CN108004220A - 提高热稳定性的碱性蛋白酶BmP突变体及其基因和应用 - Google Patents

提高热稳定性的碱性蛋白酶BmP突变体及其基因和应用 Download PDF

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CN108004220A
CN108004220A CN201711414723.6A CN201711414723A CN108004220A CN 108004220 A CN108004220 A CN 108004220A CN 201711414723 A CN201711414723 A CN 201711414723A CN 108004220 A CN108004220 A CN 108004220A
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刘丹妮
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Abstract

本发明涉及生物工程领域，具体涉及提高热稳定性的碱性蛋白酶BmP突变体及其基因和应用。所述突变体为将氨基酸序列如SEQ ID NO.6所示的碱性蛋白酶BmP的突变体的193位由氨基酸S突变为L或K；或者，288为由氨基酸S突变为K或M而得。本发明提高了莫海威芽孢杆菌(Bacillus mojavensis)碱性蛋白酶BmP的热稳定性，在未破坏该酶其他优良酶学性质的前提下，显著提高了该酶的热稳定性，为其产业化应用奠定基础。

Description

提高热稳定性的碱性蛋白酶BmP突变体及其基因和应用
技术领域
本发明涉及生物工程领域，具体涉及提高热稳定性的碱性蛋白酶BmP突变体及其基因和应用。
背景技术
蛋白酶是催化蛋白质水解的一类酶，蛋白酶作为一种重要的工业酶制剂广泛的应用于饲料加工、酿造、洗涤、食品加工等领域。蛋白酶分布广泛，目前市面上的蛋白酶主要来源于微生物，相对于动物及植物，微生物蛋白酶pH值范围广，生产成本低。芽孢杆菌属蛋白酶作为微生物蛋白酶最重要的一类，主要分为中性蛋白酶和碱性蛋白酶，目前芽孢杆菌属蛋白酶已广泛的应用于饲料、洗涤、食品等领域。
莫海威芽孢杆菌(Bacillus mojavensis)碱性蛋白酶简称BmP，前期通过实验发现其对蛋白原料分解能力强，适用于食品、饲料等工业领域。但是BmP热稳定性差，在大于70℃的高温下容易分解失活，限制了其产业化应用。因此提高BmP的热稳定性，是BmP产业化应用急需解决的问题。
发明内容
本发明对来源于莫海威芽孢杆菌(Bacillus mojavensis)碱性蛋白酶BmP进行蛋白质分子改造，从而提高BmP在高温环境下的稳定性，为BmP的产业化应用奠定基础。
本发明的目的是提供提高热稳定性碱性蛋白酶BmP的突变体。
本发明的再一目的是提供提高热稳定性碱性蛋白酶BmP突变体的编码基因。
莫海威芽孢杆菌的碱性蛋白酶BmP的核苷酸序列如SEQ ID NO.1。
SEQ ID NO.1
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTATCCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACAGCAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
根据本发明的具体实施方式，本发明采用定点饱和突变的方法对氨基酸序列如SEQ ID NO.6所示的碱性蛋白酶BmP的第193位和第288位进行突变，经过筛选确定了第193位和第288位这2个位点的有效突变氨基酸。其中193位的有效突变氨基酸分别为L或K，其中L最好；288位有效突变氨基酸分别为M或K，其中M最好。
在有效突变位点的基础上，分别进行组合，最终得到了四个热稳定性提高的碱性蛋白酶BmP突变体，分别命名为BmP1、BmP2、BmP3和BmP4。在75℃，水浴处理5分钟条件下，BmP1、BmP2、BmP3和BmP4的保留率分别为65％，60％，56％和49％，而原始酶BmP在相同条件下的保留率仅为9％。BmP1包含的突变位点为S193K和S288M；BmP2包含的突变位点为S193L和S288M；BmP3包含的突变位点为S193L和S288K；BmP4包含的突变位点为S193L和S288K，BmP1、BmP2、BmP3和BmP4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。
SEQ ID NO.2(突变体BmP1)
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAAAATTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACATGAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
核苷酸序列SEQ ID NO.3(突变体BmP2)：
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTACTCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACATGAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
核苷酸序列SEQ ID NO.4(突变体BmP3)：
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTACTCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACAAAAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
核苷酸序列SEQ ID NO.5(突变体BmP4)：
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAAAATTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACAAAAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
氨基酸酸序列SEQ ID NO.6(原始碱性蛋白酶BmP)：
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
氨基酸酸序列SEQ ID NO.7(突变体BmP1)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVKLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNMNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
氨基酸酸序列SEQ ID NO.8(突变体BmP2)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVLLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNMNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
氨基酸酸序列SEQ ID NO.9(突变体BmP3)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVLLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
氨基酸酸序列SEQ ID NO.10(突变体BmP4)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVKLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
本发明还提供了包含编码上述提高热稳定性的碱性蛋白酶BmP突变体的基因的重组载体。
本发明还提供了包含编码上述提高热稳定性的碱性蛋白酶BmP突变体的基因的宿主细胞。
根据本发明的获得提高热稳定性的碱性蛋白酶BmP突变体的方包括将氨基酸序列如SEQ ID NO.6所示的碱性蛋白酶BmP的突变体的193位由氨基酸S突变为L或K；或者，288为由氨基酸S突变为K或M的步骤。
本发明还提供了上述提高热稳定性的碱性蛋白酶BmP突变体的应用。
根据本发明的发酵制备提高热稳定性的碱性蛋白酶BmP突变体的方法包括使用上述的重组载体进行发酵的步骤。
本发明提高了莫海威芽孢杆菌(Bacillus mojavensis)碱性蛋白酶BmP的热稳定性，在未破坏该酶其他优良酶学性质的前提下，显著提高了该酶的热稳定性，为其产业化应用奠定基础。
附图说明
图1显示原始碱性蛋白酶BmP和突变体BmP1至BmP4的最适反应pH；
图2显示原始碱性蛋白酶BmP和突变体BmP1至BmP4的pH稳定性；
图3显示原始碱性蛋白酶BmP和突变体BmP1至BmP4的最适反应温度。
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、枯草芽孢杆菌WB600、表达载体phyP43L(将枯草芽孢杆菌P43启动子及碱性蛋白酶信号肽连接到载体pHY300PLK得到表达载体phyP43L)。
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)。枯草芽孢培养基为LBK，即LB培养基加卡那霉素。
实施例1、莫海威芽孢杆菌(Bacillus mojavensis)碱性蛋白酶BmP基因的克隆
根据已报道莫海威芽孢杆菌碱性蛋白酶基因的序列(Genebank:AY665611.1)直接合成目的基因。根据合成的目的基因设计两条引物(R：5'-ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3'和F：5'-TCTAGCGGCCGC TTATTGAGCGGCAGCTTCGAC-3')用于扩增莫海威芽孢杆菌碱性蛋白酶BmP基因。将扩增的PCR产物纯化回收，连接到表达载体phyP43L，得到表达载体phyP43L-BmP。
实施例2、定点饱和突变
通过饱和突变研究第193位和第288位对碱性蛋白酶BmP热稳定性的影响。定点饱和突变的过程如下：以构建好的phyP43L-BmP为模板，以相应的突变引物进行PCR扩增；将扩增好的PCR产物进行琼脂糖电泳并纯化回收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，通过电转化转入枯草芽孢杆菌WB600。
重组转化子的筛选如下：首先将在卡那霉素抗性板上生长的重组菌接入LBK培养基中，37℃，200rpm培养24小时；将培养后的菌液离心取上清，进行酶活测定，酶活测定方法参照国标2009进行；在75℃，水浴处理5分钟条件下测定突变体的热稳定性。实验结果如表1所示，由表1可知：193位饱和突变后筛选到两个突变氨基酸可以提高BmP的热稳定性，分别是S193L和S193K，保留率分别为30％和28％；288位也筛选到两个突变氨基酸可以有效提高BmP的热稳定性，分别是S288M和S288K，保留率分别为41％和38％
表1原始碱性蛋白酶BmP和突变体热稳定性
编号 保留率(％)
原始碱性蛋白酶BmP 8
S193L 30
S193K 28
S288M 41
S288K 38
实施例3、组合突变
在单点突变的基础上进行组合突变，通过实验最终得到4个组合突变分别命名为BmP1、BmP2、BmP3、BmP4。其中BmP1包含的突变位点为S193K和S288M；BmP2包含的突变位点为S193L和S288M；BmP3包含的突变位点为S193L和S288K；BmP3包含的突变位点为S193K和S288K。
实施例4、原始碱性蛋白酶BmP及突变体的热稳定性分析
为了精确比较原始碱性蛋白酶BmP和突变体的热稳定性，首先对相应的蛋白酶进行纯化，纯化方法为镍柱纯化。将纯化好的碱性蛋白酶BmP和突变体，在75℃，水浴处理5分钟条件下测定热稳定性。通过实验最终确定突变体BmP1、BmP2、BmP3和BmP4在该条件下的保留率分别为65％，60％，56％和49％，而原始碱性蛋白酶BmP为9％。
表2原始碱性蛋白酶BmP和组合突变体热稳定性
编号 保留率(％)
原始碱性蛋白酶BmP 9
BmP1：S193K/S288M 65
BmP2：S193L/S288M 60
BmP3：S193L/S288K 56
BmP3：S193K/S288K 49
实施例5、原始碱性蛋白酶BmP及突变体的最适反应pH及pH稳定性
参照国标方法测定原始碱性蛋白酶BmP及突变体BmP1、BmP2、BmP3和BmP4的最适反应pH。原始碱性蛋白酶BmP及突变体BmP1、BmP2、BmP3和BmP4的最适反应pH如图1所示。由图1可知，突变体BmP1、BmP2、BmP3和BmP4的最适pH几乎和原始碱性蛋白酶BmP一样，均为10.0。
将原始碱性蛋白酶BmP及突变体BmP1、BmP2、BmP3和BmP4分别在pH6-11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BmP1、BmP2、BmP3和BmP4的pH稳定性与原始碱性蛋白酶BmP一致。
实施例6、碱性蛋白酶BmP及突变体的最适反应温度
参照国标方法测定原始碱性蛋白酶BmP及突变体BmP1、BmP2、BmP3和BmP4的最适反应温度，结果如图3所示。由图3可知，碱性蛋白酶BmP的最适反应温度为65℃，而突变体BmP1、BmP2、BmP3和BmP4的最适反应温度为70℃。
序列表
<110> 刘丹妮
<120> 提高热稳定性的碱性蛋白酶BmP突变体及其基因和应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1140
<212> DNA
<213> 莫海威芽孢杆菌(Bacillus mojavensis)
<400> 1
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtatcct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa cagcaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 2
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtaaaat tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa catgaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 3
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtactct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa catgaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 4
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtactct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa caaaaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 5
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtaaaat tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa caaaaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 6
<211> 379
<212> PRT
<213> 莫海威芽孢杆菌(Bacillus mojavensis)
<400> 6
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Gly Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 7
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Gly Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Lys Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Met
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 8
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Gly Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Leu Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Met
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 9
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Gly Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Leu Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Lys
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 10
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Gly Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Lys Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Lys
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375

Claims (7)

1.提高热稳定性的碱性蛋白酶BmP突变体，其特征在于，所述突变体为将氨基酸序列如SEQ ID NO.6所示的碱性蛋白酶BmP的突变体的193位由氨基酸S突变为L或K；或者，288为由氨基酸S突变为K或M而得。
2.编码权利要求1所述提高热稳定性的碱性蛋白酶BmP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。
5.一种获得提高热稳定性的碱性蛋白酶BmP突变体的方法，其特征在于，所述方法包括将氨基酸序列如SEQ ID NO.6所示的碱性蛋白酶BmP的突变体的193位由氨基酸S突变为L或K；或者，288为由氨基酸S突变为K或M的步骤。
6.权利要求1所述提高热稳定性的碱性蛋白酶BmP突变体的应用。
7.一种发酵制备提高热稳定性的碱性蛋白酶BmP突变体的方法，其特征在于，所述方法包括使用权利要求3所述的重组载体进行发酵的步骤。
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