源于红树林土壤宏基因组文库的新型磷脂酶A1基因的筛选、克隆表达及酶学性质

Cloning, expression and characterization of a novel phospholipase A1 gene from the metagenomic library of mangrove soil

摘要:

【目的】利用宏基因组学的方法从红树林土壤中筛选新型酯水解酶类。【方法】构建红树林土壤宏基因组文库，采用以三丁酸甘油酯为底物的功能筛选方法，对筛选出的阳性克隆进行系统发育树分析，实现新型磷脂酶A1基因的原核表达，研究重组酶的酶学性质。【结果】筛选到一个新的磷脂酶A1编码基因phop1413 (GenBank登录号KF767097)，测序表明其全长1 413 bp，可编码470个氨基酸残基，表达蛋白约51.7 kD，表达量高达220 mg/L，NCBI中Blast比对及系统进化树分析显示该蛋白属于磷脂酶类的fAMILY Ⅵ家族；酶学性质分析表明，该重组酶的最适反应底物为对硝基苯酚己酸酯，比酶活为124 U/mg；最适反应温度为54 °C，最适pH 7.8；50 °C热处理1.5 h剩余相对酶活为44%，表现出很好的热稳定性。【结论】通过构建宏基因组文库利用功能筛选方法获得一个新型磷脂酶A1基因；研究中获得的新型磷脂酶A1性质较好，可用于植物油酶法脱胶。

Abstract:

[Objective] The aim was to isolate new hydrolytic enzymes from themetagenomic library from mangrove soil. [Methods] To isolate new hydrolytic enzymes, we constructed a metagenomic library from mangrove soil and screened clones with lipolytic activities by a function-driven approach based on a tributyrin hydrolysis. The identification of Phop1413 was based on deduced amino acid sequence comparison and phylogenetic analysis. [Results] One new phospholipase A1 gene phop1413 (GenBank KF767097) was finally identified from the library by functional screening. The result of BLASTp analysis revealed that phop1413 consisted of an open reading frame of 1 413 bp and encoded a protein of 470 amino acids. The protein showed 42% amino acid identity to phospholipase from Pseudomonas (WP 018928790.1). According to the phylogenetic analysis of Phop1413, Phop1413 belonged to FAMILY VI of lipase. Phop1413 was then subcloned to the express vector pET-32a(+), and overexpressed in E. coli BL21 with 1 mmol/L IPTG induction at 30 °C. The overexpressed protein revealed a molecular weight of 51.7 kD. A detailed analysis of the enzyme’s substrate spectrum with eight different substrates revealed that Phop1413 could hydrolyze a wide variety of substrates. Phop1413 showed the highest activity with p-Nitrophenyl caproate (C6). The optimum temperature of Phop1413 was detemined to be 54 °C, and the recombinant enzyme showed an optimum pH of 7.8. The enzyme retained 44% initial activity after incubating at 50 °C for 1.5 h. It indicated that Phop1413 had a good thermostability. [Conclusion] A new phospholipase A1 gene phop1413 was screened by function-driven approach from metagenomic library. The characterization of Phop1413 is good enough to apply to industry.