Purification and Characterization of an Alkaline Lipase from Penicillium cyclopium PG37.

ABSTRACT

An extracellular, novel alkaline lipase produced by Penicillium cyclopium PG37 was purified by centrifugation, ammonium sulfate precipitation, and phenyl-Sepharose CL-4B, DEAE Sepharose fast flow and Sephadex G-75 column chromatographies. A 16.5-fold purification of the enzyme was achieved which had a specific activity of 5 200 u/mg protein, and the recovery of the activity was 33.2%. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and polyacrylamide gel electrophoresis (PAGE). The molecular weight of the native lipase was estimated to be about 29 kD by gel filtration using Sephadex G-150, and that of the denatured lipase was determined to be about 27.5 kD by its mobility on SDS-PAGE, indicating that the lipase was a monomer. The N-terminal amino acid sequence was determined with automatic protein sequencer to be ATADAAAFPD, which has no homology with other sequences of known lipases. The optimum temperature of the action of this enzyme was 25 degrees and the lipase was stable below 30 degrees, but only 30% of its activity remained after 20 min incubation at 40 degrees. The enzyme was stable at pH from 6.5 to 10.5, and its optimal pH for activity is 10.0. Low concentration of alkaline proteinase has little effect on the lipase PG37, therefore these two enzymes can be used as ingredients that are added to commercial detergents simultaneously.