原儿茶酸的生物转化

Biotransformation of protocatechuic acid

摘要:

【背景】原儿茶酸(Protocatechuic acid，PCA)是一些植物的主要活性成分，可作为许多聚合物和药物的前体物质，目前PCA的主要来源是利用化学法从植物中提取，然而该法提取率低且对环境造成一定程度的破坏。【目的】克隆对羟基苯甲酸-3-羟化酶基因ρ-HBA-3H并进行异源表达，利用该酶催化实现原儿茶酸的生物转化。【方法】以红球菌R04基因组DNA为模板，PCR扩增得到对羟基苯甲酸-3-羟化酶基因ρ-HBA-3H，构建重组基因工程菌BL21(DE3)/pET21a(+)-ρ-HBA-3H，诱导表达对羟基苯甲酸-3-羟化酶，在底物对羟基苯甲酸(ρ-hydroxybenzoic acid，ρ-HBA)存在下进行PCA的生物转化，并对生物转化的条件进行优化。【结果】对羟基苯甲酸-3-羟化酶基因在大肠杆菌中实现了高效表达。通过生物转化PCA产量可达1.156 g/L。优化实验表明Mg2+、Triton X-100对转化效率无影响，增加反应体系的溶氧量及添加适量的吐温-80能够促进转化反应的进行。细胞连续转化基础上适量补充葡萄糖可以有效增加工程菌的转化效率，减少PCA的消耗。【结论】通过生物酶催化法实现了PCA的高效率、绿色生产，为其他重要发酵产品的工业化生产提供理论研究基础。

Abstract:

[Background] Protocatechuic acid is the main active ingredient of some plants and can be used as a precursor of many polymers as well as various drugs. At present, protocatechuic acid is mainly extracted from plants by chemical methods, with poor extraction efficiency and certain degree of damage to the environment. [Objective] The cloning and heterologous expression of ρ-hydroxybenzoic acid-3-hydroxylase gene ρ-HBA-3H for realizing the biotransformation of protocatechuic acid catalyzed by ρ-hydroxybenzoic acid-3-hydroxylase. [Methods] The ρ-hydroxybenzoic acid-3-hydroxylase gene ρ-HBA-3H was amplified by PCR using Rhodococcus sp. R04 genomic DNA as a template and then constructing recombinant genetic engineering strain BL21(DE3)/pET21a(+)-ρ-HBA-3H. The expression of ρ-hydroxybenzoic acid-3-hydroxylase was induced, and conducted biotransformated of PCA by this enzyme in the presence of the substrate ρ-hydroxybenzoic acid (ρ-HBA), besides, the conditions of biotransformation were optimized. [Results] The ρ-hydroxybenzoic acid-3-hydroxylase gene was highly expressed in E. coli. The yield of protocatechuic acid by biotransformation can reach 1.156 g/L. Optimization experiments showed that Mg2+ and Triton X-100 have no effect on the conversion efficiency. Increasing the dissolved oxygen content of the reaction system and adding appropriate amount of Tween- 80 can promote the conversion reaction. On the basis of continuous cell transformation, proper amount of glucose can effectively increase the transformation efficiency of engineering bacteria and reduce the consumption of protocatechuic acid. [Conclusion] This study realized the high-efficiency and green production of protocatechuic acid by bio-enzymatic catalysis, and provided theoretical research basis for the industrial production of other important fermentation products.