WO2018129985A1 - 提高比活的α-淀粉酶BasAmy突变体及其编码基因和应用 - Google Patents

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

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WO2018129985A1
WO2018129985A1 PCT/CN2017/107821 CN2017107821W WO2018129985A1 WO 2018129985 A1 WO2018129985 A1 WO 2018129985A1 CN 2017107821 W CN2017107821 W CN 2017107821W WO 2018129985 A1 WO2018129985 A1 WO 2018129985A1
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basamy
amylase
mutant
mutated
specific activity
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PCT/CN2017/107821
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French (fr)
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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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to the improvement of specific activity alpha-amylase BasAmy mutants and their coding genes and applications.
  • Alpha-amylase is a very important enzyme preparation that can randomly cleave ⁇ -1,4 glycosidic bonds from inside the starch molecule to form dextrin and reducing sugar. Alpha-amylase is widely used in the food industry, brewing, fermentation and textile industries.
  • Alpha-amylases are widely distributed throughout microorganisms to higher plants. Compared with ⁇ -amylases from other sources, microbial-derived ⁇ -amylases have a wide temperature range, a wide pH range, and low production cost. Therefore, microbial-derived ⁇ -amylases are widely used in various industrial fields. As the most important class of microbial ⁇ -amylase, Bacillus ⁇ -amylase is currently the most widely studied and applied.
  • BasAmy Bacillus sonorensis ⁇ -amylase, referred to as BasAmy, is a medium-temperature ⁇ -amylase with a wide pH and is suitable for use in food, paper, feed and other industrial fields. Compared with other Bacillus alpha-amylases, BasAmy has low specific activity and high production cost, which limits its application in food, paper, feed and other industrial fields. Therefore, improving the specific activity of BasAmy and reducing its production cost is an urgent problem to be solved in the industrial application of BasAmy.
  • the invention molecularly modifies the ⁇ -amylase BasAmy derived from Bacillus sinensis, thereby improving the specific activity of BasAmy and reducing the production cost, and laying a foundation for the industrial application of the ⁇ -amylase BasAmy.
  • a further object of the present invention is to provide a gene encoding a specific alpha-amylase BasAmy mutant.
  • nucleotide sequence and amino acid sequence of the ⁇ -amylase BasAmy of Bacillus sinensis are shown in SEQ ID NO. 1 and SEQ ID NO. 6, respectively.
  • the present invention adopts a method of site-directed saturation mutation to molecularly modify the 29th, 267th, 270th, 275th and 350th positions of the ⁇ -amylase BasAmy shown in SEQ ID NO. 6, after high-throughput
  • the screening identified the optimal mutant amino acids at positions 29, 267, 270, 275 and 350.
  • 29 were mutated from A to A
  • 267 was S to Q
  • 270 was S to P
  • 275 was A to Y
  • 350 was D to G.
  • nucleotide sequence of the ⁇ -amylase BasAmy shown in SEQ ID NO. 1 was modified by an error-prone PCR technique to obtain a series of mutation sites. After high-throughput screening, six effective mutants were A112R, L269F, K274S, Q278S, S279Q and L412C.
  • BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were combined one by one, and finally four Bsamy mutants with improved specific activity were named as BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4.
  • the relative specific activities of these four mutants were 130%, 160%, 180% and 125%, respectively, of BasAmy.
  • the nucleotide sequences of BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 mutants are shown in SEQ ID NO. 2 to SEQ ID NO. 5, and the amino acid sequences are SEQ ID NO. 7 to SEQ ID NO. 10 is shown.
  • BasAmy-1 contains mutation sites L29A, A112R, S267Q, K274S, Q278S, S279Q, D356G and L412C.
  • BasAmy-2 the mutation sites contained in BasAmy-2 are L29A, L269F, S270P, A275Y, Q278S, S279Q, D356G and L412C.
  • BasAmy-3 contains mutation sites L29A, A112R, L269F, K274S, A275Y, Q278S, S279Q and L412C.
  • BasAmy-4 the mutation sites contained in BasAmy-4 are S267Q, L269F, S270P, K274S, A275Y, Q278S, S279Q and L412C.
  • the invention molecularly transforms the ⁇ -amylase BasAmy of Bacillus sinensis by protein rational transformation and high-throughput screening technology, and obtains four mutants with increased specific activity. It lays the foundation for the industrial application of ⁇ -amylase from Bacillus sinensis.
  • the ⁇ -amylase of Bacillus sonorensis was purchased from China Industrial Microbial Culture Collection Management Center, strain number 10848, E. coli strain Top10, Pichia pastoris X33, vector pPICz ⁇ A, vector pGAPz ⁇ A, Zeocin purchase 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 ).
  • the B. sinensis strain was inserted into the LB medium, and after culturing for 24 hours, the genomic DNA was extracted.
  • Two primers (R: 5'-CTGAATTCATGGTTTACAAATGCAAACGG-3' and F:5'-CTTCTAGACTATCGTTGGACATAAATCGA-3') were designed for amplification based on the sequence of B. sinensis alpha-amylase (Genebank: AOFM01000005.1). Increase the B. cerevisiae alpha-amylase gene.
  • the amplified PCR products were purified and recovered, and ligated to the expression vectors pPICz ⁇ A and pPGAPz ⁇ A, respectively, to obtain expression vectors pPICz ⁇ A-Basamy and pGAPz ⁇ A-Basamy.
  • PCR amplification was carried out using the primers in the table, 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. After high-throughput screening, 5 effective mutation sites were L29A, S267Q, S270P, A275Y and D350G. The relative activities of these 5 mutants are shown in Table 1.
  • the above-mentioned pGAPz ⁇ A-Basamy was used as a template to perform random mutation amplification of error-prone PCR.
  • the specific amplification method is:
  • the first round of amplification PCR amplification using the vector promoter primers AOX5-F and AOX3-R as primers, the reaction system is as follows:
  • the first round of PCR product was recovered, and 1 ⁇ L of dilution was used 50-100 times as a template for the second round of PCR; second, the third round of error-prone PCR replaced the primers AOX5-F and AOX3 with ⁇ -amylase specific primers R and F. -R is the reaction primer and the PCR reaction is repeated.
  • the second and third rounds of the product were digested with XbaI and EcoRI and ligated between the EcoRI and XbaI sites on the pGAPz ⁇ A vector.
  • the ligation product was transformed into X33, and the mutant strain was screened in YPDZ plate culture. After high-throughput screening, six effective mutants were A112R, L269F, K274S, Q278S, S279Q and L412C. The relative activities of these six mutants are shown in Table 2.
  • BasAmy-1 contains mutation sites L29A, A112R, S267Q, K274S, Q278S, S279Q, D356G and L412C.
  • BasAmy-2 the mutation sites contained in BasAmy-2 are L29A, L269F, S270P, A275Y, Q278S, S279Q, D356G and L412C.
  • BasAmy-3 contains mutation sites L29A, A112R, L269F, K274S, A275Y, Q278S, S279Q and L412C.
  • BasAmy-4 the mutation sites contained in BasAmy-4 are S267Q, L269F, S270P, K274S, A275Y, Q278S, S279Q and L412C.
  • 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 relative specific activity of the mutant was calculated by dividing the mutant specific activity by the original alpha-amylase specific activity.
  • the relative specific activities of BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were 130%, 160%, 180% and 125%, respectively.
  • the optimum pH of the original ⁇ -amylase BasAmy and the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were determined by reference to the national standard method.
  • the optimum reaction pH of BasAmy and mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 is shown in Figure 1.
  • the optimum pH values of the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were almost the same as those of BaAmy, both of which were 6.0.
  • BasAmy and the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were respectively treated at room temperature for 2 hours under the conditions of pH 4-8, and then the enzyme activity was measured by the method of the national standard.
  • the results are shown in Fig. 2. It can be seen from Fig. 2 that the pH stability of the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 is consistent with BasAmy.
  • Example 8 Optimum reaction temperature and thermal stability of the original ⁇ -amylase and mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4
  • the optimum reaction temperature of BasAmy and mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 was determined by the national standard method. The results are shown in Fig. 3. As can be seen from Fig. 3, the optimum reaction temperature of BasAmy and mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 was 60 °C.
  • the BasAmy and the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were respectively treated in a water bath at 50 ° C to 90 ° C for 30 minutes, and then the enzyme activity was measured by the method of the national standard. The results are shown in FIG. 4 . As can be seen from Fig. 4, the thermal stability of the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 was consistent with BasAmy.

Abstract

提供了提高比活的α-淀粉酶BasAmy突变体及其编码基因和应用。所述突变位点为：氨基酸序列如SEQ ID NO.6所示的α-淀粉酶BasAmy的29位由L突变为A；267位由S突变为Q；270位由S突变为P；275位由A突变为Y；350位由D突变为G；112位由A突变为R；269位由L突变为F；274位由K突变为S；278位由Q突变为S；279位由S突变为Q；和/或412为由L突变为C。上述突变体的比活较原酶提高。

Description

提高比活的α-淀粉酶BasAmy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BasAmy突变体及其编码基因和应用。
背景技术
α-淀粉酶是一种十分重要的酶制剂，能够从淀粉分子内部随机切开α-1,4糖苷键生成糊精和还原糖。α-淀粉酶广泛应用于食品工业、酿造、发酵和纺织品工业。
α-淀粉酶分布十分广泛，遍及微生物至高等植物。相对于其他来源的α-淀粉酶，微生物来源的α-淀粉酶作用温度广，pH值范围广，生产成本低，因此微生物来源的α-淀粉酶被广泛的应用于各个工业领域。作为微生物α-淀粉酶中最重要的一类，芽孢杆菌α-淀粉酶是目前研究和应用最为广泛。
沙漠芽胞杆菌(Bacillus sonorensis)α-淀粉酶简称BasAmy，是一种中温α-淀粉酶，其作用pH广，适用于食品、造纸、饲料等工业领域。相对于其他芽孢杆菌α-淀粉酶，BasAmy比活低，生产成本高，限制了其在食品、造纸、饲料等工业领域的应用。因此，提高BasAmy的比活，降低其生产成本，是BasAmy工业化应用急需解决的问题。
发明内容
本发明对来源于索诺拉沙漠芽胞杆菌的α-淀粉酶BasAmy进行分子改造，从而提高BasAmy的比活，降低生产成本，为α-淀粉酶BasAmy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BasAmy突变体。
本发明的再一目的是提供提高比活的α-淀粉酶BasAmy突变体的编码基因。
索诺拉沙漠芽胞杆菌的α-淀粉酶BasAmy的核苷酸序列和氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.6所示。
本发明采用定点饱和突变的方法对SEQ ID NO.6所示的α-淀粉酶BasAmy的第29位、第267位、第270位、第275位和第350位进行分子改造，经过高通量筛选确定了第29位、第267位、第270位、第275位和第350位这5个位点的最优突变氨基酸。其中29位由L突变为A最好，267位则是S突变为Q，270位则是S突变为P，275位则是A突变为Y，350位则是D突变为G。
同时采用易错PCR技术对SEQ ID NO.1所示的α-淀粉酶BasAmy的核苷酸序列进行改造，从而获得一系列的突变位点。经过高通量筛选获得了6个有效突变体分别为A112R，L269F，K274S，Q278S，S279Q和L412C。
在上述有效突变位点的基础上，分别一一进行组合，最终得到了四个提高比活的Bsamy突变体分别命名为BasAmy-1、BasAmy-2、BasAmy-3和BasAmy-4。这四个突变体的相对比活分别是BasAmy的130％，160％，180％和125％。BasAmy-1、BasAmy-2、BasAmy-3和BasAmy-4突变体的核苷酸序列如SEQ ID NO.2到SEQ ID NO.5所示，氨基酸序列如SEQ ID NO.7到SEQ ID NO.10所示。
其中BasAmy-1包含的突变位点为L29A，A112R，S267Q，K274S，Q278S，S279Q，D356G和L412C。
其中BasAmy-2包含的突变位点为L29A，L269F，S270P，A275Y，Q278S，S279Q，D356G和L412C。
其中BasAmy-3包含的突变位点为L29A，A112R，L269F，K274S，A275Y，Q278S，S279Q和L412C。
其中BasAmy-4包含的突变位点为S267Q，L269F，S270P，K274S，A275Y，Q278S，S279Q和L412C。
本发明通过蛋白理性改造和高通量筛选技术对索诺拉沙漠芽胞杆菌的α-淀粉酶BasAmy进行分子改造，得到了四个比活提高的突变体。为索诺拉沙漠芽胞杆菌的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BasAmy-1至BasAmy-4的最适反应pH。
图2原始α-淀粉酶和突变体BasAmy-1至BasAmy-4的pH稳定性。
图3原始α-淀粉酶和突变体BasAmy-1至BasAmy-4的最适反应温度。
图4原始α-淀粉酶和突变体BasAmy-1至BasAmy-4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
索诺拉沙漠芽胞杆菌(Bacillus sonorensis)的α-淀粉酶购自中国工业微生物菌种保藏管理中心，菌种编号为10848、大肠杆菌菌株Topl0、毕赤酵母X33、载体pPICzαA，载体pGAPzαA，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、索诺拉沙漠芽胞杆菌(Bacillus sonorensis)的α-淀粉酶的克隆
将索诺拉沙漠芽胞杆菌接入LB培养基，培养24小时后，提取其基因组DNA。根据已报道索诺拉沙漠芽胞杆菌α-淀粉酶的序列(Genebank:AOFM01000005.1)设计两条引物(R：5'-CTGAATTCATGGTTTACAAATGCAAACGG-3'和F：5'-CTTCTAGACTATCGTTGGACATAAATCGA-3')用于扩增索诺拉沙漠芽胞杆菌α-淀粉酶基因。将扩增的PCR产物纯化回收，分别连接到表达载体pPICzαA和pPGAPzαA，得到表达载体pPICzαA-Basamy和pGAPzαA-Basamy。
实施例2、理性定点突变
以上述pPICzαA-Basamy为模板，以表中的引物进行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-Basamy(20ng) 2μL
加水至 50μL
反应程序如下：
Figure PCTCN2017107821-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。经过高通量筛选得到5个有效突变位点分别为L29A，S267Q，S270P，A275Y和D350G。这5个突变体的相对比活如表1所示。
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
L29A 115
S267Q 120
S270P 125
A275Y 119
D350G 128
实施例4、易错PCR非理性改造
以上述pGAPzαA-Basamy为模板，进行易错PCR随机突变扩增，具体地扩增方法是：
第一轮扩增：以载体启动子引物AOX5-F和AOX3-R为引物进行PCR扩增，反应体系如下：
Figure PCTCN2017107821-appb-000002
反应程序如下：
Figure PCTCN2017107821-appb-000003
回收第一轮PCR产物，去1μL稀释50-100倍用作第二轮PCR的模板；第二，第三轮易错PCR以α-淀粉酶特异性引物R及F替代引物AOX5-F和AOX3-R为反应引物，重复PCR反应。取第二、三轮的产物用XbaI和EcoRI进行双酶切，连接至pGAPzαA载体上的EcoRI和XbaI位点之间。连接产物转化X33，在YPDZ平板培养筛选突变菌株。经过高通量筛选获得了6个有效突变体分别为A112R，L269F，K274S，Q278S，S279Q和L412C。这6个突变体的相对比活如表2所示。
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
A112R 121
L269F 130
K274S 126
Q278S 131
S279Q 123
L412C 125
实施例5、组合突变
进行组合突变，通过实验最终得到4个组合突变分别命名为BasAmy-1、BasAmy-2、BasAmy-3、BasAmy-4。
其中BasAmy-1包含的突变位点为L29A，A112R，S267Q，K274S，Q278S，S279Q，D356G和L412C。
其中BasAmy-2包含的突变位点为L29A，L269F，S270P，A275Y，Q278S，S279Q，D356G和L412C。
其中BasAmy-3包含的突变位点为L29A，A112R，L269F，K274S，A275Y，Q278S，S279Q和L412C。
其中BasAmy-4包含的突变位点为S267Q，L269F，S270P，K274S，A275Y，Q278S，S279Q和L412C。
实施例6、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，来计算突变体的相对比活。最终BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的相对比活分别为130％，160％，180％和125％。
实施例7、原始α-淀粉酶及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应pH及pH稳定性
参照国标方法测定原始α-淀粉酶BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应pH。BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应pH如图1所示。由图1可知，突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适pH值几乎和BaAmy一样，均为6.0。
将BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4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，结果如图2所示。由图2可知突变体BasAmy-1、BasAmy-2、BasAmy-3和BasAmy-4的pH稳定性与BasAmy一致。
实施例8、原始α-淀粉酶及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应温度及热稳定性
参照国标方法测定BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应温度，结果如图3所示。由图3可知，BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应温度均为60℃。
将BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4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，结果如图4所示。由图4可知，突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的热稳定性与BasAmy一致。

Claims (8)

  1. 提高比活的α-淀粉酶BasAmy突变体，其特征在于，所述突变体为氨基酸序列如SEQ ID NO.6所示的α-淀粉酶BasAmy的突变体，突变位置为以下位点中的一个或多个，
    所述突变位点为：氨基酸序列如SEQ ID NO.6所示的α-淀粉酶BasAmy的29位由L突变为A；267位由S突变为Q；270位由S突变为P；275位由A突变为Y；350位由D突变为G；112位由A突变为R；269位由L突变为F；274位由K突变为S；278位由Q突变为S；279位由S突变为Q；和/或412为由L突变为C。
  2. 根据权利要求1所述的提高比活的α-淀粉酶BasAmy突变体，其特征在于，所述突变体的突变位点为：
    L29A，A112R，S267Q，K274S，Q278S，S279Q，D356G和L412C；
    L29A，L269F，S270P，A275Y，Q278S，S279Q，D356G和L412C；
    L29A，A112R，L269F，K274S，A275Y，Q278S，S279Q和L412C；或者
    S267Q，L269F，S270P，K274S，A275Y，Q278S，S279Q和L412C。
  3. 编码权利要求1所述提高比活的α-淀粉酶BasAmy突变体的基因。
  4. 包含权利要求3所述基因的重组载体。
  5. 包含权利要求3所述基因的的宿主细胞。
  6. 权利要求1所述提高比活的α-淀粉酶BasAmy突变体的应用。
  7. 提高α-淀粉酶BasAmy比活的方法，其特征在于，将氨基酸序列如SEQ ID NO.6所示的α-淀粉酶BasAmy的29位由L突变为A；267位由S突变为Q；270位由S突变为P；275位由A突变为Y；350位由D突变为G；112位由A突变为R；269位由L突变为F；274位由K突变为S；278位由Q突变为S；279位由S突变为Q；和/或412为由L突变为C。
  8. 制备提高比活的α-淀粉酶BasAmy突变体的方法，所述方法包括在宿主细胞中表达权利要求3所述基因并纯化突变体的步骤。
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