title: Improvement of the optimum temperature of lipase activity for Rhizopus niveus by random mutagenesis and its structural interpretation.
abstract: Random mutagenesis was used to improve the optimum temperature for Rhizopus niveus lipase (RNL) activity. The lipase gene was mutated using the error-prone PCR technique. One desirable mutant was isolated, and three amino acids were substituted in this mutant (P18H, A36T and E218V). The wild-type and this randomly mutated lipase were both purified and characterized. The specific activity of the mutant lipase was 80% that of the wild-type. The optimum temperature of the mutant lipase was higher by 15 degrees C than that of the wild-type. To confirm which substitution contributed to enhancing the optimum temperature for enzymic activity, two chimeric lipases from the wild-type and randomly mutated gene were constructed: chimeric lipase 1 (CL-1; P18H and A36T) and chimeric lipase 2 (CL-2; E218V). Each of the chimeric enzymes was purified, and the optimum temperature for lipase activity was measured. CL-1 had a similar optimum temperature to that of the wild-type, and CL-2 had a higher temperature like the randomly mutated lipase. The mutational effect is interpreted in terms of a three-dimensional structure for the wild-type lipase.
authors.affiliation: Central Research Institute, Tsukuba R&D Center, Fuji Oil Co. Ltd., 4-3 Kinunodai, Yawara-Mura, Tsukuba-gun, Ibaraki 300-2497, 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: J
authors.initials: J
authors.lastname: Funatsu
authors.forename: M
authors.initials: M
authors.lastname: Yoshiizumi
authors.forename: W
authors.initials: W
authors.lastname: Kugimiya
mesh_terms: D019943:Amino Acid Substitution; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D008049:Lipase