Difference between revisions of "PMID:20585771"

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'''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
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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.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=20585771 PubMed]
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Online version:[http://dx.doi.org/10.1007/s00253-010-2735-8 10.1007/s00253-010-2735-8]
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Butylene Glycols; DNA Shuffling; DNA, Bacterial; Escherichia coli K12; Hydroxybutyrates; Klebsiella pneumoniae; Methylphenazonium Methosulfate; Nitroblue Tetrazolium; Protein Engineering; Salmonella enterica; Sugar Alcohol Dehydrogenases
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==Main Points of the Paper ==
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== Materials and Methods Used ==
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==Phenotype Annotations==
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==Notes==
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==References==
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[[Category:Publication]]

Revision as of 20:43, 13 December 2011

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; DNA Shuffling; DNA, Bacterial; Escherichia coli K12; Hydroxybutyrates; Klebsiella pneumoniae; Methylphenazonium Methosulfate; Nitroblue Tetrazolium; Protein Engineering; Salmonella enterica; Sugar Alcohol Dehydrogenases

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Main Points of the Paper

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Materials and Methods Used

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Phenotype Annotations

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Phenotype of Taxon Information Genotype Information (if known) Condition Information OMP ID OMP Term Name ECO ID ECO Term Name Notes Status
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Notes

References

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