Development of a high-throughput screening method for racemase activity and its application to the identification of alanine racemase variants with activity towards L-arginine

被引：18
|
作者：
Willies, Simon C. [1 ]
White, Jemma L. [1 ]
Turner, Nicholas J. [1 ]
机构：
[1] Univ Manchester, Sch Chem, Manchester Interdisciplinary Bioctr, Manchester M1 7DN, Lancs, England
来源：
TETRAHEDRON | 2012年 / 68卷 / 37期
关键词：
Racemase; High-throughput screening; Protein engineering; Biocatalysis; Amino acid; BROAD SUBSTRATE-SPECIFICITY; AMINO-ACID RACEMASE; DIRECTED EVOLUTION; BACILLUS-STEAROTHERMOPHILUS; GALACTOSE-OXIDASE; LYSINE; 39; BIOSYNTHESIS; RACEMIZATION; GENERATION; RESISTANCE;
D O I：
10.1016/j.tet.2012.06.062
中图分类号：
O62 [有机化学];
学科分类号：
070303 ; 081704 ;
摘要：
A high-throughput solid phase screening method has been developed for alanine racemase. Conversion of an L-amino acid to the D-amino acid is detected by a D-amino acid oxidase, which is utilised as a reporter enzyme along with horseradish peroxidase and a colourimetric dye. This system has successfully been used to identify a mutant with increased activity towards arginine. Kinetic analysis and modelling studies have been performed to understand the effect of the mutations found. (C) 2012 Elsevier Ltd. All rights reserved.
引用
收藏
页码：7564 / 7567
页数：4
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