Δ+T180Δ(序列编号4)作为亲本多肽,构建包含实施例(9)中得到的多重突变和实施例(10)中得到的多重突变的各种突变体。通过实施例(6)和(7)中记载的方法,评价突变体的性能。在以下表示其结果。
[表6]
(12)多重突变体的清洗力和稳定性的评价
通过实施例(1)中记载的方法,将YR288 R178Δ+G179Δ作为亲本多肽,构建下述的突变体。通过实施例(6)和(7)中记载的方法,评价突变体的性能。在以下表示其结果。
[表7]
序列表
<110> 花王株式会社
<120> α-淀粉酶突变体
<130> KS1764
<150> JP 2020-167797
<151> 2020-10-02
<150> JP 2021-135746
<151> 2021-08-23
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1458
<212> DNA
<213> 芽孢杆菌YR288
<220>
<221> CDS
<222> (1)..(1455)
<223> 密码子优化后的寡聚核苷酸
<400> 1
gca gct caa aac ggt acg atg atg cag tac ttt gaa tgg tat gta ccg 48
Ala Ala Gln Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro
1 5 10 15
aat gat ggt cag cat tgg aat cgt ctg cgg aac gat gca gcc tac ttg 96
Asn Asp Gly Gln His Trp Asn Arg Leu Arg Asn Asp Ala Ala Tyr Leu
20 25 30
aaa tcc atc ggt gtg tca gct atc tgg aca cct cct gct tac aaa ggc 144
Lys Ser Ile Gly Val Ser Ala Ile Trp Thr Pro Pro Ala Tyr Lys Gly
35 40 45
aca agc cag aac gat gtt ggc tat gga gca tat gac ctg tac gat tta 192
Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
gga gaa ttc aac cag aaa ggc act att cgc acc aaa tat gga acg aaa 240
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
gct gaa ctg aaa agc gcg gta tcg act ctc aag tct aat ggg att caa 288
Ala Glu Leu Lys Ser Ala Val Ser Thr Leu Lys Ser Asn Gly Ile Gln
85 90 95
gtg tat gga gat gtc gtc atg aat cat aaa ggc ggt gcc gat tac aca 336
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Tyr Thr
100 105 110
gag aat gta aca gct gtg gaa gtc aat ccg agc aat cgc aat caa gaa 384
Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
aca tcc gat gag tat acg atc caa gca tgg aca gga ttc aac ttc cct 432
Thr Ser Asp Glu Tyr Thr Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140
ggg cga ggc aca acc cat agt ccg ttt aag tgg cag tgg tat cat ttt 480
Gly Arg Gly Thr Thr His Ser Pro Phe Lys Trp Gln Trp Tyr His Phe
145 150 155 160
gat gga act gac tgg gat cag tct cgg aat gcg agc aga atc ttc aaa 528
Asp Gly Thr Asp Trp Asp Gln Ser Arg Asn Ala Ser Arg Ile Phe Lys
165 170 175
ttt cgt ggg aca gga aaa gca tgg gat tgg gaa gta tca tcg gag aat 576
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn
180 185 190
ggg aat tac gac tat ctt atg tac gcc gat tta gac ttt gat cat cct 624
Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Phe Asp His Pro
195 200 205
gat gtt aga aat gag atg aag aat tgg ggc gtg tgg tac gcg aat gaa 672
Asp Val Arg Asn Glu Met Lys Asn Trp Gly Val Trp Tyr Ala Asn Glu
210 215 220
gtt ggc ttg gac gga ttt agg ctg gat gcg gtt aag cac ata aag cat 720
Val Gly Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys His
225 230 235 240
agc tat ctg ggc gat tgg gtg aac cat gtc aga act aag acg gga aaa 768
Ser Tyr Leu Gly Asp Trp Val Asn His Val Arg Thr Lys Thr Gly Lys
245 250 255
gag atg ttt aca gtt gct gaa tat tgg caa aac gac gtc aat gcc att 816
Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Val Asn Ala Ile
260 265 270
aat aac tat tta gcc aaa gtg aac tat aat cac tct gtc ttt gat gcg 864
Asn Asn Tyr Leu Ala Lys Val Asn Tyr Asn His Ser Val Phe Asp Ala
275 280 285
cct ttg cat tac aac ttt cac tat gca agc cag tca gga ggt aac tac 912
Pro Leu His Tyr Asn Phe His Tyr Ala Ser Gln Ser Gly Gly Asn Tyr
290 295 300
gac atg cgt aac tta ttg aac ggc aca gtc gtg gct gct cat ccg aca 960
Asp Met Arg Asn Leu Leu Asn Gly Thr Val Val Ala Ala His Pro Thr
305 310 315 320
aaa gca gtt acc ctt gtc gaa aat cat gac agt caa ccg gga caa tct 1008
Lys Ala Val Thr Leu Val Glu Asn His Asp Ser Gln Pro Gly Gln Ser
325 330 335
ctg gag tca gtc gtt caa ccg tgg ttt aag ccc ctt gcc tat gcg ttt 1056
Leu Glu Ser Val Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
atc ctc aca cga gca gaa ggt tat cca agt atc ttt tat ggg gat atg 1104
Ile Leu Thr Arg Ala Glu Gly Tyr Pro Ser Ile Phe Tyr Gly Asp Met
355 360 365
tat ggg act aag gga aac tca tcg tac gaa att cca gca ctc aaa acc 1152
Tyr Gly Thr Lys Gly Asn Ser Ser Tyr Glu Ile Pro Ala Leu Lys Thr
370 375 380
aaa att gaa ccg tta ctg aaa gca cgc aag gat tat gcc tat ggt acg 1200
Lys Ile Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr
385 390 395 400
cag cac aac tat atg gac cac tgg gat gtt ata ggt tgg acc aga gaa 1248
Gln His Asn Tyr Met Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu
405 410 415
ggt gac tcc acg aaa gaa aaa tct ggc ctt gcg acg ctt gtg aca gac 1296
Gly Asp Ser Thr Lys Glu Lys Ser Gly Leu Ala Thr Leu Val Thr Asp
420 425 430
ggg gca ggc ggg tct aaa tgg atg tat gta ggc aaa cag aat gct ggc 1344
Gly Ala Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln Asn Ala Gly
435 440 445
gaa gtt tgg tat gac atc aca ggc aat aga acg gat aaa atc acc att 1392
Glu Val Trp Tyr Asp Ile Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile
450 455 460
aat aca gat ggc tgg gga aac ttc caa gta aac gga ggc tca gtc tcc 1440
Asn Thr Asp Gly Trp Gly Asn Phe Gln Val Asn Gly Gly Ser Val Ser
465 470 475 480
gtg tac gtt caa cag taa 1458
Val Tyr Val Gln Gln
485
<210> 2
<211> 485
<212> PRT
<213> 芽孢杆菌YR288
<400> 2
Ala Ala Gln Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro
1 5 10 15
Asn Asp Gly Gln His Trp Asn Arg Leu Arg Asn Asp Ala Ala Tyr Leu
20 25 30
Lys Ser Ile Gly Val Ser Ala Ile Trp Thr Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Ala Glu Leu Lys Ser Ala Val Ser Thr Leu Lys Ser Asn Gly Ile Gln
85 90 95
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Tyr Thr
100 105 110
Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
Thr Ser Asp Glu Tyr Thr Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140
Gly Arg Gly Thr Thr His Ser Pro Phe Lys Trp Gln Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Gln Ser Arg Asn Ala Ser Arg Ile Phe Lys
165 170 175
Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Phe Asp His Pro
195 200 205
Asp Val Arg Asn Glu Met Lys Asn Trp Gly Val Trp Tyr Ala Asn Glu
210 215 220
Val Gly Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys His
225 230 235 240
Ser Tyr Leu Gly Asp Trp Val Asn His Val Arg Thr Lys Thr Gly Lys
245 250 255
Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Val Asn Ala Ile
260 265 270
Asn Asn Tyr Leu Ala Lys Val Asn Tyr Asn His Ser Val Phe Asp Ala
275 280 285
Pro Leu His Tyr Asn Phe His Tyr Ala Ser Gln Ser Gly Gly Asn Tyr
290 295 300
Asp Met Arg Asn Leu Leu Asn Gly Thr Val Val Ala Ala His Pro Thr
305 310 315 320
Lys Ala Val Thr Leu Val Glu Asn His Asp Ser Gln Pro Gly Gln Ser
325 330 335
Leu Glu Ser Val Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Ala Glu Gly Tyr Pro Ser Ile Phe Tyr Gly Asp Met
355 360 365
Tyr Gly Thr Lys Gly Asn Ser Ser Tyr Glu Ile Pro Ala Leu Lys Thr
370 375 380
Lys Ile Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr
385 390 395 400
Gln His Asn Tyr Met Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu
405 410 415
Gly Asp Ser Thr Lys Glu Lys Ser Gly Leu Ala Thr Leu Val Thr Asp
420 425 430
Gly Ala Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln Asn Ala Gly
435 440 445
Glu Val Trp Tyr Asp Ile Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile
450 455 460
Asn Thr Asp Gly Trp Gly Asn Phe Gln Val Asn Gly Gly Ser Val Ser
465 470 475 480
Val Tyr Val Gln Gln
485
<210> 3
<211> 1452
<212> DNA
<213> 人工序列
<220>
<223> 合成的寡聚核苷酸
<220>
<221> CDS
<222> (1)..(1449)
<400> 3
gca gct caa aac ggt acg atg atg cag tac ttt gaa tgg tat gta ccg 48
Ala Ala Gln Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro
1 5 10 15
aat gat ggt cag cat tgg aat cgt ctg cgg aac gat gca gcc tac ttg 96
Asn Asp Gly Gln His Trp Asn Arg Leu Arg Asn Asp Ala Ala Tyr Leu
20 25 30
aaa tcc atc ggt gtg tca gct atc tgg aca cct cct gct tac aaa ggc 144
Lys Ser Ile Gly Val Ser Ala Ile Trp Thr Pro Pro Ala Tyr Lys Gly
35 40 45
aca agc cag aac gat gtt ggc tat gga gca tat gac ctg tac gat tta 192
Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
gga gaa ttc aac cag aaa ggc act att cgc acc aaa tat gga acg aaa 240
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
gct gaa ctg aaa agc gcg gta tcg act ctc aag tct aat ggg att caa 288
Ala Glu Leu Lys Ser Ala Val Ser Thr Leu Lys Ser Asn Gly Ile Gln
85 90 95
gtg tat gga gat gtc gtc atg aat cat aaa ggc ggt gcc gat tac aca 336
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Tyr Thr
100 105 110
gag aat gta aca gct gtg gaa gtc aat ccg agc aat cgc aat caa gaa 384
Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
aca tcc gat gag tat acg atc caa gca tgg aca gga ttc aac ttc cct 432
Thr Ser Asp Glu Tyr Thr Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140
ggg cga ggc aca acc cat agt ccg ttt aag tgg cag tgg tat cat ttt 480
Gly Arg Gly Thr Thr His Ser Pro Phe Lys Trp Gln Trp Tyr His Phe
145 150 155 160
gat gga act gac tgg gat cag tct cgg aat gcg agc aga atc ttc aaa 528
Asp Gly Thr Asp Trp Asp Gln Ser Arg Asn Ala Ser Arg Ile Phe Lys
165 170 175
ttt ggg gga aaa gca tgg gat tgg gaa gta tca tcg gag aat ggg aat 576
Phe Gly Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190
tac gac tat ctt atg tac gcc gat tta gac ttt gat cat cct gat gtt 624
Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Phe Asp His Pro Asp Val
195 200 205
aga aat gag atg aag aat tgg ggc gtg tgg tac gcg aat gaa gtt ggc 672
Arg Asn Glu Met Lys Asn Trp Gly Val Trp Tyr Ala Asn Glu Val Gly
210 215 220
ttg gac gga ttt agg ctg gat gcg gtt aag cac ata aag cat agc tat 720
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys His Ser Tyr
225 230 235 240
ctg ggc gat tgg gtg aac cat gtc aga act aag acg gga aaa gag atg 768
Leu Gly Asp Trp Val Asn His Val Arg Thr Lys Thr Gly Lys Glu Met
245 250 255
ttt aca gtt gct gaa tat tgg caa aac gac gtc aat gcc att aat aac 816
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Val Asn Ala Ile Asn Asn
260 265 270
tat tta gcc aaa gtg aac tat aat cac tct gtc ttt gat gcg cct ttg 864
Tyr Leu Ala Lys Val Asn Tyr Asn His Ser Val Phe Asp Ala Pro Leu
275 280 285
cat tac aac ttt cac tat gca agc cag tca gga ggt aac tac gac atg 912
His Tyr Asn Phe His Tyr Ala Ser Gln Ser Gly Gly Asn Tyr Asp Met
290 295 300
cgt aac tta ttg aac ggc aca gtc gtg gct gct cat ccg aca aaa gca 960
Arg Asn Leu Leu Asn Gly Thr Val Val Ala Ala His Pro Thr Lys Ala
305 310 315 320
gtt acc ctt gtc gaa aat cat gac agt caa ccg gga caa tct ctg gag 1008
Val Thr Leu Val Glu Asn His Asp Ser Gln Pro Gly Gln Ser Leu Glu
325 330 335
tca gtc gtt caa ccg tgg ttt aag ccc ctt gcc tat gcg ttt atc ctc 1056
Ser Val Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
aca cga gca gaa ggt tat cca agt atc ttt tat ggg gat atg tat ggg 1104
Thr Arg Ala Glu Gly Tyr Pro Ser Ile Phe Tyr Gly Asp Met Tyr Gly
355 360 365
act aag gga aac tca tcg tac gaa att cca gca ctc aaa acc aaa att 1152
Thr Lys Gly Asn Ser Ser Tyr Glu Ile Pro Ala Leu Lys Thr Lys Ile
370 375 380
gaa ccg tta ctg aaa gca cgc aag gat tat gcc tat ggt acg cag cac 1200
Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
aac tat atg gac cac tgg gat gtt ata ggt tgg acc aga gaa ggt gac 1248
Asn Tyr Met Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415
tcc acg aaa gaa aaa tct ggc ctt gcg acg ctt gtg aca gac ggg gca 1296
Ser Thr Lys Glu Lys Ser Gly Leu Ala Thr Leu Val Thr Asp Gly Ala
420 425 430
ggc ggg tct aaa tgg atg tat gta ggc aaa cag aat gct ggc gaa gtt 1344
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln Asn Ala Gly Glu Val
435 440 445
tgg tat gac atc aca ggc aat aga acg gat aaa atc acc att aat aca 1392
Trp Tyr Asp Ile Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Asn Thr
450 455 460
gat ggc tgg gga aac ttc caa gta aac gga ggc tca gtc tcc gtg tac 1440
Asp Gly Trp Gly Asn Phe Gln Val Asn Gly Gly Ser Val Ser Val Tyr
465 470 475 480
gtt caa cag taa 1452
Val Gln Gln
<210> 4
<211> 483
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建体
<400> 4
Ala Ala Gln Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro
1 5 10 15
Asn Asp Gly Gln His Trp Asn Arg Leu Arg Asn Asp Ala Ala Tyr Leu
20 25 30
Lys Ser Ile Gly Val Ser Ala Ile Trp Thr Pro Pro Ala Tyr Lys Gly
35 40 45
Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Ala Glu Leu Lys Ser Ala Val Ser Thr Leu Lys Ser Asn Gly Ile Gln
85 90 95
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Tyr Thr
100 105 110
Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu
115 120 125
Thr Ser Asp Glu Tyr Thr Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140
Gly Arg Gly Thr Thr His Ser Pro Phe Lys Trp Gln Trp Tyr His Phe
145 150 155 160
Asp Gly Thr Asp Trp Asp Gln Ser Arg Asn Ala Ser Arg Ile Phe Lys
165 170 175
Phe Gly 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 Leu Asp Phe Asp His Pro Asp Val
195 200 205
Arg Asn Glu Met Lys Asn Trp Gly Val Trp Tyr Ala Asn Glu Val Gly
210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys His Ser Tyr
225 230 235 240
Leu Gly Asp Trp Val Asn His Val Arg Thr Lys Thr Gly Lys Glu Met
245 250 255
Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Val Asn Ala Ile Asn Asn
260 265 270
Tyr Leu Ala Lys Val Asn Tyr Asn His Ser Val Phe Asp Ala Pro Leu
275 280 285
His Tyr Asn Phe His Tyr Ala Ser Gln Ser Gly Gly Asn Tyr Asp Met
290 295 300
Arg Asn Leu Leu Asn Gly Thr Val Val Ala Ala His Pro Thr Lys Ala
305 310 315 320
Val Thr Leu Val Glu Asn His Asp Ser Gln Pro Gly Gln Ser Leu Glu
325 330 335
Ser Val Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350
Thr Arg Ala Glu Gly Tyr Pro Ser Ile Phe Tyr Gly Asp Met Tyr Gly
355 360 365
Thr Lys Gly Asn Ser Ser Tyr Glu Ile Pro Ala Leu Lys Thr Lys Ile
370 375 380
Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln His
385 390 395 400
Asn Tyr Met Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415
Ser Thr Lys Glu Lys Ser Gly Leu Ala Thr Leu Val Thr Asp Gly Ala
420 425 430
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln Asn Ala Gly Glu Val
435 440 445
Trp Tyr Asp Ile Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Asn Thr
450 455 460
Asp Gly Trp Gly Asn Phe Gln Val Asn Gly Gly Ser Val Ser Val Tyr
465 470 475 480
Val Gln Gln
Claims (15)
1.一种α-淀粉酶突变体,其特征在于:
其为包含与序列编号2所示的氨基酸序列的G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的1处或多处氨基酸残基的改变的亲本α-淀粉酶的突变体,所述亲本α-淀粉酶或α-淀粉酶突变体相对于序列编号4所示的氨基酸序列具有至少90%的序列同一性。
2.如权利要求1所述的突变体,其特征在于:
所述与G5、S38、T49、Q96、N126、T129、G140、F153、Q167、G179、W186、E187、N192、M199、Y200、L203、Y205、D206、R211、K215、H240、S241、Y242、G244、E257、F259、K278、H283、S284、A288、H295、Y296、N303、T320、S331、L348、Y360、W408、L429、V430、G433、A434、W439、N471、G476和G477的各位置相当的位置的氨基酸残基的改变分别为G5E/D/P/R/K、S38N、T49Q、Q96R/K、N126Y、T129I、G140Y/F/W、F153W、Q167E、G179D/H、W186L、E187P、N192F、M199L/T/A/N/Q/S/V/I、Y200G、L203Y/M/F、Y205F、D206R/E/N/T/G、R211V/L/I、K215F、H240F、S241A/Q/D/L/Y/P/H、Y242F、G244K/W/L/R、E257T、F259W、K278L/D/W/I/H/S/T/N/Q/V/A/Y/F、H283Q、S284W、A288F、H295Y、Y296A、N303R/E/S/G/V/D/T/A、T320D/E、S331T、L348I、Y360C/M/L/V、W408P、L429V、V430M、G433GS、A434V、W439R、N471T、G476A/P/E/S/F/R/K和G477E。
3.如权利要求1或2所述的突变体,其特征在于:
氨基酸残基的改变为2处以上的改变。
6.如权利要求1或2所述的突变体,其特征在于:
突变体至少包含选自下述表3所示的突变组合中的突变,
。
7.一种多核苷酸,其特征在于:
编码权利要求1~6中任一项所述的突变体。
8.一种载体或DNA片段,其特征在于:
包含权利要求7所述的多核苷酸。
9.一种转化细胞,其特征在于:
含有权利要求8所述的载体或DNA片段。
10.如权利要求9所述的转化细胞,其特征在于:
其为微生物。
11.一种清洗剂组合物,其特征在于:
包含权利要求1~6中任一项所述的突变体。
12.如权利要求11所述的清洗剂组合物,其特征在于:
其为衣物清洗剂或餐具清洗剂。
13.如权利要求12所述的清洗剂组合物,其特征在于:
其为粉末或液体。
14.如权利要求12或13所述的清洗剂组合物,其特征在于:
其在低温下使用。
15.如权利要求14所述的清洗剂组合物,其特征在于:
其在5~40℃的温度下使用。
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JP2021135746A JP2022060158A (ja) | 2020-10-02 | 2021-08-23 | α-アミラーゼ変異体 |
JP2021-135746 | 2021-08-23 | ||
PCT/JP2021/036011 WO2022071451A1 (ja) | 2020-10-02 | 2021-09-29 | α-アミラーゼ変異体 |
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WO1994026881A1 (en) | 1993-05-19 | 1994-11-24 | Kao Corporation | LIQUEFYING ALKALINE α-AMYLASE, PROCESS FOR PRODUCING THE SAME, AND DETERGENT COMPOSITION CONTAINING THE SAME |
US6486113B1 (en) | 1997-03-31 | 2002-11-26 | Kao Corporation | Mutant α-amylases |
JP3512981B2 (ja) | 1997-05-19 | 2004-03-31 | 花王株式会社 | 耐熱性アルカリセルラ−ゼ、それを生産する微生物及びその製造方法 |
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JP3872373B2 (ja) | 2002-04-18 | 2007-01-24 | 花王株式会社 | 粉末洗浄剤組成物 |
CA2854912A1 (en) | 2004-07-05 | 2006-01-12 | Novozymes A/S | Alpha-amylase variants with altered properties |
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US20160017305A1 (en) | 2013-03-11 | 2016-01-21 | Danisco Us Inc. | Alpha-amylase combinatorial variants |
JP5973112B1 (ja) | 2015-12-10 | 2016-08-23 | 花王株式会社 | 界面活性剤組成物 |
EP3577219B1 (en) * | 2017-02-01 | 2023-08-23 | Novozymes A/S | Alpha-amylase variants |
RU2019123965A (ru) * | 2017-02-01 | 2021-02-01 | Дзе Проктер Энд Гэмбл Компани | Очищающие композиции, содержащие варианты амилазы |
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