Improvement of the optimum temperature of lipase activity for Rhizopus niveus by random mutagenesis and its structural interpretation.
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.
Kohno
M
M
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
Enatsu
M
M
Funatsu
J
J
Yoshiizumi
M
M
Kugimiya
W
W
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
D011994:Recombinant Proteins; D008049:Lipase