Separation of Functional Domains for the α-1,4 and α-1,6 Hydrolytic Activities of a Bacillus Amylopullulanase by Limited Proteolysis with Papain

Katsutoshi Ara, Kazuaki Igarashi, Hiroshi Hagihara, Kazuhisa Sawada, Tohru Kobayashi, Susumu Ito

研究成果: Article査読

16 被引用数 (Scopus)

抄録

An amylopullulanase (APase) from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both α-1,6 linkages in pullulan and α-1,4 linkages in other polysaccharides, each being maximally active at an alkaline pH, to generate oligosaccharides. We analyzed proteolytic fragments that were produced by exposing pure APase to various proteases, to identify its catalytic domain(s). The intact, pure 210-kDa APase was partially digested with papain for a short time, yielding simultaneously two smaller non-overlapping active fragments, designated amylose-hydrolyzing fragment (AHF114,114 kDa) and pullulan-hydrolyzing fragment (PHF102, 102 kDa). The two truncated protein fragments, each containing a single catalytic domain, were purified to homogeneity. The purified AHF114 and PHF102 had similar enzymatic properties to the amylase and pullulanase activities, respectively, of intact APase. The partial amino-terminal sequences of APase and AHF114 were both Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and that of PHF102 was Thr-Val-Pro-Leu-Ala-Leu-Val-Ser-Gly-Glu-Val-Leu-Ser-Asp-Lys-Leu. These results were direct evidence that the α-1,6 and α-1,4 hydrolytic activities were associated with two different active sites in this novel enzyme. Our alkaline APase is obviously a “biheaded enzyme”.

本文言語English
ページ(範囲)634-639
ページ数6
ジャーナルBioscience, Biotechnology and Biochemistry
60
4
DOI
出版ステータスPublished - 1996 1
外部発表はい

ASJC Scopus subject areas

  • バイオテクノロジー
  • 分析化学
  • 生化学
  • 応用微生物学とバイオテクノロジー
  • 分子生物学
  • 有機化学

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