CN108570461B - 一种提高比活力的碱性蛋白酶BmP突变体及其编码基因 - Google Patents

一种提高比活力的碱性蛋白酶BmP突变体及其编码基因 Download PDF

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CN108570461B
CN108570461B CN201810343751.1A CN201810343751A CN108570461B CN 108570461 B CN108570461 B CN 108570461B CN 201810343751 A CN201810343751 A CN 201810343751A CN 108570461 B CN108570461 B CN 108570461B
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刘丹妮
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Abstract

本发明涉及蛋白质分子改造领域，具体涉及一种提高比活力的碱性蛋白酶BmP突变体及其编码基因。本发明碱性蛋白酶BmP突变体的突变位点为：氨基酸序列如SEQ ID NO.11所示的碱性蛋白酶BmP的氨基酸序列的236位由N突变为G或C或D，且293位为由N突变为S或K或M。实验证实，本发明获得的突变体比原始酶具有更高的的比活，降低了碱性蛋白酶的生产成本，为BmP的工业化应用奠定基础。

Description

一种提高比活力的碱性蛋白酶BmP突变体及其编码基因
技术领域
本发明涉及蛋白质分子改造领域，具体涉及一种提高比活力的碱性蛋白酶BmP突变体及其编码基因。
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。根据微生物蛋白酶最适反应pH环境可将蛋白酶分为酸性蛋白酶、中性蛋白酶和碱性蛋白酶。碱性蛋白酶是一类在碱性条件下水解蛋白质的酶类，目前碱性蛋白酶主要来源于地衣芽孢杆菌、枯草芽孢杆菌以及环状芽孢杆菌，其他芽孢杆菌碱性蛋白酶报道相对较少。
在前期的实验中，本发明克隆、表达以及分析了来自莫海威芽孢杆菌(Bacillusmojavensis)的碱性蛋白酶BmP，BmP对蛋白原料分解能力强，适用于食品、洗涤、饲料等工业领域。但是BmP比活力相对较低，生产成本高，限制了其产业化应用。因此提高BmP的比活力，是BmP产业化应用急需解决的问题。
申请号为201310394552.0的中国专利申请，报道了一种蛋白酶及突变体及其应用，该发明公开了一种碱性蛋白酶，其氨基酸序列如SEQIDNO.1所示,其还公开了基于上述蛋白酶突变的对LAS-Na耐受性提升的突变蛋白酶，其氨基酸序列如SEQIDNO.2和SEQIDNO.3所示。此外，还公开了上述蛋白酶DNA的克隆/表达载体的构建，突变及突变酶的筛选方法，并研究了重组突变酶在液体洗涤剂中的应用性能。申请号为201510554970.0的中国专利申请，公开了一种新型碱性蛋白酶突变体，以来源于克劳氏芽孢杆菌的碱性蛋白酶aprE为基础，通过易错PCR技术获得了三个碱性蛋白酶突变体aprE-M1、aprE-M2和aprE-M3，并分别构建了表达上述突变体的重组菌枯草芽孢杆菌BS-4，BS-5和BS-10。
发明内容
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所示，氨基酸序列如SEQ ID NO.11所示。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，研究证实，本发明的碱性蛋白酶BmP突变体显著提高了BmP的比活力，降低其生产成本，为BmP的工业化应用奠定基础。
本发明的目的是提供一种提高比活力的碱性蛋白酶BmP的突变体。所述的突变位点为第236位和293位。具体地，所述突变体为氨基酸序列如SEQ ID NO.11所示的碱性蛋白酶BmP突变体的突变位点为236位由N突变为G或C或D；293位由N突变为S或K或M。
本发明选定第236位和293位作为定点饱和突变目标位点。采用定点饱和突变的方法对SEQ ID NO.11所示的碱性蛋白酶BmP的第236位和293位进行突变，经过筛选得到9个突变体可以有效提高碱性蛋白酶BmP的比活力。这9个突变体分别命名为P1、P2、P3、P4、P5、P6、P7、P8和P9。
相对于原始碱性蛋白酶BmP，P1、P2、P3、P4、P5、P6、P7、P8和P9的比活力分别提高了41％、51％、48％、38％、67％、53％、65％、36％和48％。突变体P1、P2、P3、P4、P5、P6、P7、P8和P9的核苷酸序列如SEQ ID NO.2到SEQ ID NO.10所示，氨基酸序列如SEQ ID NO.12到SEQID NO.20所示。其中P1包含的突变位点为N236G和N293S；P2包含的突变位点为N236G和N293K；P3包含的突变位点为N236G和N293M；P4包含的突变位点为N236C和N293S；P5包含的突变位点为N236C和N293K；P6包含的突变位点为N236C和N293M；P7包含的突变位点为N236D和N293S；P8包含的突变位点为N236D和N293K；P9包含的突变位点为N236D和N293M。
根据已报道莫海威芽孢杆菌碱性蛋白酶基因序列(Genebank:AY665611.1)直接合成目的基因，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；通过饱和突变碱性蛋白酶BmP第236位和293位获得所述的提高比活力碱性蛋白酶BmP的突变体。
其中定点饱和突变具体包括如下步骤：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。
本发明的再一目的是提供提高比活力碱性蛋白酶BmP突变体的编码基因。所述基因的核苷酸序列为SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、EQ ID NO.6、SEQID NO.7、SEQ ID NO.8、SEQ ID NO.9或SEQ ID NO.10所示。
本发明还请求保护一种包含所述提高比活力碱性蛋白酶BmP突变体编码基因的重组载体。
本发明还请求保护一种包含提高比活力碱性蛋白酶BmP突变体编码基因的宿主细胞。
本发明还请求保护一种所述提高比活的碱性蛋白酶BmP突变体在饲料加工、酿造、洗涤或食品加工领域的应用。
本发明所述突变体为氨基酸序列如SEQ ID NO.11所示的碱性蛋白酶BmP突变体的突变位点为236位由N突变为G或C或D；293位由N突变为S或K或M。通过突变提高了莫海威芽孢杆菌(Bacillusmojavensis)碱性蛋白酶BmP的比活力，降低其生产成本，为其工业化应用奠定基础。
附图说明
图1原始碱性蛋白酶BmP和突变体P1至P9的最适反应pH。
图2原始碱性蛋白酶BmP和突变体P1至P9的pH稳定性。
图3原始碱性蛋白酶BmP和突变体P1至P9的最适反应温度。
图4原始碱性蛋白酶BmP和突变体P1至P9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
大肠杆菌菌株Topl0、枯草芽孢杆菌WB600、表达载体phyP43L均由本实验室保藏。
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为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'-TCTAGCGGCCGCTTATTGAGCGGCAGCTTCGAC-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、通过饱和突变研究第236位和293位对碱性蛋白酶BmP比活力的影响
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GBT/28715-2012进行。
实施例3、原始碱性蛋白酶BmP及单突变体比活力分析
采用镍柱纯化对碱性蛋白酶BmP和单突变体进行纯化。将纯化后的碱性蛋白酶BmP和突变体进行比活力测定，实验结果如表1所示，由表1可知第236位和293是影响碱性蛋白酶BmP水解活性的关键氨基酸位点，当236位突变为G、C和D时，相对比活力分别提高了12％，15％和18％；当293位突变为S、K和M时，相对比活力分别提高了21％，28％和31％。
表1原始碱性蛋白酶BmP和突变体比活力分析
Figure BDA0001631430830000041
Figure BDA0001631430830000051
实施例4、原始碱性蛋白酶BmP及组合突变体比活力分析
将有效的单突变体，进行组合突变，通过实验最终得到9个组合突变分别命名为P1、P2、P3、P4、P5、P6、P7、P8和P9。其中P1包含的突变位点为N236G和N293S；P2包含的突变位点为N236G和N293K；P3包含的突变位点为N236G和N293M；P4包含的突变位点为N236C和N293S；P5包含的突变位点为N236C和N293K；P6包含的突变位点为N236C和N293M；P7包含的突变位点为N236D和N293S；P8包含的突变位点为N236D和N293K；P9包含的突变位点为N236D和N293M。采用镍柱纯化对碱性蛋白酶BmP和组合突变体进行纯化。将纯化后的碱性蛋白酶BmP和组合突变体进行比活力测定，实验结果如表2所示。由表2可知，相对于原始突变体P1、P2、P3、P4、P5、P6、P7、P8和P9比活分别提高41％、51％、48％、38％、67％、53％、65％、36％和48％。
表2原始碱性蛋白酶BmP和突变体比活力分析
编号 相对比活力(％)
原始碱性蛋白酶BmP 100
P1(N236G和N293S) 141
P2(N236G和N293K) 151
P3(N236G和N293M) 148
P4(N236C和N293S) 138
P5(N236C和N293K) 167
P6(N236C和N293M) 153
P7(N236D和N293S) 165
P8(N236D和N293K) 136
P9(N236D和N293M) 148
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P1至P9的最适反应pH，原始碱性蛋白酶BmP及突变体P1至P9的最适反应pH如图1所示。由图1可知，突变体P1至P9的最适pH值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P1至P9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P1至P9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及热稳定性
参照国标方法测定原始碱性蛋白酶BmP及突变体P1至P9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、P1、P4和P8的最适反应温度均为65℃，突变体P2、P3、P5、P6、P7和P9的最适反应温度则下降为60℃。
将原始碱性蛋白酶BmP及突变体B1至B5分别在70℃，水浴处理5分钟后，参照国标测定剩余酶活。由图4可知，突变体P1、P4、P8和碱性蛋白酶BmP热稳定性相当，剩余酶活保留率均为40％左右。而突变体P2、P3、P5、P6、P7和P9均有所下降，剩余酶活保留率分别为32％，35％，27％，29％、36％和33％。
序列表
<110> 横琴仲泰生物医药有限公司
<120> 一种提高比活力的碱性蛋白酶BmP突变体及其编码基因
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 1
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaacg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 2
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 2
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaggcac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgagtg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 3
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 3
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaggcac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaaag gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 4
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 4
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaggcac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgatgg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 5
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 5
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggatgtac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgagtg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 6
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 6
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggatgtac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaaag gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 7
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 7
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggatgtac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgatgg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 8
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 8
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggagatac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgagtg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 9
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 9
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggagatac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaaag gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 10
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 10
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggagatac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgatgg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taa 1053
<210> 11
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 11
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 12
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 12
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Gly Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 13
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 13
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Gly Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Lys Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 14
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 14
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Gly Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Met Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 15
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 15
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Cys Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 16
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 16
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Cys Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Lys Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 17
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 17
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Cys Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Met Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 18
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 18
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asp Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 19
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 19
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asp Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Lys Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350
<210> 20
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 20
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asp Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Met Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
340 345 350

Claims (8)

1.一种提高比活力的碱性蛋白酶BmP突变体，其特征在于，所述突变体为氨基酸序列如SEQ ID NO.11所示的蛋白酶BmP的突变体，其中突变位点为第236位和293位，所述的突变体包括4个组合突变体，分别命名为P2、P3、P8和P9，其中P2包含的突变位点为N236G和N293K；P3包含的突变位点为N236G和N293M；P8包含的突变位点为N236D和N293K；P9包含的突变位点为N236D和N293M。
2.根据权利要求1所述的提高比活力的碱性蛋白酶BmP突变体，其特征在于，所述的碱性蛋白酶BmP突变体P2、P3、P8和P9的氨基酸序列分别如SEQ ID NO.13、SEQ ID NO.14、SEQID NO.19和SEQ ID NO.20所示。
3.一种编码权利要求1所述提高比活的碱性蛋白酶BmP突变体的基因，其特征在于，所述碱性蛋白酶BmP突变体P2、P3、P8和P9的编码基因的核苷酸序列为SEQ ID NO.3、SEQ IDNO.4、SEQ ID NO.9和SEQ ID NO.10所示。
4.一种获得权利要求1-2任一所述提高比活力的碱性蛋白酶BmP突变体的方法，其包括如下步骤：
1)碱性蛋白酶BmP基因合成及克隆：根据莫海威芽孢杆菌碱性蛋白酶基因序列直接合成目的基因，然后以目的基因为模板进行PCR扩增，将扩增得到的片段克隆到载体phyP43L，得到表达载体phyP43L-BmP；2)定点饱和突变：以构建好的phyP43L-BmP载体为模板进行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。
5.根据权利要求4所述的突变方法，其特征在于，所述步骤1)中PCR扩增引物为：
R：5'-ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3'；
F：5'-TCTAGCGGCCGC TTATTGAGCGGCAGCTTCGAC-3'。
6.一种包含权利要求3所述基因的重组载体。
7.一种包含权利要求3所述基因的宿主细胞。
8.一种权利要求1所述提高比活的碱性蛋白酶BmP突变体在饲料加工、酿造、洗涤或食品加工领域的应用。
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