Studies on the lipase from proteus vulgaris K80

Abstract

A facultatively anaerobic bacterium producing an extracellular alkaline lipase was isolated from the soil collected near a sewage disposal plant and identified to be a strain of Proteus vulgaris. The lipase of P. vulgaris was purified by ammonium sulfate precipitation and CM-Sepharose chromatography. The molecular mass of the purified enzyme was estimated to be 31 kDa by SDS-PAGE. This lipase was found to be an alkaline enzyme having maximum hydrolytic activity at pH 10, and yet it is fairly stable in a wide pH range from 5 to 11. The gene for the enzyme was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 861 bp coding for a polypeptide of 287 amino acid residues. The amino acid sequence of the lipase deduced from the nucleotide sequence of the gene has 46.3%, 41.5%, and 39.7% of sequence homolgies with the lipases from Pseudomonas fragi, P. aeruginosa, and P. glumae, respectively. Inspection of the sequence alignment with these lipases indicated that Ser79, Asp232, and His254 form a catalytic triad of the enzyme. This lipase is similar to group I lipases with respect to the molecular size and amino acid sequence. However, this enzyme is also similar to group II lipases including P. fluorescens and S. marcescens lipases in that it (i) does not contain an N-terminal signal sequence and (ii) it does not require any additional protein for active form. Therefore, P. vulgaris K80 lipase may be considered to be a new lipase with some properties similar to those of group I lipases and with some other characteristics to group II lipases. An expression plasmid, pKLE carrying the lipase gene was constructed. The lipase content in E. coli cells harboring the plasmid pKLE estimated from the specific activity of E. coli cell extract was more than 22% of total soluble protein, 4000-fold higher than that of E. coli JM83/pKLB. The enzyme displayed a partial interfacial activation toward p-nitrophenyl butyrate, indicating that this enzyme is able...