Purification and characterization of an alkaline isoamylase from an alkalophilic strain of Bacillus

K. Ara, K. Saeki, S. Ito

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Alkaline isoamylase (glycogen 6-glucanohydrolase, EC 3.2.1.68) activity was detected in the culture medium of an alkalophilic strain of Bacillus sp., designated KSM-3309, which was isolated from a soil sample. This novel enzyme was purified to homogeneity from the culture filtrate by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and DEAE-Bio-Gel A, and gel filtration on Sephacryl S-200. The purified enzyme had a pH optimum of approximately 9.0, and displayed maximum catalytic activity at 55°C. The enzyme had a molecular mass of 65 kDa, as determined by both SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200. The isoelectric point was 4.2. This enzyme cleaved the branching points of both amylopectin and glycogen, and incubation of the enzyme with these glucans caused large increases in coloration of the iodine reagent. Amylose, pullulan and maltose were practically insensitive to the enzyme. The enzyme activity was inhibited by Hg 2+ ions and by N-bromosuccinimide, but the thiol inhibitors iodoacetate, 4-chloromercuribenzoate and N-ethylmaleimide had either no effect or a slightly inhibitory effect. β-Cyclodextrin, an inhibitor of pullulanase, was not inhibitory.

Original languageEnglish
Pages (from-to)781-786
Number of pages6
JournalJournal of General Microbiology
Volume139
Issue number4
DOIs
Publication statusPublished - 1993 Jan 1
Externally publishedYes

Fingerprint

Isoamylase
Bacillus
Enzymes
Gel Chromatography
1,4-alpha-Glucan Branching Enzyme
Bromosuccinimide
Chloromercuribenzoates
Iodoacetates
Amylopectin
DEAE-Cellulose Chromatography
Amylose
Ethylmaleimide
Maltose
Glucans
Isoelectric Point
Cyclodextrins
Ammonium Sulfate
Glycogen
Sulfhydryl Compounds
Iodine

ASJC Scopus subject areas

  • Microbiology

Cite this

Purification and characterization of an alkaline isoamylase from an alkalophilic strain of Bacillus. / Ara, K.; Saeki, K.; Ito, S.

In: Journal of General Microbiology, Vol. 139, No. 4, 01.01.1993, p. 781-786.

Research output: Contribution to journalArticle

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