Expression, Purification and Enzymatic Characterization of Klebsiella sp. glycerol dehydrogenase in E. coli

China Biotechnology

2008, Vol. 28

Issue (12): 30-35 DOI:

Expression, Purification and Enzymatic Characterization of Klebsiella sp. glycerol dehydrogenase in E. coli

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Abstract

The dhaD gene encoding glycerol dehydrogenase (GDH) from Klebsiella sp. was amplified. and was inserted into expression vector pET-28a(+), the plasmid pET-28a-dhaD was constructed and was transformed into Escherichia coli BL21 (DE3). SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21. Then GDH was purified by Ni-NTA affinity chromatography, the results showed a single band about 39kDa on SDS-PAGE gel, and the specified activity was about 156U/mg. The special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%. The optimum reaction pH was 11.0, and the GDH activity have little changed when incubated in the buffer of pH7.0～11.0. The optimum reactive temperature was 30℃，and the GDH was more stable on the temperature of 25℃～45℃. The Km value was 0.54mmol/L and Vmax was 0.49 μmol/(mL·min) in the glycerol.

Key words： Klebsiella sp glycerol dehydrogenase (GDH) purification characterization

Received: 19 June 2008 Published: 20 April 2009

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	XU Mei-Juan
	RAO Zhi-Meng
	CHEN Wei
	LIU Zhi-Guo
	LI Zhang-Gao
	CHU Ge-Bin
	FANG Hui-Yang
	CHU Ge-Jian

Cite this article:

. Expression, Purification and Enzymatic Characterization of Klebsiella sp. glycerol dehydrogenase in E. coli. China Biotechnology, 2008, 28(12): 30-35.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2008/V28/I12/30

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