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

Abstract

A novel ␣-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60°C. The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction. The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM. Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H 2 O 2 (1.8 M).Starch, a main component of our daily diet, is frequently found not only in food residues on dishes but also in food stains on clothes (36). Enzymatic hydrolysis of starch is catalyzed by ␣-amylase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.1), ␤-amylase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.2), glucoamylase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.3), ␣-glucosidase (1,4-␣-D-glucan glucanohydrolase; EC 3.2.1.20), and debranching enzymes such as pullulanase (pullulan 6-glucanohydrolase; EC 3.2.1.41) and isoamylase (glycogen 6-glucanohydrolase; EC 3.2.1.68). 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). In particular, the alkaline amylopullulanase is unique in that it hydrolyzes ␣-1,6 and ␣-1,4 linkages in various carbohydrates at different active sites (3, 12).␣-Amylases are used widely in technical applications, such as in bread making, production of glucose and/or fructose syrups and fuel ethanol from starches, and desizing of textiles and paper. The demand for ␣-amylase in laundry and automatic dishwashing detergents has also been growing for several years (36). However, most of the Bacillus ␣-amylases, such as the enzymes from Bacillus licheniformis (BLA) (29), Bacillus amyloliquefaciens (BAA) (38), and Bacillus stearothermophilus (BSA) (23), are acid or neutral enzymes having pH optima at around 6.5. These neutral enzyme...

Alkaliphiles

“…The four conserved regions were found in the deduced amino acid sequence. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C. Furthermore, they 37) improved the thermostability of the amylase by deleting an arginineglycine residue in that molecule.…”

Partial characterization of amylases of two indigenous Central Amazonian rhizobia strains

“…The amylases produced by several bacterial sources, including Bacillus sp., have a variety of pH profiles. The maximum activity of most of the enzymes earlier reported has been in the pH range 5.0-12.0 (Kim et al, 1995;Leveque et al, 2000;Hagihara et al, 2001;Gomes et al, 2003;Goyal et al, 2005). In spite of earlier reports on the amylase activity at low pH for some amylases (Buonocore et al, 1976;Ohdan et al, 1999), there are few reports on amylases that have maximum activity at pH lower than 5.0 (Vihinen and Mäntsälä, 1989;Matzke et al, 1997;Jorgensen et al, 1997).…”

“…Inhibition or no effect in the presence of some mono and trivalent cations (1 mM, 5 mM, 10 mM or 50 mM) were also documented for amylases from Lactobacillus manihotivorans LMG 18010T (Aguilar et al, 2000), Bacillus amyloliquefaciens (Sarikaya and Gürgün, 2000), Bacillus sp. KSM-K39 (Hagihara et al, 2001) and Bacillus sp. (Cordeiro et al, 2002).…”

“…However, 1 mM Hg 2+ had no inhibitory effect on amylase activity of Bacillus sp. strain KSM-K38 (Hagihara et al, 2001). These results suggested that the effect of Hg 2+ could vary among the microbial amylases, as observed for other heavy metal ions such as Cd 2+ , Co 2+ , Fe 2+ and Ni 2+ (Pandey et al, 2000;Lo et al, 2001;Dey et al, 2003).…”

Purification and characterization of extracellular α-amylase from a thermophilic Anoxybacillus thermarum A4 strain

“…These findings are in line with earlier reports showing that calcium cation is essential for enzyme folding 27, Aspergillus oryzae strain S2 28, Bacillus stearothermophilus 29 . The effect of EDTA alkaliphilic Bacillus species varies considerably, some being unaffected at EDTA concentrations as high as 100 mM 30 , while others were completely inhibited in the presence of low EDTA concentration, e.g. the α-amylase of Bacillus sp.…”