Atypical Ca2+-independent, raw-starch hydrolysing [alpha]-amylase from Bacillus sp. GRE1: characterization and gene isolation

Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca2+-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l-1. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca2+ produced similar temperature optima of 65-70°C. The optimum pH was in the range of 5.5-6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0-9.0. The Vmax and K m values for soluble starch were 42 mg reducing sugar min-1 and 4.98 mg starch ml-1, respectively. Strong inhibitors of enzyme activity included Cu2+, Zn2+ and Fe2+. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme. [PUBLICATION ABSTRACT]