Improvement of the optimum temperature of lipase activity for Rhizopus niveus by random mutagenesis and its structural interpretation

Journal of Biotechnology
M KohnoW Kugimiya

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.

References

Jul 1, 1995·Protein Science : a Publication of the Protein Society·T P Creamer, G D Rose
Sep 1, 1996·Journal of Biochemistry·M KohnoY Morita
Sep 16, 2000·Journal of Biotechnology·M KohnoW Kugimiya

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Citations

Aug 21, 2008·Proteins·Ki Hyun NamKwang Yeon Hwang
Jan 11, 2005·Journal of Biotechnology·Go KagiyaKazutaka Yamamoto
Apr 7, 2009·Biochemical and Biophysical Research Communications·Ki Hyun NamKwang Yeon Hwang
Apr 12, 2002·Biochemical and Biophysical Research Communications·Zhaozhong HanMichael R Boyd

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