Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization

Abstract

Different bacterial isolates with amylolytic activity were insulated from various honey samples. The most active isolate was identified by the molecular 16SrRNA sequence technique as Bacillus atrophaeus NRC1. The bacterium showed maximum amylase production under optimum culture conditions at pH 6.0, 40 °C and after 24 h incubation. Two amylase isoenzymes (AmyI and AmyII) from Bacillus atrophaeus NRC1 have been purified to homogeneity by using ammonium sulfate precipitation, Sephacryl S-200 and DEAE-Sepharose chromatography. The major isoenzyme, AmyI, had a specific activity 4635 U/mg proteins with molecular weight of 61 kDa using SDS-PAGE electrophoresis. The maximum activity of AmyI against starch was determined at pH 6.0 and 50 °C. AmyI was stable up to 50 °C after incubation for 30 min, retained 65 and 23% of its activity at 60 and 70 °C, respectively. Pre-incubation with Ca²⁺, Mg²⁺ and Ba²⁺ cations for 30 min enhanced the enzyme activity; while it was completely inhibited by Hg²⁺. Varied inhibition degree of the enzyme activity was determined with K⁺, Ni²⁺, Zn²⁺, Na²⁺ and Cu²⁺ ions. AmyI was inhibited by EDTA, PMSF and SDS, while it was activated by l-Cysteine-HCl and DTT. AmyI had the ability to degrade starch, amylopectin, glycogen, amylose and lacked the affinity towards β-1,4-linked xyloses.

Keywords: Bacillus atrophaeus; Characterization; Purification; α-amylase.