Lysophospholipase L1 from Escherichia coli K-12 overproducer.
After screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain. Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies. The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography. The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate. The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E. coli strains bearing its cloned structural gene, pldB [Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S. (1985) J. Biochem, 98, 1117-1125]. The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C). Its restriction enzyme map was also different from that of cloned pldB. These results further confirmed that, in E. coli, there are two lysophospholipases with distinct characteristics.
Karasawa
K
K
Faculty of Pharmaceutical Sciences, University of Tokyo.
Kudo
I
I
Kobayashi
T
T
Homma
H
H
Chiba
N
N
Mizushima
H
H
Inoue
K
K
Nojima
S
S
D000595:Amino Acid Sequence; D003086:Bacteriocin Plasmids; D002462:Cell Membrane / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000201:enzymology; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008245:Lysophospholipase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010641:Phenotype; D010743:Phospholipids / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility; D013379:Substrate Specificity
D010743:Phospholipids; D004247:DNA; D008245:Lysophospholipase