北京大学学报（自然科学版）
北京大學學報（自然科學版）
북경대학학보（자연과학판）
ACTA SCIENTIARUM NATURALIUM UNIVERSITATIS PEKINENSIS
2007年
6期
828-833
,共6页
青枯雷尔氏菌 GMI1000菌株%龙胆酸1,2-双加氧酶%酶性质
青枯雷爾氏菌 GMI1000菌株%龍膽痠1,2-雙加氧酶%酶性質
청고뢰이씨균 GMI1000균주%룡담산1,2-쌍가양매%매성질
Ralstonia solanacearum GMI1000%gentisate 1,2-dioxygenase%characterization
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, 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.0.1～1mmol/L Fe2+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.
本研究剋隆、錶達瞭青枯雷爾氏菌GMI1000菌株中編碼龍膽痠1,2-雙加氧酶的基因, 併通過親和層析對該酶進行瞭純化, SDS-PAGE結果錶明該酶亞基約為38kDa.該酶的最適反應溫度和最適pH分彆為30℃和7.5.該酶的Km為56μmol/L,pI為4.6～4.8.純化後的龍膽痠1,2-雙加氧高度不穩定:4℃下放置72h即失去85%的酶活. 甘油和β-巰基乙醇可穩定該酶酶活,在添加10%(體積比)甘油至酶液(50mmol/L, pH 7.3的燐痠鹽緩遲液保存)後,-20℃保藏2週後均能檢齣明顯的酶活.0.1～1mmol/L Fe2+可以激活或者穩定該酶的酶活.Na+,K+,Mg2+和Ca2+(分彆為1～2mmol/L)對該酶的酶活無明顯的影響.Mn2+,Zn2+和Fe3+的添加量超過2mmol/L時,酶活急劇下降.1mmol/L的Cu2+即使該酶失去酶活.
본연구극륭、표체료청고뢰이씨균GMI1000균주중편마룡담산1,2-쌍가양매적기인, 병통과친화층석대해매진행료순화, SDS-PAGE결과표명해매아기약위38kDa.해매적최괄반응온도화최괄pH분별위30℃화7.5.해매적Km위56μmol/L,pI위4.6～4.8.순화후적룡담산1,2-쌍가양고도불은정:4℃하방치72h즉실거85%적매활. 감유화β-구기을순가은정해매매활,재첨가10%(체적비)감유지매액(50mmol/L, pH 7.3적린산염완충액보존)후,-20℃보장2주후균능검출명현적매활.0.1～1mmol/L Fe2+가이격활혹자은정해매적매활.Na+,K+,Mg2+화Ca2+(분별위1～2mmol/L)대해매적매활무명현적영향.Mn2+,Zn2+화Fe3+적첨가량초과2mmol/L시,매활급극하강.1mmol/L적Cu2+즉사해매실거매활.
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 38kDa. 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 56μmol/L. The pI was 4.6-4.8. The active site of the gentisate 1,2-dioxygenase with gentisate was also modeled.
