Cloning and expression of the gene encoding phospholipase A1 from Serratia sp. MK1 in Escherichia coli

Abstract

The gene encoding extracellular phospholipase A(1) of Serratia sp. MKI was cloned from a genomic DNA library. Formation of transparent halos on the PCY agar plates was used to identify E. coli carrying the phospholipase A(1) gene. A 4.2 kb EcoRI fragment was isolated and sequenced. From nucleotide sequences and expression of various plasmids, two open reading frames (plaA and plaS) involved in efficient expression of phospholipase A(1) in natural and recombinant host were identified. Extracellular phospholipase A(1) activity was identified as the gene product of plaA encoding 321 amino acids with a predicted M-W of 33 400. Analysis of the amino acid sequence revealed significant homology (around 70%) to phospholipase A(1) of Serratia liquefaciens and Yersinia enterocolitica. The sequence, -Gly-X-1-Ser-X-2-Gly-, known as a lipase-specific consensus sequence was also found in the bacterial phospholipase A(1). PlaS encoding a protein of 224 amino acids showed no enzymatic activity, but might be necessary for the efficient expression of phospholipase A(1) in E. coil. To further improve the production of phospholipase A(1) as a soluble and active form in E, coli, the effect of some parameters was examined. Surprisingly, a higher yield of soluble and active phospholipase A(1) could be obtained under the combined conditions of a lower temperature, an enriched medium, and a lower-strength promoter. (C) 1999 Elsevier Science B.V. All rights reserved.