Purification, Characterization, and Crystallization of Two Types of Lipase from Rhizopus niveus

Abstract

The purification and some properties of two types of lipase (Lipase I and Lipase II) from Rhizopus niveus are described. The enzymes were purified to homogeneity by column chromatographies on DEAE-Toyopearl (1 pass) and CM-Toyopearl (2 passes). Lipase I consists of two polypeptide chains [a small peptide with sugar moiety (A-chain) and a large peptide of molecular weight 34, 000 (B-chain)]. Lipase II has a molecular weight of 30, 000 consisting of a single polypeptide chain. Lipase I appeared to be converted to Lipase II by limited proteolysis by a specific protease a small amount of which is in the culture supernatant from Rh. niveus, because one of the peptides formed has the same N-terminal sequence and C-terminal amino acid as Lipase II, as well as the molecular mass estimated by SDS-PAGE. Lipase I had a pH optimum of 6. 0-6. 5 and a temperature optimum of 35°C, while, for Lipase II these values were pH 6. 0 and 40°C. Both enzymes were obtained in the crystalline state using the hanging drop method of vapor diffusion and PEG as the precipitating agents.