Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and <i>Bacillus amyloliquefaciens</i>

Abstract

This study reports utilization of mixture of wheat and barley bran (1:1) for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1) was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL), moisture 80%, pH 7±0.02, NaCl (3%), temperature 38±1°C, incubation for 72 h, maltose (1%) and tryptone (1%). After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C), NaCl concentration (0.5-4 M), and pH (6-10) ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS), CuCl3 and ethylenediaminetetraacetic acid (EDTA) strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.