Abstract: 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 K_m of the enzyme was determined to be 56 μmol/L. The pI was 4.6-4.8. The active site of the gentisate 1,2-dioxygenase with gentisate was also modeled.

Key words: Ralstonia solanacearum GMI1000, gentisate 1, 2-dioxygenase, characterization

摘要： 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38 kDa。该酶的最适反应温度和最适pH分别为30 ℃和7.5。该酶的K_m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72 h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（50 mmol/L， pH 7.3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。0.1～1 mmol/L Fe²⁺可以激活或者稳定该酶的酶活。Na⁺，K⁺，Mg²⁺和Ca²⁺（分别为1～2 mmol/L）对该酶的酶活无明显的影响。Mn²⁺，Zn²⁺和Fe³⁺的添加量超过2 mmol/L时，酶活急剧下降。1 mmol/L的Cu²⁺即使该酶失去酶活。

关键词: 青枯雷尔氏菌GMI1000菌株, 龙胆酸1, 2-双加氧酶, 酶性质