Cloning and expression of phenolic acid decarboxylase from Bacillus amyloliquefaciens

Abstract:

[Background] 4-vinylphenols produced by phenolic acid decarboxylase catalytic degradation of phenolic acid can be used in food additives, flavor and fragrance industries. The expression level of phenolic acid decarboxylase is relatively low, so high levels of phenolic acid decarboxylase are a prerequisite for the industrial scale production of 4-vinylphenols. [Objective] The gene of phenolic acid decarboxylase from Bacillus amyloliquefaciens was cloned to achieve efficient heterologous expression in E. coli. The substrate specificity of phenolic acid decarboxylase was analyzed and its expression conditions were optimized. [Methods] The gene of phenolic acid decarboxylase was constructed using PCR to construct a recombinant genetically engineered bacterium. The sequencing results were compared with the phenolic acid decarboxylase family, and IPTG was used to induce protein expression. It was reacted with four different substrates, and response surface experiments were designed to optimize the inducing conditions. [Results] The specific enzyme activity ratios of phenolic acid decarboxylase to p-coumaric acid, ferulic acid, caffeic acid, and sinapic acid were: 100, 23.33, 15.39, 10.51. Combined with the comparison results with other phenolic acid decarboxylase, it was found that the amino acid sequence of the C-terminal region had the highest rate of variation, which was related to the substrate specificity and catalytic mechanism of the phenolic acid decarboxylase. The optimal conditions for expression of phenolic acid decarboxylase by designing response surface experiments were: 2×YT medium, inducting temperature 30 °C, inoculation amount 1.78%, inducing point 3.8 h, IPTG 1.25 mmol/L , inducing time 18 h, the predicted value and actual enzyme activity were 47.61 IU/mL and 47.55 IU/mL, respectively. [Conclusion] It is feasible to optimize the induced expression of phenolic acid decarboxylase by response surface methodology. This study provides an important theoretical basis for the production of stable and high-yield phenolic acid decarboxylase and understanding of its catalytic mechanism.

Key words:Bacillus amyloliquefaciens, Phenolic acid decarboxylase, Cloning and expression, Substrate specificity