Optimization of Alpha-Amylase Production Using Bacillus Subtilis PTCC 1720 under Solid-State Fermentation and Partial Purification of the Enzyme

Abstract

Introduction: Alpha-amylase is one of the most widely-used amylase types in the industry. Bacterial amylase production is less expensive and easier. Also, solid-state fermentation is an efficient method for the production of industrial amylase due to its advantages such as significant cost reduction and recycling of nutrient-rich wastes. Agricultural residues containing nutrients such as wheat bran are more economical substrates for solid-state fermentation. The aim of the present study was to optimize the production of alpha-amylase from Bacillus subtilis using wheat bran under solid-fermentation and partial purification of this enzyme. Materials and Methods: In this study, alpha-amylase production by Bacillus subtilis PTCC 1720 in solid-state fermentation (SSF) was investigated and optimized. The produced alpha-amylase in solid-state fermentation was precipitated, separated, concentrated, and dialyzed with 75% ammonium sulfate. Alpha-amylase activity was measured by the DNS method. The protein content after dialysis was estimated by the Bradford method and the purity of the enzyme was calculated. The molecular weight and partial purity of the enzyme were determined by polyacrylamide-sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). Results: The results of the study showed optimal enzyme production was achieved when 10 gr of wheat bran mixed with 10 ml tap water and inoculated with 3 ml of bacterial inoculum with OD=1.0 (109 CFU/mL) and incubated at 37 °C for 72 h. The alpha-amylase activity after precipitation with 75% ammonium sulfate solutions was 3.563 U/mL. As a result, the purity of alpha-amylase precipitated with 75% saturated ammonium sulfate was estimated to be 26.8% and its molecular weight was 63 KDa Discussion and Conclusion: Based on the results of the study, solid-state fermentation is a new and valuable way for amylase production using Bacillus subtilis.

Keywords

• alpha-amylase
• bacillus subtilis
• wheat bran
• solid-state fermentation (ssf)
• enzyme purification