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-red fluorescent plaques on rhodamine B-triolein plates was used to identify phages carrying the lipase gene. A 2.8-kb SalI fragment was subcloned into a plasmid, and lipase was expressed in Escherichia coli. Extracellular lipase was detected in the presence of the secretion plasmid pGSD6 carrying the genes prtD, -E, and -F, which guide the secretion of protease from Erwinia chrysanthemi. Determination of the nucleotide sequence of the entire cloned fragment revealed an open reading frame coding for a 613-amino-acid protein with a predicted M(r) of 64,800. Analysis of the amino acid sequence revealed significant homology (around 70%) to lipases of Pseudomonas fluorescens strains. The lipase-specific consensus sequence G-X1-S-X2-G resided in the amino-terminal 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, respectively, which are both found in proteins secreted by the class I secretion pathway. Lipase was purified from the supernatant of a culture carrying a lipase expression vector, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an M(r) of 64,000 for the purified protein. Our results suggest that the lipase of S. marcescens belongs to the group of extracellular enzyme proteins secreted by the class I secretion pathway.
Li
X
X
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934, USA.
Tetling
S
S
Winkler
U K
UK
Jaeger
K E
KE
Benedik
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012706:Serratia marcescens / Q000201:enzymology*
D011994:Recombinant Proteins; D008049:Lipase