Separation and characterization of two molecular forms of Geotrichum candidum lipase.
Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA. As expected, two forms of lipase (lipases I and II) were actually isolated by hydrophobic interaction chromatography after a multistep procedure including ammonium sulfate fractionation, anion exchange chromatography, and gel filtration of the culture filtrate. Lipase I, the first eluted fraction, was the predominant form, and more than 80% of the total activity was attributed to this form. Amino acid sequence analysis of the amino and carboxyl termini of these two enzyme preparations indicated that lipase I was the product of the lipase gene whose cDNA had previously been cloned and sequenced [Shimada et al. (1989) J. Biochem. 106, 383-388]. Lipase II, on the other hand, had similar amino acid composition, but different terminal sequences which were not found in the primary structure of lipase I deduced from the cDNA sequence. These results gave lines of evidence for the expression of truely different lipase genes and ruled out the possibility that the observed multiple forms are caused by proteolytic digestion. The molecular mass estimated by SDS-PAGE and the isoelectric point of lipase I were 64 kDa and 4.3, while those of lipase II were 66 kDa and 4.3, respectively. The two lipases had essentially the same specific activities, substrate specificities, pH stabilities, and optimal temperatures, but different pH optima and thermal stabilities.
Sugihara
A
A
Osaka Municipal Technical Research Institute.
Shimada
Y
Y
Tominaga
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D015139:Blotting, Southern; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005848:Geotrichum / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification; D008049:Lipase / Q000032:analysis / Q000302:isolation & purification*; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D011489:Protein Denaturation
D000596:Amino Acids; D004271:DNA, Fungal; D007527:Isoenzymes; D008049:Lipase