Expression, purification and enzymatic characteristics of 2-hydroxyacid dehydrogen-ase from Ketogulonigenium vulgare Y25
10.7644/j.issn.1674-9960.2014.07.013
- VernacularTitle:酮古龙酸菌Y25 D-2-羟酸脱氢酶的表达、纯化与酶学性质
- Author:
Yuying LIANG
;
Xianghua XIONG
;
Bin ZHU
;
Nan ZHAO
;
Jianhua WANG
;
Weicai ZHANG
- Publication Type:Journal Article
- Keywords:
Ketogulonigenium vulgare;
2-hydroxyacid dehydrogenase;
ketogulonate reductase;
expression;
purification
- From:
Military Medical Sciences
2014;(7):523-526,541
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone 2-hydroxyacid dehydrogenase (HADH) gene from Ketogulonigenium vulgare(KGV) Y25 and investigate its expression , purification, and enzymatic characterics .Methods The hadh gene was amplified from ketogulonigenium vulgare Y 25 and cloned into the expression plasmid pITG .The recombinant plasmid was transformed into Escherichia coli BL21(DE3).HADH was then successfully expressed with induction .To explore its enzymatic characteris-tics,HADH was purified by Ni +exchange chromatography .Results HADH constituted more than 50% of the total cell proteins analyzed by SDS-PAGE,with a relative molecular mass of about 35 ×103.With 2-keto-gulonic acid(2-KGA) as substrate, the optimal pH of HADH was at 8.0 ,while the optimal temperature of the purified HADH was at 45℃.Mean-while, such metal ions and chelating agents as Cu 2+, Ca2+, Mg2+, EDTA, and DEPC exerted little effect on enzymatic activities.The maximum initial activity of the enzyme towards 2-KGA reached 27 U/mg, and the Km was calculated as 2.6 mmol/L.The results of in vivo enzyme activity assay showed that HADH could metabolize 2-KGA.Conclusion The HADH gene form Y25 is successfully expressed in E.coli BL21 ( DE3 ) and the enzymatic characteristics of HADH are explored, which will facilitate subsequent studies on sorbose metabolic pathways and sugar acid conversion .
