CN106754825B - 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 PDFInfo
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Abstract
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。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％，为酸居芽胞杆菌酸性α‑淀粉酶的工业化应用奠定基础。
Description
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 IDNO.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(Bacillusacidicola)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25ug/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 | 23uL |
| 对应突变体引物 | 1uL |
| 对应突变体引物 | 1uL |
| pPICzαA-BaAmy(20ng) | 2uL |
| 加水至 | 50uL |
反应程序如下：
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/T 24401-2009》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。
<110> 广东溢多利生物科技股份有限公司
<120> 提高比活的α-淀粉酶BaAmy突变体及其编码基因和应用
<160> 14
<210> 1
<211> 1446
<212> DNA
<213> 酸居芽胞杆菌
<400> 1
ccgtttaacg gcaccatgat gcagtatttt gaatggtact tgccggatga tggcacgtta 60
tggaccaaag tggccaatga agccaacaac ttatccagcc ttggcatcac cgctctttgg 120
ctgccgcccg cttataaagg aacaagccgc agcgacgtag ggtacggagt atacgacttg 180
tatgacctcg gcgaattcaa tcaaaaaggg accgtccgca caaaatacgg aacaaaagct 240
caatatcttc aaaccattca agccgcccac gccgctggaa tgcaagtgta cgccgatgtc 300
gtgttcgacc ataaaggcgg cgccgacggc acggaatggg tggacgccgt cgaagtcaat 360
ccgtccgacc gcaaccaaga aatctcgggc acctatcaaa tccaagcatg gacgaaattt 420
gattttcccg ggcggggcaa cacctactcc agctttaagt ggcgctggta ccattttgac 480
ggcgttgatt gggacgaaag ccgaaaattg agccgcattt acaaattccg cggcatcggc 540
aaagcgtggg attgggaagt agacacggaa aacggaaact acgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
gtcaacacaa cgaacattga tgggttccgg cttgatgccg tcaagcatat taagttcagt 720
tcttttcctg attggttatc gtatgtgcgt tctcagactg gcaagccgct atttaccgtc 780
ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacaaacgga 840
acgatgtctt tgtttgatgc cccgttacac agcaaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca gccgacattg 960
gccgtcacct tcgttgataa tcatgacacc gaacccgtcc aagcgctgca gtcatgggtc 1020
gacccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatc ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
cttgatcact ccgacatcat cgggtggaca agggaaggcg tcactgaaaa accaggatcc 1260
ggactggccg cactgatcac cgatgggccg ggaggaagca aatggatgta cgttggcaaa 1320
caacacgccg gaaaagtgtt ctatgacctt accggcaacc ggagtgacac cgtcaccatc 1380
aacagtgatg gatggggaga attcaaagtc aatggtggtt cggtttcggt ttgggttcct 1440
agatag 1446
<210> 2
<211> 1446
<212> DNA
<213> 人工序列
<400> 2
ccgtttaacg gcaccatgat gcagtatttt gaatggtact tgccggatga tggcacgtta 60
tggaccaaag tggccaatga agccaacaac ttatccagcc ttggcatcct cgctctttgg 120
ctgccgcccg cttataaagg aacaagccgc agcgacgtag ggtacggagt atacgacttg 180
tatgacctcg gcgaattcaa tcaaaaaggg accgtccgca caaaatacgg aacaaaagct 240
caatatcttc aaggtattca agccgcccac gccgctggaa tgcaagtgta cgccgatgtc 300
gtgttcgacc ataaaggcgg cgccgacggc acggaatggg tggacgccgt cgaagtcaat 360
ccgtccgacc gcaaccaaga aatctcgggc acctatcaaa tccaagcatg gacgaaattt 420
gattttcccg ggcggggcaa cacctactcc agctttaagt ggcgctggta ccattttgac 480
ggcgttgatt gggacgaaag ccgaaaattg agccgcattt acaaattccg cggcatcggc 540
aaagcgtggg attgggaagt agacacggaa cctggaaact acgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
gtcaacacaa cgaacattga tgggttccgg cttgatgccg tcaagcatat taagttcagt 720
gcttttcctg attggttatc gtatgtgcgt tctcagactg gcaagccgct atttaccgtc 780
ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacagatgga 840
acgatgtctt tgtttgatgc cccgttacac aataaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca gccgcatttg 960
gccgtcacct tcgttgataa tcatgacacc gaacccggtc aagcgctgca gtcatgggtc 1020
gacccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatc ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
cttgatcact ccgacatcat cgggtggaca agggaaggcg tcactgaaaa accaggatcc 1260
ggactggccg cactgatcac cgatgggccg ggaggaagca aatggatgta cgttggcaaa 1320
caacacgccg gaaaagtgtt ctatgacctt accggcaacc ggagtgacac cgtcaccatc 1380
aacagtgatg gatggggaga attcaaagtc aatggtggtt cggtttcggt ttgggttcct 1440
agatag 1446
<210> 3
<211> 1446
<212> DNA
<213> 人工序列
<400> 3
ccgtttaacg gcaccatgat gcagtatttt gaatggtact tgccggatga tggcacgtta 60
tggaccaaag tggccaatga agccaacaac ttatccagcc ttggcatcaa cgctctttgg 120
ctgccgcccg cttacaaagg aacaagccgc agcgacgtag ggtacggagt atacgacttg 180
tatgacctcg gcgagtttaa tcaaaaaggg accgtccgca caaaatatgg aacaaaagct 240
caatatcttc aagccattca agccgcccac gccgctggaa tgcaagtgta cgccgatgtc 300
gtgttcgacc ataaaggcgg cgctgacggc acggaatggg tggacgccgt cgaagtcaat 360
ccgtccgacc gcaaccaaga aatctcgggc acctatcaaa tccaagcatg gacgaaattt 420
gattttcccg ggcggggcaa cacctactcc agctttaagt ggcgctggta ccattttgac 480
ggcgttgatt gggacgaaag ccgaaaatta agccgcattt acaaattccg cggcatcggc 540
aaagcgtggg attgggaagt agacacagaa ttcggaaact atgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
gtcaacacaa cgaacattga tgggttccgg cttgatgccg tcaagcatat taagttcagt 720
ttttttcctg attggttgtc gtatgtgcgt tctcagactg gcaagccgct atttaccgtc 780
ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacagatgga 840
acgatgtctt tgtttgatgc cccgttacac aacaaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca accgcatttg 960
gccgtcacct tcgttgataa tcatgacacc gaacccggcc aagcgctgca gtcatgggtt 1020
gatccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatt ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
cttgatcact ccgacatcat cgggtggaca agggaagggg tcactgaaaa accaggctca 1260
gggctggccg cactgatcac cgatgggccg ggaggaagca aatggatgta cgttggcaaa 1320
caacacgctg gaaaagtgtt ctatgacctt accggcaacc ggagtgacac cgtcaccatc 1380
aacagtgatg gatggggaga attcaaagtc aatggtggtt cggtttcggt ttgggttcct 1440
agatag 1446
<210> 4
<211> 1446
<212> DNA
<213> 人工序列
<400> 4
ccgtttaacg gcaccatgat gcagtatttt gaatggtact tgccggatga tggcacgtta 60
tggaccaaag tggccaatga agccaacaac ttatccagcc ttggcatccc tgctctttgg 120
ctgccgcccg cttataaagg aacaagccgc agcgacgtag ggtacggagt atacgacttg 180
tatgacctcg gcgaattcaa tcaaaaaggg accgtccgca caaaatacgg aacaaaagct 240
caatatcttc aacgtattca agccgcccac gccgctggaa tgcaagtgta cgccgatgtc 300
gtgttcgacc ataaaggcgg cgccgacggc acggaatggg tggacgccgt cgaagtcaat 360
ccgtccgacc gcaaccaaga aatctcgggc acctatcaaa tccaagcatg gacgaaattt 420
gattttcccg ggcggggcaa cacctactcc agctttaagt ggcgctggta ccattttgac 480
ggcgttgatt gggacgaaag ccgaaaattg agccgcattt acaaattccg cggcatcggc 540
aaagcgtggg attgggaagt agacacggaa cttggaaact acgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
gtcaacacaa cgaacattga tgggttccgg cttgatgccg tcaagcatat taagttcagt 720
gtttttcctg attggttatc gtatgtgcgt tctcagactg gcaagccgct atttaccgtc 780
ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacagttgga 840
acgatgtctt tgtttgatgc cccgttacac aagaaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca gccgtgtttg 960
gccgtcacct tcgttgataa tcatgacacc gaaccccccc aagcgctgca gtcatgggtc 1020
gacccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatc ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
cttgatcact ccgacatcat cgggtggaca agggaaggcg tcactgaaaa accaggatcc 1260
ggactggccg cactgatcac cgatgggccg ggaggaagca aatggatgta cgttggcaaa 1320
caacacgccg gaaaagtgtt ctatgacctt accggcaacc ggagtgacac cgtcaccatc 1380
aacagtgatg gatggggaga attcaaagtc aatggtggtt cggtttcggt ttgggttcct 1440
agatag 1446
<210> 5
<211> 1446
<212> DNA
<213> 人工序列
<400> 5
ccgtttaacg gcaccatgat gcagtatttt gaatggtact tgccggatga tggcacgtta 60
tggaccaaag tggccaatga agccaacaac ttatccagcc ttggcatctg ggctctttgg 120
ctgccgcccg cttataaagg aacaagccgc agcgacgtag ggtacggagt atacgacttg 180
tatgacctcg gcgaattcaa tcaaaaaggg accgtccgca caaaatacgg aacaaaagct 240
caatatcttc aacaaattca agccgcccac gccgctggaa tgcaagtgta cgccgatgtc 300
gtgttcgacc ataaaggcgg cgccgacggc acggaatggg tggacgccgt cgaagtcaat 360
ccgtccgacc gcaaccaaga aatctcgggc acctatcaaa tccaagcatg gacgaaattt 420
gattttcccg ggcggggcaa cacctactcc agctttaagt ggcgctggta ccattttgac 480
ggcgttgatt gggacgaaag ccgaaaattg agccgcattt acaaattccg cggcatcggc 540
aaagcgtggg attgggaagt agacacggaa gttggaaact acgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
gtcaacacaa cgaacattga tgggttccgg cttgatgccg tcaagcatat taagttcagt 720
acttttcctg attggttatc gtatgtgcgt tctcagactg gcaagccgct atttaccgtc 780
ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacacccgga 840
acgatgtctt tgtttgatgc cccgttacac aaaaaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca gccggaattg 960
gccgtcacct tcgttgataa tcatgacacc gaacccgctc aagcgctgca gtcatgggtc 1020
gacccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatc ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
cttgatcact ccgacatcat cgggtggaca agggaaggcg tcactgaaaa accaggatcc 1260
ggactggccg cactgatcac cgatgggccg ggaggaagca aatggatgta cgttggcaaa 1320
caacacgccg gaaaagtgtt ctatgacctt accggcaacc ggagtgacac cgtcaccatc 1380
aacagtgatg gatggggaga attcaaagtc aatggtggtt cggtttcggt ttgggttcct 1440
agatag 1446
<210> 6
<211> 1446
<212> DNA
<213> 人工序列
<400> 6
ccgtttaacg gcaccatgat gcagtatttt gaatggtact tgccggatga tggcacgtta 60
tggaccaaag tggccaatga agccaacaac ttatccagcc ttggcatcaa ggctctttgg 120
ctgccgcccg cttataaagg aacaagccgc agcgacgtag ggtacggagt atacgacttg 180
tatgacctcg gcgaattcaa tcaaaaaggg accgtccgca caaaatacgg aacaaaagct 240
caatatcttc aagaaattca agccgcccac gccgctggaa tgcaagtgta cgccgatgtc 300
gtgttcgacc ataaaggcgg cgccgacggc acggaatggg tggacgccgt cgaagtcaat 360
ccgtccgacc gcaaccaaga aatctcgggc acctatcaaa tccaagcatg gacgaaattt 420
gattttcccg ggcggggcaa cacctactcc agctttaagt ggcgctggta ccattttgac 480
ggcgttgatt gggacgaaag ccgaaaattg agccgcattt acaaattccg cggcatcggc 540
aaagcgtggg attgggaagt agacacggaa attggaaact acgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
gtcaacacaa cgaacattga tgggttccgg cttgatgccg tcaagcatat taagttcagt 720
aattttcctg attggttatc gtatgtgcgt tctcagactg gcaagccgct atttaccgtc 780
ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacagttgga 840
acgatgtctt tgtttgatgc cccgttacac tggaaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca gccgtatttg 960
gccgtcacct tcgttgataa tcatgacacc gaaccccccc aagcgctgca gtcatgggtc 1020
gacccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatc ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
cttgatcact ccgacatcat cgggtggaca agggaaggcg tcactgaaaa accaggatcc 1260
ggactggccg cactgatcac cgatgggccg ggaggaagca aatggatgta cgttggcaaa 1320
caacacgccg gaaaagtgtt ctatgacctt accggcaacc ggagtgacac cgtcaccatc 1380
aacagtgatg gatggggaga attcaaagtc aatggtggtt cggtttcggt ttgggttcct 1440
agatag 1446
<210> 7
<211> 1446
<212> DNA
<213> 人工序列
<400> 7
ccgtttaacg gcaccatgat gcagtatttt gaatggtact tgccggatga tggcacgtta 60
tggaccaaag tggccaatga agccaacaac ttatccagcc ttggcatctg tgctctttgg 120
ctgccgcccg cttataaagg aacaagccgc agcgacgtag ggtacggagt atacgacttg 180
tatgacctcg gcgaattcaa tcaaaaaggg accgtccgca caaaatacgg aacaaaagct 240
caatatcttc aacaaattca agccgcccac gccgctggaa tgcaagtgta cgccgatgtc 300
gtgttcgacc ataaaggcgg cgccgacggc acggaatggg tggacgccgt cgaagtcaat 360
ccgtccgacc gcaaccaaga aatctcgggc acctatcaaa tccaagcatg gacgaaattt 420
gattttcccg ggcggggcaa cacctactcc agctttaagt ggcgctggta ccattttgac 480
ggcgttgatt gggacgaaag ccgaaaattg agccgcattt acaaattccg cggcatcggc 540
aaagcgtggg attgggaagt agacacggaa gttggaaact acgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
gtcaacacaa cgaacattga tgggttccgg cttgatgccg tcaagcatat taagttcagt 720
aactttcctg attggttatc gtatgtgcgt tctcagactg gcaagccgct atttaccgtc 780
ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacacatgga 840
acgatgtctt tgtttgatgc cccgttacac aaaaaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca gccgtatttg 960
gccgtcacct tcgttgataa tcatgacacc gaacccggtc aagcgctgca gtcatgggtc 1020
gacccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatc ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
cttgatcact ccgacatcat cgggtggaca agggaaggcg tcactgaaaa accaggatcc 1260
ggactggccg cactgatcac cgatgggccg ggaggaagca aatggatgta cgttggcaaa 1320
caacacgccg gaaaagtgtt ctatgacctt accggcaacc ggagtgacac cgtcaccatc 1380
aacagtgatg gatggggaga attcaaagtc aatggtggtt cggtttcggt ttgggttcct 1440
agatag 1446
<210> 8
<211> 481
<212> PRT
<213> 酸居芽胞杆菌
<400> 8
PFNGTMMQYF EWYLPDDGTL WTKVANEANN LSSLGITALW LPPAYKGTSR SDVGYGVYDL 60
YDLGEFNQKG TVRTKYGTKA QYLQTIQAAH AAGMQVYADV VFDHKGGADG TEWVDAVEVN 120
PSDRNQEISG TYQIQAWTKF DFPGRGNTYS SFKWRWYHFD GVDWDESRKL SRIYKFRGIG 180
KAWDWEVDTE NGNYDYLMYA DLDMDHPEVV TELKNWGKWY VNTTNIDGFR LDAVKHIKFS 240
SFPDWLSYVR SQTGKPLFTV GEYWSYDINK LHNYITKTNG TMSLFDAPLH SKFYTASKSG 300
GAFDMRTLMT NTLMKDQPTL AVTFVDNHDT EPVQALQSWV DPWFKPLAYA FILTRQEGYP 360
CVFYGDYYGI PQYNIPSLKS KIDPLLIARR DYAYGTQHDY LDHSDIIGWT REGVTEKPGS 420
GLAALITDGP GGSKWMYVGK QHAGKVFYDL TGNRSDTVTI NSDGWGEFKV NGGSVSVWVP 480
R 481
<210> 9
<211> 481
<212> PRT
<213> 人工序列
<400> 9
PFNGTMMQYF EWYLPDDGTL WTKVANEANN LSSLGILALW LPPAYKGTSR SDVGYGVYDL 60
YDLGEFNQKG TVRTKYGTKA QYLQGIQAAH AAGMQVYADV VFDHKGGADG TEWVDAVEVN 120
PSDRNQEISG TYQIQAWTKF DFPGRGNTYS SFKWRWYHFD GVDWDESRKL SRIYKFRGIG 180
KAWDWEVDTE PGNYDYLMYA DLDMDHPEVV TELKNWGKWY VNTTNIDGFR LDAVKHIKFS 240
AFPDWLSYVR SQTGKPLFTV GEYWSYDINK LHNYITKTDG TMSLFDAPLH NKFYTASKSG 300
GAFDMRTLMT NTLMKDQPHL AVTFVDNHDT EPGQALQSWV DPWFKPLAYA FILTRQEGYP 360
CVFYGDYYGI PQYNIPSLKS KIDPLLIARR DYAYGTQHDY LDHSDIIGWT REGVTEKPGS 420
GLAALITDGP GGSKWMYVGK QHAGKVFYDL TGNRSDTVTI NSDGWGEFKV NGGSVSVWVP 480
R 481
<210> 10
<211> 481
<212> PRT
<213> 人工序列
<400> 10
PFNGTMMQYF EWYLPDDGTL WTKVANEANN LSSLGINALW LPPAYKGTSR SDVGYGVYDL 60
YDLGEFNQKG TVRTKYGTKA QYLQAIQAAH AAGMQVYADV VFDHKGGADG TEWVDAVEVN 120
PSDRNQEISG TYQIQAWTKF DFPGRGNTYS SFKWRWYHFD GVDWDESRKL SRIYKFRGIG 180
KAWDWEVDTE FGNYDYLMYA DLDMDHPEVV TELKNWGKWY VNTTNIDGFR LDAVKHIKFS 240
FFPDWLSYVR SQTGKPLFTV GEYWSYDINK LHNYITKTDG TMSLFDAPLH NKFYTASKSG 300
GAFDMRTLMT NTLMKDQPHL AVTFVDNHDT EPGQALQSWV DPWFKPLAYA FILTRQEGYP 360
CVFYGDYYGI PQYNIPSLKS KIDPLLIARR DYAYGTQHDY LDHSDIIGWT REGVTEKPGS 420
GLAALITDGP GGSKWMYVGK QHAGKVFYDL TGNRSDTVTI NSDGWGEFKV NGGSVSVWVP 480
R 481
<210> 11
<211> 481
<212> PRT
<213> 人工序列
<400> 11
PFNGTMMQYF EWYLPDDGTL WTKVANEANN LSSLGIPALW LPPAYKGTSR SDVGYGVYDL 60
YDLGEFNQKG TVRTKYGTKA QYLQRIQAAH AAGMQVYADV VFDHKGGADG TEWVDAVEVN 120
PSDRNQEISG TYQIQAWTKF DFPGRGNTYS SFKWRWYHFD GVDWDESRKL SRIYKFRGIG 180
KAWDWEVDTE LGNYDYLMYA DLDMDHPEVV TELKNWGKWY VNTTNIDGFR LDAVKHIKFS 240
VFPDWLSYVR SQTGKPLFTV GEYWSYDINK LHNYITKTVG TMSLFDAPLH KKFYTASKSG 300
GAFDMRTLMT NTLMKDQPCL AVTFVDNHDT EPPQALQSWV DPWFKPLAYA FILTRQEGYP 360
CVFYGDYYGI PQYNIPSLKS KIDPLLIARR DYAYGTQHDY LDHSDIIGWT REGVTEKPGS 420
GLAALITDGP GGSKWMYVGK QHAGKVFYDL TGNRSDTVTI NSDGWGEFKV NGGSVSVWVP 480
R 481
<210> 12
<211> 481
<212> PRT
<213> 人工序列
<400> 12
PFNGTMMQYF EWYLPDDGTL WTKVANEANN LSSLGIWALW LPPAYKGTSR SDVGYGVYDL 60
YDLGEFNQKG TVRTKYGTKA QYLQQIQAAH AAGMQVYADV VFDHKGGADG TEWVDAVEVN 120
PSDRNQEISG TYQIQAWTKF DFPGRGNTYS SFKWRWYHFD GVDWDESRKL SRIYKFRGIG 180
KAWDWEVDTE VGNYDYLMYA DLDMDHPEVV TELKNWGKWY VNTTNIDGFR LDAVKHIKFS 240
TFPDWLSYVR SQTGKPLFTV GEYWSYDINK LHNYITKTPG TMSLFDAPLH KKFYTASKSG 300
GAFDMRTLMT NTLMKDQPEL AVTFVDNHDT EPAQALQSWV DPWFKPLAYA FILTRQEGYP 360
CVFYGDYYGI PQYNIPSLKS KIDPLLIARR DYAYGTQHDY LDHSDIIGWT REGVTEKPGS 420
GLAALITDGP GGSKWMYVGK QHAGKVFYDL TGNRSDTVTI NSDGWGEFKV NGGSVSVWVP 480
R 481
<210> 13
<211> 481
<212> PRT
<213> 人工序列
<400> 13
PFNGTMMQYF EWYLPDDGTL WTKVANEANN LSSLGIKALW LPPAYKGTSR SDVGYGVYDL 60
YDLGEFNQKG TVRTKYGTKA QYLQEIQAAH AAGMQVYADV VFDHKGGADG TEWVDAVEVN 120
PSDRNQEISG TYQIQAWTKF DFPGRGNTYS SFKWRWYHFD GVDWDESRKL SRIYKFRGIG 180
KAWDWEVDTE IGNYDYLMYA DLDMDHPEVV TELKNWGKWY VNTTNIDGFR LDAVKHIKFS 240
NFPDWLSYVR SQTGKPLFTV GEYWSYDINK LHNYITKTVG TMSLFDAPLH WKFYTASKSG 300
GAFDMRTLMT NTLMKDQPYL AVTFVDNHDT EPPQALQSWV DPWFKPLAYA FILTRQEGYP 360
CVFYGDYYGI PQYNIPSLKS KIDPLLIARR DYAYGTQHDY LDHSDIIGWT REGVTEKPGS 420
GLAALITDGP GGSKWMYVGK QHAGKVFYDL TGNRSDTVTI NSDGWGEFKV NGGSVSVWVP 480
R 481
<210> 14
<211> 481
<212> PRT
<213> 人工序列
<400> 14
PFNGTMMQYF EWYLPDDGTL WTKVANEANN LSSLGIWALW LPPAYKGTSR SDVGYGVYDL 60
YDLGEFNQKG TVRTKYGTKA QYLQQIQAAH AAGMQVYADV VFDHKGGADG TEWVDAVEVN 120
PSDRNQEISG TYQIQAWTKF DFPGRGNTYS SFKWRWYHFD GVDWDESRKL SRIYKFRGIG 180
KAWDWEVDTE VGNYDYLMYA DLDMDHPEVV TELKNWGKWY VNTTNIDGFR LDAVKHIKFS 240
NFPDWLSYVR SQTGKPLFTV GEYWSYDINK LHNYITKTHG TMSLFDAPLH KKFYTASKSG 300
GAFDMRTLMT NTLMKDQPYL AVTFVDNHDT EPGQALQSWV DPWFKPLAYA FILTRQEGYP 360
CVFYGDYYGI PQYNIPSLKS KIDPLLIARR DYAYGTQHDY LDHSDIIGWT REGVTEKPGS 420
GLAALITDGP GGSKWMYVGK QHAGKVFYDL TGNRSDTVTI NSDGWGEFKV NGGSVSVWVP 480
R 481
Claims (6)
1.提高比活的酸性α-淀粉酶BaAmy突变体，其特征在于，通过对氨基酸序列如SEQ IDNO.8所示的酸性α-淀粉酶BaAmy的位点组合进行以下突变获得所述突变体，
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。
2.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。
3.包含权利要求2所述基因的重组载体。
4.包含权利要求2所述基因的宿主细胞。
5.提高酸性α-淀粉酶BaAmy比活的方法，其特征在于，所述方法包括对氨基酸序列如SEQ ID NO.8所示的酸性α-淀粉酶BaAmy的位点组合进行以下突变的步骤：
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。
6.权利要求1所述提高比活的酸性α-淀粉酶BaAmy突变体用于水解淀粉的应用。
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|---|
| AEW07376.1;Sharma,A.et al;《GENBANK》;20121005;全文 * |
| α-淀粉酶的基因改造与菌种选育研究进展;封棣等;《食品工业科技》;20141231;第35卷(第15期);381-385 * |
| 地芽孢杆菌Geobacillus sp.GXS1α-淀粉酶的饱和突变及酶学性质研究;薛蓓等;《生物技术通报》;20131016(第10期);153-159 * |
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Application publication date: 20170531 Assignee: Zhuhai Hengqin Yitai Biotechnology Co.,Ltd. Assignor: GUANGDONG VTR BIO-TECH Co.,Ltd. Contract record no.: X2023980053875 Denomination of invention: Improve specific activity a- Amylase BaAmy Mutant and Its Encoding Genes and Applications Granted publication date: 20201106 License type: Common License Record date: 20231226 |