Cloning and expression of acidstable, high maltose-forming, Ca2+-independent α-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis

Abstract

The α-amylase encoding gene from acidophilic bacterium Bacillus acidicola was cloned into pET28a(+) vector and expressed in Escherichia coli BL21 (DE3). The recombinant E. coli produced a 15-fold higher α-amylase than B. acidicola strain. The recombinant α-amylase was purified to homogeneity by one-step nickel affinity chromatography using Ni(2+)-NTA resin with molecular mass of 62 KDa. It is active in the pH range between 3.0 and 7.0 and 30 and 100 °C with optimum at pH 4.0 and 60 °C. The enzyme is Ca(2+)-independent with K (m) and k (cat) values (on soluble starch) of 1.6 mg ml(-1) and 108.7 s(-1), respectively. The α-amylase of B. acidicola is acidstable, high maltose forming and Ca(2+)-independent, and therefore, is a suitable candidate for starch hydrolysis and baking.

The optimized production, purification, characterization, and application in the bread making industry of three acid‐stable alpha‐amylases isoforms from a new isolated Bacillus subtilis strain US586

“…Findings showed that maltotriose is the main product involved after a 24 hr of incubation, whereas maltose was the major end product after 48 hr of incubation (Figure ). These products were similar to those produced by Bacillus acidiocola (Sharma & Satyanarayana, ).…”

“…B. licheniformis ATCC 9945a was also observed to generate six amylase isoforms (Natasa, Josep, & Zoran, ), while only two isoforms with the same physicochemical properties were delivered by Bacillus subtilis X‐23 through carboxyl truncation (Kohji et al, ). Bacterial amylases were chosen over other origins because of their specific properties (Goesaert et al, ) including an optimum acidic pH and an intermediate thermostability (Sharma & Satyanarayana, ). B. subtilis strains, were considered as an important cell factory producing α‐amylase (Messaoud, Mabrouk, Jemli, & Bejar, ; Villaverde & Miralles, ) and are endotoxin‐free (Jan & Michael, ; Liu et al, ).…”

“…B. licheniformis ATCC 9945a was also observed to generate six amylase isoforms (Natasa, Josep, & Zoran, 2011), while only two isoforms with the same physicochemical properties were delivered by Bacillus subtilis X-23 through carboxyl truncation (Kohji et al, 1999). Bacterial amylases were chosen over other origins because of their specific properties (Goesaert et al, 2009) including an optimum acidic pH and an intermediate thermostability (Sharma & Satyanarayana, 2012).…”

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

“…Woodward’s reagent K is a negatively charged reagent that covalently and irreversibly modifies carboxyl-containing amino acid residues (Adsul et al, 2009; Kumar and Satyanarayana, 2013). Strong inhibitory effect of NBS indicated structural and catalytic role of tryptophan residues in Ba-Gt-amy (Sharma and Satyanarayana, 2012). …”

Genome Sequence of Tumebacillus flagellatus GST4, the First Genome Sequence of a Species in the Genus Tumebacillus

“…Therefore, acid-resistant amylases are the preferred enzymes for starch hydrolysis in several industrial processes (6, 7). For example, ethanol industries mainly use commercially available acid-resistant glucoamylase, α-amylase, and pullulanase (8).…”