Cloning and expression of a gene with phospholipase B activity from Pseudomonas fluorescens in Escherichia coli

► This is the first report on the cloning of a plb gene from the genus Pseudomonas. ► Pf-PLB from P. fluorescens BIT-18 is a member of a new phospholipase B family. ► The overproduction of Pf-PLB is of interest for the applications in many industries. A gene from Pseudomonasfluorescens BIT-18 encoding a protein with phospholipase B activity (Pf-PLB) was cloned in E. coli BL21 (DE3). The open reading frame consists of 1272bp and potentially encodes a protein of 423 amino acid residues with a calculated molecular mass of 45.8kDa. The nucleotide sequence of Pf-PLB is 45%, 42%, 41%, 40%, 33%, and 31% identical to that of Bifidobacteriumanimals, Mycobacteriumparascrofulaceum, Acidobacteriumcapsulatum, Lactobacillusjohnsonii, Moraxellabovis, and Moraxellacatarrhalis, respectively. The His-tagged protein was purified by affinity chromatography and the eluted protein hydrolyzed both the 1- and 2-ester bond of phosphatidylcholine. The recombinant Pf-PLB had optimal activity at pH 6.0 and 30°C, and it showed 20.1% higher efficiency in the conversion rate of the phosphorus content than the wild-type.