Production and partial characterization of thermostable and calcium-independent α-amylase of an extreme thermophile Bacillus thermooleovorans NP54

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Aim:An investigation was carried out on the production of α-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization.Methods and Results:The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 °C and pH 7.0, agitated at 200 rev min−1 with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml−1 and 250 μmol mg−1 protein min−1, respectively. The enzyme was optimally active at 100 °C and pH 8.0 with a half-life of 3 h at 100 °C. Both α-amylase activity and production were Ca2+ independent.Conclusions:Bacillus thermooleovorans NP54 produced calcium-independent and thermostable α-amylase.Significance and Impact of the Study:The calcium-independent and thermostable α-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the α-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.

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