Production, purification and characterization of an extracellular lipase from Mucor hiemalis f. hiemalis

Abstract

Mucor hiemalis f. hiemalis is a major contaminant of cameroonian palm fruit and produces an inducible extracellular lipase in batch fermentation. Rape oil was the best inducer for enzyme production, with the highest activity being achieved after 6 days of incubation. The enzyme was purified 2200-fold by ultrafiltration, ammonium sulfate fractionation, Sephadex G75 chromatography, Q-Sepharose chromatography, and Sephacryl S-200 chromatography. The purified enzyme showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the lipase was estimated to be 49 kDa using gel filtration on Sephadex G75, and 49 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was identified as a glycoprotein with pI of 4.6. The N-terminal amino acid sequence data (19 residues) and the amino acid composition were determined. The optimum pH and temperature for activity of the enzyme were 7.0 and 40°C, respectively. The lipase was stable in the pH range of 4–9 and at 45°C for 15 min. It hydrolyzed both synthetic and natural triglycerides with optimal activities recorded on tricaprylin and rape oil, respectively. Ca 2+, Mg 2+, Co 2+, Mn 2+, and Na +-enhanced lipase activity, whereas Fe 2+, Cu 2+, Ba 2+, and surfactants—such as taurocholic acid, triton X-100, and Tween 20—strongly reduced lipase activity. The enzyme activity was not affected by EDTA (ethylenediaminetetraacetic acid disodium dihydrate), PMSF (phenylmethylsulfonylfluoride), (p-chloromercuribenzoic), and Benzamidine.