A jacalin-related lectin domain-containing lipase from chestnut (Castanea crenata): Purification, characterization, and protein identification

Abstract

A novel lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was discovered from Korean chestnut (Castanea crenata). The lipase was isolated and purified by ammonium sulfate precipitation and a fast protein liquid chromatography system equipped with HiTrap DEAE-Sepharose Fast Flow, HiTrap Q-Sepharose Fast Flow, and HiPrep Sephacryl S-100 Hi-Resolution columns. The purified C. crenata lipase showed a 15.8% yield, purification fold number of 465.8, and specific activity against triolein of 88.5 mU/mg. The enzyme exhibited hydrolytic activity toward tributyrin, trilaurin, and triolein, and was maximally active at pH 8.0 and 35 °C, with triolein used as the substrate. The activation energy (Ea) and deactivation energy (Ed) of triolein hydrolysis were 38.41 and 83.35 kJ/mol, respectively. In the enzyme kinetic study, Vmax, Km, and kcat were 110.58 mU/mg, 0.11 mM, and 0.221 min−1, respectively. The relatively low Km value indicated that the lipase has high affinity for its substrate. Moreover, Mg2+ and Ca2+ increased the lipase activity to 115.4% and 108.3%, respectively. The results of peptide fingerprinting revealed that the C. crenata lipase with a molecular weight of 33.3 kDa was structurally similar to the mannose-binding lectin of the jacalin-related lectin domain superfamily, implying that it has potential as a therapeutic agent for use in the biomedical industry.