CN114457058B - 一种饲用α淀粉酶的突变改良方法及应用 - Google Patents
一种饲用α淀粉酶的突变改良方法及应用 Download PDFInfo
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- CN114457058B CN114457058B CN202111216119.9A CN202111216119A CN114457058B CN 114457058 B CN114457058 B CN 114457058B CN 202111216119 A CN202111216119 A CN 202111216119A CN 114457058 B CN114457058 B CN 114457058B
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- amylase
- asn
- gly
- ala
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Abstract
本发明公开了L型α淀粉酶变体及其应用。所述的α淀粉酶变体由亲代α淀粉酶氨基酸序列中依次将四个氨基酸残基突变，仍然保持了亲代水解α‑1,4糖苷键的能力的α淀粉酶。本发明提供的α淀粉酶变体，在80℃～100℃以及pH2.5～5.5酸性条件下保温2小时和90℃以上高温条件下有较高的催化活力。淀粉酶是淀粉消化的关键酶，在饲料中添加淀粉酶，可以提高提高饲料的利用率。本发明提供的α淀粉酶突变体在pH2.7，温度41℃，在不同加酶量、酶解时间、固液比下，体外酶解蛋鸡饲料(淀粉含量高达57％)效果均优于原淀粉酶。本发明提供的α淀粉酶突变体具有良好的耐酸性、热稳定性，以及优良的饲料酶解效果，可应用于动物饲料行业。
Description
技术领域
本发明属于酶工程领域，涉及α淀粉酶变体。
背景技术
α-淀粉酶是动物饲料中常用的—种淀粉酶,它以随机方式水解淀粉分子中的α-1,4糖苷键,是一种内切型淀粉酶,水解效率较高。α-淀粉酶虽然对淀粉分子链内部的α-1,4糖苷键具有良好的水解作用,却不能水解支链淀粉分支点处的α-1，6糖苷键和紧靠α-1,6糖苷键的α-1,4糖苷键,但能跨越α-1,6糖苷键而继续切开支链内部的α-1,4糖苷键。因此,α-淀粉酶作用于直链淀粉时,其终产物是麦芽糖和葡萄糖；作用于支链淀粉时,其终产物除麦芽糖和葡萄糖外,还有α-极限糊精。由于该酶作用于淀粉时所产生的葡萄糖残基C1碳原子为α-构型,故称α-淀粉酶。
饲料中使用的α-淀粉酶有细菌α-淀粉酶和真菌α-淀粉酶两种,枯草芽孢杆菌BF7658α-淀粉酶是我国工业化深层发酵生产的第一个微生物酶制剂,也是目前饲料中普通使用的淀粉酶:该酶是一种中温α-淀粉酶,最适作用温度较高。真菌α-淀粉酶主要由霉菌发酵产生,因其最适作用温度较中温α-淀粉酶低20～30℃，亦称为低温α-淀粉酶。
在本发明中，为适应饲料行业发展的需要，我们利用B.velezensisα-amylase为模板构建了一系列新的α-淀粉酶突变体，提高了该酶种的应用效率与体外酶解蛋鸡饲料的效率。
发明内容
本发明的目的是提供一种B.velezensisα-淀粉酶变体，该系列变体可以提高淀粉酶的耐酸性，并能应用于饲料行业。特别是在pH为2.5～2.8的条件下。
本发明的目的是提供编码该α淀粉酶变体的基因。
本发明的又一目的是提供该α淀粉酶变体的生产方法和应用。
本发明的目的可通过以下技术方案实现：
一种α淀粉酶变体，是通过B.velezensis的α淀粉酶蛋白序列的第546、572、614、622号位突变，分别为由脯氨酸突变为谷氨酸,组氨酸突变为天冬氨酸,丙氨酸突变为谷氨酸,赖氨酸突变为谷氨酸。
所述的B.velezensis的α淀粉酶的全长编码基因序列见SEQ ID NO.1；对应的氨基酸序列见SEQ ID NO.2。
所述的α淀粉酶变体的氨基酸序列如序列表中SEQ ID NO.4所示。
所述的α淀粉酶变体的核苷酸编码序列优选自如SEQ ID NO.3所示。
用于表达本发明所述的α淀粉酶变体的表达载体，其中含有本发明所述的编码α淀粉酶变体的基因。
一种本发明所述的α淀粉酶变体的生产方法，包括在适宜α淀粉酶变体表达的条件下对含有编码α淀粉酶变体基因序列的重组子进行培养，并从重组子或者其培养上清液中获得α淀粉酶变体。
本发明所述的α淀粉酶变体在水解多糖的α-1,4糖苷键中的应用；优选在高温和/或低pH条件下水解多糖的α-1,4糖苷键中的应用；所述的高温优选40℃～100℃；所述的低pH优选2.5～5.5。
有益效果
本发明提供的一种新的α淀粉酶变体，在pH2.5-5.5酸性条件下和80℃以上高温条件下有较高的催化活度。本发明提供的α淀粉酶变体在体外酶解蛋鸡饲料时，效果均优于原淀粉酶。这些α淀粉酶变体具有良好的耐酸性、热稳定性，以及优良的饲料酶解效果，可用于动物饲料行业。
附图说明
图1为pET-28a载体，包括一个卡那霉素决定基因(ErmC)——在E.coli中可以耐受100μg/mL的卡那霉素。
图2为pET-28a-amy载体的示意图。
图3淀粉酶在BL21(DE3)表达菌株中表达情况以及纯化后的SDS-PAGE图。
图4为不同底物(玉米淀粉)浓度下，淀粉酶活力比较
图5为在不同pH条件下，40℃下保温两小时，淀粉酶活力比较
图6为在pH4.5时，不同温度下保温两小时，淀粉酶活力比较
图7为在pH2.7时，41℃，摇床转速为180rpm时，不同加酶量、不同添饲量、不同酶解时间下体外酶解蛋鸡饲料的比较
本发明的详细描述
本发明中，α淀粉酶指的是能够水解多糖的α-1,4糖苷键的酶。例如，α淀粉酶能将淀粉水解成糊精。
本发明中，亲代α淀粉酶是指天然的α淀粉酶。天然的α淀粉酶是细菌α淀粉酶，来源包括但不局限于Bacillus subtilis,B.licheniformis，B.amyloliquefaciens，G.stearothermophilus和Bacillus cereus。
根据本发明的创新，天然α淀粉酶是来源于B.velezensis(本实验室从玉米籽粒中分离得到)的全长编码序列见SEQ ID NO.1；对应的氨基酸序列见SEQ ID NO.2。
本发明中，术语“α淀粉酶变体”是指非天然存在的，在亲代的α淀粉酶氨基酸序列的有效点位进行一个或数个氨基酸残基的增加、删除和/或替代，同时仍然保持了亲代水解α-1,4糖苷键的能力的α淀粉酶。
本发明中“液化”一般是指将碳水化合物分解为小分子的多糖的过程。当加入α淀粉酶或者α淀粉酶变体时，“液化”特指水解碳水化合物的α-1,4糖苷键。
本发明中，“α-1,4糖苷键”指将前一个葡萄糖的C1与后一个葡萄糖的C4连接起来的键，即为α-1,4糖苷键。
本发明涉及将亲代α淀粉酶做序列改造获得的“α淀粉酶变体”。亲代α淀粉酶是天然的α淀粉酶，特别来源于细菌的天然α淀粉酶。根据本发明的实施例，α淀粉酶变体是将亲代的α淀粉酶的氨基酸序列的有效点位进行一个或数个氨基酸残基的突变、增加或删除。
本发明包括一系列的α淀粉酶变体。根据本发明的实施例，这一系列的α淀粉酶变体的氨基酸序列的同源性至少达到95％。作为说明性和非限制性的本发明示例，α淀粉酶变体是由B.velezensis的亲代α淀粉酶蛋白序列的第546、572、614、622号位突变，分别为由脯氨酸突变为谷氨酸,组氨酸突变为天冬氨酸,丙氨酸突变为谷氨酸,赖氨酸突变为谷氨酸，见SEQ ID NO.4。
本发明中的α淀粉酶变体保持了水解α-1,4糖苷键的能力。另外，这些α淀粉酶的性能使用上符合工业生产要求，比如，液化效率的提高，酸性pH或高温条件下催化活性稳定。根据本发明的实施例，一个α淀粉酶变体在pH5.0以下的酸性条件或者温度高于80℃的条件下，催化活性稳定。α淀粉酶变体这些提高的性能更适应饲料行业。因为在饲料行业中，胃环境是一个低pH、高温的环境。
本发明的所有α淀粉酶变体都可以用于液化反应。在优选的实施例中，α淀粉酶变体来源于亲代α淀粉酶，是指来源于B.velezensis亲代的α淀粉酶。
根据本发明，任何含有α-1,4糖苷键的碳水化合物都可以用于液化反应。含有一个或多个α-1,4糖苷键的碳水化合物包括但不仅限于淀粉，支链淀粉，直链淀粉和葡萄聚糖。
本发明提供了一种α淀粉酶变体能够在任何适合工业化生产的温度和pH条件下水解α-1，4糖苷键进行糖化反应的方法。根据本发明，液化反应可以在80℃至100℃的高温下反应，比如80℃，90℃，100℃。
根据本发明的实施例，α淀粉酶变体催化的液化反应在酸性pH和80℃以上温度条件下催化活性稳定。在pH2.7，41℃时，体外酶解蛋鸡饲料优于对照组。
根据本发明实施例，重组宿主细胞可以基因工程改造包含一个或多个α淀粉酶变体基因表达的核酸序列。任何技术均可用于基因工程改造宿主细胞包含一个或多个本发明中的α淀粉酶变体编码合成的核酸序列，例如，染色体整合。含有温度敏感起源和抗性筛选标记的载体可以用于整合步骤。这些载体通过坎贝尔机制与基因组的特定区域整合，通过抗性筛选得到重组菌，重组菌在随后的培养过程中通过同源重组去掉抗性筛选标记。
本发明提供了一种α淀粉酶变体的生产方法。根据本发明的实施例，该方法包括在适宜α淀粉酶变体表达的条件下对含有编码α淀粉酶变体核苷酸序列的重组宿主细胞进行培养，并从重组宿主细胞或者其上清液中获得α淀粉酶变体。
本发明的所有重组宿主细胞都能够生产α淀粉酶变体。重组宿主细胞包含了至少一个拷贝的编码α淀粉酶变体的核苷酸序列。这些编码α淀粉酶变体的核苷酸序列能够在适宜的条件下表达α淀粉酶变体。从重组宿主细胞中分泌的α淀粉酶变体能够从重组子或上清液中收集到。收集的方法包括但不仅限于过滤，离心等。
本发明中下列的例子进一步阐述了本发明的本质。应当理解，下面的例子不限制本发明，本发明的范围是由所附的权利要求确定。
具体实施方式
实施例1:pET-28a质粒的构建
pET-28a载体带有一个N端的His/Thrombin/T7蛋白标签，同时含有一个可以选择的C端His标签。pET-28a载体的单一的多克隆位点见上面的环状质粒图谱。注意：载体序列是以pBR322质粒的编码规矩进行编码的，所以T7蛋白表达区在质粒图谱上面是反向的。T7RNA聚合酶启动的克隆和表达区域在质粒图谱中也被标注了出来。质粒的F1复制子是被定向的，所以在T7噬菌体聚合酶的作用下，包含有蛋白编码序列的病毒粒子能够产生，并启动蛋白表达，同时蛋白表达将被T7终止子序列(Cat.No.69337-3)的作用下终止蛋白翻译，用卡那霉素进行筛选。
pet28a质粒的构建过程为：将质粒pet28a(本实验室保存)用EcorI和XhoI双酶切，回收纯化5.3k的片段，-20℃保存备用。
在本发明中，为了获得α淀粉酶基因(1980bp)，从B.velezensisD1总基因组DNA中克隆扩增得到。用于PCR反应的引物是由Tsingke公司合成的。引物序列如下：
Amy1980s：GATATCGGGGATCCGAATTCATGTTTGAAAAACGATTCAA
Amy1980anti：GTGGTGGTGGTGGTGCTCGAGATGCGGAAGATAACCATTC
PCR扩增体系为50ul，反应程序如下：
(1)贝莱斯芽孢杆菌基因组dna预变性95℃，10分钟；
(2)95℃，15秒；
(3)58℃，30秒；
(4)72℃，60秒；重复2-4步骤25-30次；
(5)终延伸72℃，2分钟。
(6)1％琼脂糖凝胶跑胶，用genstar试剂盒进行胶回收
(7)用EcorI和XhoI双酶切双酶切，回收纯化约2.0k的片段，-20℃保存备用。
将胶回收后的线性质粒和淀粉酶基因片段用T4 DNA ligase(莫纳生物)进行连接，连接完成后用DH5α大肠杆菌感受态进行转化，在含50ug/ml的卡那霉素的LB固体培养基上过夜培养，挑取阳性克隆进行菌落pcr鉴定并送至通用公司测序，得到转化子T1980。用转化子T1980转化BL21(DE3)大肠杆菌感受态，在含50ug/ml的卡那霉素的LB固体培养基上过夜培养，取阳性克隆进行菌落pcr鉴定并送至通用公司测序，得到该淀粉酶表达菌株BLO-amy。
菌落pcr鉴定程序：
用于PCR反应的引物是由Tsingke公司合成的。引物序列如下：
Amy1047s：CCTCTTTACTGCCGTTATT
Amy47anti：ATCTTACACCATTTCCTCC
PCR扩增体系为15ul，反应程序如下：
(1)单菌落培养液预变性95℃，10分钟；
(2)95℃，15秒；
(3)58℃，30秒；
(4)72℃，30秒；重复2-4步骤25-30次；
(5)终延伸72℃，2分钟。
实施例2:pet28a-amy第一次定点突变
提取转化子T1980质粒pet28a-amy，分步进行定点突变。
用于PCR反应的引物是由Tsingke公司合成的。引物序列如下：
546s：CGAACGGCGAGGGTCAAGCGAGAACCCAAG
546anti：GTTCTCGCTTGACCCTCGCCGTTCGTTCCG
PCR扩增体系为50ul，反应程序如下：
(1)质粒pet28a-amy预变性95℃，10分钟；
(2)95℃，60秒；
(3)62℃，30秒；
(4)72℃，2分30秒；重复2-4步骤15-20次；
(5)终延伸72℃，2分钟。
dpnI消化pcr产物，直接转化DH5α大肠杆菌感受态，在含50ug/ml的卡那霉素的LB固体培养基上过夜培养，挑取阳性克隆进行菌落pcr鉴定并送至通用公司测序，得到转化子MTF1980。
实施例3:pet28a-amy第二次定点突变
提取转化子MTF1980质粒,进行PCR第二次定点突变。
用于PCR反应的引物是由Tsingke公司合成的。引物序列如下：
572s：TGGCTATCAAAACCCGGACGATTGGG
572anti：TGGCCCCAATCGTCCGGGTTTTGATA
PCR扩增体系为50ul，反应程序如下：
(1)质粒预变性95℃，10分钟；
(2)95℃，60秒；
(3)62℃，30秒；
(4)72℃，2分30秒；重复2-4步骤15-20次；
(5)终延伸72℃，2分钟。
dpnI消化pcr产物，直接转化DH5α大肠杆菌感受态，在含50ug/ml的卡那霉素的LB固体培养基上过夜培养，挑取阳性克隆进行菌落pcr鉴定并送至通用公司测序，得到转化子MTS1980。
实施例4:pet28a-amy第三次定点突变
提取转化子MTS1980质粒,进行PCR第三次定点突变。
用于PCR反应的引物是由Tsingke公司合成的。引物序列如下：
614s：CTGACGCTGCCTGAGAATACGGATACGGCCAATGCCGAAGTGATTT
614anti：TGTACATTCCATTTGCATTCTTAGTCATTGCTTTCCCCGGCCACGA
PCR扩增体系为50ul，反应程序如下：
(1)质粒预变性95℃，10分钟；
(2)95℃，60秒；
(3)65℃，30秒；
(4)72℃，2分30秒；重复2-4步骤15-20次；
(5)终延伸72℃，2分钟。
dpnI消化pcr产物，直接转化DH5α大肠杆菌感受态，在含50ug/ml的卡那霉素的LB固体培养基上过夜培养，挑取阳性克隆进行菌落pcr鉴定并送至通用公司测序，得到转化子MTT1980。
实施例5:质粒转化
提取转化子MTT1980的质粒，转化BL21(DE3)大肠杆菌感受态，在含50ug/ml的卡那霉素的LB固体培养基上过夜培养，取阳性克隆进行菌落pcr鉴定并送至通用公司测序，得到该淀粉酶表达菌株BLM-amy。菌落PCR鉴定程序：
用于PCR反应的引物是由Tsingke公司合成的。引物序列如下：
Amy1047s：CCTCTTTACTGCCGTTATT
Amy47anti：ATCTTACACCATTTCCTCC
PCR扩增体系为15ul，反应程序如下：
(1)单菌落培养液预变性95℃，10分钟；
(2)95℃，15秒；
(3)58℃，30秒；
(4)72℃，30秒；重复2-4步骤25-30次；
(5)终延伸72℃，2分钟。
产α淀粉酶变体的BL21(DE3)工程菌株在-80℃保存。
实施例6:α淀粉酶变体生产的液体培养
取一个活化的细菌单克隆(含有α淀粉酶变体表达框)，接种到20ml液体培养基中(LB液体培养基以及0.25％的卡那霉素，LB液体培养基配方：蛋白胨1％，酵母提取物0.5％，NaCl1％)37℃培养到OD值在0.4～0.6之间，加入终浓度为0.8mmol/L的IPTG(异丙基-β-D-硫代半乳糖苷)，25℃，在往复摇床中180rpm振荡培养12小时。在超声破碎仪上用功率300w，超声破碎30分钟，冷冻离心取上清液做SDS-PAGE分析，结果得知该α淀粉酶变体分子量约为66kD。
实施例7:淀粉酶的纯化
1.将实施例中的上清液分批次转移到含2mLProteinIso Ni-NTAResin的12mL亲和层析柱中，每次都在冰上孵育1h，每隔10min上下颠倒混匀，直至上清液被Ni-NTA Resin完全亲和。收集流出液命名为流穿液。
2.用不同浓度的咪唑溶液分批次冲洗Ni柱。
3.用1.5mL 500mM的咪唑溶液冰上孵育Ni柱20min，每隔2min震荡混匀，收集洗脱液。
4.上述样品分别取20uL后，分别加入4μL6xSDS-PAGE Loading Buffer,煮沸6min，然后进SDS-PAGE凝胶电泳。
5.电泳结束后，用考马斯亮蓝染色液染色3h后再用脱色液脱色直至看到清晰的蛋白条带。
6.选择无杂蛋白的蛋白溶液加入透析袋中，将透析袋放入透析液中，于4℃搅拌透析8h。
实施例8:淀粉酶活力测定
淀粉酶活力单位定义：在pH7.0、60℃条件下，1min将淀粉水解生成1μmoL的葡萄糖所需的酶量为1个活力单位(U/mL)。
酶活测定如下操作：0.5ml2％可溶性淀粉溶液与0.5ml pH7.0磷酸缓冲液混合，先在60℃预热8min，加入1.0ml稀释后的酶液，准确反应10分钟，加入2mlDNS试剂(酒石酸钾钠18.2g，溶于50ml蒸馏水中，加热，于热溶液中依次加入3，5-二硝基水杨酸0.63g，NaOH.1g，苯酚0.5g，搅拌至溶，冷却后用蒸馏水定容至100ml)，并以1mlpH7.0磷酸缓冲液溶液、1ml淀粉溶液和2mlDNS试剂为空白，沸水浴5分钟，冷却至室温并定容至25ml于540nm波长下，迅速测定其吸光值，根据葡萄糖标准曲线，获得测试样品的酶活。
实施例9:淀粉酶的酶学性质
如表1所示，使用本发明α淀粉酶变体液化液和原淀粉酶液化液，突变体并没有降低酶活性且优于原淀粉酶液化液。结果见表1。
表1:不同底物浓度条件下，淀粉酶活力比较
表2：不同pH下，40℃保温2小时，淀粉酶活力比较
其次，我们测定了淀粉酶的耐酸性，同时进行不同pH条件下的淀粉酶活力。液化反应条件同上所述，pH分别为2.5、3.5、4.5和5.5，加酶量为50ul，以BLO-amy为对照，结果见表2。在pH4.5条件下，本发明α淀粉酶变体仍能正常液化，说明本发明α淀粉酶变体对低pH具有较强的耐受性，而原淀粉酶BLO-amy耐受低pH能力较弱。
表3:在pH4.5时，不同温度下保温2小时，淀粉酶活力比较
然后我们测定了两种淀粉酶的热稳性。在pH4.5条件下，分别在30℃、40℃、50℃、60℃、70℃、80℃、90℃、95℃和100℃保温2小时。结果见表3淀粉酶突变体BLM-amy仍具有较高的酶活性，而原淀粉酶BLO-amy耐受高温能力不超过80℃。
实施例8:淀粉酶在饲料中的应用
通过突变前和突变后淀粉酶酶解蛋鸡饲料，饲料配方如下表4，比较酶解饲料中还原糖释放量。设置两组实验，都为3因素3水平正交试验，另一组为突变淀粉酶实验组，优选温度41℃，pH2.5～2.8，进一步优选pH2.7，转速180rpm。
表4：蛋鸡饲料配方
| 原料Ingredients | 含量Content(kg) |
| 玉米Corn | 57.84 |
| 豆粕Soybean meal | 27.30 |
| 豆油Soybean oil | 1.86 |
| 石粉Limestone | 8.00 |
| 预混料Premix | 5.00 |
| 合计Total | 100.00 |
取不同质量的蛋鸡饲料溶于20ml模拟胃液中，并用2mol/L盐酸调到特定pH,在不同加酶量(100U,200U,400U)、不同酶解时间(4h，5h，6h)和固液比(固：粉碎的蛋鸡饲料；液：模拟胃液)(0.3g/ml，0.5g/ml，0.7g/ml)条件下，比较酶解效果(上清液还原糖的释放量)。结果如表5。该结果表明突变淀粉酶体酶解效益均高于原淀粉酶体。并且，在加酶量为200U,固液比为0.3g/ml，酶解时间为6小时，酶解效益较其他组高。
综上所述，依照本发明中的实验结果，该系列L型淀粉酶变体有较好的耐热性、耐酸性，良好的饲料酶解效果，可应用与饲料行业。
表5:体外饲料酶解效果
本发明的实施例除了应用了本领域内的技术方法外，更离不开发明主旨的指导。因此，本发明不仅限于所公开的具体实施例，更要覆盖本发明精神和范围内的修订条款，详见权利要求。
序列表
<110> 安徽农业大学
<120> 一种饲用α淀粉酶的突变改良方法及应用
<140> 202111216119.9
<141> 2021-10-19
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1980
<212> DNA
<213> 贝莱斯芽孢杆菌(B. velezensis)
<400> 1
atgtttgaaa aacgattcaa aacctcttta ctgccgttat tcgccggatt tttattgctg 60
tttcatttgg ttttgtcagg cccggcggct gcaaacgctg aaactgcaaa caaatcgaat 120
aaggtgaccg cgtcatcggt caaaaacggg accatccttc atgcatggaa ttggtcgttc 180
aatacgttaa cacaaaatat gaaagatatt cgtgatgcgg gctatgcagc cattcagacg 240
tctccgatta accaagtaaa ggaagggaac caaggagata aaagcatgag gaactggtac 300
tggctgtatc agccgacatc gtaccaaatc ggcaaccgtt acttaggcac tgaacaagaa 360
tttaaggaca tgtgtgcagc cgcggaaaag tatggcgtaa aagtcattgt cgatgcggtt 420
atcaatcata ctaccagcga ttatggcgcg atttctgacg agattaagcg tattccaaac 480
tggacccatg gaaacacaca aattaaaaat tggtcggatc gatgggacgt cactcaaaat 540
tccttgcttg ggctgtatga ttggaataca cagaatactg aggtgcaggc ctatctgaaa 600
cgtttcttgg aaagagcatt gaatgacgga gcagacggat tccgctatga tgccgccaag 660
catatagagc ttccggatga tgggaattac ggcagccagt tttggccgaa tatcacaaat 720
acatcggcgg agttccaata cggagaaatc ctgcaagaca gcgcgtccag agatactgct 780
tatgcgaatt atatgaatgt gacggcttct aactacgggc attccatcag atccgcttta 840
aagaaccgta atctgagtgt gtcgaatatc tcccgttatg catctgacgt gtctgcggac 900
aagttagtca catgggtgga atcacatgat acgtatgcca atgatgatga agagtccaca 960
tggatgagtg atgacgatat tcgtttaggc tgggcagtga ttggttcccg ctcaggaagc 1020
acgcctcttt tcttttccag acctgagggc ggaggaaatg gtgtaagatt tcccggaaaa 1080
agtcaaatag gagatcgcgg gagcgcctta tttaaagatc aggcgattac tgcggtcaat 1140
acatttcaca atgtaatggc cgggcagcct gaggaactct cgaatccgaa tgggaacaac 1200
caaatcttta tgaatcagcg cggctcaaaa ggcgttgtgc tggcaaatgc aggatcgtct 1260
tctgtcacca tcaatacttc aacgaaatta cctgacggca ggtatgataa tagggccggc 1320
gccggttcat ttcaagtagc gaacggcaaa ctgacaggta cgatcaatgc cagatcggcg 1380
gctgttcttt atcctgatga tattggaaat acgcctcatg tctttcttga gaattaccaa 1440
acgggggcag tccattcttt caatgatcag ctgacggtca ccctgcgtgc aaatgcgaaa 1500
acaacaaaag ccgtttacca aatcaataat gggcagcaga cagcatttaa ggatggagac 1560
cgactaacga tcgggaaagg agatccaatc ggcacgacat acaacatcaa attaaccgga 1620
acgaacggcg agggtccagc gagaacccaa gaatacacat ttgtcaaaaa agacccgtcc 1680
caaaccaaca tcattggcta tcaaaacccg gaccattggg gccaggtaaa tgcttatatc 1740
tataaacatg atggaggcag ggccatagaa ttaaccggat cgtggccggg gaaagcaatg 1800
actaagaatg caaatggaat gtacacgctg acgctgcctg cgaatacgga tacggccaat 1860
gccaaagtga tttttaacaa tggcagcgcc caagtgcccg gacagaacca gcccggcttt 1920
gattatgtgc agaatggttt gtataacaac tccggtttga atggttatct tccgcattaa 1980
<210> 2
<211> 659
<212> PRT
<213> 贝莱斯芽孢杆菌(B. velezensis)
<400> 2
Met Phe Glu Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly
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Phe Leu Leu Leu Phe His Leu Val Leu Ser Gly Pro Ala Ala Ala Asn
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Ala Glu Thr Ala Asn Lys Ser Asn Lys Val Thr Ala Ser Ser Val Lys
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Asn Gly Thr Ile Leu His Ala Trp Asn Trp Ser Phe Asn Thr Leu Thr
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Gln Asn Met Lys Asp Ile Arg Asp Ala Gly Tyr Ala Ala Ile Gln Thr
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Ser Pro Ile Asn Gln Val Lys Glu Gly Asn Gln Gly Asp Lys Ser Met
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Arg Asn Trp Tyr Trp Leu Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn
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Arg Tyr Leu Gly Thr Glu Gln Glu Phe Lys Asp Met Cys Ala Ala Ala
115 120 125
Glu Lys Tyr Gly Val Lys Val Ile Val Asp Ala Val Ile Asn His Thr
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Thr Ser Asp Tyr Gly Ala Ile Ser Asp Glu Ile Lys Arg Ile Pro Asn
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Trp Thr His Gly Asn Thr Gln Ile Lys Asn Trp Ser Asp Arg Trp Asp
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Val Thr Gln Asn Ser Leu Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn
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Thr Glu Val Gln Ala Tyr Leu Lys Arg Phe Leu Glu Arg Ala Leu Asn
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Asp Gly Ala Asp Gly Phe Arg Tyr Asp Ala Ala Lys His Ile Glu Leu
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Pro Asp Asp Gly Asn Tyr Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn
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Thr Ser Ala Glu Phe Gln Tyr Gly Glu Ile Leu Gln Asp Ser Ala Ser
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Arg Asp Thr Ala Tyr Ala Asn Tyr Met Asn Val Thr Ala Ser Asn Tyr
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Gly His Ser Ile Arg Ser Ala Leu Lys Asn Arg Asn Leu Ser Val Ser
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Asn Ile Ser Arg Tyr Ala Ser Asp Val Ser Ala Asp Lys Leu Val Thr
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Trp Val Glu Ser His Asp Thr Tyr Ala Asn Asp Asp Glu Glu Ser Thr
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Trp Met Ser Asp Asp Asp Ile Arg Leu Gly Trp Ala Val Ile Gly Ser
325 330 335
Arg Ser Gly Ser Thr Pro Leu Phe Phe Ser Arg Pro Glu Gly Gly Gly
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Asn Gly Val Arg Phe Pro Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser
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Ala Leu Phe Lys Asp Gln Ala Ile Thr Ala Val Asn Thr Phe His Asn
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Val Met Ala Gly Gln Pro Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn
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Gln Ile Phe Met Asn Gln Arg Gly Ser Lys Gly Val Val Leu Ala Asn
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Ala Gly Ser Ser Ser Val Thr Ile Asn Thr Ser Thr Lys Leu Pro Asp
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Gly Arg Tyr Asp Asn Arg Ala Gly Ala Gly Ser Phe Gln Val Ala Asn
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Gly Lys Leu Thr Gly Thr Ile Asn Ala Arg Ser Ala Ala Val Leu Tyr
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Pro Asp Asp Ile Gly Asn Thr Pro His Val Phe Leu Glu Asn Tyr Gln
465 470 475 480
Thr Gly Ala Val His Ser Phe Asn Asp Gln Leu Thr Val Thr Leu Arg
485 490 495
Ala Asn Ala Lys Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn Gly Gln
500 505 510
Gln Thr Ala Phe Lys Asp Gly Asp Arg Leu Thr Ile Gly Lys Gly Asp
515 520 525
Pro Ile Gly Thr Thr Tyr Asn Ile Lys Leu Thr Gly Thr Asn Gly Glu
530 535 540
Gly Pro Ala Arg Thr Gln Glu Tyr Thr Phe Val Lys Lys Asp Pro Ser
545 550 555 560
Gln Thr Asn Ile Ile Gly Tyr Gln Asn Pro Asp His Trp Gly Gln Val
565 570 575
Asn Ala Tyr Ile Tyr Lys His Asp Gly Gly Arg Ala Ile Glu Leu Thr
580 585 590
Gly Ser Trp Pro Gly Lys Ala Met Thr Lys Asn Ala Asn Gly Met Tyr
595 600 605
Thr Leu Thr Leu Pro Ala Asn Thr Asp Thr Ala Asn Ala Lys Val Ile
610 615 620
Phe Asn Asn Gly Ser Ala Gln Val Pro Gly Gln Asn Gln Pro Gly Phe
625 630 635 640
Asp Tyr Val Gln Asn Gly Leu Tyr Asn Asn Ser Gly Leu Asn Gly Tyr
645 650 655
Leu Pro His
<210> 3
<211> 1980
<212> DNA
<213> 贝莱斯芽孢杆菌(B. velezensis)
<400> 3
atgtttgaaa aacgattcaa aacctcttta ctgccgttat tcgccggatt tttattgctg 60
tttcatttgg ttttgtcagg cccggcggct gcaaacgctg aaactgcaaa caaatcgaat 120
aaggtgaccg cgtcatcggt caaaaacggg accatccttc atgcatggaa ttggtcgttc 180
aatacgttaa cacaaaatat gaaagatatt cgtgatgcgg gctatgcagc cattcagacg 240
tctccgatta accaagtaaa ggaagggaac caaggagata aaagcatgag gaactggtac 300
tggctgtatc agccgacatc gtaccaaatc ggcaaccgtt acttaggcac tgaacaagaa 360
tttaaggaca tgtgtgcagc cgcggaaaag tatggcgtaa aagtcattgt cgatgcggtt 420
atcaatcata ctaccagcga ttatggcgcg atttctgacg agattaagcg tattccaaac 480
tggacccatg gaaacacaca aattaaaaat tggtcggatc gatgggacgt cactcaaaat 540
tccttgcttg ggctgtatga ttggaataca cagaatactg aggtgcaggc ctatctgaaa 600
cgtttcttgg aaagagcatt gaatgacgga gcagacggat tccgctatga tgccgccaag 660
catatagagc ttccggatga tgggaattac ggcagccagt tttggccgaa tatcacaaat 720
acatcggcgg agttccaata cggagaaatc ctgcaagaca gcgcgtccag agatactgct 780
tatgcgaatt atatgaatgt gacggcttct aactacgggc attccatcag atccgcttta 840
aagaaccgta atctgagtgt gtcgaatatc tcccgttatg catctgacgt gtctgcggac 900
aagttagtca catgggtgga atcacatgat acgtatgcca atgatgatga agagtccaca 960
tggatgagtg atgacgatat tcgtttaggc tgggcagtga ttggttcccg ctcaggaagc 1020
acgcctcttt tcttttccag acctgagggc ggaggaaatg gtgtaagatt tcccggaaaa 1080
agtcaaatag gagatcgcgg gagcgcctta tttaaagatc aggcgattac tgcggtcaat 1140
acatttcaca atgtaatggc cgggcagcct gaggaactct cgaatccgaa tgggaacaac 1200
caaatcttta tgaatcagcg cggctcaaaa ggcgttgtgc tggcaaatgc aggatcgtct 1260
tctgtcacca tcaatacttc aacgaaatta cctgacggca ggtatgataa tagggccggc 1320
gccggttcat ttcaagtagc gaacggcaaa ctgacaggta cgatcaatgc cagatcggcg 1380
gctgttcttt atcctgatga tattggaaat acgcctcatg tctttcttga gaattaccaa 1440
acgggggcag tccattcttt caatgatcag ctgacggtca ccctgcgtgc aaatgcgaaa 1500
acaacaaaag ccgtttacca aatcaataat gggcagcaga cagcatttaa ggatggagac 1560
cgactaacga tcgggaaagg agatccaatc ggcacgacat acaacatcaa attaaccgga 1620
acgaacggcg agggtcaagc gagaacccaa gaatacacat ttgtcaaaaa agacccgtcc 1680
caaaccaaca tcattggcta tcaaaacccg gacgattggg gccaggtaaa tgcttatatc 1740
tataaacatg atggaggcag ggccatagaa ttaaccggat cgtggccggg gaaagcaatg 1800
actaagaatg caaatggaat gtacacgctg acgctgcctg agaatacgga tacggccaat 1860
gccgaagtga tttttaacaa tggcagcgcc caagtgcccg gacagaacca gcccggcttt 1920
gattatgtgc agaatggttt gtataacaac tccggtttga atggttatct tccgcattaa 1980
<210> 4
<211> 659
<212> PRT
<213> 贝莱斯芽孢杆菌(B. velezensis)
<400> 4
Met Phe Glu Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly
1 5 10 15
Phe Leu Leu Leu Phe His Leu Val Leu Ser Gly Pro Ala Ala Ala Asn
20 25 30
Ala Glu Thr Ala Asn Lys Ser Asn Lys Val Thr Ala Ser Ser Val Lys
35 40 45
Asn Gly Thr Ile Leu His Ala Trp Asn Trp Ser Phe Asn Thr Leu Thr
50 55 60
Gln Asn Met Lys Asp Ile Arg Asp Ala Gly Tyr Ala Ala Ile Gln Thr
65 70 75 80
Ser Pro Ile Asn Gln Val Lys Glu Gly Asn Gln Gly Asp Lys Ser Met
85 90 95
Arg Asn Trp Tyr Trp Leu Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn
100 105 110
Arg Tyr Leu Gly Thr Glu Gln Glu Phe Lys Asp Met Cys Ala Ala Ala
115 120 125
Glu Lys Tyr Gly Val Lys Val Ile Val Asp Ala Val Ile Asn His Thr
130 135 140
Thr Ser Asp Tyr Gly Ala Ile Ser Asp Glu Ile Lys Arg Ile Pro Asn
145 150 155 160
Trp Thr His Gly Asn Thr Gln Ile Lys Asn Trp Ser Asp Arg Trp Asp
165 170 175
Val Thr Gln Asn Ser Leu Leu Gly Leu Tyr Asp Trp Asn Thr Gln Asn
180 185 190
Thr Glu Val Gln Ala Tyr Leu Lys Arg Phe Leu Glu Arg Ala Leu Asn
195 200 205
Asp Gly Ala Asp Gly Phe Arg Tyr Asp Ala Ala Lys His Ile Glu Leu
210 215 220
Pro Asp Asp Gly Asn Tyr Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn
225 230 235 240
Thr Ser Ala Glu Phe Gln Tyr Gly Glu Ile Leu Gln Asp Ser Ala Ser
245 250 255
Arg Asp Thr Ala Tyr Ala Asn Tyr Met Asn Val Thr Ala Ser Asn Tyr
260 265 270
Gly His Ser Ile Arg Ser Ala Leu Lys Asn Arg Asn Leu Ser Val Ser
275 280 285
Asn Ile Ser Arg Tyr Ala Ser Asp Val Ser Ala Asp Lys Leu Val Thr
290 295 300
Trp Val Glu Ser His Asp Thr Tyr Ala Asn Asp Asp Glu Glu Ser Thr
305 310 315 320
Trp Met Ser Asp Asp Asp Ile Arg Leu Gly Trp Ala Val Ile Gly Ser
325 330 335
Arg Ser Gly Ser Thr Pro Leu Phe Phe Ser Arg Pro Glu Gly Gly Gly
340 345 350
Asn Gly Val Arg Phe Pro Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser
355 360 365
Ala Leu Phe Lys Asp Gln Ala Ile Thr Ala Val Asn Thr Phe His Asn
370 375 380
Val Met Ala Gly Gln Pro Glu Glu Leu Ser Asn Pro Asn Gly Asn Asn
385 390 395 400
Gln Ile Phe Met Asn Gln Arg Gly Ser Lys Gly Val Val Leu Ala Asn
405 410 415
Ala Gly Ser Ser Ser Val Thr Ile Asn Thr Ser Thr Lys Leu Pro Asp
420 425 430
Gly Arg Tyr Asp Asn Arg Ala Gly Ala Gly Ser Phe Gln Val Ala Asn
435 440 445
Gly Lys Leu Thr Gly Thr Ile Asn Ala Arg Ser Ala Ala Val Leu Tyr
450 455 460
Pro Asp Asp Ile Gly Asn Thr Pro His Val Phe Leu Glu Asn Tyr Gln
465 470 475 480
Thr Gly Ala Val His Ser Phe Asn Asp Gln Leu Thr Val Thr Leu Arg
485 490 495
Ala Asn Ala Lys Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn Gly Gln
500 505 510
Gln Thr Ala Phe Lys Asp Gly Asp Arg Leu Thr Ile Gly Lys Gly Asp
515 520 525
Pro Ile Gly Thr Thr Tyr Asn Ile Lys Leu Thr Gly Thr Asn Gly Glu
530 535 540
Gly Gln Ala Arg Thr Gln Glu Tyr Thr Phe Val Lys Lys Asp Pro Ser
545 550 555 560
Gln Thr Asn Ile Ile Gly Tyr Gln Asn Pro Asp Asp Trp Gly Gln Val
565 570 575
Asn Ala Tyr Ile Tyr Lys His Asp Gly Gly Arg Ala Ile Glu Leu Thr
580 585 590
Gly Ser Trp Pro Gly Lys Ala Met Thr Lys Asn Ala Asn Gly Met Tyr
595 600 605
Thr Leu Thr Leu Pro Glu Asn Thr Asp Thr Ala Asn Ala Glu Val Ile
610 615 620
Phe Asn Asn Gly Ser Ala Gln Val Pro Gly Gln Asn Gln Pro Gly Phe
625 630 635 640
Asp Tyr Val Gln Asn Gly Leu Tyr Asn Asn Ser Gly Leu Asn Gly Tyr
645 650 655
Leu Pro His
Claims (7)
1.一种α淀粉酶变体，其特征在于所述α淀粉酶变体氨基酸序列如序列表中 SEQ IDNO.4所示。
2.编码权利要求1所述的α淀粉酶变体的基因。
3.根据权利要求2所述的基因，其特征在于α淀粉酶变体核苷酸序列如序列表中SEQ IDNO.3所示。
4.一种用于表达权利要求1所述的α淀粉酶变体的重组子，其特征在于包含权利要求3所述的编码α淀粉酶变体的基因。
5.一种权利要求1所述的α淀粉酶变体的生产方法，其特征在于包括在适宜α淀粉酶变体表达的条件下对含有编码α淀粉酶变体基因序列的重组子进行培养，并从重组子或者其培养上清液中获得α淀粉酶变体。
6.权利要求1所述的α淀粉酶变体在水解多糖的α-1,4糖苷键中的应用。
7.根据权利要求6所述的应用，其特征在于所述的α淀粉酶变体在高温、低pH条件下水解多糖的α-1,4糖苷键中的应用，所述高温为40℃~90℃,低pH为2.5～5.5。
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