Purification and characterization of an alkaline amylopullulanase with both α-1,4 and α-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378

Katsutoshi Ara, Katsuhisa Saeki, Kazuaki Igarashi, Mikio Takaiwa, Takaaki Uemura, Hiroshi Hagihara, Shuji Kawai, Susumu Ito

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

The novel alkaline amylopullulanase produced by alkalophilic Bacillus sp. KSM-1378 was purified to an electrophoretically homogeneous state from culture medium. The purified enzyme was a glycoprotein with an apparent molecular mass of about 210 kDa and an isoelectric point of pH 4.8. The N-terminal amino acid sequence was Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and showed no homology to the N-terminal regions of other amylopullulanases reported to date. The enzyme was able to attack specifically the α-1,6 linkages in pullula.n to generate maltotriose as the major end product, as well as the α-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and α-amylase activities were pH 9.5 and 50°C and pH 8.5 and 50°C respectively. Both activities were strongly inhibited by well characterized inhibitors, such as diethyl pyrocarbonate and N-bromosuccinimide. The pullulanase activity was specifically inactivated by Hg2+ ions, α-cyclodextrin and β-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of α-1,4-linked substrates and one for the cleavage of α-1,6-linked substrates.

Original languageEnglish
Pages (from-to)315-324
Number of pages10
JournalBBA - General Subjects
Volume1243
Issue number3
DOIs
Publication statusPublished - 1995 Apr 13

Keywords

  • (Bacillus)
  • Amylase
  • Amylopullulanase
  • Pullulanase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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