来源于Pseudomonas多巴脱羧酶的异源表达、纯化及酶学性质研究

Heterologous expression,purification and enzymatic properties of dopa decarboxylase from Pseudomonas

将来源于Pseudomonas的多巴脱羧酶(DOPA decarboxylase,DODC)基因序列经过PCR扩增,双酶切,连接到载体CV6-pGEX-6P-1上,经验证、测序成功构建表达载体CV6-pGEX-6P-1-DODC。转入大肠杆菌BL21(DE3)重组表达,表达条件为OD值达到0.6~0.8,异丙基β-D-1-硫代半乳糖苷(IPTG)终浓度为0.1 mmol/L,16℃过夜培养12~16 h。结果表明:在BL21(DE3)大肠杆菌中经诱导表达得到较高表达量的DODC融合蛋白;经过GST-亲和层析、3C蛋白酶切、离子交换层析得到纯度95%以上的DODC纯化蛋白;对DODC的酶学性质进行研究,该酶的最适反应温度为40℃,对温度的影响比较敏感,在20~30℃酶活在80%以上,超过30℃酶活大幅度降低,最适缓冲液为PBS缓冲液,最适反应pH值为7.5,最适底物为左旋多巴(L-DOPA),金属阳离子Ca^(2+)对酶活力有促进作用。序列同源性分析表明,来源于Pseudomonas的DODC属于AAT-Ι超家族,并且预测出该酶的保守催化活性位点为Thr 241。

The dopamine decarboxylase(DODC)gene sequence from Pseudomonas was PCR amplified,double digested and ligated to the vector CV6-pGEX-6P-1,and the expression vector CV6-pGEX-6P-1-DODC was successfully constructed by validation and sequencing.Escherichia coli BL21(DE3)was transferred for recombinant expression.The OD value was 0.6-0.8,the final concentration of isopropylβ-D-1-thiogalactoside(IPTG)was 0.1 mmol/L,and cultured at 16℃overnight for 12-16 h.The results showed that DODC fusion protein with high expression was obtained in E.coli BL21(DE3)by induction expression.DODC purified protein with purity above 95%was obtained by GST-affinity chromatography,3C protease digestion and ion exchange chromatography.The enzymatic properties of DODC were studied.The optimum reaction temperature of the enzyme was 40℃,which was sensitive to the effect of temperature,and the enzyme activity was more than 80%at 20-30℃.The enzyme activity decreased substantially above 30℃.The optimal buffer solution was PBS buffer solution,the optimal reaction pH was 7.5,the optimal substrate was L-DOPA.The metal cation Ca^(2+)promoted the enzyme activity.The sequence homology analysis showed that DODC from Pseudomonas belongs to the AAT-Ⅰsuperfamily,and the conserved catalytic active site of the enzyme was predicted to be Thr 241.