Site-directed Mutagenesis of Catalytic Active-site Residues of Taka-amyiase A

Tadashi Nagashima, Setsuzo Tada, Katsuhiko Kitamoto, Katsuya Gomi, Chieko Kumagai, Hiroko Toda

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no α-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.

Original languageEnglish
Pages (from-to)207-210
Number of pages4
JournalBioscience, Biotechnology, and Biochemistry
Volume56
Issue number2
DOIs
Publication statusPublished - 1992 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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