Optimization of BMW-amylase refolding by response surface methodology

Abstract

Introduction: Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies (IBs), which becomes the major bottleneck in the preparation of recombinant proteins and their applications. Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. α-Amylases (1,4-α-D-glucan glucanohydrolase; EC 3.2.1.1) are enzymes that catalyze the hydrolysis of the internal α-1,4-glucosidic linkage in starch and related oligosaccharides. Enzyme produced by Bacillus has been studied for several important industrial applications such as sugar, brewing, food, and detergents. Because of their high thermostability, the Bacillus α-amylases have found widespread use in industrial processes, and much attention has been devoted to optimizing these enzymes for the very harsh conditions. Material and methods: In this study, an experimental design was employed to optimize the refolding condition of a recombinant α-amylase from Bacillus megaterium WHO (BMW) which was expressed in Escherichia coli as inclusion bodies. Half-fractional factorial design (H-FFD) was employed for initial screening of the factors, potentially influencing the response. Then the selected factors were subjected to Response surface methodology in order to obtain the optimal values for individual factor. The effects of several variables on the refolding have been studied using dilution method. Results: The optimized condition for the refolding was obtained in 50 mM Tris buffer (pH 7) containing unfolded α-amylase (3 mg/ml) in combination with glycerol (20%) and CaCl2 (25 mM). Conclusion: By using this method, yield of refolding has been increased (8 fold).