一株对羟基苯甲酸酯海洋降解菌的关键酶基因克隆与表达

Cloning and expression of genes encoding key enzymes of a marine paraben-degrading strain

摘要:

【背景】对羟基苯甲酸及其酯类常作为合成多种芳香族化合物的前体物质广泛应用于多个领域，但其难以自然降解给环境造成了污染问题，同时这些污染物随着洋流迁移到海洋中破坏海洋生态环境。【目的】从海洋环境中筛选对羟基苯甲酸酯高效降解菌，通过全基因组测序及注释分析，预测对羟基苯甲酸酯代谢通路，确定其代谢过程中的关键酶并进行功能研究。【方法】通过富集培养从海洋环境中分离对羟基苯甲酸酯降解菌，利用基因克隆技术将降解对羟基苯甲酸酯关键酶基因在大肠杆菌中高效表达，探究重组蛋白活性及酶学特征。【结果】从海底泥沙中筛选到一个菌株，经16s rRNA基因测序鉴定为硝化柠檬球菌(Citricoccus nitrophenolicus)；该菌株能够利用多种对羟基苯甲酸酯类物质进行生长，在甲酯为碳源条件下生长状态最好；将羧酸酯酶基因和单加氧酶基因在大肠杆菌中进行高效表达，重组表达的羧酸酯酶最适反应条件为：pH 8.0，30 ℃反应30 min；重组表达的单加氧酶活性表达依赖于辅酶，Mg²⁺、Mn²⁺、Zn²⁺和Fe³⁺可增强该酶活性；经荧光定量PCR进一步确定z13175、z09075为编码羧酸酯酶的基因。【结论】羧酸酯酶和单加氧酶是对羟基苯甲酸酯类转化为原儿茶酸的关键酶，在对羟基苯甲酸酯类生物降解过程中起到重要作用。

关键词:对羟基苯甲酸酯;羧酸酯酶;单加氧酶;异源表达

Abstract:

[Background] 4-hydroxybenzoic acid and its esters are precursors for various aromatic compounds. They are, however, difficult to be decomposed in the nature, causing environmental pollution. These pollutants migrate to the ocean along with ocean current, damaging the marine ecological environment.[Objective] To screen a 4-hydroxybenzoic acid esters-degrading strain from the marine environment, predict the metabolic pathways of 4-hydroxybenzoic acid esters by whole-genome sequencing and annotation analysis, and analyze the key enzymes in the pathways and the functions of the enzymes. [Methods] We isolated the 4-hydroxybenzoic acid esters-degrading strain through enrichment culture. We used gene cloning technology to express the genes encoding key enzymes in the metabolic pathways of 4-hydroxybenzoic acid esters in Escherichia coli and explored the activities and enzymological characteristics of the recombinant proteins. [Results] A strain (B1 strain) was screened out from the seabed sediment and identified as Citricoccus nitrophenolicus by 16S rRNA sequencing. B1 can grow with diverse 4-hydroxybenzoic acid esters, and the growth status was the best when methyl ester was the only carbon source. Genes encoding carboxylesterase and monooxygenase were successfully expressed in E. coli. The optimal reaction conditions of recombinant carboxylesterase were pH 8.0 and 30℃ (30 min). The activity of recombinant monooxygenase depended on coenzyme, and Mg²⁺, Mn²⁺, Zn²⁺, and Fe³⁺ can enhance the activity of monooxygenase. As further identified by fluorescent quantitative PCR, z13175 and z09075 were genes encoding carboxylesterase. In the absence of oxygen, B1 strain carried out anaerobic respiration and reduced nitrate to nitrite.[Conclusion] Carboxylesterase and monooxygenase are key enzymes in the conversion of 4-hydroxybenzoic acid esters to protocatechuic acid, which play an important role in the biodegradation of the esters.

Key words:4-hydroxybenzoic acid esters;carboxylesterase;monooxygenase;heterologous expression