WO2018129981A1 - 比活提高的α-淀粉酶JcAmy突变体及其编码基因和应用 - Google Patents

比活提高的α-淀粉酶JcAmy突变体及其编码基因和应用 Download PDF

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WO2018129981A1
WO2018129981A1 PCT/CN2017/107576 CN2017107576W WO2018129981A1 WO 2018129981 A1 WO2018129981 A1 WO 2018129981A1 CN 2017107576 W CN2017107576 W CN 2017107576W WO 2018129981 A1 WO2018129981 A1 WO 2018129981A1
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amylase
jcamy
mutant
amino acid
acid sequence
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PCT/CN2017/107576
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French (fr)
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李阳源
黄江
王建荣
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
    • 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)

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  • the invention relates to the field of genetic engineering, in particular to an alpha-amylase JcAmy mutant with increased specific activity and its coding gene and application.
  • Alpha-amylase system name 1,4- ⁇ -D-glucan hydrolase, is an endohydrolase whose main function is to catalyze the reduction of 1,4- ⁇ -D-glucan from starch.
  • Dextrin and sugar play an important role in the fields of starch, detergents, beverages and textiles.
  • the processing of starchy raw materials generally undergoes two stages of liquefaction and saccharification.
  • the enzymes used in the liquefaction and saccharification processes are mainly alpha-amylases and saccharification enzymes.
  • Commercially available 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.
  • JcAmy The salt-seasoned sea bacillus (Jeotgalibacillus campisalis) alpha-amylase is referred to as JcAmy.
  • JcAmy is an acid-resistant amylase with an optimum pH of 5.0 and excellent stability in the pH range of 4 to 8, allowing it to perform well under acidic liquefaction conditions.
  • JcAmy has good pH characteristics, its low specific activity and high production cost limit its industrial application. Therefore, improving the specific enzyme activity of JcAmy and reducing its production cost is an urgent problem to be solved in the industrial application of JcAmy.
  • the invention improves the specific activity of Bacillus aeruginosa ⁇ -amylase JcAmy by site-directed mutagenesis technology, greatly reduces the production cost thereof, and lays a foundation for further industrial application.
  • the object of the present invention is to make the modified ⁇ -amylase have higher specific activity and lower the production cost by molecularly modifying the B. sphaeroides ⁇ -amylase JcAmy derived from salt and salt, and the Bacillus alkaloidis
  • the industrial application of alpha-amylase lays the foundation.
  • a further object of the present invention is to provide a gene encoding the above ⁇ -amylase JcAmy mutant.
  • nucleotide sequence and amino acid sequence of the B. sphaeroides alpha-amylase JcAmy are as shown in SEQ ID NO. 1, and the amino acid sequence thereof is shown in SEQ ID NO.
  • the present invention adopts a method of site-directed saturation mutation to molecularly modify the 6th, 53rd, 173th, 245th and/or 281th positions of the ⁇ -amylase JcAmy shown in SEQ ID NO. Flux screening resulted in increased specific activity alpha-amylase mutants.
  • the amino acid sequences of these mutants are shown in SEQ ID NO. 3 to SEQ ID NO. 10, and the nucleotide sequences encoding the mutants are shown in SEQ ID NO. 11 to SEQ ID NO.
  • An optimized modified ⁇ -amylase JcAmy mutant according to a specific embodiment of the present invention, the amino acid sequence of which is the 6th, 53rd, 173th, and 245th position of the ⁇ -amylase JcAmy of SEQ ID NO. And/or at least one amino acid in position 281 is correspondingly substituted with one of the following amino acids:
  • the 6th position of the ⁇ -amylase JcAmy was replaced by G6F, G6M, G6P, G6N or G6S;
  • the 53rd position of the ⁇ -amylase JcAmy was replaced by N35S or N35A;
  • the 173th position of the ⁇ -amylase JcAmy was replaced by N173K or N173S;
  • the 245th position of the ⁇ -amylase JcAmy was replaced by Q245G, Q245P or Q245R;
  • the 281th position of the ⁇ -amylase JcAmy was replaced with G281N, G281D, G281S or G281K.
  • the optimized ⁇ -amylase JcAmy mutant JcAmy-1 has the mutation sites: G6F, N35S, N173K, Q245G, G281N, and the amino acid sequence is shown in SEQ ID NO.
  • the optimized ⁇ -amylase JcAmy mutant JcAmy-2 has the mutation sites: G6M, N35S, N173S, Q245P, G281D, and the amino acid sequence is shown in SEQ ID NO.
  • the optimized ⁇ -amylase JcAmy mutant JcAmy-3 has the mutation sites: G6P, N35A, N173K, Q245R, G281K, and the amino acid sequence is shown in SEQ ID NO.
  • the optimized ⁇ -amylase JcAmy mutant JcAmy-4 has mutation sites: G6N, N35S, N173K, Q245P, G281S, and the amino acid sequence is shown in SEQ ID NO.
  • Optimized modified alpha-amylase JcAmy mutant JcAmy-5 according to a specific embodiment of the invention,
  • the mutation sites are: G6S, N35A, N173S, Q245P, G281K, and the amino acid sequence is shown in SEQ ID NO.
  • the optimized ⁇ -amylase JcAmy mutant JcAmy-6 has mutation sites: G6P, N35A, N173K, Q245G, G281S, and the amino acid sequence is shown in SEQ ID NO.
  • the optimized modified ⁇ -amylase JcAmy mutant JcAmy-7 according to a specific embodiment of the present invention has mutation sites: G6S, N35A, N173K, Q245P, G281N, and the amino acid sequence is shown in SEQ ID NO.
  • the optimized modified ⁇ -amylase JcAmy mutant JcAmy-8 according to a specific embodiment of the present invention has mutation sites: G6M, N35A, N173K, Q245G, G281S, and the amino acid sequence is shown in SEQ ID NO.
  • the invention molecularly transforms the a-amylase JcAmy of Zeotgalibacillus campisalis by protein rational transformation and high-throughput screening technology. Compared with the specific activity of the original ⁇ -amylase, the specific activity of ⁇ -amylase after mutation increased by 21%-92%, which laid a foundation for the industrial application of B. auriculata ⁇ -amylase.
  • Figure 1 shows the optimum pH of the original alpha-amylase and alpha-amylase mutant JcAmy1-8, in accordance with an embodiment of the present invention.
  • FIG 2 shows the pH stability of the original alpha-amylase and alpha-amylase mutant JcAmy1-8, in accordance with an embodiment of the present invention.
  • E. coli strain Topl0, Pichia pastoris X33, vector pPICz ⁇ A, Zeocin was purchased from Invitrogen.
  • Enzyme and kit Q5 high-fidelity Taq enzyme MIX was purchased from NEB, plasmid extraction, gel purification, restriction endonuclease and kit were purchased from Shanghai Shenggong Company.
  • E. coli medium is LB, formula: 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 is YPD, and the formula is: 1% yeast extract, 2% peptone, 2% glucose.
  • the yeast screening medium was YPDZ and the formulation was YPD + 100 mg/L zeocin.
  • BMGY Yeast induction medium
  • BMGY formulated as 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V) and BMMY, divided by 0.5% methanol instead of glycerol, the remaining components are BMGY is the same.
  • Two primers (F: 5'-GATCGAATTCGCTACTCCTCAAAACGGTACTATGA-3' and R: 5'-TAGCGCGGCCGCCTACTCACCATAAATGGAAACAGAA-3') were designed based on the sequence of the synthetic a-amylase JcAmy gene for amplification of B. sphaeroides alpha-amylase gene.
  • the amplified PCR product was purified and ligated, and ligated into the expression vector pPICz ⁇ A to obtain the expression vector pPICz ⁇ A-JcAmy.
  • G6F G6M, G6P, G6N or G6S; or
  • 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. A series of single-site mutants with increased specific activity were obtained by screening, and the relative activities of these mutants are shown in Table 1.
  • 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/T 24401-2009.
  • No. 281 G281N, G281D, G281S, G281K.
  • JcAmy-1 contains mutation sites: G6F, N35S, N173K, Q245G, G281N.
  • JcAmy-2 The mutation sites contained in JcAmy-2 are: G6M, N35S, N173S, Q245P, G281D.
  • JcAmy-3 contains mutation sites: G6P, N35A, N173K, Q245R, G281K.
  • JcAmy-4 contains mutation sites: G6N, N35S, N173K, Q245P, G281S.
  • JcAmy-5 contains mutation sites: G6S, N35A, N173S, Q245P, G281K.
  • JcAmy-6 the mutation sites contained in JcAmy-6 are: G6P, N35A, N173K, Q245G, G281S.
  • JcAmy-7 contains mutation sites: G6S, N35A, N173K, Q245P, G281N.
  • JcAmy-8 The mutation sites contained in JcAmy-8 are: G6M, N35A, N173K, Q245G, G281S.
  • the original ⁇ -amylase and the mutant ⁇ -amylase were separately purified by a nickel column purification method.
  • 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 JcAmy, the specific activity of the mutant JcAmy increased by 21%-92% (see Table 2 for the specific results).
  • the optimum pH of the original alpha-amylase and the alpha-amylase mutant JcAmy1-8 was determined by reference to the national standard method.
  • the optimum pH of the original alpha-amylase and alpha-amylase mutant JcAmy1-8 is shown in Figure 1. As can be seen from Fig. 1, the optimum pH of the mutant JcAmy1-8 did not change much, almost the same as the original ⁇ -amylase.
  • the original ⁇ -amylase and the ⁇ -amylase mutant JcAmy1-8 were each treated at room temperature for 3 hours under the conditions of pH 4-8, and then the enzyme activity was measured by the method of the national standard.
  • the pH stability of the original alpha-amylase and alpha-amylase mutant JcAmy1-8 is shown in Figure 2. It can be seen from Fig. 2 that the mutants JcAmy1, JcAmy3 and JcAmy7 are more stable under the pH 4 condition than the original ⁇ -amylase, while the pH stability of the mutants JcAmy2, JcAmy4, JcAmy5, JcAmy6, JcAmy8 and the original ⁇ -amylase Consistent.

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Abstract

提供一种α-淀粉酶JcAmy突变体及其编码基因和应用，所述突变体的氨基酸序列为SEQ ID No.2所示的氨基酸序列的第6，53，173，245和/或281位中的一个或多个位置被取代。所述突变体的α-淀粉酶比活比原始α-淀粉酶比活提高了21%-92%。

Description

比活提高的α-淀粉酶JcAmy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，具体涉及比活提高的α-淀粉酶JcAmy突变体及其编码基因和应用。
背景技术
α-淀粉酶，系统名称为1,4-α-D-葡聚糖水解酶，是一种内切水解酶，其主要作用是催化淀粉的1,4-α-D-葡聚糖生成还原性糊精和糖类，在淀粉、清洁剂、饮料和纺织等领域具有重要作用。
目前在酒精酿造、淀粉糖等领域中，淀粉质原料加工过程中一般要经过液化和糖化两个阶段。在液化和糖化过程中所使用的酶主要为α-淀粉酶和糖化酶。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。
盐地咸海鲜芽胞杆菌(Jeotgalibacillus campisalis)α-淀粉酶简称JcAmy。JcAmy是一种耐酸性淀粉酶，其最适pH为5.0，在pH4到8范围内具有很好的稳定性，使其能够在酸性液化条件下发挥很好的作用。虽然JcAmy具有很好的pH特性，但是其比活低，生产成本高，限制了其工业化应用。因此，提高JcAmy的比酶活，降低其生产成本，是JcAmy工业化应用急需解决的题。
近年来，一系列的微生物源α-淀粉酶实现在大肠杆菌或者酵母中的异源表达。但是由于从自然界筛选得到的野生菌产的α-淀粉酶的活力一般都比较低，不能直接运用于工业化的发酵生产。现有技术一般是通过对野生菌株进行诱变、杂交育种等技术手段来提高菌株的产酶能力，但是诱变杂交等技术的工作量大并且不可控地出现负突变的几率比较大。
本发明通过定点突变技术，提高了盐地咸海鲜芽胞杆菌α-淀粉酶JcAmy的比活力，大大降低了其生产成本，为其进一步工业化应用奠定基础。
发明内容
本发明的目的是通过对来源于盐地咸海鲜芽胞杆菌α-淀粉酶JcAmy进行分子改造，使改造后的α-淀粉酶具有更高的比活，降低生产成本，为盐地咸海鲜芽胞杆菌α-淀粉酶的工业化应用奠定基础。
本发明的目的是提供比活提高的α-淀粉酶JcAmy突变体。
本发明的的再一目的是提供上述α-淀粉酶JcAmy突变体的编码基因。
盐地咸海鲜芽胞杆菌α-淀粉酶JcAmy的核苷酸序列和氨基酸序列如SEQ ID NO.1，其氨基酸序列如SEQ ID NO.2所示。
本发明采用定点饱和突变的方法对SEQ ID NO.2所示的α-淀粉酶JcAmy的第6位，第53位，第173位，第245位和/或第281位进行分子改造，经过高通量筛选得到提高比活的α-淀粉酶突变体。这些突变体的氨基酸序列如SEQ ID NO.3到SEQ ID NO.10所示，编码突变体的核苷酸序列如SEQ ID NO.11到SEQ ID NO.18所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体，其氨基酸序列为SEQ ID NO.2的α-淀粉酶JcAmy的第6位，第53位，第173位，第245位和/或第281位中至少一个氨基酸被相应的置换为以下氨基酸之一：
α-淀粉酶JcAmy的第6位被替换为G6F，G6M，G6P，G6N或G6S；
α-淀粉酶JcAmy的第53位被替换为N35S或N35A；
α-淀粉酶JcAmy的的第173位被替换为N173K或N173S；
α-淀粉酶JcAmy的第245位被替换为Q245G，Q245P或Q245R；
α-淀粉酶JcAmy的第281位被替换为G281N，G281D，G281S或G281K。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-1，其突变位点为：G6F，N35S，N173K，Q245G，G281N，氨基酸序列如SEQ ID NO.3所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-2，其突变位点为：G6M，N35S，N173S，Q245P，G281D，氨基酸序列如SEQ ID NO.4所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-3，其突变位点为：G6P，N35A，N173K，Q245R，G281K，氨基酸序列如SEQ ID NO.5所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-4，其突变位点为：G6N，N35S，N173K，Q245P，G281S，氨基酸序列如SEQ ID NO.6所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-5，其 突变位点为：G6S，N35A，N173S，Q245P，G281K，氨基酸序列如SEQ ID NO.7所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-6，其突变位点为：G6P，N35A，N173K，Q245G，G281S，氨基酸序列如SEQ ID NO.8所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-7，其突变位点为：G6S，N35A，N173K，Q245P，G281N，氨基酸序列如SEQ ID NO.9所示。
根据本发明的具体实施方式的优化改良的α-淀粉酶JcAmy突变体JcAmy-8，其突变位点为：G6M，N35A，N173K，Q245G，G281S，氨基酸序列如SEQ ID NO.10所示。
本发明通过蛋白理性改造和高通量筛选技术对盐地咸海鲜芽胞杆菌(Jeotgalibacillus campisalis)a-淀粉酶JcAmy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21％-92％，为盐地咸海鲜芽胞杆菌α-淀粉酶的工业化应用奠定基础。
附图说明
图1显示根据本发明具体实施方式的原始α-淀粉酶及α-淀粉酶突变体JcAmy1-8的最适pH。
图2显示根据本发明具体实施方式的原始α-淀粉酶及α-淀粉酶突变体JcAmy1-8的pH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：大肠杆菌菌株Topl0、毕赤酵母X33、载体pPICzαA，Zeocin购自Invitrogen公司。
2、基因：将已公布的盐地咸海鲜芽胞杆菌(Jeotgalibacillus campisalis)α-淀粉酶JcAmy(Sequence ID:WP_052476631.1)，根据毕赤酵母密码子优化后进行合成基因。
3、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。
4、培养基：大肠杆菌培养基为LB，配方：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，配方为：1％酵母提取物，2％蛋白胨，2％葡萄糖。酵母筛选培养基为YPDZ，配方为YPD+100mg/L zeocin。
酵母诱导培养基BMGY，配方为1％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、盐地咸海鲜芽胞杆菌(Jeotgalibacillus campisalis)α-淀粉酶JcAmy的克隆
根据合成的a-淀粉酶JcAmy基因的序列设计两条引物(F：5'-GATCGAATTCGCTACTCCTCAAAACGGTACTATGA-3'和R：5'-TAGCGCGGCCGCCTACTCACCATAAATGGAAACAGAA-3')用于扩增盐地咸海鲜芽胞杆菌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-JcAmy。
实施例2、定点突变
单突变位点为
G6F，G6M，G6P，G6N或G6S；或者
N35S，N35A，N173K或N173S；或者
Q245G，Q245P或Q245R；或者
G281N，G281D，G281S或G281K
以上述pPICzαA-JcAmy为模板，以相应的引物进行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-JcAmy(20ng) 2μL
加水至 50μL
反应程序如下：
Figure PCTCN2017107576-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。通过筛选得到一系列提高比活的单位点突变体，这些突变体的相对比活如表1所示。
表1原始α-淀粉酶和单点突变体α-淀粉酶相对比活
编号 相对比活(％)
原始α-淀粉酶 100
G6F 115
G6M 121
G6P 130
G6N 119
G6S 116
N35S 135
N35A 141
N173K 126
N173S 136
Q245G 123
Q245P 128
Q245R 121
G281N 142
G281D 136
G281S 115
G281K 127
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1.6mLBMMY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/T 24401-2009》进行测定。
实施例4、组合突变
第6位：G6F，G6M，G6P，G6N，G6S；
第53位：N35S，N35A；
第173位：N173K，N173S；
第245位：Q245G，Q245P，Q245R；
第281位：G281N，G281D，G281S，G281K。
将实施例2中酶比活提高的单突变位点G6F，G6M，G6P，G6N，G6S，N35S，N35A，N173K，N173S，Q245G，Q245P，Q245R，G281N，G281D，G281S，G281K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8个组合突变分别命名为JcAmy-1、JcAmy-2、JcAmy-3、JcAmy-4、JcAmy-5、JcAmy-6、JcAmy-7、JcAmy-8
其中JcAmy-1包含的突变位点为：G6F，N35S，N173K，Q245G，G281N。
其中JcAmy-2包含的突变位点为：G6M，N35S，N173S，Q245P，G281D。
其中JcAmy-3包含的突变位点为：G6P，N35A，N173K，Q245R，G281K。
其中JcAmy-4包含的突变位点为：G6N，N35S，N173K，Q245P，G281S。
其中JcAmy-5包含的突变位点为：G6S，N35A，N173S，Q245P，G281K。
其中JcAmy-6包含的突变位点为：G6P，N35A，N173K，Q245G，G281S。
其中JcAmy-7包含的突变位点为：G6S，N35A，N173K，Q245P，G281N。
其中JcAmy-8包含的突变位点为：G6M，N35A，N173K，Q245G，G281S。
实施例5、原始α-淀粉酶及α-淀粉酶突变体的比活分析
分别将原始α-淀粉酶和突变体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JcAmy，突变后的JcAmy比活提高幅度为21％-92％(具体结果见表2)。
表2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
JcAmy-1 135
JcAmy-2 159
JcAmy-3 142
JcAmy-4 121
JcAmy-5 130
JcAmy-6 150
JcAmy-7 192
JcAmy-8 160
实施例6、原始α-淀粉酶及α-淀粉酶突变体JcAmy1-8的最适pH及pH稳定性
参照国标方法测定原始α-淀粉酶及α-淀粉酶突变体JcAmy1-8的最适pH。原始α-淀粉酶及α-淀粉酶突变体JcAmy1-8的最适pH如图1所示。由图1可知，突变体JcAmy1-8的最适pH并没有发生太大变化，几乎和原始α-淀粉酶一样。
将原始α-淀粉酶及α-淀粉酶突变体JcAmy1-8分别在pH4-8条件下室温处理3小时，然后参照国标的方法测定酶活。原始α-淀粉酶及α-淀粉酶突变体JcAmy1-8的pH稳定性如图2所示。由图2可知，突变体JcAmy1、JcAmy3和JcAmy7在pH4条件下的稳定性要好于原始α-淀粉酶，而突变体JcAmy2、JcAmy4、JcAmy5、、JcAmy6、JcAmy8的pH稳定性与原始α-淀粉酶一致。

Claims (9)

  1. α-淀粉酶JcAmy突变体，其特征在于，所述突变体的氨基酸序列为如SEQ ID No.2所示的氨基酸序列的第6位，第53位，第173位，第245位和/或第281位中的任何一个或更多个位置为取代基团。
  2. 根据权利要求1所述的α-淀粉酶JcAmy突变体，其特征在于，所述突变体的氨基酸序列为如SEQ ID No.2所示的氨基酸序列的第6位被替换为G6F，G6M，G6P，G6N或G6S。
  3. 根据权利要求1所述的α-淀粉酶JcAmy突变体，其特征在于，所述突变体的氨基酸序列为如SEQ ID No.2所示的氨基酸序列的第53位被替换为N35S或N35A。
  4. 根据权利要求1所述的α-淀粉酶JcAmy突变体，其特征在于，所述突变体的氨基酸序列为如SEQ ID No.2所示的氨基酸序列的第173位被替换为N173K或N173S。
  5. 根据权利要求1所述的α-淀粉酶JcAmy突变体，其特征在于，所述突变体的氨基酸序列为如SEQ ID No.2所示的氨基酸序列的第245位被替换为Q245G，Q245P或Q245R。
  6. 根据权利要求1所述的α-淀粉酶JcAmy突变体，其特征在于，所述突变体的氨基酸序列为如SEQ ID No.2所示的氨基酸序列的第281位被替换为G281N，G281D，G281S或G281K。
  7. 根据权利要求1所述的α-淀粉酶JcAmy突变体，其特征在于，所述突变体的突变位点为：G6F，N35S，N173K，Q245G，G281N；
    G6M，N35S，N173S，Q245P，G281D；
    G6P，N35A，N173K，Q245R，G281K；
    G6N，N35S，N173K，Q245P，G281S；
    G6S，N35A，N173S，Q245P，G281K；
    G6P，N35A，N173K，Q245G，G281S；
    G6S，N35A，N173K，Q245P，G281N；或
    G6M，N35A，N173K，Q245G，G281S。
  8. 编码权利要求1-8中任意一项所述α-淀粉酶JcAmy突变体的基因。
  9. 权利要求1-8中任意一项所述α-淀粉酶JcAmy突变体的应用。
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