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[1]汪芳,杨套伟,周俊平,等.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):1411-1417.[doi:10.19675/j.cnki.1006-687x.2018.01022]
 WANG Fang,et al..Molecular modification of L-aspartate β-decarboxylase to improve its catalytic activity under acidic condition[J].Chinese Journal of Applied & Environmental Biology,2018,24(06):1411-1417.[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:
1411-1417
栏目:
研究简报
出版日期:
2018-12-25

文章信息/Info

Title:
Molecular modification of L-aspartate β-decarboxylase to improve its catalytic activity under acidic condition
作者:
汪芳 杨套伟 周俊平 徐美娟 张显 饶志明
江南大学生物工程学院,工业微生物教育部重点实验室 无锡 214122
Author(s):
WANG Fang et al.
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 β-decarboxylase enzymatic property substrate entrance tunnel site mutation
分类号:
Q789
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-脱羧酶基因(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. 为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. 本研究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 参24)
Abstract:
L-Aspartate beta-decarboxylase gene (Asd) from Pseudomonas dacunhae was heterologously expressed in Escherichia coli. However, it showed low activity under acidic condition, thereby limiting its wide application in industrial process. In this study, we aimed to improve the activity of Asd under acidic condition by using site mutation. The non-conservative residues located in the substrate entrance tunnel were selected, and the enzymatic properties of variants were investigated. Finally, the whole-cell transformation was performed with variants. The activities of most variants were significantly lower than those of wild type (WT, 65.95 U/mg), except N34D, which showed higher activity of 71.67 U/mg. The mutant N34D showed higher activity at the pH range from 3.5 to 5.5, and the relative activity was over 82% at pH 5.0, which was considerably higher than that of the wild-type (50%). The activity of mutant N34D at pH range of 5.0-8.5 was the same with the wild-type. Under the optimal catalytic conditions, the production of L-alanine by N34D was 1.5 times higher than that of the wild-type. The activity of variant N34D was successfully increased under acidic condition, which shows a promising application for the production of L-alanine at an industrial scale.

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更新日期/Last Update: 2018-12-25