Expression,Purification and Enzymatic Characterization of Klebsiella sp.Glycerol Dehydrogenase in E.coli
- VernacularTitle:克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究
- Author:
Mei-Juan XU
;
Tao-Wei YANG
;
Zhi-Ming RAO
;
Wei SHEN
;
Chuan-Zhi ZHANG
;
Bin ZHUGE
;
Hui-Ying FANG
;
Jian ZHUGE
;
- Publication Type:Journal Article
- Keywords:
Klebsiella sp Glycerol dehydrogenase(GDH) Purification Characterization
- From:
China Biotechnology
2006;0(12):-
- CountryChina
- Language:Chinese
-
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 ?mol/ml?min in the glycerol.
