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Engineering of glycerol dehydrogenase for improved activity towards 1, 3-butanediol

  1. Author:
    Zhang, H. F.
    Lountos, G. T.
    Ching, C. B.
    Jiang, R. R.

  2. Author Address

    [Zhang, Hongfang; Ching, Chi Bun; Jiang, Rongrong] Nanyang Technol Univ, Sch Chem & Biomed Engn, Singapore 637459, Singapore. [Lountos, George T.] NCI, Macromol Crystallog Lab, Frederick, MD 21702 USA.;Jiang, RR, Nanyang Technol Univ, Sch Chem & Biomed Engn, 62 Nanyang Dr, Singapore 637459, Singapore.;rrjiang@ntu.edu.sg
    1. Year: 2010
    2. Date: Sep
  2. Journal: Applied Microbiology and Biotechnology
    1. 88
    2. 1
    3. Pages: 117-124
  4. Type of Article: Article
  5. ISSN: 0175-7598
  1. 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.

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External Sources

  1. View Full Text
  2. DOI: 10.1007/s00253-010-2735-8
  3. View record in Web of Science®
  4. WOS: 000280914900013

Library Notes

  1. Fiscal Year: FY2009-2010
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