Purification, Characterization, and cDNA Cloning of sn 1-Specific Triacylglycerol Lipase from Cordyceps militaris
Chang Woo Kwon, Jung-Ha Park, Min-Je Jo, Seon-Joo Lee, Kyung-Min Park, Pahn-Shick Chang
Dept. of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea

Based on the preliminary screening, Cordyceps militaris, known as a traditional medicinal ingredient in Eastern Asia, was selected as a promising candidate with high lipolytic activity and sn-1 positional specificity. The lipase from Cordyceps militaris was purified through sequential operation of ammonium sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography, and gel permeation chromatography. As a result, the purified lipase represented 94.54-purification fold with 2.47% yield and the monomeric structure with a molecular mass of approx. 60 kDa. From the enzymological charaterization of the lipase from Cordyceps militaris, the optimum pH and temperature were found to be 9.0 and 40°C, respectively. Aside from the environment at pH  2.0,  the purified lipase had remarkable stability at the broad range of pH 3.0-12.0 and retained above 84.48% of its original activity during 24 hr. In relation to kinetic parameters, Km, Vmax, and kcat/Km values were determined as 0.07 mM, 4.86 µmolmin-1mg-1, and 0.07 sec-1mM-1 with p-nitrophenyl palmitate as a substrate, respectively. Interestingly, it was revealed that Cordyceps militaris lipase preferred the cleavege at sn-1 position of triolein (sn-1 regiospecificity) by the thin layer chromatography showing the release of 1,2-diolein as main products. From the ESI-Q-TOF MS analysis and BLAST searching, the fragmented peptides indicated a high degree of homology to the domain of putative extracellar lipase in the genome sequence of Cordyceps militaris. The cDNA sequence of Cordyceps militaris lipase was obtained from the polymerase chain reaction with the gene specific primers designed according to the genome sequencing. An open reading frame (1,743 bp) was found to encode a putative protein comprising 581 amino acids and the recombinant protein was expressed in Escherichia coli systems.