Purification, characterization, and crystallization of two types of lipase from Rhizopus niveus.
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 degrees C, while, for Lipase II these values were pH 6.0 and 40 degrees C. Both enzymes were obtained in the crystalline state using the hanging drop method of vapor diffusion and PEG as the precipitating agents.
Kohno
M
M
Central Research Institute Tsukuba R & D Center, Fuji Oil Co., Ltd., Ibaraki, Japan.
Kugimiya
W
W
Hashimoto
Y
Y
Morita
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004795:Enzyme Stability; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D012233:Rhizopus / Q000201:enzymology*; D013816:Thermodynamics
D000596:Amino Acids; D006020:Glycopeptides; D006023:Glycoproteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D008049:Lipase