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

A gene from Pseudomonas fluorescens BIT-18 encoding a protein with phospholipase B activity (Pf-PLB) was cloned in E. coli BL21 (DE3). The open reading frame consists of 1272 bp and potentially encodes a protein of 423 amino acid residues with a calculated molecular mass of 45.8 kDa. The nucleotide sequence of Pf-PLB is 45%, 42%, 41%, 40%, 33%, and 31% identical to that of Bifidobacterium animals, Mycobacterium parascrofulaceum, Acidobacterium capsulatum, Lactobacillus johnsonii, Moraxella bovis, and Moraxella catarrhalis, 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 degrees C, and it showed 20.1% higher efficiency in the conversion rate of the phosphorus content than the wild-type. (C) 2011 Elsevier Ltd. All rights reserved.