Site-specific mutagenesis at positions 272 and 273 of the Bacillus sp. SAM1606 α-glucosidase to screen mutants with altered specificity for oligosaccharide production by transglucosylation

Maki Okada, Toru Nakayama, Akio Noguchi, Masayasu Yano, Hisashi Hemmi, Tokuzo Nishino, Takashi Ueda

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14 Citations (Scopus)

Abstract

The Bacillus sp. SAM1606 α-glucosidase catalyzes the transglucosylation of sucrose to produce theanderose (6-OG-glucosylsucrose) as the major transfer product along with the other di-, tri-, and tetrasaccharides. To obtain an α-glucosidase variant(s) producing theanderose more abundantly, we carried out site-specific mutagenesis studies, in which an amino acid residue (Gly273 or Thr272) near the putative catalytic site (Glu271) of this α-glucosidase was replaced by all other naturally-occurring amino acids. Each mutant, whose concentration was set at 2.6 U/ml (sucrose-hydrolyzing units), was reacted at 60°C and pH 6.0 with 1.75 M sucrose, and the course of the oligosaccharide production was monitored by HPLC to systematically analyze the effects of amino acid substitutions on the specificity of transglucosylation. The analysis clearly showed site- and residue-dependent differential effects of substitution near the catalytic site on the specificity of oligosaccharide production. For example, mutants with substitution at position 273 by aromatic amino acids or His virtually lost the ability to produce oligosaccharides by transglucosylation. Mutants with substitution at position 272 by amino acids that were bulkier than the wild-type Thr showed enhanced production of tetrasaccharides; whereas, mutants with substitution at position 273 by Lys and Arg exclusively produced disaccharidal transfer products. The highest specificity for theanderose formation (i.e. the highest content of theanderose in the reaction product) was obtained with the T272I mutant, which showed 1.74 times higher productivity (per sucrose-hydrolyzing unit) of theanderose than that of the wild-type enzyme.

Original languageEnglish
Pages (from-to)265-274
Number of pages10
JournalJournal of Molecular Catalysis - B Enzymatic
Volume16
Issue number5-6
DOIs
Publication statusPublished - 2002 Feb 18

Keywords

  • Mutagenesis
  • Oligosaccharide
  • Theanderose
  • Transglucosylation
  • α-glucosidase

ASJC Scopus subject areas

  • Catalysis
  • Bioengineering
  • Biochemistry
  • Process Chemistry and Technology

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