Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme

Mitsuo Yamashita, Takuya Kinoshita, Michiko Ihara, Tomomi Mikawa, Yoshikatu Murooka

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

To study the structure and function of pullulanase from Klebsiella aerogenes, a method involving random mutagenesis of the entire gene for pullulanase was used. Out of 50, 000 clones screened at high temperature, seven genes for mutant proteins were identified by DNA sequencing. The amino acid substitutions in the seven mutant proteins were clustered on the NH2-terminal side of the four conserved regions found in α-amylases. These mutant pullulanases were classified into two types: those whose catalytic activity was altered and those whose thermal stability was increased. The results presented here and in previous reports suggest that pullulanase from K. aerogenes has similar active sites to those of α-amylases with the four conserved regions, as well as another substrate-binding site closer to the NH2-terminus. The plate assay method used for isolation of thermostable variants may be applicable to the generation of useful variants of other enzymes.

Original languageEnglish
Pages (from-to)1233-1246
Number of pages14
JournalJournal of Biochemistry
Volume116
Issue number6
Publication statusPublished - 1994 Dec 1
Externally publishedYes

Fingerprint

Mutagenesis
Amylases
Enzymes
Genes
Mutant Proteins
Proteins
Binding sites
Amino acids
Assays
Catalyst activity
Thermodynamic stability
DNA
Substitution reactions
Substrates
Binding Sites
Amino Acids
Temperature
pullulanase
Amylase
Protein

Keywords

  • Pullulanase
  • Random mutagenesis

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Radiology Nuclear Medicine and imaging
  • Molecular Biology
  • Biochemistry

Cite this

Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme. / Yamashita, Mitsuo; Kinoshita, Takuya; Ihara, Michiko; Mikawa, Tomomi; Murooka, Yoshikatu.

In: Journal of Biochemistry, Vol. 116, No. 6, 01.12.1994, p. 1233-1246.

Research output: Contribution to journalArticle

Yamashita, Mitsuo ; Kinoshita, Takuya ; Ihara, Michiko ; Mikawa, Tomomi ; Murooka, Yoshikatu. / Random mutagenesis of pullulanase from Klebsiella aerogenes for studies of the structure and function of the enzyme. In: Journal of Biochemistry. 1994 ; Vol. 116, No. 6. pp. 1233-1246.
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