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

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 Hg2+ ions and by N-bromosuccinimide, but the thiol inhibitors iodoacetate, 4chloromercuribenzoate and N-ethylmaleimide had either no effect or a slightly inhibitory effect. B-Cyclodextrin, an inhibitor of pullulanase, was not inhibitory.

Novel α-Amylase That Is Highly Resistant to Chelating Reagents and Chemical Oxidants from the Alkaliphilic Bacillus Isolate KSM-K38

“…These amylolytic enzymes, especially ␣-amylase and pullulanase, are very important, particularly in the food and detergent industries (1,28). We have found and characterized some unique debranching enzymes, such as a highalkaline pullulanase (4), an alkali-resistant neopullulanase (16), an alkaline isoamylase (6), and an alkaline amylopullulanase (5) from cultures of alkaliphilic Bacillus strains. These alkaline amylolytic enzymes can be used as effective additives in laundry and automatic dishwashing detergents operating under high alkalinity, as we also reported alkaline cellulases and a highly alkaline protease from alkaliphilic Bacillus strains (21).…”

Extra-cellular isoamylase production by Rhizopus oryzae in solid-state fermentation of agro wastes

“…This rapid rate of growth conferred with the similar growth rate of 70-72 h taken by other isoamylase producer such as Bacillus sp (Ara et al, 1993), Flavobacterium sp, Micrococcus sp and Arthrobacter sp (Yamada et al, 1994). Enzyme production did not further increase after 72 h of growth in SSF (Fig.…”

“…5), which was higher than that from the mould Hendersonula toruloidea, cultivated at pH 5.5 (Odibo et al, 1992), Xanthomonas maltophila showing a broad range of pH preference 6-8 (Yamada et al, 1994), but lower than Bacillus sp (Ara et al, 1993). …”

“…But with the increase in temperature, under the influence of thermal dimorphism (Morrow and Fraser, 2009) it was converted to pellet form with drastic reduction in isoamylase synthesis. Although a lower temperature of 25°C was preferred by Lipomyces kononenkoae (Spancer Martins, 1982), the cultivation temperature for almost all of the reported isoamylase producer ranged between 28°-30°C (Ueda and Nanri, 1967;Ara et al, 1993;Fang et al, 1994;Yamada et al, 1994;Takahashi et al, 1996;Olempska Beer, 2007).…”

Pullulanase: A Potential Enzyme for Industrial Application

“…Glycogen and amylopectin-debranching enzyme such as pullulanase and isoamylase can be used as an effective additive in dish washing and laundry detergent as reported by for alkaline endo glucanase from some strain of Bacillus the properties of which also fulfill the essential requirements for enzyme to be used for such purposes (Ara 1993). …”