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CN110699337B - 提高比活的α-淀粉酶突变体BasAmy-4及其编码基因和应用 - Google Patents

提高比活的α-淀粉酶突变体BasAmy-4及其编码基因和应用 Download PDF

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CN110699337B
CN110699337B CN201911068186.3A CN201911068186A CN110699337B CN 110699337 B CN110699337 B CN 110699337B CN 201911068186 A CN201911068186 A CN 201911068186A CN 110699337 B CN110699337 B CN 110699337B
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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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Guangdong Vtr Bio Tech Co ltd
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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发生以下突变：S267Q，L269F，S270P，K274S，A275Y，Q278S，S279Q和L412C。本发明获得的突变体的比活较原酶提高。

Description

提高比活的α-淀粉酶突变体BasAmy-4及其编码基因和应用
本申请是以下发明专利申请的分案申请(申请号：2017100308497，申请日：2017-01-16，发明名称：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-淀粉酶突变体BasAmy-3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SEQID 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 IDNO.5所示，氨基酸序列如SEQ IDNO.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25ug/mLZeocin。
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(Bacillussonorensis)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 23uL
对应突变体引物 1uL
对应突变体引物 1uL
pPICzαA-Basamy(20ng) 2uL
加水至 50uL
反应程序如下：
Figure BDA0002260070190000041
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1.6mLBMMY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。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 BDA0002260070190000051
反应程序如下：
Figure BDA0002260070190000052
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。
序列表
<110> 广东溢多利生物科技股份有限公司
<120> 提高比活的α-淀粉酶突变体BasAmy-4及其编码基因和应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1536
<212> DNA
<213> 索诺拉沙漠芽胞杆菌(Bacillus sonorensis)
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acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaactggagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210> 2
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
gcttctaaaa catatgcggc aagcgctaac ggcacgctga tgcagtattt tgaatggaat 120
ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180
attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240
ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300
acgaaatacg ggatgaaaac agagcttcag tcaagagtcg gttcgcttca ttcccagaac 360
atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420
gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600
tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660
tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttcca gtttctttct gattggttgt cggctgtgcg ttctcagact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960
caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatggtac acagccgggg 1080
caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140
acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210> 3
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
gcttctaaaa catatgcggc aagcgctaac ggcacgctga tgcagtattt tgaatggaat 120
ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180
attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240
ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300
acgaaatacg ggatgaaaac agagcttcag tcagctgtcg gttcgcttca ttcccagaac 360
atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420
gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600
tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660
tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttctc tttttttcct gattggttga agtatgtgcg ttctcagact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960
caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatggtac acagccgggg 1080
caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140
acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210> 4
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
gcttctaaaa catatgcggc aagcgctaac ggcacgctga tgcagtattt tgaatggaat 120
ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180
attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240
ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300
acgaaatacg ggatgaaaac agagcttcag tcaagagtcg gttcgcttca ttcccagaac 360
atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420
gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600
tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660
tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttctc ttttttttct gattggttgt cgtatgtgcg ttctcagact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960
caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatgacac acagccgggg 1080
caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140
acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210> 5
<211> 1536
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
gcttctaaaa catatgcggc aagcctgaac ggcacgctga tgcagtattt tgaatggaat 120
ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180
attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240
ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300
acgaaatacg ggatgaaaac agagcttcag tcagctgtcg gttcgcttca ttcccagaac 360
atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420
gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600
tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660
tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttcca gttttttcct gattggttgt cgtatgtgcg ttctcagact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960
caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatgacac acagccgggg 1080
caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140
acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210> 6
<211> 511
<212> PRT
<213> 索诺拉沙漠芽胞杆菌(Bacillus sonorensis)
<400> 6
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Leu Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Ala
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Ser Asp Trp
260 265 270
Leu Lys Ala Val Arg Gln Ser Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Leu Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210> 7
<211> 511
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Arg
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe Leu Ser Asp Trp
260 265 270
Leu Ser Ala Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Gly Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210> 8
<211> 511
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Ala
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Pro Asp Trp
260 265 270
Leu Lys Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Gly Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210> 9
<211> 511
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Arg
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Ser Asp Trp
260 265 270
Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210> 10
<211> 511
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Leu Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Ala
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe Phe Pro Asp Trp
260 265 270
Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510

Claims (7)

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SEQID NO. 6所示的α-淀粉酶BasAmy的突变体，突变位点为：
S267Q，L269F，S270P，K274S，A275Y，Q278S，S279Q和L412C。
2.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。
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.权利要求1所述提高比活的α-淀粉酶BasAmy突变体在水解淀粉中的应用。
6.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267位由S突变为Q； 269位由L突变为F；274位由K突变为S；270位由S突变为P; 275位由A突变为Y；278位由Q突变为S；279位由S突变为Q； 412位由L突变为C。
7.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2所述基因并纯化突变体的步骤。
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