Purification and
Characterization of an Alkaline Lipase from Penicillium
cyclopium PG37
WU Min-Chen, WANG Shu, HUANG Wei-Da and SUN Chong-Rong
( Department of Biochemistry, Fudan University, Shanghai 200433,
China )
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 ℃ and the lipase was stable below 30 ℃, but only 30% of its
activity remained after 20 min incubation at 40 ℃. 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.
Key Words Penicillium cyclopium； alkaline lipase； purification and
characterization； N-terminal amino acid sequence
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