CN114591935B - 一种热稳定性提高的蛋白酶突变体blapr3及其编码基因和应用 - Google Patents

一种热稳定性提高的蛋白酶突变体blapr3及其编码基因和应用 Download PDF

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CN114591935B
CN114591935B CN202210237401.3A CN202210237401A CN114591935B CN 114591935 B CN114591935 B CN 114591935B CN 202210237401 A CN202210237401 A CN 202210237401A CN 114591935 B CN114591935 B CN 114591935B
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肖志壮
方安然
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Abstract

本发明提供了一种热稳定性提高的蛋白酶突变体BLAPR3及其编码基因和应用，本发明使用易错PCR方法对Bacillus licheniformis来源的蛋白酶基因进行突变，再通过高通量筛选，获得蛋白酶突变体BLAPR3，相比于未突变的蛋白酶，本发明得到的蛋白酶突变体的热稳定性得到显著提高，具有良好的市场应用前景和工业价值。

Description

一种热稳定性提高的蛋白酶突变体BLAPR3及其编码基因和 应用
技术领域
本发明属于基因工程和酶工程领域，具体涉及一种热稳定性提高的蛋白酶突变体BLAPR3及其编码基因和应用。
背景技术
蛋白酶是催化水解蛋白质中肽键的一类酶，广泛存在于动物、植物和微生物中，有着许多不同的生理功能，是酶学研究开展的较早，也是最为成熟的一种。蛋白酶在各个领域中的应用愈加广泛，例如食品工业、酿酒酿造、洗涤剂行业、饲料工业、制革工业、丝绸行业和医药行业等，与我们的生活息息相关，这种环境下对蛋白酶的产量和特性改良方面都提出了更高的要求。
目前市场上大多数蛋白酶70℃处理后仅剩余酶活20%不到，总体耐温性能差，限制了蛋白酶的广泛使用。例如在饲料生产时，酶制剂与饲料混匀后要经过高温造粒，在此过程中酶易变性失活。因此提高蛋白酶的热稳定性至关重要。
易错PCR技术是在采用DNA聚合酶进行PCR反应扩增目的片段时，通过调整反应条件，增加扩增过程中的突变频率，从而以一定的频率向目的基因中随机引入突变，构建突变体文库，从而筛选需要的正向突变体。易错PCR技术可以很好地应用于蛋白质的分子改造中。
发明内容
本发明提供了一种热稳定性提高的蛋白酶突变体BLAPR3及其编码基因和应用，通过构建突变体文库和定向筛选的方法，对Bacillus licheniformis WX-02来源的蛋白酶基因进行改良，筛选得到热稳定性提高的突变体，借此有助于提升其在饲料、食品或洗涤领域中的应用效果。
为了实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种热稳定性提高的蛋白酶突变体BLAPR3，其氨基酸序列如SEQ IDNO：7所示，其编码基因的核苷酸序列如SEQ ID NO：8所示。
本发明还提供了包含所述蛋白酶突变体编码基因的重组表达载体。
本发明提供了包含所述蛋白酶突变体编码基因的的基因工程菌，所述基因工程菌为枯草芽胞杆菌和地衣芽胞杆菌。
本发明提供了所述蛋白酶突变体的制备方法，包括以下步骤：
1）重组基因工程菌构建：将所述蛋白酶突变体的编码基因连接到pUB110载体上，再将重组载体转化入枯草芽胞杆菌中，利用抗性标记筛选阳性克隆；
2）重组基因工程菌摇瓶发酵：将验证正确的阳性克隆接种到摇瓶中发酵，震荡培养，发酵产生蛋白酶突变体；
3）重组菌株放大发酵：将表达蛋白酶突变体的基因工程菌株接种到发酵罐中，从而发酵产生蛋白酶突变体BLAPR3。
进一步的：所述步骤（3）发酵培养基包括以下成分：豆粕5-10%、玉米粉1-5%、PPG-20000 0.1-1.0%、蛋白酶0.1-1.0%、淀粉酶0.1-1.0%、磷酸氢二钠0.2-0.5%，以质量比计。
本发明提供了所述蛋白酶突变体在用于制备饲料添加剂、食品添加剂和洗涤剂中的应用。
与现有技术相比，本发明的优点和技术效果是：本发明以Bacillus licheniformis WX-02来源的蛋白酶基因为基础，分别提供了包含A196C的单点突变体BLAPR1，分别包含I140C/A196C、K49E/A196C的双位点突变体BLAPR2和BLAPR3，以及包含S191C/A196C/G308E的三位点突变体BLAPR4。
本发明改造后的突变体BLAPR1，BLAPR2，BLAPR3和BLAPR4在75℃处理3分钟时的热稳定性比原始蛋白酶分别提高了30.2％、64.0％、45.9％、39.0％。热稳定性得到了显著的提升。
所以通过本发明技术方案得到的蛋白酶突变体的热稳定性较野生型有较大提高，使其在饲料、食品或洗涤等领域有很好的应用潜力。具有良好的市场应用前景。
附图说明
图1是本发明中蛋白酶在30L发酵罐中的发酵数据。
图2是本发明中蛋白酶突变体在75℃水浴3min耐热性比较。
图3是本发明中蛋白酶突变体对豆粕抗性蛋白的降解效果（1、19是蛋白Marker；2、8、12、18是豆粕没有加酶；3-7分别是加入200U/g的BLAPR0，BLAPR1，BLAPR2，BLAPR3和BLAPR4； 9-13分别是加入400U/g的BLAPR0，BLAPR1，BLAPR2，BLAPR3和BLAPR4；13-17分别是加入600U/g的BLAPR0，BLAPR1，BLAPR2，BLAPR3和BLAPR4）。
具体实施方式
为了便于理解本发明，以下将结合附图及实施例对本文发明做更全面、详细地说明，但本发明的保护范围并不限于以下具体实施例。
以下实施例中未作具体说明的分子生物学实验方法，可以参照《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。具体实施例中使用的试剂和生物材料如无特殊说明均可从商业途径获得。
1 菌株和载体
枯草芽胞杆菌WB600，质粒pUB110，大肠杆菌BL21，质粒pET-21a(+)购自Invitrogen公司。
2 试剂与培养基
质粒提取试剂盒，片段纯化回收试剂盒，限制性内切酶等购自宝生物工程(大连)有限公司；GeneMorph II随机突变PCR试剂盒购自Stratagene公司；氨苄青霉素，IPTG等购自生工生物工程(上海)股份有限公司；蛋白Marker：Blue Plus II Protein Marker (14-120 kDa) 购自北京全式金生物技术有限公司。LB培养基：1％胰蛋白胨，0 .5％酵母提取物，1％NaCl。
实施例1：易错PCR构建蛋白酶突变体文库
参考Bacillus licheniformis WX-02来源的蛋白酶氨基酸序列(SEQ ID NO：1)，和DNA序列(SEQ ID NO：2)，设计引物，在5’端设计Xba I限制性酶切位点，3’端设计BamH I限制性酶切位点。
SEQ ID NO：1
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID NO：2
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATGCGGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
用GeneMorph II随机突变PCR试剂盒，以SEQ ID NO：2的基因为模版，进行随机突变，使用的引物序列如下：
BLAPR-F：GCTCTAGAATGATGAGGAAGAAATC（SEQ ID NO：11）；
BLAPR-R：CGCGGATCCTTACTGTGCAGCTGCTTCGA（SEQ ID NO：12）。
将扩增好的随机突变PCR产物用Xba I和BamH I双酶切，纯化回收后连接到pET-21a (+)载体上，转化大肠杆菌BL21-DE3，氨苄青霉素抗性LB平板筛选阳性克隆，得到pET-BLAPRx。同样方法将合成的原始基因连接到pET-21a(+) 载体上并转化大肠杆菌BL21-DE3，得到pET-BLAPR0。
将筛选的单菌落接种到96孔深孔板。每个平板接入2个表达BLAPR0的单菌落为对照。每孔装入300uL LB液体培养基(含有100μg/mL氨苄青霉素)， 37℃200rpm震荡培养4小时后，转移50uL菌液到新的96孔平板保种，在平板剩余菌液中添加200uL含有IPTG的LB-Amp培养基，使IPTG终浓度为1mM，氨苄青霉素终浓度为100μg/mL，37℃200rpm摇床培养10h诱导表达蛋白酶。
将诱导完成的菌液反复冻融进行破碎，将破碎后的细胞液离心取上清，进行热处理(75℃水浴3min)，然后检测蛋白酶的剩余活性。将剩余酶活高于对照的突变体基因进行测序。
筛选到以BLAPR0为出发模板的耐热性提高的突变体A196C (命名为BLAPR1)，氨基酸序列如SEQ ID NO：3所示，其编码的核苷酸序列如 SEQ ID NO：4所示。
SEQ ID NO：3
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID NO：4
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
实施例2：第二轮易错PCR构建蛋白酶BLAPR1的突变体文库
以实施例1中筛选到的蛋白酶基因BLAPR1为模版，进行第二轮随机突变，突变库构建过程以及使用材料试剂和操作条件等均同实施例1；突变体培养和筛选时以BLAPR1为对照，经过热处理(75℃水浴3min)后检测蛋白酶突变体的剩余活性，将剩余酶活高于BLAPR1的突变体基因进行测序。
最终筛选到热稳定性提高的以下突变体：
BLAPR2突变方式为I140C/A196C，氨基酸序列如SEQ ID No：5所示，基因序列如SEQID No：6所示；
BLAPR3突变方式为K49E/A196C，氨基酸序列如SEQ ID No：7所示，基因序列如SEQID No：8所示；
BLAPR4突变方式为S191C/A196C/G308E，氨基酸序列如SEQ ID No：9所示，基因序列如SEQ ID No：10所示。
SEQ ID No：5
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGCQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID No：6
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGATGC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
SEQ ID No：7
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVETASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID No：8
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTGAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
SEQ ID No：9
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPCVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAEVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID No：10
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTGCGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGAAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
实施例3：热稳定性提高的蛋白酶突变体在枯草芽胞杆菌中的表达验证
分别将上述优良突变体克隆入质粒pUB110的Xba I和BamH I位点，参考Spizizen创立的枯草芽胞杆菌转化方，将重组质粒转化入枯草芽胞杆菌WB600中，获得重组菌。按照发酵培养基：豆粕粉50-80g/L，玉米淀粉60-100g/L，磷酸氢二钠2-4g/L，碳酸钠1-2g/L， pH自然，进行摇瓶发酵78h后，将培养液离心获得上清，测定每个突变体发酵液上清的平均酶活，取每个突变体中酶活最高的转化子发酵上清液，在75℃水浴处理3min后比较酶活保留率，结果如图2。突变后得到的蛋白酶BLAPR1，BLAPR2，BLAPR3和BLAPR4在75℃处理3分钟时的热稳定性比原始蛋白酶分别提高了30.2％、64.0％、45.9％、39.0％。
以上结果表明将BLAPR0第196位的Ala突变为Cys能在保持原有酶活力的同时提高其耐热性。在此基础上将其第140位的Ile突变为Cys得到的突变体，或将其49位Lys突变为Glu得到的突变体；或将其第191位的Ser突变为Cys，同时将其第308位的 Gly突变为Glu得到的突变体，其耐热性有进一步提高。
实施例4：蛋白酶突变体在30L发酵罐中发酵和制备
将表达上述实施例中的蛋白酶突变体BLAPR0、BLAPR1、BLAPR2、BLAPR3、BLAPR4的基因工程菌分别划线于含有卡那霉素抗性的（终浓度为20μg/mL）LB平板上，37℃培养至长出单菌落，挑取长势良好的单菌落继续在含有卡那霉素抗性的（终浓度为20μg/mL）LB平板上划线培养，如此活化三代得到的重组枯草芽胞杆菌菌落接种于50mL含有卡那霉素终浓度为20μg/mL的LB培养基中，37℃、200rpm 培养24h。以2％的接种量接种到1L含有卡那霉素终浓度为20μg/mL的LB培养基中，37℃、200rpm培养至 OD600为5左右，用作种子液接种发酵罐。
发酵生产工艺：豆粕5-10%、玉米粉1-5%、PPG-20000 0.1-1.0%、蛋白酶0.1-1.0%、淀粉酶0.1-1.0%、磷酸氢二钠（12水）0.2-0.5%、pH自然、温度37℃、搅拌速率600rpm、通风量1 .5(v/v)，溶氧控制在20％以上。发酵过程pH自然，发酵24h后开始测定酶活力，待发酵结束后（一般48h），发酵液通过板框过滤机处理得到粗酶液经喷塔喷干成粉制剂，用于应用测试。
实施例5：蛋白酶突变体对抗性蛋白的降解实验
（1）豆粕抗性蛋白酶解反应
豆粕底物：取2g过60目筛的粉碎豆粕，加入20mL缓冲液（pH 8.0 0.02M Tris-Hcl缓冲液）配制成悬浮液；
实验组：将蛋白酶突变体BLAPR0、BLAPR1、BLAPR2、BLAPR3、BLAPR4基因工程菌的发酵液，进行离心，获得发酵液上清。准确测定酶活力，按照相同酶活力（200 U/g、400U/g、600U/g）进行添加；
对照组：不添加酶，处理过程与试验组一致；
反应条件：将酶液和底物混合均匀后，于水浴摇床上，37℃，120rpm/min酶解反应2h；
反应完成后，取0.5mL豆粕酶解液加入4.5mL ELISA提取液，25℃，200 rpm/min，提取16h。
（2）SDS-PAGE 考察抗原蛋白去除情况
取步骤（1）中经过反应和提取的样品100μL 加入100μL Loading Buffer，煮沸10min，取20μL进行上样。
电泳条件：浓缩胶时电压80-90mv，电流30mA附近；分离胶时，电压110-120mv，电流40mA附近。样品前言离胶边沿1cm处，停止电泳。剥胶，染色2h，脱色24h，期间更换脱色液。
试验结果如图3所示：在相同条件，相同酶活力单位的添加量时，蛋白酶突变体BLAPR0、BLAPR1、BLAPR2、BLAPR3、BLAPR4对豆粕的降解效果，基本一致；当蛋白酶突变体的添加量为400U/g时，降解豆粕效果显著，只剩很浅的一条带约50KD（β亚基）；当蛋白酶突变体的添加量为600U/g时，豆粕都能被彻底降解。
该实验表明蛋白酶突变体BLAPR1、BLAPR2、BLAPR3、BLAPR4保持了原有高效的降解抗性蛋白的性质，同时其热稳定性有显著提高，所以蛋白酶突变体BLAPR1、BLAPR2、BLAPR3、BLAPR4具有很好应用潜力。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 青岛尚德生物技术有限公司
青岛红樱桃生物技术有限公司
<120> 一种热稳定性提高的蛋白酶突变体BLAPR3及其编码基因和应用
<160> 12
<170> SIPOSequenceListing 1.0
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<213> 地衣芽孢杆菌(Bacillus licheniformis)
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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 Ser Asp Ser Ala Ser Ala Ala Gln Pro
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Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
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Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
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Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
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Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
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Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
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Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
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Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
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Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
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atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
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aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
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caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
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Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
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Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
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Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
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Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
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Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
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Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
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Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
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Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
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Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
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Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
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Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
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Ser Leu Tyr Cys Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
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Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
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Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
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Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
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Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
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Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
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<213> 地衣芽孢杆菌(Bacillus licheniformis)
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atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
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gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
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aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
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caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
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gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
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<210> 5
<211> 379
<212> PRT
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<400> 5
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 Ser 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 Cys 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 Cys 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> 6
<211> 1140
<212> DNA
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<400> 6
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggatgc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 7
<211> 379
<212> PRT
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<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 Ser 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
Glu 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 Cys 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> 8
<211> 1140
<212> DNA
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<400> 8
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttgaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 9
<211> 379
<212> PRT
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<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 Ser 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 Cys Val
180 185 190
Ser Leu Tyr Cys 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 Glu 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> 1140
<212> DNA
<213> 地衣芽孢杆菌(Bacillus licheniformis)
<400> 10
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tgcgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tgaagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gctctagaat gatgaggaag aaatc 25
<210> 12
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cgcggatcct tactgtgcag ctgcttcga 29

Claims (7)

1.一种热稳定性提高的蛋白酶突变体BLAPR3，其特征在于，其氨基酸序列如SEQ IDNO：7所示，其编码基因的核苷酸序列如SEQ ID NO：8所示。
2.包含权利要求1所述蛋白酶突变体编码基因的重组表达载体。
3.包含权利要求1所述蛋白酶突变体编码基因的基因工程菌，其特征在于所述基因工程菌为枯草芽胞杆菌和地衣芽胞杆菌。
4.权利要求1所述蛋白酶突变体的制备方法，其特征在于包括以下步骤：
1）重组基因工程菌构建：将所述蛋白酶突变体的编码基因连接到pUB110载体上，再将重组载体转化入枯草芽胞杆菌中，利用抗性标记筛选阳性克隆；
2）重组基因工程菌摇瓶发酵：将验证正确的阳性克隆接种到摇瓶中发酵，震荡培养，发酵产生蛋白酶突变体；
3）重组菌株放大发酵：将表达蛋白酶突变体的基因工程菌株接种到发酵罐中，从而发酵产生蛋白酶突变体BLAPR3。
5.根据权利要求4所述蛋白酶突变体的制备方法，其特征在于所述步骤（3）发酵培养基包括以下成分：豆粕5-10%、玉米粉1-5%、PPG-20000 0.1-1.0%、蛋白酶0.1-1.0%、淀粉酶0.1-1.0%、磷酸氢二钠0.2-0.5%，以质量比计。
6.权利要求1所述蛋白酶突变体在用于制备饲料添加剂和食品添加剂中的应用。
7.权利要求1所述蛋白酶突变体在用于制备洗涤剂中的应用。
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