CN117660418A - 一种酶活性提高的芒果几丁质酶突变体Chitinase及其应用 - Google Patents
一种酶活性提高的芒果几丁质酶突变体Chitinase及其应用 Download PDFInfo
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Abstract
本发明公开了一种酶活提高的几丁质酶突变体Chitinase及其应用。几丁质酶的突变体chitinaseK172A,K173A，其氨基酸序列如SEQ ID NO.3所示。本发明所述突变体chitinaseK172A,K173A是对序列如SEQ ID NO.2所示的氨基酸序列进行定点突变，将第172和173位的Lys突变成Ala，从而提高几丁质酶的活性。本发明所得到的几丁质酶突变体chitinaseK172A,K173A可以作为一种可以显著提高酶催化活力的突变体材料，为几丁质酶的进一步研究提供参考。
Description
技术领域：
本发明属于生物技术领域，具体涉及一种酶活性提高的几丁质酶突变体chitinaseK172A,K173A及其应用。
背景技术：
几丁质酶要存在于虾、蟹、昆虫等甲壳类动物的外壳与软体动物的器官(例如乌贼的软骨)，以及真菌类的细胞壁中。而几丁质酶可催化几丁质水解，具有抵御真菌侵染的作用，成为抗真菌病害的研究热点。几丁质酶降解几丁质得到的中间产物被广泛应用于医药、食品加工、化妆品、饲料和造纸等行业，尤其是在农作物病虫害的生物防治中有显著的效果。
发明内容：
本发明的第一个目的是提供几丁质酶的突变体chitinaseK172A,K173A，其氨基酸序列如SEQ ID NO.3所示。
本发明的第二个目的是提供编码上述突变体chitinaseK172A,K173A的基因。
本发明的第三个目的是提供一种含有编码突变体chitinaseK172A,K173A的基因的重组表达质粒。
优选，所述的表达质粒是表达载体pMAL-c2x。
本发明的第四个目的是提供一种含有上述重组表达质粒的宿主表达细胞。所述的宿主表达细胞是大肠杆菌。
本发明的第五个目的是突变体chitinaseK172A,K173A在催化几丁质水解反应中的应用。
本发明的第六个目的是提供一种获得突变体chitinaseK172A,K173A的编码基因的方法，其包括以下步骤：
(1)以chitinase(XP_044463598.1)基因为模板，以SEQ ID NO.4和SEQ ID NO.5所示序列为引物进行PCR，得第一PCR产物，所述的chitinase(XP_044463598.1)基因的核苷酸序列如SEQ ID NO.1所示；
(2)以chitinase(XP_044463598.1)基因为模板，以SEQ ID NO.6和SEQ ID NO.7所示序列为引物进行PCR，得第二PCR产物，所述的chitinase(XP_044463598.1)基因的核苷酸序列如SEQ ID NO.1所示；
(3)以第一和第二PCR产物(1:1)为模板，以SEQ ID NO.4和SEQ ID NO.7所示序列为引物进行PCR，得含酶切位点的突变体chitinaseK172A,K173A的编码基因。本发明对所述PCR的体系并没有特殊限定，利用本领域的常规PCR体系即可。
目前对于几丁质酶通过定点突变提高酶催化活力尚未有相关报道，本发明通过定点突变将第172位和173位的赖氨酸(Lys)突变成丙氨酸(Ala)，获得几丁质酶的突变体chitinaseK172A,K173A，通过原核表达和蛋白纯化得到几丁质酶及其突变体chitinaseK172A ,K173A蛋白，通过酶活检测发现，突变体chitinaseK172A,K173A酶活性明显提高。本发明可以为生物化学及分子生物学方面针对几丁质酶的进一步研究提供技术参考。
附图说明：
图1是本发明实施例中MiChitinase-MBP及其突变体MiChitinaseK172A,K173A-MBP纯化蛋白SDS-PAGE；
图2是本发明实施例中96孔酶标仪测定酶活对比图。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
本发明的几丁质酶的突变体chitinaseK172A,K173A，其氨基酸序列如SEQ ID NO.3所示。本发明所述突变体chitinaseK172A,K173A是将Michitinase氨基酸序列中第172位和173位的Lys突变成Ala；所述Michitinase的氨基酸序列如SEQ ID NO.2所示。本发明对所述突变的方法并没有具体限定，优选为点突变。本发明所述Michitinase优选来源于芒果，基因序列如SEQ ID NO.1所示，共837个碱基序列，基因编码的蛋白中含有278个氨基酸，在NCBI中对应的蛋白编号是XP_044463598.1。
下面详述本发明构建几丁质酶的突变体chitinaseK172A,K173A的方法和活性试验。
实施例1
几丁质酶的突变体chitinaseK172A,K173A基因是通过PCR方式获得，所述PCR用引物包括含酶切位点的引物对和含突变位点的引物；所述含酶切位点的引物对的序列如SEQ IDNO.4和7所示；所述含突变位点的引物对的序列如SEQ IDNO.5和6所示(突变位点氨基酸加粗)。
1、样品：所选用的芒果品种是桂七
2、Chitinase基因的克隆
通过华越洋RNA提取试剂盒对芒果果实中总RNA进行提取，将提取的总RNA按照试剂盒PrimeScriptTM RT Master Mix(Perfect Real Time，RR036A，TaKara)提供的方法将RN A反转录成cDNA，反应体系：500ng RNA、2μL 5×Mix、RNase Free water补齐至10μL反应体系，混匀，然后PCR仪中进行以下反应程序：37℃，15min，85℃，5s。根据NCBI中Chitinase基因序列设计含EcoRI和BamHI酶切位点的引物以及突变位点的引物，引物序列A和 所示；
以芒果cDNA为模板，先分别用含酶切位点的上游引物 与中间突变下游引物进行PCR得到产物1，然后以芒果cDNA为模板，先分别用含酶切位点的下游引物/>与中间突变上游引物 进行PCR得到产物2；将产物1和产物2等比例混合为模板，用含酶切位点基因的上游引物 与含突变位点的下游引物/>进行PCR，得到含酶切位点的突变体chitinaseK172A,K173APCR产物。
以芒果cDNA为模板，用含酶切位点的上游引物 和下游引物/>进行PCR扩增获得chitinase基因的扩增产物。
PCR条件为：98℃预变性3min，然后进行35个循环(98℃变性10s、60℃退火20s、72℃延伸30s)，72℃延伸5min。PCR反应结束后，用1％琼脂糖凝胶电泳检测。
3、Chitinase表达菌株的构建
分别将Chitinase基因的扩增产物和ChitinaseK172A,K173A的PCR产物回收，与pMAL-c2x载体连接构建重组表达质粒，本发明所述连接选用的反应体系为In-fusion反应体系(Vazyme)优选：纯化的PCR产物，100ng；酶切后的线性化载体：200ng；5×In-Fusion EnzymePremix，2μL，将反应体系用超纯水补齐至10μL。
本发明所述连接的程序：将以上反应体系轻轻混匀后放置于50℃孵育15min，然后置于冰上，即得连接产物。将连接产物转化大肠杆菌DH5α感受态细胞，在1mL LB液体培养基中培养1h(37℃，200rpm)，取200μL菌液涂布于含100μg/mL氨苄抗生素的固体LB平板，37℃过夜培养，挑取阳性菌落PCR鉴定，并将阳性菌液送上海生工测序公司进行测序，将测序正确的质粒转化大肠杆菌Rosetta(DE3)感受态细胞，通过PCR鉴定阳性菌液按1:1加入50％灭菌甘油低温(-80℃)保存，分别得到Chitinase和ChitinaseK172A,K173A表达菌株。
4、Chitinase和ChitinaseK172A,K173A的原核表达和纯化
(1)原核表达
分别将chitinase和chitinaseK172A,K173A表达菌株进行扩大培养(37℃，200rpm)，当菌液浓度达到OD600为0.4-0.6时，将菌液降温至18℃后加入终浓度为1mM的蛋白诱导剂IPTG进行低温诱导(18℃，100rpm)，培养18-20h后收集菌体。
(2)蛋白纯化
先加入无菌水将菌体悬浮，4℃6000g离心6min除去上清，然后加入适量体积的蛋白提取液(20mM Tris-HCl，500mM NaCl，pH 7.5)将菌体悬浮，根据菌液体积按1：1000加入TritonX-100和1mM的PMSF以提高蛋白的提取效率和抑制蛋白降解。充分悬浮菌体后，利用低温超高压连续流细胞破碎机将菌体破碎1h(超声30s，停30s)，超声期间要将盛有菌液的容器要保持充分冰浴，以免超声过程中探头发热导致蛋白活性降低。超声破碎完毕后加入终浓度为1mM的PMSF抑制蛋白降解。4℃9000g离心30min，将上清液过膜(0.45μm)，然后将已用蛋白提取液平衡好的MBP Dextrin Beads(SA077025，Smart-lifesciences)加入到蛋白溶液中,于4℃低温使蛋白与填料旋转结合2.5h。将含填料蛋白提取液加入到重力柱中，当上清液全部流过重力柱后，加入蛋白提取液(含20mM Tris-HCl，500mM NaCl，pH 7.5)洗杂蛋白，直至流下的溶液用考马斯亮蓝G250(碧云天)检测不变蓝为止。然后用10mM麦芽糖(含20mM Tris-HCl，500mM NaCl，pH 7.5)洗脱目标蛋白。最后将纯化后的目标蛋白保存在-80℃超低温冰箱，chitinase-MBP及其突变体chitinaseK172A,K173A-MBP蛋白的SDS-PAGE如附图1所示。
5、采用酶标仪法对蛋白活性进行检测。
选用索莱宝生物技术有限公司的试剂盒测定chitinase-MBP及其突变体chitinaseK172A,K173A-MBP蛋白的活性，具体的操作步骤见几丁质酶活性检测试剂盒说明书(BC0825)。
活性单位定义：37℃下，每mg蛋白每小时分解几丁质产生1μmol N-乙酰氨基葡萄糖的酶量为一个酶活性单位。计算公式如下：
几丁质酶活性(U/mgprot)＝x×V样÷(V样×Cpr)÷T＝x÷Cpr
结果如图2所示，从图2可以看出突变体chitinaseK172A,K173A-MBP的活性显著高于chitinase-MBP。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
SEQ ID NO.1(chitinase基因的核苷酸序列)
ATGGCTTTCAACATGAGAAAAAATCTACTTACGTTTGCTCTTCTGGGACTTTTTTCCTTGGCTATTGTTCCTAAAAATGTCATGTCCCAAAACTGTGACTGTGCTCCCAACTTGTGTTGCAGTCAGTTTGGTTACTGTGGCACCGGCGAAGCCTACTGTGGATTGGGGTGTAAGGGGGGTCCTTGCACCTCGACGCCATCGACACCGTCACCTACACCAACCGGTGGTGGTTCAGTTGCCAGTATTGTTACGGCTGATTTCTTTGATGGGATAAAGAATCAAGCTGCTGCAAGCTGTGCTGGAAAGAGCTTCTACACAAGAGATGGATTTCTTAATGCAGCCAATTCGTTTCCTCAGTTTGGATCAGGCTCTGCCGACGAATCCAAGCGTGAGATTGCTGCATTTTTTGCCCACGTTACTCATGAAACTGGACATTTATGCTACACCGAAGAGATTGACAAGTCAAATGCCTACTGTGACCAATCAAACACACAGTACCCATGTGTCCCCGGAAAGAAGTATTACGGGCGTGGACCGATGCAGCTCACCTGGAACTACAACTACGGTGCCTGTGGAACAGCCGTCGGGTTCGATGGACTCAACGCTCCCGAAACAGTGTCTAATGACCCTGCTGTCTCCTTTAAGGCTGCCTTGTGGTTCTGGATGACCAATGTTCACTCAGTCATGAACCAAGGCTTTGGGGCTACCATTCAGAAAATTAATGGCGCTCTTGAATGCGGTGGAAAGCAACCTGATAAAGTTAATGCTCGTATTGGTTATTACACTGATTATTGCCAGAAATTTGGTGTTGATCCCGGCCAGAATTTGTCCTGCTAG
SEQ ID NO.2(chitinase的氨基酸序列)
MAFNMRKNLLTFALLGLFSLAIVPKNVMSQNCDCAPNLCCSQFGYCGTGEAYCGLGCKGGPCTSTPSTPSPTPTGGGSVASIVTADFFDGIKNQAAASCAGKSFYTRDGFLNAANSFPQFGSGSADESKREIAAFFAHVTHETGHLCYTEEIDKSNAYCDQSNTQYPCVPGKKYYGRGPMQLTWNYNYGACGTAVGFDGLNAPETVSNDPAVSFKAALWFWMTNVHSVMNQGFGATIQKINGALECGGKQPDKVNARIGYYTDYCQKFGVDPGQNLSC
SEQ ID NO.3(chitinaseK172A,K173A的氨基酸序列)
MAFNMRKNLLTFALLGLFSLAIVPKNVMSQNCDCAPNLCCSQFGYCGTGEAYCGLGCKGGPCTSTPSTPSPTPTGGGSVASIVTADFFDGIKNQAAASCAGKSFYTRDGFLNAANSFPQFGSGSADESKREIAAFFAHVTHETGHLCYTEEIDKSNAYCDQSNTQYPCVPGAAYYGRGPMQLTWNYNYGACGTAVGFDGLNAPETVSNDPAVSFKAALWFWMTNVHSVMNQGFGATIQKINGALECGGKQPDKVNARIGYYTDYCQKFGVDPGQNLSC。
Claims (9)
1.芒果几丁质酶的突变体chitinaseK172A,K173A，其特征在于，氨基酸序列如SEQ ID NO.3所示。
2.一种编码权利要求1所述的突变体chitinaseK172A,K173A的基因。
3.一种含有权利要求2所述的编码突变体chitinaseK172A,K173A的基因的重组表达质粒。
4.根据权利要求3所述的重组表达质粒，其特征在于，所述的表达质粒是表达载体pMAL-c2x。
5.一种含有权利要求3或4所述的重组表达质粒的宿主表达细胞。
6.根据权利要求5所述的宿主表达细胞，其特征在于，所述的宿主表达细胞是大肠杆菌。
7.权利要求1所述的突变体chitinaseK172A,K173A在催化几丁质水解中的应用。
8.一种获得突变体chitinaseK172A,K173A的编码基因的方法，其特征在于，包括以下步骤：
(1)以chitinase基因为模板，以SEQ ID NO.4和SEQ ID NO.5所示序列为引物进行PCR，得第一PCR产物，所述的chitinase基因的核苷酸序列如SEQ ID NO.1所示；
(2)以chitinase基因为模板，以SEQ ID NO.6和SEQ ID NO.7所示序列为引物进行PCR，得第二PCR产物；
(3)以第一和第二PCR产物为模板，以SEQ ID NO.4和SEQ ID NO.7所示序列为引物进行PCR，得含酶切位点的突变体chitinaseK172A,K173A的编码基因。
9.根据权利要求8所述的方法，其特征在于，步骤(1)和(2)的PCR的程序为：98℃预变性3min；98℃变性10s、60℃退火20s、72℃延伸30s，35个循环；72℃延伸5min。
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