青枯雷尔氏菌龙胆酸1,2-双加氧酶基因的克隆、表达和酶性质的研究(英文)

Characterization of a Gentisate 1,2-dioxygenase from Ralstonia solanacearum GMI1000

本研究克隆、表达了青枯雷尔氏菌GMI1000菌株中编码龙胆酸1,2-双加氧酶的基因,并通过亲和层析对该酶进行了纯化,SDS-PAGE结果表明该酶亚基约为38kDa。该酶的最适反应温度和最适pH分别为30℃和7.5。该酶的Km为56μmol/L,pI为4.6～4.8。纯化后的龙胆酸1,2-双加氧高度不稳定:4℃下放置72h即失去85%的酶活。甘油和β-巯基乙醇可稳定该酶酶活,在添加10%(体积比)甘油至酶液(50mmol/L,pH7.3的磷酸盐缓冲液保存)后,-20℃保藏2周后均能检出明显的酶活。0.1～1mmol/LFe2+可以激活或者稳定该酶的酶活。Na+,K+,Mg2+和Ca2+(分别为1～2mmol/L)对该酶的酶活无明显的影响。Mn2+,Zn2+和Fe3+的添加量超过2mmol/L时,酶活急剧下降。1mmol/L的Cu2+即使该酶失去酶活。

The gene encoding gentisate 1,2-dioxygenase from a soil-borne Gram-negative bacterium, Ralstonia solanacearum GMI1000, was cloned and overexpressed in E. coli. The resulting product incorporated a （His） 6 tag was purified to homogeneity from the harvested cell extracts by affinity chromatography. SDS-PAGE showed that the polypeptide exhibited an approximate molecular mass of 38 kDa. The optimal temperature and pH for gentisate cleavage catalyzed by the enzyme were 30 ℃ and 7.5, respectively. The Km of the enzyme was determined to be 56 μmol/L. The pl was 4.6-4.8. The active site of the gentisate 1,2- dioxygenase with gentisate was also modeled.