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

研究成果: Article査読

51 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)315-324
ページ数10
ジャーナルBBA - General Subjects
1243
3
DOI
出版ステータスPublished - 1995 4 13
外部発表はい

ASJC Scopus subject areas

  • 生物理学
  • 生化学
  • 分子生物学

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