WO2018129986A1 - 提高比活的α-淀粉酶BaAmy突变体及其编码基因和应用 - Google Patents

提高比活的α-淀粉酶BaAmy突变体及其编码基因和应用 Download PDF

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WO2018129986A1
WO2018129986A1 PCT/CN2017/107823 CN2017107823W WO2018129986A1 WO 2018129986 A1 WO2018129986 A1 WO 2018129986A1 CN 2017107823 W CN2017107823 W CN 2017107823W WO 2018129986 A1 WO2018129986 A1 WO 2018129986A1
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baamy
amylase
mutant
specific activity
alpha
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李阳源
王建荣
黄江
聂金梅
陈丽芝
何小梅
杨玲
黄佳乐
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广东溢多利生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to the improvement of specific activity alpha-amylase BaAmy mutants and their coding genes and applications.
  • the alpha-amylase is capable of randomly cleaving the alpha-1,4 glycosidic linkage from within the starch molecule across the alpha-1,6 linkage that does not cleave the branching point.
  • ⁇ -amylase is widely used in food processing, alcohol brewing, pharmaceutical, textile desizing, paper making, washing liquid and feed.
  • alpha-amylases and saccharification enzymes currently widely used in the industry have optimum pH values of about 6.5 and 4.5, so that acid and base are required to adjust pH during liquefaction and saccharification.
  • the large amount of acid and alkali added during liquefaction and saccharification not only complicates the processing process, but also increases production costs. If it is possible to develop an ⁇ -amylase that is stable under acidic conditions, it is not necessary to add an acid or a base for pH adjustment during liquefaction and saccharification, which not only reduces reagent consumption, simplifies the processing, but also reduces production costs and saves food. . It is of great significance in the field of starch processing.
  • BaAmy Bacillus acidophilus ⁇ -amylase, abbreviated as BaAmy, has good acid stability, but its specific activity is low and its production cost is high, which limits its industrial application. Therefore, improving the specific enzyme activity of BaAmy and reducing its production cost is an urgent problem to be solved in the industrial application of BaAmy.
  • the object of the present invention is to carry out molecular modification of the acid ⁇ -amylase BaAmy derived from Bacillus acidola, so that the modified acid ⁇ -amylase has higher specific activity and lowers production cost, and is acid.
  • the industrial application of Bacillus subtilis acid alpha-amylase lays the foundation.
  • the nucleotide sequence and amino acid sequence of the acid alpha-amylase BaAmy of Bacillus acidola are shown in SEQ ID NO. 1 and SEQ ID NO.
  • the present invention adopts a method of site-directed saturation mutation at positions 37, 85, 191, 279, 291, 319 and 333 of the ⁇ -amylase BaAmy shown in SEQ ID NO.
  • the nucleotide sequences of these mutants are shown in SEQ ID NO. 2 to SEQ ID NO. 7, and the amino acid sequences are shown in SEQ ID NO. 9 to SEQ ID NO.
  • the invention molecularly transforms the acid alpha-amylase BaAmy of Bacillus acidola by protein rational transformation and high-throughput screening technology. Compared with the specific activity of the original ⁇ -amylase, the specific activity of the ⁇ -amylase after the mutation increased by 45%-110%, which laid a foundation for the industrial application of the acid ⁇ -amylase of Bacillus acidophilus.
  • Bacillus acidola was purchased from China Industrial Microbial Culture Collection Management Center, strain number 21144, E. coli strain Top10, Pichia pastoris X33, vector pPICz ⁇ A, and Zeocin was purchased from Invitrogen.
  • Q5 high-fidelity Taq enzyme MIX was purchased from NEB, plasmid extraction, gel purification, restriction endonuclease, and kit purchased from Shanghai Shenggong Company.
  • the E. coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LBZ was added to 25 ⁇ g/mL Zeocin in LB medium.
  • the yeast medium was YPD (1% yeast extract, 2% peptone, 2% glucose).
  • the yeast screening medium was YPDZ (YPD + 100 mg/L zeocin).
  • BMGY Yeast induction medium
  • BMGY I% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V)
  • BMMY BMMY (divided by 0.5% methanol instead of glycerol, the remaining components are the same as BMGY ).
  • Bacillus acidophilus was introduced into LB medium, and after culturing for 24 hours, its genomic DNA was extracted.
  • Two primers (R: 5'-ATGAATTCAACGGCACCATGATGCAGTAT-3' and F: 5'-GCTCTAGACTAAACCGAACCACCATTGACTTTG-3') were designed based on the sequence of Bacillus acidophilus alpha-amylase (Genebank: JN680873) for amplification of acid-based spores. Bacillus alpha-amylase gene.
  • the amplified PCR product was purified and recovered, and ligated into the expression vector pPICz ⁇ A to obtain the expression vector pPICz ⁇ A-BaAmy.
  • PCR amplification was carried out using the corresponding primers, and the specific amplification reaction system was as follows:
  • the PCR amplification results were detected by agarose electrophoresis, and the PCR product was purified and recovered.
  • the original plasmid was decomposed by restriction endonuclease DpnI, and the decomposed product was transferred into E. coli Top10 by heat shock method.
  • the recombinant transformant was verified by bacterial PCR, and the plasmid of the correct transformant was extracted and sequenced to confirm Corresponding mutants.
  • the correct mutant will be sequenced, linearized with SacI, and transferred to Pichia pastoris X33.
  • the yeast recombinant transformants in Example 2 were picked one by one with a toothpick to a 24-well plate, and 1 mL of BMGY-containing medium was added to each well, and cultured at 30 ° C, 220 rpm for about 24 hours, and the supernatant was centrifuged. Then, 1.6 mL of BMMY medium was separately added for induction culture. After culturing for 24 hours, the supernatant was centrifuged, and the supernatant was taken out to 200 ⁇ L to a 96-well plate to measure the ⁇ -amylase activity.
  • the ⁇ -amylase enzyme activity assay was carried out in accordance with the National Standard of the People's Republic of China, GB/T24401-2009.
  • BaAmy-1 contains mutation sites: T37L, T85G, N191P, S241A, N279D, S291N, T319H, V333G.
  • BaAmy-2 contains mutation sites: T37N, T85A, N191F, S241F, N279D, S291N, T319H, V333G.
  • BaAmy-3 contains mutation sites: T37P, T85R, N191L, S241V, N279V, S291K, T319C, V333P.
  • BaAmy-4 contains mutation sites: T37W, T85Q, N191V, S241T, N279P, S291K, T319E, V333A.
  • BaAmy-5 contains mutation sites: T37K, T85E, N191I, S241N, N279V, S291W, T319Y, V333P.
  • BaAmy-6 contains mutation sites: T37W, T85Q, N191V, S241N, N279H, S291K, T319Y, V333G.
  • Example 5 Specific activity analysis of original ⁇ -amylase BaAmy and ⁇ -amylase mutants
  • the original ⁇ -amylase BaAmy and the mutant ⁇ -amylase were separately purified by a nickel column.
  • the purified ⁇ -amylase and the mutant ⁇ -amylase were each measured for the corresponding enzyme activity and the specific activity was calculated.
  • the specific activity of the mutant was calculated by dividing the mutant specific activity by the original alpha-amylase specific activity. Compared with the original BaAmy, the specific activity of the modified BaAmy increased by 45%-110% (see Table 1 for specific results).
  • the optimum pH of the original ⁇ -amylase BaAmy and the ⁇ -amylase mutant BaAmy-1 to BaAmy-6 was determined by reference to the national standard method.
  • the optimum reaction pH of BaAmy and BaAmy-1 to BaAmy-6 is shown in Figure 1. It can be seen from Fig. 1 that the optimum pH of the mutant BaAmy-1 to BaAmy-6 did not change much, and the optimum pH value was 4.5, almost the same as BaAmy.
  • the BaAmy and ⁇ -amylase mutants BaAmy-1 to BaAmy-6 were treated at room temperature for 2 hours at pH 4-8, respectively, and then the enzyme activity was measured by the method of the national standard.
  • the pH stability of BaAmy and ⁇ -amylase mutants BaAmy-1 to BaAmy-6 is shown in Figure 2.
  • the mutants BaAmy-1 and BaAmy-5 have better acid resistance than BaAmy, and the residual enzyme activities are 95% and 96%, respectively, at pH 4.
  • the pH stability of the mutants BaAmy-2, BaAmy-3, BaAmy-4 and BaAmy-6 was consistent with BaAmy.
  • the optimum reaction temperature of BaAmy and mutant BaAmy-1 to BaAmy-6 was determined by the national standard method.
  • the optimum reaction temperature for BaAmy and mutants BaAmy-1 to BaAmy-6 is shown in Figure 3. As can be seen from Fig. 3, the optimum reaction temperature of BaAmy and mutants BaAmy-1 to BaAmy-6 was 60 °C.
  • BaAmy and the mutants BaAmy-1 to BaAmy-6 were respectively subjected to a water bath treatment at 50 ° C to 90 ° C for 30 minutes, and then the enzyme activity was measured by the method of the national standard.
  • the thermal stability of BaAmy and mutant BaAmy-1 to BaAmy-6 is shown in Figure 4. As can be seen from Figure 4, the mutants BaAmy-2 and BaAmy-6 have better thermal stability than BaAmy. After 30 minutes of water bath treatment at 90 ° C, the residual enzyme activities of the mutant BaAmy-2 and BaAmy-6 were 85% and 80%, respectively, while the remaining enzyme activity of BaAmy was 50%.
  • the thermal stability of the mutants BaAmy-1, BaAmy-3, BaAmy-4 and BaAmy-5 is consistent with BaAmy.

Abstract

提供提高比活的α-淀粉酶BaAmy突变体及其编码基因和应用，所述突变体为氨基酸序列如SEQ ID NO.8所示的酸性α-淀粉酶BaAmy的突变体，其中突变位点为第37,85,191,241,279,291,319和/或及333位氨基酸中任何的1个或更多个。相对于原始α-淀粉酶的比活，突变后α-淀粉酶比活的提高幅度为45%-110%。

Description

提高比活的α-淀粉酶BaAmy突变体及其编码基因和应用 技术领域
本发明涉及基因工程领域，具体涉及提高比活的α-淀粉酶BaAmy突变体及其编码基因和应用。
背景技术
α-淀粉酶能够能跨越不能切分支点的α-1,6键从淀粉分子内部随机地切开α-1,4糖苷键。α-淀粉酶作为一种重要的水解酶广泛的应用于食品加工、酒精酿造、制药、纺织退浆、造纸、洗涤液及饲料等多种领域。
目前工业上广泛使用的商品化α-淀粉酶和糖化酶的最适pH值约为6.5和4.5，因此在液化和糖化过程中期间需要加入酸碱来调节pH。在液化和糖化过程中大量加入酸碱不仅使加工工艺复杂化，而且增加了生产成本。如果能够开发出在酸性条件下稳定的α-淀粉酶，则在液化和糖化过程中不需要额外添加酸碱进行pH调节，不仅可以减少试剂消耗，简化加工工艺，而且可以降低生产成本，节约粮食。对淀粉加工领域具有重大意义。
酸居芽胞杆菌α-淀粉酶简称BaAmy，虽然具有很好的酸稳定性，但是其比活低，生产成本高，限制了其工业化应用。因此，提高BaAmy的比酶活，降低其生产成本，是BaAmy工业化应用急需解决的问题。
发明内容
本发明的目的是通过对来源于酸居芽胞杆菌(Bacillus acidicola)的酸性α-淀粉酶BaAmy进行分子改造，使改造后的酸性α-淀粉酶具有更高的比活，降低生产成本，为酸居芽胞杆菌酸性α-淀粉酶的工业化应用奠定基础。
本发明的目的是提供提高比活的α-淀粉酶BaAmy突变体的编码基因。
酸居芽胞杆菌(Bacillus acidicola)的酸性α-淀粉酶BaAmy的核苷酸序列和氨基酸序列如SEQ ID NO.1和SEQ ID NO.8所示。
本发明采用定点饱和突变的方法对SEQ ID NO.8所示的α-淀粉酶BaAmy的第37位、第85位、第191位、第279位、第291位、第319位和第333位进行分子改造，经过高通量筛选得到一系列提高比活的α-淀粉酶突变体。这些突变体的核苷酸序列如SEQ ID NO.2到SEQ ID NO.7所示，氨基酸序列如SEQ ID NO.9到SEQ ID NO.14所示。
本发明通过蛋白理性改造和高通量筛选技术对酸居芽胞杆菌(Bacillus acidicola)的酸性α-淀粉酶BaAmy进行分子改造。相对于原始α-淀粉酶的比活，突变后α-淀粉酶比活的提高幅度为45％-110％，为酸居芽胞杆菌酸性α-淀粉酶的工业化应用奠定基础。
附图说明
图1原始α-淀粉酶和突变体BaAmy-1至BaAmy-6的最适反应pH。
图2原始α-淀粉酶和突变体BaAmy-1至BaAmy-6的pH稳定性。
图3原始α-淀粉酶和突变体BaAmy-1至BaAmy-6的最适反应温度。
图4原始α-淀粉酶和突变体BaAmy-1至BaAmy-6的热稳定性。
具体实施方式
以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实验材料和试剂：
1、菌株与载体
酸居芽胞杆菌(Bacillus acidicola)购自中国工业微生物菌种保藏管理中心，菌种编号为21144、大肠杆菌菌株Topl0、毕赤酵母X33、载体pPICzαA，Zeocin购自Invitrogen公司。
2、酶与试剂盒
Q5高保真Taq酶MIX购自NEB公司，质粒提取，胶纯化，限制性内切酶、试剂盒购自上海生工公司。
3、培养基
大肠杆菌培养基为LB(1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0)。LBZ为LB培养基加25μg/mL Zeocin。
酵母培养基为YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖)。酵母筛选培养基为YPDZ(YPD+100mg/L zeocin)。
酵母诱导培养基BMGY(I％酵母提取物、2％蛋白胨、1.34％YNB、0.00004％Biotin、1％甘油(V/V))和BMMY(除以0.5％甲醇代替甘油，其余成份相与BMGY相同)。
实施例1、酸居芽胞杆菌(Bacillus acidicola)的酸性α-淀粉酶的克隆
将酸居芽胞杆菌接入LB培养基，培养24小时后，提取其基因组DNA。根据已报道酸居芽胞杆菌α-淀粉酶的序列(Genebank:JN680873)设计两条引物(R：5'-ATGAATTCAACGGCACCATGATGCAGTAT-3'和F：5'-GCTCTAGACTAAACCGAACCACCATTGACTTTG-3')用于扩增酸居芽胞杆菌α-淀粉酶基因。将扩增的PCR产物纯化回收，连接到表达载体pPICzαA，得到表达载体pPICzαA-BaAmy。
实施例2、定点突变
以上述pPICzαA-BaAmy为模板，用相应的引物进行PCR扩增，具体地扩增反应体系如下：
Q5高保真Taq酶MIX 23μL
对应突变体引物 1μL
对应突变体引物 1μL
pPICzαA-BaAmy(20ng) 2μL
加水至 50μL
反应程序如下：
Figure PCTCN2017107823-appb-000001
琼脂糖电泳检测PCR扩增结果，纯化回收PCR产物。用限制性内切酶DpnI将原始质粒分解，将分解完的产物才用热激法转入大肠杆菌Top10，通过菌液PCR验证重组转化子，提取验证正确的转化子的质粒进行测序，从而确定相应的突变体。将测序正确的突变体，用SacI线性化，转入毕赤酵母X33。
实施例3、高通量筛选高比活突变菌株
将实施例2中的酵母重组转化子用牙签逐个挑至24孔板，每个孔中加入1mL含有BMGY培养基，30℃，220rpm培养24h左右，离心去上清。再分别加入1.6mL BMMY培养基进行诱导培养。培养24h后，离心取上清，将上述上清液分别取出200μL至96孔板，进行α-淀粉酶酶活测定。α-淀粉酶酶活检测参照中华人民共和国国家标准《GB/T24401-2009》进行测定。
实施例4、组合突变
将实施例2中酶比活提高的单突变位点T37N，T37L，T37P，T37W，T37K，T85R，T85G，T85Q，T85E，T85A，N191F，N191L，N191P，N191V，N191I， S241F，S241A，S241T，S241V，S241N，N279D，N279V，N279P，N279Q，N279F，S291N，S291K，S291I，S291W，S291R，T319H，T319C，T319G，T319E，T319Y，V333G，V333P，V333N，V333A，V333D，分别进行组合，实验过程同实施例2相同。通过实验最终得到6个组合突变分别命名为BaAmy-1、BaAmy-2、BaAmy-3、BaAmy-4、BaAmy-5、BaAmy-6
其中BaAmy-1包含的突变位点为：T37L，T85G，N191P，S241A，N279D，S291N，T319H，V333G。
其中BaAmy-2包含的突变位点为：T37N，T85A，N191F，S241F，N279D，S291N，T319H，V333G。
其中BaAmy-3包含的突变位点为：T37P，T85R，N191L，S241V，N279V，S291K，T319C，V333P。
其中BaAmy-4包含的突变位点为：T37W，T85Q，N191V，S241T，N279P，S291K，T319E，V333A。
其中BaAmy-5包含的突变位点为：T37K，T85E，N191I，S241N，N279V，S291W，T319Y，V333P。
其中BaAmy-6包含的突变位点为：T37W，T85Q，N191V，S241N，N279H，S291K，T319Y，V333G。
实施例5、原始α-淀粉酶BaAmy及α-淀粉酶突变体的比活分析
分别将原始α-淀粉酶BaAmy和突变体α-淀粉酶进行纯化，纯化方法为镍柱纯化。将纯化好的α-淀粉酶和突变体α-淀粉酶分别测定相应的酶活并计算出比活。以突变体比活除以原始α-淀粉酶比活，来计算突变体比活的提高幅度。相比原始BaAmy，突变后的BaAmy比活提高幅度为45％-110％(具体结果见表1)。
表1原始α-淀粉酶和突变体α-淀粉酶相对比活
编号 相对比活(％)
原始α-淀粉酶 100
BaAmy-1 145
BaAmy-2 150
BaAmy-3 180
BaAmy-4 175
BaAmy-5 210
BaAmy-6 195
实施例6、原始α-淀粉酶BaAmy及α-淀粉酶突变体BaAmy-1至BaAmy-6最适反应pH及pH稳定性
参照国标方法测定原始α-淀粉酶BaAmy及α-淀粉酶突变体BaAmy-1至BaAmy-6的最适反应pH。BaAmy及BaAmy-1至BaAmy-6的最适反应pH如图1所示。由图1可知，突变体BaAmy-1至BaAmy-6的最适pH并没有发生太大变化，最适反应pH值均为4.5，几乎和BaAmy一样。
将BaAmy及α-淀粉酶突变体BaAmy-1至BaAmy-6分别在pH4-8条件下室温处理2小时，然后参照国标的方法测定酶活。BaAmy及α-淀粉酶突变体BaAmy-1至BaAmy-6的pH稳定性如图2所示。相比BaAmy，突变体BaAmy-1和BaAmy-5具有更好的耐酸性，在pH4条件下，剩余酶活分别为95％和96％。而突变体BaAmy-2、BaAmy-3、BaAmy-4和BaAmy-6的pH稳定性与BaAmy一致。
实施例7、原始α-淀粉酶BaAmy及α-淀粉酶突变体BaAmy-1至BaAmy-6最适反应温度及热稳定性
参照国标方法测定BaAmy及突变体BaAmy-1至BaAmy-6的最适反应温度。BaAmy及突变体BaAmy-1至BaAmy-6的最适反应温度如图3所示。由图3可知，BaAmy及突变体BaAmy-1至BaAmy-6的最适反应温度均为60℃。
将BaAmy及突变体BaAmy-1至BaAmy-6分别在50℃-90℃条件下水浴处理30分钟，然后参照国标的方法测定酶活。BaAmy及突变体BaAmy-1至BaAmy-6的热稳定性如图4所示。由图4可知，突变体BaAmy-2和BaAmy-6热稳定性要好于BaAmy。在90℃条件下水浴处理30分钟后，突变体BaAmy-2和BaAmy-6的剩余酶活分别为85％和80％，而BaAmy的剩余酶活为50％。突变体BaAmy-1、BaAmy-3、BaAmy-4和BaAmy-5的热稳定性与BaAmy一致。

Claims (8)

  1. 提高比活的酸性α-淀粉酶BaAmy突变体，其特征在于，所述突变体为氨基酸序列如SEQ ID NO.8所示的酸性α-淀粉酶BaAmy的突变体，其中，突变位点为第37，85，191，241，279，291，319和/或及333位氨基酸中任何的1个或更多个。
  2. 根据权利要求1所述的提高比活的酸性α-淀粉酶BaAmy突变体，其特征在于，氨基酸序列如SEQ ID NO.8所示的酸性α-淀粉酶BaAmy的突变位点为：
    第37位：T37N，T37L，T37P，T37W和/或T37K；
    第85位：T85R，T85G，T85Q，T85E和/或T85A；
    第191位：N191F，N191L，N191P，N191V和/或N191I；
    第241位：S241F，S241A，S241T，S241V和/或S241N；
    第279位：N279D，N279V，N279P，N279Q和/或N279F；
    第291位：S291N，S291K，S291I，S291W和/或S291R；
    第319位，T319H，T319C，T319G，T319E，T319Y；和/或
    第333位，V333G，V333P，V333N，V333A和/或V333D。
  3. 根据权利要求1所述的提高比活的酸性α-淀粉酶BaAmy突变体，其特征在于，氨基酸序列如SEQ ID NO.8所示的酸性α-淀粉酶BaAmy的突变位点为：
    T37L，T85G，N191P，S241A，N279D，S291N，T319H，V333G；或
    T37N，T85A，N191F，S241F，N279D，S291N，T319H，V333G；或
    T37P，T85R，N191L，S241V，N279V，S291K，T319C，V333P；或
    T37W，T85Q，N191V，S241T，N279P，S291K，T319E，V333A；或
    T37K，T85E，N191I，S241N，N279V，S291W，T319Y，V333P；或
    T37W，T85Q，N191V，S241N，N279H，S291K，T319Y，V333G。
  4. 编码权利要求1所述提高比活的酸性α-淀粉酶BaAmy突变体的基因。
  5. 包含权利要求4所述基因的重组载体。
  6. 包含权利要求4所述基因的宿主细胞。
  7. 提高酸性α-淀粉酶BaAmy比活的方法，其特征在于，突变氨基酸序列如SEQ ID NO.8所示的酸性α-淀粉酶BaAmy第37，85，191，241，279，291，319和/或及333位，将上述位点的氨基酸突变为：
    第37位：T37N，T37L，T37P，T37W和/或T37K；
    第85位：T85R，T85G，T85Q，T85E和/或T85A；
    第191位：N191F，N191L，N191P，N191V和/或N191I；
    第241位：S241F，S241A，S241T，S241V和/或S241N；
    第279位：N279D，N279V，N279P，N279Q和/或N279F；
    第291位：S291N，S291K，S291I，S291W和/或S291R；
    第319位，T319H，T319C，T319G，T319E，T319Y；和/或
    第333位，V333G，V333P，V333N，V333A和/或V333D。
  8. 权利要求1所述提高比活的酸性α-淀粉酶BaAmy突变体的应用。
PCT/CN2017/107823 2017-01-16 2017-10-26 提高比活的α-淀粉酶BaAmy突变体及其编码基因和应用 WO2018129986A1 (zh)

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