An active-site mutation causes enhanced reactivity and altered regiospecificity of transglucosylation catalyzed by the Bacillus sp. SAM 1606 α-glucosidase

Misa Inohara-Ochiai, Maki Okada, Toru Nakayama, Hisashi Hemmi, Takashi Ueda, Takashi Iwashita, Yukiko Kan, Yuji Shibano, Toshihiko Ashikari, Tokuzo Nishino

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

Bacillus sp. SAM1606 α-glucosidase catalyzes the transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-O(G)-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were allowed to react with 1.75 M sucrose at 60°C, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6- glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6- O(F)-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that the amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the Bacillus cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.

Original languageEnglish
Pages (from-to)431-437
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume89
Issue number5
DOIs
Publication statusPublished - 2000

Keywords

  • Bacillus
  • Regiospecificity
  • Transglucosylation
  • α-amylase family
  • α-glucosidase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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