Thermostable, Solvent, Surfactant, Reducing Agent and Chelator Resistant α-Amylase from Bacillus Strain IBT108: A Suitable Candidate Enables One-Step Fermentation of Waste Potato for High Butanol and Hydrogen Production

Abstract

α-Amylase is the major biocatalyst involved in saccharification process of starch-based biofuel production. Most of the α-amylases reported in literature are not stable and catalytically active in presence of metabolites (biosolvents, and bioacids), and other media components such as salts and reducing agents, hence starch-based biofuels are produced by two-step process of separate hydrolysis by α-amylase, and later the fermentation of biofuel production. Finding new α-amylase with special properties can assist to integrate the process of saccharification and biofuel fermentation. We report a new α-amylase produced from wild type Bacillus strain IBT108. This α-amylase (10.5 U/ml) can be produced from wheat bran (5%) based medium. It can be purified by diethylaminoethyl weak anion exchange-based fast protein liquid chromatography. Purified α-amylase has molecular mass of 68 kDa with a high specific activity (734.8 U/mg), and significant Vmax (1428.6 U/mg) and Km (2.8 mg/ml). Protein fingerprinting analysis reveals that it is a unique member of super-families of AmyAc and MaltAmyC which can act on both α-1,4 and α-1,6-glycosidic linkages of starch. It shows optimal activity at pH 7 and 70 °C, retains 72–92% of its activity in acidic pH range between 4 and 6.5, and stable at 60 °C for 2 h. Notably, it is resistant to 5 mM concentration of urea, dithiothreitol, β-mercaptoethanol, surfactants and EDTA, also shows > 70% of its activity in presence of 1 M solvents. Consequently, adding this α-amylase (2.5–3 U/ml) assists Clostridia to effectively utilize 375 g/l waste potato, and produce high concentration of butanol (20.41–23.74 g/l) and hydrogen (3.20–4.38 l/l).