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

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.