Amino acid residues specific for the catalytic action towards α-1,6- glucosidic linkages in Klebsiella pullulanase

Mitsuo Yamashita, Dai Matsumoto, Yoshikatsu Murooka

Research output: Contribution to journalArticlepeer-review

34 Citations (Scopus)


Mutations were introduced at residues His607, Asp677, His682, and His833 in pullulanase from Klebsiella aerogenes in order to probe the role of these amino acid residues, which are located in the four conserved regions of the α-amylase family, in the action of the enzyme towards a-1,6-glucosidic linkages. For the mutations, His was replaced by Ash and Ala, and Asp by Asn and Ser. Amino acid substitutions for His607, Asp677, or His833 resulted in complete loss of enzyme activity. In contrast, the mutations at His682 still retained their activities. The binding affinity of these variants for α- or β-cyclodextrine (CD), which are competitive inhibitors for pullulanase, was measured using an α-CD Sepharose column. The mutations at His833 did not change the binding affinity for α-CD, whereas the mutations at His607 or Asp677 resulted in these two variants losing their binding ability towards pullulan. These results suggest that in Klebsiella pullulanase, His607 and Asp677 participate in substrate binding and His833 is involved in catalysis, but His682 may be not in the active site. We also found new amino acid consensus sequences specific for starch debranching enzymes in two oligo- 1,6-glucosidases, several pullulanases, and an isoamylase. Two amino acid residues in the predicted consensus region of Klebsiella pullulanase, Tyr559 and Tyr564, were replaced by Ala or Phe. The Tyr559 variants resulted in complete loss of pullulanase activity without seriously affecting the binding affinities for α-CD and pullulan. The mutations at Tyr564 did not completely inactivate the enzymes, but dramatically decreased the activity. Thus, the region in Klebsiella pullulanase that includes Tyr559-Tyr564 probably participates in catalysis specific towards α-1,6-glucosidic linkages in starch debranching enzymes.

Original languageEnglish
Pages (from-to)283-290
Number of pages8
JournalJournal of Fermentation and Bioengineering
Issue number4
Publication statusPublished - 1997 Jan 1
Externally publishedYes


  • Active site
  • Site-directed mutagenesis
  • Starch- debranching enzyme
  • Substrate-binding site

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology


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