Abstract: 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 isopropyl β-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 Ca2+ promoted the enzyme activity. The sequence homology analysis showed that DODC from Pseudomonas belongs to the AAT-Ⅰ superfamily, and the conserved catalytic active site of the enzyme was predicted to be Thr 241.

Key words: DOPA decarboxylase, heterologous expression, protein purification, enzymatic properties, substrate specificity