GENE CLONING, SEQUENCE-ANALYSIS, PURIFICATION, AND SECRETION BY ESCHERICHIA-COLI OF AN EXTRACELLULAR LIPASE FROM SERRATIA-MARCESCENS

The gene encoding extracellular lipase of Serratia marcescens has been identified from a phage lambda genomic library. Formation of orange-r ed fluorescent plaques on rhodamine B-triolein plates was used to iden tify phages carrying the lipase gene. A 2.8-kb SalI fragment was subcl oned into a plasmid, and lipase was expressed in Escherichia coli. Ext racellular lipase was detected in the presence of the secretion plasmi d pGSD6 carrying the genes prtD, -E, and -F, which guide the secretion of protease from Erwinia chrysanthemi. Determination of the nucleotid e sequence of the entire cloned fragment revealed an open reading fram e coding for a 613-amino-acid protein with a predicted M(r) of 64,800. Analysis of the amino acid sequence revealed significant homology (ar ound 70%) to lipases of Pseudomonas fluorescens strains. The lipase-sp ecific consensus sequence G-X(1)-S-X(2)-G resided in the amino-termina l part of the protein, and carboxyl-terminal consensus sequences were an L-X-G-G-B-G-B-B-X repeat motif and a so-called aspartate box, respe ctively, which are both found in proteins secreted by the class I secr etion pathway. Lipase was purified from the supernatant of a culture c arrying a lipase expression vector, and analysis by sodium dodecyl sul fate-polyacrylamide gel electrophoresis revealed an M(r) of 64,000 for the purified protein. Our results suggest that the lipase of S. marce scens belongs to the group of extracellular enzyme proteins secreted b y the class I secretion pathway.