CN105647892A - 一种D-丙氨酰-D-丙氨酸羧肽酶dacA基因及应用 - Google Patents

一种D-丙氨酰-D-丙氨酸羧肽酶dacA基因及应用 Download PDF

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CN105647892A
CN105647892A CN201610123620.3A CN201610123620A CN105647892A CN 105647892 A CN105647892 A CN 105647892A CN 201610123620 A CN201610123620 A CN 201610123620A CN 105647892 A CN105647892 A CN 105647892A
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alanyl
daca
alanine carboxypeptidase
alanine
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许菲
杨海泉
胡金远
强书敏
沈微
陈献忠
郑虹宁
宁璐璐
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Jiangnan University
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Abstract

本发明涉及基因工程领域，具体地，一种D-丙氨酰-D-丙氨酸羧肽酶dacA基因及应用。本发明提供了一种来源于大肠杆菌(Escherichia？coli)BL21(DE3)的D-丙氨酰-D-丙氨酸羧肽酶dacA，其氨基酸序列如序列1所示，且本发明提供了编码上述dacA的编码基因dacA。本发明的D-丙氨酰-D-丙氨酸羧肽酶能够从肽聚糖五肽上移除最后一个末端D-丙氨酸(D-Ala)，导致转肽反应中供体不可用，从而控制肽聚糖的网状交联水平，可以提高E.coli蛋白胞外分泌水平。

Description

一种D-丙氨酰-D-丙氨酸羧肽酶dacA基因及应用
技术领域
发明涉及基因工程领域，具体地，本发明涉及一种D-丙氨酰-D-丙氨酸羧肽酶dacA及其基因和应用。
背景技术
肽聚糖是由双糖单位，四肽尾还有肽桥聚合而成得多层网状大分子结构。肽聚糖层的骨架由N-乙酰葡萄糖胺和N-乙酰胞壁酸通过β-1，4糖苷键连接而成，糖链间由肽链交联，构成稳定的网状结构，这样构成了整个细菌表面的细胞壁。作为细胞壁的主要成分，肽聚糖的组成与细胞结构的完整性、外膜的通透性的维持、细菌的抗干燥能力和耐热性等均密切相关。dacA基因表达的D-丙氨酰-D-丙氨酸羧肽酶能够从肽聚糖五肽上移除最后一个末端D-Ala，导致转肽反应中供体不可用，可以控制肽聚糖的网状交联水平，从而改变细胞的通透性。
大肠杆菌(Escherichiacoli)表达系统是目前基因工程中最常用的蛋白表达系统，有操作简单、成本低廉等优点，可以快速大规模地生产目的蛋白。但是在E.coli的表达过程中，大部分蛋白在信号肽的引导下分泌到周质空间，会导致中间体大量积聚，对蛋白质的生产造成阻碍。
发明内容
本发明的目的是提供一种可以提高E.coli胞外分泌水平的D-丙氨酰-D-丙氨酸羧肽酶、其氨基酸序列、编码该D-丙氨酰-D-丙氨酸羧肽酶的基因、含有该基因的重组质粒、含有该重组载体的重组菌株、该D-丙氨酰-D-丙氨酸羧肽酶的应用。
具体包括：
本发明提供了一种D-丙氨酰-D-丙氨酸羧肽酶dacA，其氨基酸序列如序列1所示。
序列1：
MNTIFSARIMKRLALTTALCTAFISAAHADDLNIKTMIPGVPQIDAESYILIDYNSGKVLAEQNADVRRDPASLTKMMTSYVIGQAMKAGKFKETDLVTIGNDAWATGNPVFKGSSLMFLKPGMQVPVSQLIRGINLQSGNDACVAMADFAAGSQDAFVGLMNSYVNALGLKNTHFQTVHGLDADGQYSSARDMALIGQALIRDVPNEYSIYKEKEFTFNGIRQLNRNGLLWDNSLNVDGIKTGHTDKAGYNLVASATEGQMRLISAVMGGRTFKGREAESKKLLTWGFRFFETVNPLKVGKEFASEPVWFGDSDRASLGVDKDVYLTIPRGRMKDLKASYVLNSSELHAPLQKNQVVGTINFQLDGKTIEQRPLVVLQEIPEGNFFGKIIDYIKLMFHHWFG
其中，该酶基因编码403个氨基酸，其理论分子量为44.45kDa。
本发明提供了编码上述D-丙氨酰-D-丙氨酸羧肽酶的基因dacA，具体的，该酶的基因序列如序列2所示。
序列2：
ATGAATACCATTTTTTCCGCTCGTATCATGAAGCGCCTGGCGCTCACCACGGCTCTTTGCACAGCCTTTATCTCTGCTGCACATGCCGATGACCTGAATATCAAAACTATGATCCCGGGTGTACCGCAGATCGATGCGGAGTCCTACATCCTGATTGACTATAACTCCGGCAAAGTGCTCGCCGAACAGAACGCAGATGTCCGCCGCGATCCTGCCAGCCTGACCAAAATGATGACCAGTTACGTTATCGGCCAGGCAATGAAAGCCGGTAAATTTAAAGAAACTGATTTAGTCACTATCGGCAACGACGCATGGGCCACCGGTAACCCGGTGTTTAAAGGTTCTTCGCTGATGTTCCTCAAACCGGGCATGCAGGTTCCGGTTTCTCAGCTGATCCGCGGTATTAACCTGCAATCGGGTAACGATGCTTGTGTCGCCATGGCTGATTTTGCCGCTGGTAGCCAGGACGCTTTTGTTGGCTTGATGAACAGCTACGTTAACGCCCTGGGCCTGAAAAACACCCACTTCCAGACGGTACATGGTCTGGATGCTGATGGTCAGTACAGCTCCGCGCGCGATATGGCGCTGATCGGCCAGGCGTTGATCCGTGACGTACCGAATGAATACTCGATCTATAAAGAAAAAGAATTTACGTTTAACGGTATTCGCCAGCTGAACCGTAACGGCCTGTTATGGGATAACAGCCTGAATGTCGACGGCATCAAAACCGGACACACTGACAAAGCAGGTTACAACCTTGTTGCTTCTGCGACTGAAGGCCAGATGCGCTTGATCTCTGCGGTGATGGGCGGACGTACTTTTAAAGGCCGTGAAGCCGAAAGTAAAAAACTGCTGACCTGGGGCTTCCGTTTCTTCGAAACCGTTAACCCACTGAAAGTAGGTAAAGAGTTCGCCTCTGAACCGGTTTGGTTTGGTGATTCTGATCGCGCTTCGTTAGGGGTTGATAAAGACGTGTACCTGACCATTCCGCGTGGCCGCATGAAAGATCTGAAAGCCAGCTATGTGCTGAACAGCAGTGAATTGCATGCGCCGCTGCAAAAGAATCAGGTCGTCGGTACTATCAACTTCCAGCTTGATGGCAAAACGATCGAACAACGCCCGTTGGTTGTACTGCAAGAAATCCCGGAAGGTAACTTCTTCGGCAAAATCATTGATTACATTAAATTAATGTTCCATCACTGGTTTGGTTAA
本发明通过PCR方法克隆了D-丙氨酰-D-丙氨酸羧肽酶基因dacA，DNA全序列分析结果表明，D-丙氨酰-D-丙氨酸羧肽酶的编码基因dacA全长1212bp。
将上述D-丙氨酰-D-丙氨酸羧肽酶基因dacA序列及推导出的氨基酸序列在GenBank中进行BLAST比对，该基因与来源于E.colistr.K-12substr.MG1655的D-丙氨酰-D-丙氨酸羧肽酶序列相似性最高(99％)。
本发明还提供了包含上述D-丙氨酰-D-丙氨酸羧肽酶基因dacA的重组质粒，为pRSFDuet-dacA。将本发明的D-丙氨酰-D-丙氨酸羧肽酶基因插入至表达载体相应的限制酶切位点，使其核苷酸序列与表达调控序列相连接，作为本发明的一个最优选实验方案，优选D-丙氨酰-D-丙氨酸羧肽酶基因插入到质粒pRSFDuet，使该核苷酸序列位于T7lac启动子的下游并受其调控，得到重组E.coli表达质粒pRSFDuet-dacA。
本发明还提供了上述D-丙氨酰-D-丙氨酸羧肽酶基因dacA的应用菌株，优选所述菌株为E.coli。
本发明能够增强E.coli胞内蛋白到达胞外，促进其分泌，降低其在胞内积累，提供一种可提高E.coli蛋白胞外分泌水平的D-丙氨酰-D-丙氨酸羧肽酶、及其氨基酸序列、基因序列。
附图说明
图1为D-丙氨酰-D-丙氨酸羧肽酶重组质粒pRSFDuet-dacA图谱。
图2为D-丙氨酰-D-丙氨酸羧肽酶的过量表达
图3为过量表达D-丙氨酰-D-丙氨酸羧肽酶对E.coli胞外分泌α-半乳糖苷酶的影响。
具体实施方式
实施例1：E.coliBL21(DE3)D-丙氨酰-D-丙氨酸羧肽酶基因dacA的克隆
基于PCR获得D-丙氨酰-D-丙氨酸羧肽酶dacA的DNA序列，将D-丙氨酰-D-丙氨酸羧肽酶的基因片段与表达载体pRSFDuet连接，获得重组质粒pRSFDuet-dacA(图1)。然后转化到E.coliBL21(DE3)感受态细胞中，获得重组菌株E.coliBL21(DE3)/dacA。
实施例2：D-丙氨酰-D-丙氨酸羧肽酶酶活
将已构建的重组菌，接种250mL摇瓶培养，经诱导剂(IPTG)诱导后，收集E.coli细胞，采用超声破碎法破壁细胞。高速离心后，收集破壁上清，取样测定破壁上清中的羧肽酶酶活力。通过测定酶活力计算出过量表达后，羧肽酶dacA-Δp在胞内的比酶活力为5.83U/g(图2)，对照组羧肽酶比酶活力仅为1.51U/g。
实施例3：过量表达dacA对E.coli胞外分泌α-半乳糖苷酶的影响
在E.coli中过量表达羧肽酶dacA时，诱导10h后，E.coli胞外分泌α-半乳糖苷酶的水平显著提高，胞外α-半乳糖苷酶酶活力达到10.64U/g(图3)。但没有过量表达任何羧肽酶的对照菌株诱导发酵10h后，在E.coli胞外测得的α-半乳糖苷酶的酶活力仅为1.82U/g。

Claims (5)

1.一种D-丙氨酰-D-丙氨酸羧肽酶dacA，其特征在于：由序列1所示氨基酸序列组成的蛋白质。
2.一种D-丙氨酰-D-丙氨酸羧肽酶基因dacA，其特征在于，编码权利要求1所述的D-丙氨酰-D-丙氨酸羧肽酶dacA。
3.根据权利要求2所述D-丙氨酰-D-丙氨酸羧肽酶基因dacA，其特征在于：其DNA序列如序列2所示。
4.包含权利要求2或3所述的D-丙氨酰-D-丙氨酸羧肽酶基因dacA的重组质粒。
5.根据权利要求4所述的重组质粒，其特征在于，所述载体为pRSFDuet。
CN201610123620.3A 2016-03-04 2016-03-04 一种D-丙氨酰-D-丙氨酸羧肽酶dacA基因及应用 Pending CN105647892A (zh)

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