PMID:20585771
| Citation |
Zhang, H, Lountos, GT, Ching, CB and Jiang, R (2010) Engineering of glycerol dehydrogenase for improved activity towards 1, 3-butanediol. Appl. Microbiol. Biotechnol. 88:117-24 |
|---|---|
| Abstract |
The objective of this study was to use protein engineering techniques to enhance the catalytic activity of glycerol dehydrogenase (GlyDH) on racemic 1, 3-butanediol (1, 3-BDO) for the bioproduction of the important pharmaceutical intermediate 4-hydroxy-2-butanone. Three GlyDH genes (gldA) from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae MGH78578 were shuffled to generate a random mutagenesis library. The nitroblue tetrazolium/phenazine methosulfate high throughput screening protocol was used to select four chimeric enzymes with up to a 2.6-fold improved activity towards 1, 3-BDO. A rational design method was also employed to further improve the enzyme activity after DNA shuffling. Based on the homology model of GlyDH (Escherichia coli), Asp121 was predicted to influence 1, 3-BDO binding and replaced with Ala by site-directed mutagenesis. Combination of the mutations from both DNA shuffling and rational design produced the best mutant with a V (max) value of 126.6 U/mg, a 26-fold activity increase compared with that of the wild type GlyDH from E. coli. |
| Links |
PubMed Online version:10.1007/s00253-010-2735-8 |
| Keywords |
Butylene Glycols/metabolism; DNA Shuffling; DNA, Bacterial/genetics; Escherichia coli K12/enzymology; Escherichia coli K12/genetics; Hydroxybutyrates/metabolism; Klebsiella pneumoniae/enzymology; Klebsiella pneumoniae/genetics; Methylphenazonium Methosulfate/metabolism; Nitroblue Tetrazolium/metabolism; Protein Engineering; Salmonella enterica/enzymology; Salmonella enterica/genetics; Sugar Alcohol Dehydrogenases/genetics; Sugar Alcohol Dehydrogenases/metabolism |
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