CN113462677A - 一种α亚基突变的腈水合酶突变体及其应用 - Google Patents
一种α亚基突变的腈水合酶突变体及其应用 Download PDFInfo
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- CN113462677A CN113462677A CN202110867862.4A CN202110867862A CN113462677A CN 113462677 A CN113462677 A CN 113462677A CN 202110867862 A CN202110867862 A CN 202110867862A CN 113462677 A CN113462677 A CN 113462677A
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Abstract
本发明公开了一种α亚基突变的腈水合酶突变体及其应用。本发明通过蛋白质分子改造，显著提高了源于博得特氏菌DSM 12804(Bordetella petrii DSM12804)的腈水合酶在基因工程菌E.coli BL21(DE3)中活性表达能力。以丙烯腈为底物，本发明的腈水合酶突变体的催化活力显著提高，单位LB摇瓶发酵液的酶活最高为突变体NHAB‑A2M，为初始型NHAB的1.7倍；突变体NHAB‑A14M的酶活提高了1.4倍，其热稳定性更是显著提高，在45℃条件下热处理1h后，残余酶活为78％，是野生型的4.8倍。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种α亚基突变的腈水合酶突变体及其应用。
背景技术
腈水合酶(Nitrile hydratase，NHase)是一类可催化腈类化合物水合生成酰胺类物质的生物催化剂。微生物生产的腈水合酶可以高效催化丙烯腈水合生成丙烯酰胺；丙烯酰胺聚合生成的聚丙烯酰胺作为降阻剂、絮凝剂和增稠剂在石油开采、水处理以及造纸等方面广泛应用。
生物酶催化法制备丙烯酰胺相较于传统的化学催化法，具有转化效率高、产物纯度高、反应条件温和以及环境友好等优点，有逐步取代化学法的趋势。日本三菱公司利用含有腈水合酶的玫瑰色红球菌J1(CN91101323.7)催化丙烯腈生产丙烯酰胺，年产量达20万吨以上。但红球菌是一种野生菌，其生长周期较长、生产效率较低，并且仅在10～30℃条件下稳定，因此在实际生产应用中还是有一定限制。目前，生物酶催化法生产丙烯酰胺的研究重点在于高产腈水合酶菌株的发现以及对腈水合酶本身性能的改造提高。腈水合酶在工业应用过程中普遍存在热稳定性差的问题，而腈类的水合过程属于放热反应，反应后期催化效率严重降低，致使整个生产过程效率不高；所以在工业催化过程中，需要引入冷却装置以及其他工序，但这又会造成能源浪费，增加生产成本。
通过构建具有腈水合酶催化活性的基因工程菌株，并对腈水合酶进行蛋白质工程改造可在一定程度上解决解决丙烯酰胺生产中的实际问题。基因工程菌适应性强，发酵周期短，有利于实现大规模培养和工业化生产，且可以有效地降低生产成本。专利文献(CN104498466)公开了一种来源于博得特氏菌DSM 12804(Bordetella petrii DSM12804)的腈水合酶(NHAB)基因序列，其对丙烯腈有较高的催化活力，但其酶活和热稳定性仍不能满足工业化。因此，选择NHAB进行蛋白质工程改造，可获得酶活和热稳定性均有提高的腈水合酶突变体，更好地应用于丙烯酰胺的大规模生产。
发明内容
本发明针对来源于博得特氏菌DSM 12804(Bordetella petrii DSM12804)的腈水合酶，对其催化活性中心所在的α亚基进行理性设计，提供了3个腈水合酶突变体，不仅提高了其表达酶活，并且热稳定性也大大提高。
一种腈水合酶的α亚基突变体，由野生型α亚基序列经突变所得，野生型α亚基的氨基酸序列如SEQ ID NO.1所示，突变位点为以下任一组：
(1)两点突变：第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸；
(2)三点突变：第71位丙氨酸突变为天冬氨酸、第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸；
(3)十四点突变：第30位丝氨酸突变为苏氨酸、第46位缬氨酸突变为异亮氨酸、第71位丙氨酸突变为天冬氨酸、第74位丙氨酸突变为天冬氨酸、第78位丙氨酸突变为精氨酸、第79位天冬酰胺突变为天冬氨酸、第81位丝氨酸突变为苏氨酸、第92位缬氨酸突变为精氨酸、第96位天冬氨酸突变为组氨酸、第97位苏氨酸突变为甲硫氨酸、第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸、第133位丙氨酸突变为脯氨酸、第179位丙氨酸突变为谷氨酸。
本发明又公开了一种α亚基突变的腈水合酶突变体，包括α亚基和β亚基，其中，所述α亚基为所述的α亚基突变体。β亚基的氨基酸序列如SEQ ID NO.2所示。
本发明又公开了一种用于表达所述腈水合酶突变体的基因簇，所述基因簇包括分别编码α亚基突变体、β亚基以及调控蛋白p14K的基因序列。
所述的基因簇，编码α亚基突变体的基因序列如SEQ ID NO.7～9任一所示，编码β亚基的基因序列如SEQ ID NO.10所示，编码调控蛋白p14K的基因序列如SEQ ID NO.11。
所述的基因簇，编码α亚基突变体的基因序列与编码β亚基的基因序列之间具有第一连接序列，第一连接序列的序列为：GGAGATCATC；编码β亚基的基因序列与编码调控蛋白p14K的基因序列之间具有第二连接序列，第二连接序列的序列为：TC。α亚基、β亚基及调控蛋白p14K对应的编码基因均是独立表达的，各自均有起始密码子和终止密码子，而第一连接序列和第二连接序列为非编码序列，属于RBS结合位点。其中，α亚基两点突变体腈水合酶为NHAB-A2M，α亚基三点突变体腈水合酶为NHAB-A3M，α亚基十四点突变体腈水合酶为NHAB-A14M。
本发明又提供了包含所述基因簇的重组表达载体。
本发明又提供了包含所述重组表达载体的基因工程菌。
本发明还提供了所述的腈水合酶突变体在催化丙烯腈生产丙烯酰胺中的应用。
本发明还提供了所述的基因工程菌在催化丙烯腈生产丙烯酰胺中的应用。
本发明的有益效果在于：通过蛋白质分子改造，显著提高了源于博得特氏菌DSM12804(Bordetella petrii DSM12804)的腈水合酶在基因工程菌E.coli BL21(DE3)中活性表达能力。以丙烯腈为底物，本发明的腈水合酶突变体的催化活力显著提高，单位LB摇瓶发酵液的酶活最高为突变体NHAB-A2M，为野生型NHAB的1.7倍；突变体NHAB-A14M的酶活提高了1.4倍，其热稳定性更是显著提高，在45℃条件下热处理1h后，残余酶活为78％，是野生型的4.8倍。
附图说明
图1为腈水合酶NHAB催化丙烯腈生成丙烯酰胺的气相检测图谱。
图2为腈水合酶野生型NHAB和突变体的初始相对酶活以及45℃水浴热处理1h后残余酶活的结果图。
具体实施方式
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参见J.萨姆布鲁克等编的《分子克隆实验指南》。
本发明所涉及带有腈水合酶基因的重组大肠杆菌，所用载体为pET-30a(+)，所用宿主为大肠杆菌E.coli BL21(DE3)。制备化转感受态细胞的试剂盒购自TAKARA公司。
下游催化工艺所用试剂：丙烯腈购自国药集团化学试剂有限公司；乙酰胺购自上海麦克林生化科技有限公司；丙烯酰胺购自阿拉丁试剂有限公司；其他常用试剂购自国药集团化学试剂有限公司。在本申请文本中所使用的氨基酸三字母或单字母表达方式，采用IUPAC规定的氨基酸代码(Eur.J.Biochem.，138：9-37，1984)。
酶活的定义(U/mL)：每分钟催化底物丙烯腈生成1μmol丙烯酰胺所需的酶量。
腈水合酶酶活标准检测体系：适量的酶液、50g/L底物，总体系为5mL，反应介质为pH 7.5的0.25M磷酸盐缓冲液。28℃反应5min，气相检测产物丙烯酰胺的生成量。
丙烯酰胺的检测：气相色谱法分析测定，载气为氮气，流速0.24mL/min，柱炉温度210℃，检测温度250℃，气化温度250℃。色谱柱为Propack Q填充柱。
热稳定性的测定：将初始型NHAB和突变体均重悬于pH＝7.5的PB缓冲液中，成为OD600＝2的菌液，置于45℃水浴热处理1h后测定残余酶活，未经热处理的酶的酶活定义为100％，得到热稳定性结果。
实施例1：构建初始型NHAB及突变体重组菌
来源于博得特氏菌DSM 12804(Bordetella petrii DSM12804)的野生型腈水合酶NHAB，由α亚基、β亚基以及调控蛋白p14K组成，其氨基酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。针对其催化活性中心所在的α亚基进行蛋白质工程改造：将该野生型酶的α亚基第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸得到突变体NHAB-A2M，其α亚基氨基酸序列如SEQ ID NO.4所示。
进一步地，将该野生型酶的α亚基的第71位丙氨酸突变为天冬氨酸、第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸得到突变体NHAB-A3M，其α亚基氨基酸序列如SEQ ID NO.5所示。
更进一步地，将该野生型酶的α亚基的第30位丝氨酸突变为苏氨酸、第46位缬氨酸突变为异亮氨酸、第71位丙氨酸突变为天冬氨酸、第74位丙氨酸突变为天冬氨酸、第78位丙氨酸突变为精氨酸、第79位天冬酰胺突变为天冬氨酸、第81位丝氨酸突变为苏氨酸、第92位缬氨酸突变为精氨酸、第96位天冬氨酸突变为组氨酸、第97位苏氨酸突变为甲硫氨酸、第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸、第133位丙氨酸突变为脯氨酸、第179位丙氨酸突变为谷氨酸得到突变体NHAB-A14M，其α亚基氨基酸序列如SEQ IDNO.6所示。
然后，将分别编码SEQ ID NO.4、5、6的基因序列SEQ ID NO.7、SEQ ID NO.8、SEQID NO.9，分别与来源于博得特氏菌DSM 12804(Bordetella petrii DSM12804)的腈水合酶的野生型β亚基基因(SEQ ID NO.10)以及调控蛋白p14k基因(SEQ ID NO.11)组合(用于在同一载体上)，得到腈水合酶基因簇α-β-p14k，其中，α亚基与β亚基之间的连接序列为GGAGATCATC，β亚基与调控蛋白p14k之间的连接序列为TC；将该基因簇构建入表达载体pET-30a(+)上，转入大肠杆菌基因工程菌E.coli BL21(DE3)中，获得表达NHAB-A2M菌株E.coliBL21(DE3)-pET-30a(+)-NHAB-A2M，表达NHAB-A3M菌株E.coli BL21(DE3)-pET-30a(+)-NHAB-A3M，表达NHAB-A14M菌株E.coli BL21(DE3)-pET-30a(+)-NHAB-A14M。
委托北京擎科新业生物技术有限公司提供密码子优化和基因合成服务，将来源于Bordetella petrii DSM12804的腈水合酶NHAB野生型及突变体的基因克隆于pET-30a(+)质粒上，将目的基因置于酶切位点EcoR I和Hind III之间。获得pET-30a(+)-NHAB和pET-30a(+)-NHAB-A2M、pET-30a(+)-NHAB-A3M、pET-30a(+)-NHAB-A14M重组质粒，并将其分别转化进入基因工程菌E.coliBL21(DE3)。获得表达NHAB-A2M菌株E.coli BL21(DE3)-pET-30a(+)-NHAB-A2M，表达NHAB-A3M菌株E.coli BL21(DE3)-pET-30a(+)-NHAB-A3M，表达NHAB-A14M菌株E.coli BL21(DE3)-pET-30a(+)-NHAB-A14M。
实施例2：微生物培养与酶活测定
(1)微生物的培养
LB液体培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl10g/L，用去离子水溶解后定容，121℃灭菌20min，待用。LB固体培养基(平板培养皿)：在LB液体培养基基础上加入20g/L琼脂粉，121℃灭菌、降温后导入培养皿，制作成平板。
将含有相关基因的工程菌E.coli BL21(DE3)(实施例1中获得)接种至含50μμg/mL卡那霉素的5mL LB液体培养基中，37℃震荡培养12h。转接至500mL同样含50μg/mL Kan的新鲜LB液体培养基中，37℃震荡培养至OD600达到0.8左右时，加入IPTG至其浓度为0.5mM，加入氯化钴至其终浓度0.4mM，18℃下诱导培养16-18h。培养结束后，将培养液4000rpm离心10min，弃上清，收集菌体细胞，放到-70℃超低温冰箱中保存，待用。
(2)酶活力的测定
将培养结束后收集到的菌体细胞，用0.25M PB缓冲液(pH 7.5)洗涤菌体两次。之后将菌体重悬于2倍发酵液体积的0.25M PB缓冲液(pH 7.5)中，该细胞悬液用于后续测定。
酶活定义：1961年国际酶学会议规定，1个酶活力单位是指在特定条件下，在1分钟内能转化1微摩尔底物的酶量，或是转化底物中1微摩尔的有关基团的酶量。
腈水合酶酶活测定体系：总体系为5mL，反应介质为pH 7.5的0.25M磷酸盐缓冲液，底物为4320μL 50g/L丙烯腈，加入430μL OD600＝2的菌液，28℃下反应5min后，加入250μL4mol/L HCl终止反应。
取反应液，12000rpm离心2min，吸取上清加入等量内标(20g/L乙酰胺溶液)，使用气相内标法测定产物丙烯酰胺的生成量。气相分析图谱如图1所示。图1为腈水合酶NHAB催化丙烯腈生成丙烯酰胺的气相检测图谱，出峰顺序依次是底物、内标物和产物，其中底物丙烯腈的保留时间为1.2min，内标乙酰胺的保留时间为4.4min，产物丙烯酰胺保留时间为7.7min。
野生型NHAB的发酵液粗酶活定义为100％，突变体NHAB-A2M的相较于野生型提高了1.7倍，是三个突变体中最优；而NHAB-A14M的粗酶活提高了1.4倍。
实施例3：NHAB野生型及突变体的热稳定性测定
酶的热稳定性表征：酶液分别置于45℃热水浴处理60min，然后利用酶活检测体系测定残余酶活，未经过处理的酶的酶活定义为100％。NHAB野生型在45℃下60min后残余酶活仅为16％，热稳定性最好的是突变体NHAB-A14M，在45℃下60min残余酶活为76％，是野生型的4.75倍。野生型NHAB和所有突变体的热稳定性如图2所示。图2为腈水合酶野生型NHAB和突变体的初始相对酶活以及45℃水浴热处理1h后残余酶活的结果图，其中，实心柱形表示初始相对酶活，以NHAB野生型为100％；条纹柱形表示45℃，1h热处理后的残余酶活。
实施例4：NHAB-A2M催化丙烯腈生成丙烯酰胺
取实施例2构建的基因工程菌E.coli BL21(DE3)-pET-30a(+)-NHAB-A2M的发酵液，4000rpm，10min离心收集菌体，称取9.2g湿菌体用200mL0.25M磷酸盐缓冲液(pH7.5)重悬，20℃进行水合反应，前2h以0.22g/min的流速滴加丙烯腈，后2h以0.18g/min的流速。停止滴加，共反应8h。之后用气相色谱检测反应体系中丙烯腈、丙烯酰胺和丙烯酸的含量。底物转化率大于89％，丙烯酰胺浓度为285g/L，在反应体系中无丙烯酸的生成。
实施例5：NHAB-A3M催化丙烯腈生成丙烯酰胺
取实施例2构建的基因工程菌E.coli BL21(DE3)-pET-30a(+)-NHAB-A3M的发酵液，4000rpm，10min离心收集菌体，称取9.2g湿菌体用200mL 0.25M磷酸盐缓冲液(pH 7.5)重悬，20℃进行水合反应，前2h以0.22g/min的流速滴加丙烯腈，后2h以0.18g/min的流速滴加。停止滴加，共反应8h。之后用气相色谱检测反应体系中丙烯腈、丙烯酰胺和丙烯酸的含量。底物转化率大于87％，丙烯酰胺浓度为280g/L，在反应体系中无丙烯酸的生成。
实施例6：NHAB-A14M催化丙烯腈生成丙烯酰胺
取实施例2构建的基因工程菌E.coli BL21(DE3)-pET-30a(+)-NHAB-A14M的发酵液，4000rpm，10min离心收集菌体，称取9.2g湿菌体用200mL 0.25M磷酸盐缓冲液(pH 7.5)重悬，20℃进行水合反应，前2h以0.22g/min的流速滴加丙烯腈，后2h以0.18g/min的流速滴加。停止滴加，共反应8h。之后用气相色谱检测反应体系中丙烯腈、丙烯酰胺和丙烯酸的含量。底物转化率大于90％，丙烯酰胺浓度为290g/L，在反应体系中无丙烯酸的生成。
对比例1：NHAB催化丙烯腈生成丙烯酰胺
取实施例2构建的基因工程菌E.coli BL21(DE3)-pET-30a(+)-NHAB的发酵液，4000rpm，10min离心收集菌体，称取9.2g湿菌体用200mL 0.25M磷酸盐缓冲液(pH 7.5)重悬，20℃进行水合反应，前2h以0.22g/min的流速滴加丙烯腈，后2h以0.18g/min的流速滴加。停止滴加，共反应8h。之后用气相色谱检测反应体系中丙烯腈、丙烯酰胺和丙烯酸的含量。底物转化率大于78％，丙烯酰胺浓度为250g/L，在反应体系中无丙烯酸的生成。
序列表
<110> 浙江大学杭州国际科创中心
<120> 一种α亚基突变的腈水合酶突变体及其应用
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Pro Pro Glu Asp Ile Ala Leu Arg Val Lys Ala Leu Glu Ser Leu Leu
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Val Glu Lys Gly Leu Val Asp Pro Ala Ala Met Asp Ala Val Val Gln
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Thr Tyr Glu His Lys Val Gly Pro Arg Asn Gly Ala Lys Val Val Ala
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Lys Ala Trp Val Asp Pro Ala Tyr Lys Ala Arg Leu Leu Ala Asn Gly
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Thr Val Ile Leu Glu Asn Thr Pro Ala Val His Asn Val Phe Val Cys
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Gly Val Leu Ala Glu Phe Gly Leu Val Ile Pro Thr Asn Lys Glu Ile
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Arg Val Trp Asp Thr Thr Ala Glu Leu Arg Tyr Met Val Leu Pro Glu
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Val Thr Arg Asp Ser Met Ile Gly Thr Gly Leu Pro Thr Gln Pro Lys
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Met Asn Gly Ile His Asp Thr Gly Gly Ala His Gly Tyr Gly Pro Val
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Tyr Arg Glu Pro Asn Glu Pro Ile Leu His Gly Glu Trp Glu Gly Arg
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Val Leu Ala Leu Phe Pro Ala Leu Phe Ala Asn Gly Asn Phe Asn Ile
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Asp Glu Phe Arg His Gly Ile Glu Arg Met Asn Pro Ile Asp Tyr Leu
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Lys Gly Thr Tyr Tyr Glu His Trp Ile His Ser Ile Glu Thr Leu Leu
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Val Glu Lys Gly Val Leu Thr Ala Thr Glu Leu Ala Thr Gly Lys Ala
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Asp Leu Trp Asp Asp Tyr Leu Glu Pro Ala
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Lys His Glu Pro Gln Arg Ser Pro Ile Lys Val Val Ala Ala Met Ala
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<212> PRT
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Val Glu Lys Gly Leu Val Asp Pro Ala Ala Met Asp Ala Val Val Gln
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Lys Ala Trp Val Asp Pro Ala Tyr Lys Ala Arg Leu Leu Ala Asn Gly
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Met Gly Gln Ser His Thr His Asp His His His Asp Gly Tyr Gln Ala
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Pro Pro Glu Asp Ile Ala Leu Arg Val Lys Ala Leu Glu Ser Leu Leu
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Val Glu Lys Gly Leu Val Asp Pro Ala Ala Met Asp Ala Val Val Gln
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Thr Tyr Glu His Lys Val Gly Pro Arg Asn Gly Ala Lys Val Val Ala
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Ser Ala Gly Ile Ala Glu Leu Gly Phe Ser Gly Val Gln Gly Glu Asp
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Thr Val Ile Leu Glu Asn Thr Pro Asp Val His Asn Val Phe Val Cys
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Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Ala
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Trp Tyr Lys Ala Ala Pro Tyr Arg Ser Arg Met Val Ser Asp Pro Arg
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Gly Val Leu Ala Glu Phe Gly Leu Val Ile Pro Thr Asn Lys Glu Ile
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Arg Pro Ala Gly Thr Glu Gly Tyr Ser Glu Glu Gln Leu Ala Glu Leu
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Val Thr Arg Asp Ser Met Ile Gly Thr Gly Leu Pro Thr Gln Pro Lys
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Pro Ser His
210
<210> 6
<211> 211
<212> PRT
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Met Gly Gln Ser His Thr His Asp His His His Asp Gly Tyr Gln Ala
1 5 10 15
Pro Pro Glu Asp Ile Ala Leu Arg Val Lys Ala Leu Glu Thr Leu Leu
20 25 30
Val Glu Lys Gly Leu Val Asp Pro Ala Ala Met Asp Ala Ile Val Gln
35 40 45
Thr Tyr Glu His Lys Val Gly Pro Arg Asn Gly Ala Lys Val Val Ala
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Lys Ala Trp Val Asp Pro Asp Tyr Lys Asp Arg Leu Leu Arg Asp Gly
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Thr Ala Gly Ile Ala Glu Leu Gly Phe Ser Gly Arg Gln Gly Glu His
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Met Val Ile Leu Glu Asn Thr Pro Asp Val His Asn Val Phe Val Cys
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Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Ala
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Trp Tyr Lys Ala Pro Pro Tyr Arg Ser Arg Met Val Ser Asp Pro Arg
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Gly Val Leu Ala Glu Phe Gly Leu Val Ile Pro Thr Asn Lys Glu Ile
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Arg Val Trp Asp Thr Thr Ala Glu Leu Arg Tyr Met Val Leu Pro Glu
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<210> 7
<211> 636
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggggcaat cacacacaca cgaccaccat cacgacgggt accaggcacc gcctgaagac 60
attgcgctgc gggtgaaggc cttggagtct ctgctcgtcg agaaaggttt ggtcgacccg 120
gcggccatgg acgctgtggt ccaaacctat gaacacaagg tgggccctcg gaacggcgcc 180
aaggttgttg ccaaggcctg ggtggacccg gcatacaagg cgcgcttgct ggcgaatggc 240
agcgctggca ttgccgaact gggcttctct ggagtgcagg gagaagacac agtcattctg 300
gaaaacaccc ccgacgtgca caacgtcttc gtctgcaccc tgtgctcttg ctacccatgg 360
ccggtgctgg gcttgccgcc ggcctggtac aaggccgcac cctaccggtc gcgcatggtg 420
agcgacccgc gtggggtcct ggcggagttc ggtttggtga tccccaccaa caaggaaatc 480
cgcgtctggg acaccacagc cgaattgcgc tacatggtgc tgccggaaag gcccgcagga 540
accgaaggct acagcgaaga acaactggcc gaactcgtca cccgcgattc gatgatcggc 600
actggcctgc ccacccaacc caaaccttcc cactaa 636
<210> 8
<211> 636
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggggcaat cacacacaca cgaccaccat cacgacgggt accaggcacc gcctgaagac 60
attgcgctgc gggtgaaggc cttggagtct ctgctcgtcg agaaaggttt ggtcgacccg 120
gcggccatgg acgctgtggt ccaaacctat gaacacaagg tgggccctcg gaacggcgcc 180
aaggttgttg ccaaggcctg ggtggacccg gactacaagg cgcgcttgct ggcgaatggc 240
agcgctggca ttgccgaact gggcttctct ggagtgcagg gagaagacac agtcattctg 300
gaaaacaccc ccgacgtgca caacgtcttc gtctgcaccc tgtgctcttg ctacccatgg 360
ccggtgctgg gcttgccgcc ggcctggtac aaggccgcac cctaccggtc gcgcatggtg 420
agcgacccgc gtggggtcct ggcggagttc ggtttggtga tccccaccaa caaggaaatc 480
cgcgtctggg acaccacagc cgaattgcgc tacatggtgc tgccggaaag gcccgcagga 540
accgaaggct acagcgaaga acaactggcc gaactcgtca cccgcgattc gatgatcggc 600
actggcctgc ccacccaacc caaaccttcc cactaa 636
<210> 9
<211> 636
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggggcaat cacacacaca cgaccaccat cacgacgggt accaggcacc gcctgaagac 60
attgcgctgc gggtgaaggc cttggagacc ctgctcgtcg agaaaggttt ggtcgacccg 120
gcggccatgg acgctatcgt ccaaacctat gaacacaagg tgggccctcg gaacggcgcc 180
aaggttgttg ccaaggcctg ggtggacccg gactacaagg accgcttgct gcgcgacggc 240
accgctggca ttgccgaact gggcttctct ggacgccagg gagaacacat ggtcattctg 300
gaaaacaccc ccgacgtgca caacgtcttc gtctgcaccc tgtgctcttg ctacccatgg 360
ccggtgctgg gcttgccgcc ggcctggtac aaggccccgc cctaccggtc gcgcatggtg 420
agcgacccgc gtggggtcct ggcggagttc ggtttggtga tccccaccaa caaggaaatc 480
cgcgtctggg acaccacagc cgaattgcgc tacatggtgc tgccggaaag gcccgaagga 540
accgaaggct acagcgaaga acaactggcc gaactcgtca cccgcgattc gatgatcggc 600
actggcctgc ccacccaacc caaaccttcc cactaa 636
<210> 10
<211> 657
<212> DNA
<213> 博得特氏菌(Bordetella petrii)
<400> 10
atgaacggca ttcacgacac tggcggagca catggttatg gcccggttta cagggagccg 60
aatgagccca tccttcatgg cgagtgggag ggtcgggtcc tggcattgtt tccggcgctt 120
ttcgcaaacg gcaacttcaa catcgatgag tttcgacacg gcatcgagcg catgaacccc 180
atcgactacc tgaagggaac ctactacgaa cactggatcc attccatcga aaccttgctg 240
gtcgaaaagg gtgtgctcac ggcaacggaa ctcgcgaccg gcaaggcatc tggcaagaca 300
gcgacaccgg tgctgacgcc ggtcatggtg gacggactgc tcagtaacgg agcttctgcc 360
gcccgcaagg agggggtgca ggcgcggttc gctgtgggcg acaaggttcg cgtcctcaac 420
aagcacccgg tgggccatac ccgcatgccg cgctacacgc ggggcaaagt ggggacagtg 480
gtcatcgacc atggtgtgtt cgtgacgccg gacaccgcgg cacacggaaa gggcgagcac 540
ccccagcacg tttacaccgt gagtttcacg tcggtcgaac tgtgggggca agacgcttcc 600
tcgccgaagg acacgattcg cgtcgacttg tgggatgact acctggagcc agcgtga 657
<210> 11
<211> 435
<212> DNA
<213> 博得特氏菌(Bordetella petrii)
<400> 11
atgaaagacg aacggtttcc attgccagag ggttcgctga aggacctcga tggccctgtg 60
tttgacgagc cttggcagtc ccaggcgttt gccttggtgg tcagcatgca caaggccggt 120
ctctttcagt ggaaagactg ggccgagacc ttcaccgccg aaatcgacgc ttccccggct 180
ctgcccggcg aaagcgtcaa cgacacctac taccggcaat gggtgtcggc gctggaaaag 240
ttggtggcgt cgctggggct tgtgacgggt ggagacgtca actcgcgcgc acaggagtgg 300
aaacaggccc acctcaacac cccacatggg cacccgatcc tgctggccca tgcgctttgc 360
ccgccagcga tcgaccccaa gcacaagcac gagccacaac gctcaccgat caaggtcgtt 420
gccgcaatgg cttga 435
<210> 12
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggagatcatc 10
Claims (10)
1.一种腈水合酶的α亚基突变体，其特征在于，由野生型α亚基序列经突变所得，野生型α亚基的氨基酸序列如SEQ ID NO.1所示，突变位点为以下任一组：
(1)第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸；
(2)第71位丙氨酸突变为天冬氨酸、第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸；
(3)第30位丝氨酸突变为苏氨酸、第46位缬氨酸突变为异亮氨酸、第71位丙氨酸突变为天冬氨酸、第74位丙氨酸突变为天冬氨酸、第78位丙氨酸突变为精氨酸、第79位天冬酰胺突变为天冬氨酸、第81位丝氨酸突变为苏氨酸、第92位缬氨酸突变为精氨酸、第96位天冬氨酸突变为组氨酸、第97位苏氨酸突变为甲硫氨酸、第105位丙氨酸突变为天冬氨酸、第122位丝氨酸突变为缬氨酸、第133位丙氨酸突变为脯氨酸、第179位丙氨酸突变为谷氨酸。
2.一种α亚基突变的腈水合酶突变体，其特征在于，包括α亚基和β亚基，其中，所述α亚基为权利要求1所述的α亚基突变体。
3.如权利要求2所述的腈水合酶突变体，其特征在于，β亚基的氨基酸序列如SEQ IDNO.2所示。
4.一种用于表达权利要求2所述腈水合酶突变体的基因簇，其特征在于，所述基因簇包括分别编码α亚基突变体、β亚基以及调控蛋白p14K的基因序列。
5.如权利要求4所述的基因簇，其特征在于，编码α亚基突变体的基因序列如SEQ IDNO.7～9任一所示，编码β亚基的基因序列如SEQ ID NO.10所示，编码调控蛋白p14K的基因序列如SEQ ID NO.11。
6.如权利要求5所述的基因簇，其特征在于，编码α亚基突变体的基因序列与编码β亚基的基因序列之间具有第一连接序列，第一连接序列的序列为：GGAGATCATC；编码β亚基的基因序列与编码调控蛋白p14K的基因序列之间具有第二连接序列，第二连接序列的序列为：TC。
7.包含权利要求4～6任一所述基因簇的重组表达载体。
8.包含权利要求7所述重组表达载体的基因工程菌。
9.如权利要求3所述的腈水合酶突变体在催化丙烯腈生产丙烯酰胺中的应用。
10.如权利要求8所述的基因工程菌在催化丙烯腈生产丙烯酰胺中的应用。
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