摘要
从土壤中分离睾酮丛毛单胞菌 ,提取其基因组DNA ,PCR扩增 3α 羟类固醇脱氢酶 (3α HSD)基因。以质粒pET 1 5b为载体 ,构建 3α HSD的原核表达系统。经IPTG诱导后 ,得到了具有酶活性的融合蛋白 ,细菌总蛋白的表达量为 0 73g L ,其中融合酶蛋白占 1 6 4 % ,活性达 1 38× 1 0 5U L。利用融合蛋白N末端的His tag,经金属螯合亲和层析纯化后 ,得到了纯度较高的融合酶蛋白 ,酶蛋白的得率为 6 8% ,比活性为 1 94 7U mg。凝血酶切除His tag后 ,经质谱鉴定 ,重组蛋白的分子量为 2 6 5kD ,与理论推测值基本相同。 2 5℃以雄酮为底物 ,以NAD+ 和thio NAD+ 为辅因子时 ,融合蛋白参与酶促反应的最适pH分别为 1 0 5和 9 4。 37℃条件下酶促反应较快 ,2 5℃时的反应速度只有 37℃时的 5 2 % ,Q1 0 ( 2 5℃～ 35℃ ) 为 1 7。 2 5℃、pH1 0 5、以雄酮和各级胆汁酸为底物、NAD+ 为辅因子时 ,融合酶蛋白的动力学常数Km位于 4 2～ 5 1 1 μmol L范围内。融合酶蛋白发挥催化作用并不需要金属离子的参与 ,而Fe3+ 、Fe2 + 、Zn2 + 则抑制酶活性 ,反应体系中加入EDTA也并不影响酶的活性。活性和纯度均较高的融合蛋白 3α HSD的获得及其生物学性质的研究 。
To clone and overexpress the 3α hydroxysteroid dehydrogenase (3α HSD) in E.coli and to study its characterization, the 3α hydroxysteroid dehydrogenase gene was amplified by PCR from the genomic DNA of Comamonas testosteroni which was isolated from pond mud. The 3α HSD prokaryotic expression system was constructed with plasmid pET15b as the vector. The fusion protein with enzyme activity was overexpressed in the host bacteria of E.coli BL21(DE3) pLysS after IPTG induction. The fusion protein with an N terminal His tag sequence was purified in one step using metal chelate affinity chromatography to homogeneity on a Ni 2+ Sepharose column. The overall yield of the fusion protein was 64% and the relative activity was 194 7U/mg. After thrombin cleavage of the His tag, the molecular weight of the recombinant protein was 26 5kD according to the mass spectrum, which was identical to that predicted from the genomic sequence. With androsterone as the substrate and NAD + and thio NAD + as the cofactor, the optimum pH for the enzymatic reaction at 25℃ was 10 5 and 9 4 respectively. The rate of the enzymatic reaction was more quickly at 37℃ than at 25℃ and Q 10(25℃～35℃) was 1 7 The fusion 3α HSD could efficiently catalyze 3α hydroxyl dehydrogenation of androsterone and bile acids series with K m values in the range of 4 2～51 1μmol/L. The enzyme activity didn't need the participation of metal ions, but was inhibited by Fe 3+ , Fe 2+ and Zn 2+ . And EDTA did not affect its activity. The obtainment of fusion 3α HSD with high purity and activity and the investigation on its characterization laid a necessary foundation for the construction of a enzymatic cycling method to determine the human serum total bile acids.
出处
《微生物学报》
CAS
CSCD
北大核心
2004年第4期496-499,共4页
Acta Microbiologica Sinica
关键词
3α-羟类固醇脱氢酶
表达
纯化
酶学性质
睾酮丛毛单胞菌
Comamonas testosteroni ,3α Hydroxysteroid dehydrogenase,Gene expression, Enzyme properties,Bile acids
