Heterologous expression,purification and enzymatic properties of dopa decarboxylase from Pseudomonas
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 sequen-cing.Escherichia coli BL21(DE3)was transferred for recombinant expression.The OD value was 0.6-0.8,the final concentration of isopropyl β-D-l-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 tem-perature,and the enzyme activity was more than 80％at 20-30 ℃.The enzyme activity decreased substantially above 30 ℃.The opti-mal buffer solution was PBS buffer solution,the optimal reaction pH was 7.5,the optimal substrate was L-DOPA.The metal cation Ca2+promoted the enzyme activity.The sequence homology analysis showed that DODC from Pseudomonas belongs to the AAT-I superfam-ily,and the conserved catalytic active site of the enzyme was predicted to be Thr 241.
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维普
