Directed evolution of the epoxide hydrolase from Aspergillus niger

被引：30
|
作者：
Cedrone, F
Niel, S
Roca, S
Bhatnagar, T
Ait-abdelkader, N
Torre, C
Krumm, H
Maichele, A
Reetz, MT
Baratti, JC
机构：
[1] Fac Sci Luminy, CNRS, UMR 6111, F-13288 Marseille 9, France
[2] Max Planck Inst Kohlenforsch, D-45470 Mulheim, Germany
来源：
BIOCATALYSIS AND BIOTRANSFORMATION | 2003年 / 21卷 / 06期
关键词：
directed evolution; epoxide hydrolase; expression system; enzyme engineering; catalytic efficiency; enantioselectivity;
D O I：
10.1080/102420310001630137
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
An efficient and genetically stable expression system for the directed evolution of epoxide hydrolase from Aspergillus niger (ANEH) has been constructed. Error prone polymerase chain reaction (PCR) with defined mutation rates was used to create biodiversity in two libraries of mutants. Screening for activity allowed the isolation of clones with improved properties. One of these clones shows an expression level 3.4 higher than the original wild type clone in E. coli SG13009 and a 3.3 fold increased catalytic efficiency on 4-(p-nitrophenoxy)-1,2-epoxybutane. In addition, a screening assay for determining the enantioselectivity in the kinetic resolution of styrene oxide has been established using mass spectrometry.
引用
收藏
页码：357 / 364
页数：8
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