Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation

Yoshiaki Nishiya, Naoko Harada, Shin Ichi Teshima, Mitsuo Yamashita, Isao Fujii, Noriaki Hirayama, Yoshikatsu Murooka

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Random mutagenesis was used to enhance the thermal stability of Streptomyces cholesterol oxidase. Four thermostable mutants were isolated and the following amino acid substitutions were identified: Ser103 to Thr (mutant S103T), Val121 to Ala (mutant V121A), Arg135 to His (mutant R135H) and Val145 to Glu (mutant V145E). The wild-type and mutant enzymes were purified and characterized. The properties of mutants S103T, V121A and R135H were similar to those of the wild type but they showed improved thermal stability. When the V145E mutation was introduced, the thermal stability of the enzyme was markedly increased and the optimum pH was desirably changed to encompass a broad range from acid to alkali. Analysis of multiple mutants constructed by site-directed mutagenesis showed that all the mutations except that of R135H had an additive influence on the other mutations. These mutational effects are discussed in terms of a three-dimensional structural model of the enzyme constructed on the basis of homology modelling.

Original languageEnglish
Pages (from-to)231-235
Number of pages5
JournalProtein Engineering
Volume10
Issue number3
Publication statusPublished - 1997 Mar
Externally publishedYes

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Cholesterol Oxidase
Mutagenesis
Cholesterol
Thermodynamic stability
Enzymes
Alkalies
Amino acids
Substitution reactions
Amino Acids
Acids
Oxidoreductases

Keywords

  • Cholesterol oxidase
  • Random mutagenesis
  • Streptomyces
  • Structural characterization
  • Thermostable mutants

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Nishiya, Y., Harada, N., Teshima, S. I., Yamashita, M., Fujii, I., Hirayama, N., & Murooka, Y. (1997). Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation. Protein Engineering, 10(3), 231-235.

Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation. / Nishiya, Yoshiaki; Harada, Naoko; Teshima, Shin Ichi; Yamashita, Mitsuo; Fujii, Isao; Hirayama, Noriaki; Murooka, Yoshikatsu.

In: Protein Engineering, Vol. 10, No. 3, 03.1997, p. 231-235.

Research output: Contribution to journalArticle

Nishiya, Y, Harada, N, Teshima, SI, Yamashita, M, Fujii, I, Hirayama, N & Murooka, Y 1997, 'Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation', Protein Engineering, vol. 10, no. 3, pp. 231-235.
Nishiya, Yoshiaki ; Harada, Naoko ; Teshima, Shin Ichi ; Yamashita, Mitsuo ; Fujii, Isao ; Hirayama, Noriaki ; Murooka, Yoshikatsu. / Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation. In: Protein Engineering. 1997 ; Vol. 10, No. 3. pp. 231-235.
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