辅因子
NAD+激酶
生物催化
生物化学
定向进化
生物转化
酶
脱氢酶
饱和突变
化学
蛋白质工程
突变
突变体
生物
发酵
反应机理
催化作用
基因
作者
Ryan D. Woodyer,Wilfred A. van der Donk,Huimin Zhao
出处
期刊:Combinatorial Chemistry & High Throughput Screening
[Bentham Science]
日期:2006-05-01
卷期号:9 (4): 237-245
被引量:39
标识
DOI:10.2174/138620706776843246
摘要
Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important properties of phosphite dehydrogenase for NAD(P)H regeneration. A two-tiered sorting method of selection and screening was used in conjunction with random and rational mutagenesis. Following six rounds of directed evolution, soluble expression in E. coli was increased more than 3-fold, while the turnover rate was increased about 2-fold, effectively lowering the cost of the enzyme by >6-fold. Large-scale production of the final mutant enzyme by fermentation resulted in approximately 6-times higher yield (Units/Liter) than the WT enzyme. The enhancements of PTDH were independent of expression vector and E. coli strain utilized. The advantage of the final mutant over the WT enzyme was demonstrated using the industrially relevant bioconversion of trimethylpyruvate to L-tert-leucine. The mutations discovered are discussed in the context of a three dimensional structural model and the resulting changes in kinetics and soluble expression. The engineered phosphite dehydrogenase has great potential for NAD(P)H regeneration in industrial biocatalysis.
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