Abstract
The recombinant acidstable α-amylase (Ba-amy) of acidophilic bacterium Bacillus acidicola TSAS1 has been produced extracellularly using a combination of cloning (E. coli and P. pastoris) and physico-chemical treatment strategies. A total of 150,000U/L of Ba-amy were attained under constitutive promoter in P. pastoris, which is 15-fold higher than that of the wild strain B. acidicola (10,000U/L). The recombinant P. pastoris integrated two copies of Ba-amy under GAP promoter. The pure Ba-amy expressed in P. pastoris is a glycoprotein of 66kDa, which is optimally active at pH 4.0 and 60°C with a T 1/2 of 25min at 70°C. The K m, V max and K cat values of the recombinant Ba-amy are 1.66mg/mL, 53.6µmol/mg/min and 106.8/s, respectively. The enzyme generates maltose (30%), maltotriose (20%) and other higher maltooligosaccharides from starch, thus, useful in baking as an antistale. This is the first report on the optimization of extracellular production of recombinant acidic α-amylase of an acidophilic bacterium.
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