title: Thermal stability of Rhizopus niveus lipase expressed in a kex2 mutant yeast.
abstract: Lipase from Rhizopus niveus (RNL) has a complex structure, and recombinant RNL, has even more complex structural properties in the yeast, Saccharomyces cerevisiae. These properties are due to the processing and to the size of the glycosylated sugar chain. The processing site was presumed to be that for the proteinase product of the KEX2 gene in yeast. We therefore, constructed an expression system in which the KEX2 gene was disrupted to produce a non-processed type of lipase with high thermal stability. This type of lipase was thermally stable to a temperature 15 degrees C higher than that of each processed type of lipase. This non-processed lipase had 50% residual activity after 2 h at 50 degrees C, while the residual activity of the processed lipases was only 10% after 30-45 min of incubation at 50 degrees C. The CD spectrum of the non-processed type of lipase at 222 nm was almost unchanged by heating, suggesting that this group of lipases had a very rigid structure and that the peptide bond between the A- and B-chain contributed to maintain this rigid structure. On the other hand, the length of the sugar chain bound to the lipase had no effect on the thermal stability.
authors.affiliation: Central Research Institute, Tsukuba R&D Center, Fuji Oil Co. Ltd., 4-3 Kinunodai, Yawara-Mura, Tsukuba-gun, 300-2497, Ibaraki, Japan. kouno@mail.tkc.fujioil.co.jp
authors.forename: M
authors.initials: M
authors.lastname: Kohno
authors.forename: M
authors.initials: M
authors.lastname: Enatsu
authors.forename: R
authors.initials: R
authors.lastname: Takee
authors.forename: W
authors.initials: W
authors.lastname: Kugimiya
mesh_terms: D004795:Enzyme Stability; D006031:Glycosylation; D006358:Hot Temperature; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D043484:Proprotein Convertases; D012233:Rhizopus / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000235:genetics; D014162:Transfection
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D008049:Lipase; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae