源于解淀粉芽胞杆菌酚酸脱羧酶的克隆与表达

Cloning and expression of phenolic acid decarboxylase from Bacillus amyloliquefaciens

摘要:

【背景】酚酸脱羧酶催化分解酚酸产生的4-乙烯基酚类物质可用于食品添加剂及香精香料行业，而酚酸脱羧酶的表达水平相对较低，因此，高水平的酚酸脱羧酶是工业规模生产4-乙烯基酚类物质的先决条件。【目的】克隆解淀粉芽胞杆菌的酚酸脱羧酶基因，实现在大肠杆菌中的高效异源表达，分析酚酸脱羧酶的底物特异性，并对其表达条件进行优化。【方法】通过PCR技术获得酚酸脱羧酶的基因，构建重组基因工程菌，将测序结果与其他酚酸脱羧酶序列进行比对，利用IPTG诱导方法高效表达蛋白。将重组酚酸脱羧酶与4种不同的底物进行反应，设计响应面试验对诱导条件进行优化。【结果】酚酸脱羧酶对对香豆酸、阿魏酸、咖啡酸、芥子酸的比酶活比率为：100:23.33:15.39:10.51。结合与其他酚酸脱羧酶比对结果发现酚酸脱羧酶家族的C末端区域氨基酸序列的变异率最高，这与酚酸脱羧酶的底物特异性和催化机制有关。通过单因素和响应面试验得到酚酸脱羧酶诱导表达的最佳条件为：2×YT培养基，诱导温度30 °C，接种量1.78%，诱导时机3.8 h，IPTG 1.25 mmol/L，诱导时间18 h，此时预测酶活和实际酶活分别为47.61 IU/mL和47.55 IU/mL。【结论】应用响应面法优化酚酸脱羧酶的诱导表达是可行的，本试验为以后生产稳定、高产的酚酸脱羧酶以及了解其催化机理提供了重要的理论基础。

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.