esterase for aqueous-organic solvents

来自 Nature

阅读量:

214

作者:

JC MooreFH Arnold

展开

摘要:

Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibioticester in aqueous-organic solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleusester was not feasible, theester was employed. Catalytic performance on the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. Oneesterase variant performs as well in 30% dimethylformamide as the wildtype enzyme in water, reflecting a 16-fold increase in esterase activity. Random pairwise gene recombination of two positive variants led to a further two-fold improvement in activity. Considering also the increased expression level achieved during these experiments, the net result of four sequential generations of random mutagenesis and the one recombination step is a 50–60-fold increase in total activity. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (<2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model developed based on its homology to acetylcholinesterase and triacylglycerol lipase. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

展开

DOI:

10.1038/nbt0496-458

被引量:

844

年份:

1996

通过文献互助平台发起求助,成功后即可免费获取论文全文。

相似文献

参考文献

引证文献

来源期刊

引用走势

2001
被引量:62

辅助模式

0

引用

文献可以批量引用啦~
欢迎点我试用!

引用