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[1]汪 芳 杨套伟** 周俊平 徐美娟 张 显 饶志明**.提高天冬氨酸β-脱羧酶在酸性环境中催化活力的分子改造*[J].应用与环境生物学报,2018,24(06):1-9.[doi:10.19675/j.cnki.1006-687x.2018.01022]
 WANG Fang,YANG Taowei**,ZHOU Jun ping,et al.Molecular modification of L-aspartate -decarboxylase to improve its catalytic activity in acidic condition*[J].Chinese Journal of Applied & Environmental Biology,2018,24(06):1-9.[doi:10.19675/j.cnki.1006-687x.2018.01022]
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提高天冬氨酸β-脱羧酶在酸性环境中催化活力的分子改造*()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
24卷
期数:
2018年06期
页码:
1-9
栏目:
简报
出版日期:
2018-12-25

文章信息/Info

Title:
Molecular modification of L-aspartate -decarboxylase to improve its catalytic activity in acidic condition*
文章编号:
201801022
作者:
汪 芳 杨套伟** 周俊平 徐美娟 张 显 饶志明**
工业微生物教育部重点实验室,江南大学生物工程学院 无锡 214122
Author(s):
WANG Fang YANG Taowei** ZHOU Jun ping XU Meijuan ZHANG Xian & RAO Zhiming**?
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
关键词:
德阿昆哈假单胞菌L-天冬氨酸β-脱羧酶酶学性质底物通道定点突变
Keywords:
Pseudomonas dacunhae L -aspartate -decarboxylase enzymatic property substrate entrance tunnel site mutation
DOI:
10.19675/j.cnki.1006-687x.2018.01022
摘要:
克隆了德阿昆哈假单胞菌(Pseudomonas dacunhae)来源的L-天冬氨酸β-脱羧酶(Asd),实现其在Escherichia coli 中的异源表达,但该酶在酸性环境中活性较低,不利于工业生产,本研究拟通过定点突变技术提高该酶在酸性环境中的活力。选择底物通道区域内的5个氨基酸残基作为突变位点,构建6个突变体,随后分析突变体的酶学特性。结果表明,相比于野生型Asd,大多数突变体的比酶活显著下降,只有突变体N34D比酶活(71.67 U/mg)比野生型(65.95 U/mg )略高;另外,突变体N34D在3.5 < pH < 5.5酸性环境中的酶活力与野生型Asd相比均得到了提高,其中pH 5.0条件下突变体N34D相对酶活达到82%,而野生型Asd相对酶活只有50%左右;而在pH 5.5-8.5范围内突变体N34D的酶活力与野生型相当。最后将突变体N34D应用于催化合成L-丙氨酸,在最适催化条件(pH 5.5)下,突变体N34D催化合成L-丙氨酸的产量是野生型Asd的1.5倍。通过对德阿昆哈假单胞菌来源的N34位点进行突变,成功提高了天冬氨酸β-脱羧酶在酸性环境中的酶活力,并提高了其合成L-丙氨酸的能力,这对酶法生产L-丙氨酸具有重要的指导意义。(图 4 表 4 参23)
Abstract:
Objectives: L-Aspartate beta-decarboxylase gene (Asd) from Pseudomonas dacunhae was heterologously expressed in Escherichia coli. However, it showed low activity in the acidic condition, limit their wide application in industrial process. In this study, we aimed to improve the activity of Asd in in the acidic condition by molecular modification. Methods: The Asd gene from P. dacunhae was cloned and expressed in E. coli. The non-conservative residues located in the substrate entrance tunnel were selected, and the enzymatic properties of variants were studied. Finally, the whole-cell transformation was performed with variants. Results: The activities of most variants were significantly lower than that of wild type (WT, 8.33 U/mg), except the N34D showed a relative equivalent activity-71.67 U/mg. The mutant N34D showed a higher actively at the pH range from 3.5 to 5.5, and the relative activity was over 82% at pH 5.0, which was much higher than that of the wild-type (50%). The activity of mutant N34D at pH range of 5.0-8.5 was same with the wild-type. Under the optimal catalytic conditions, the production of L - alanine by N34D was1.5 times higher than that of the wild-type WT. Conclusions: The activity of variant N34D was successfully increased in the acidic condition, which shows a promising application for production of L - alanine in industrial scale. ? ??

备注/Memo

备注/Memo:
收稿日期: 2018-01-17 接受日期: 2018-04-04*国家自然科学基金(31300028)、中国博士后科学基金资助项目(2017M620189)、江苏高校优势学科建设工程资助项目、江苏省杰出青年科学基金(BK20150002)资助 **通讯作者(E-mail:yangtw@jiangnan.edu.cn; raozhm@jiangnan.edu.cn)点击摘要页题目后的“PDF”可下载阅读全文;本文为已录用的作者修定稿,尚未经编辑全面修改。引用本文请注明出处本刊;发表刊期和页码将以正式出版时的安排为准,但DOI确定不变。
更新日期/Last Update: 2018-04-16