Isolation and molecular identification of microorganisms producing protease and amylase enzymes from honey in west of Iran and optimizing the growth of microorganisms

Abstract

Alpha-amylase and Protease are enzymes with applications in medicine and industry. One of the enzyme's limitations in industry is a lack of tolerance for harsh conditions such as high temperature, pH, and low water conditions. Objective: This study aimed to screen alpha-amylase and protease producing microorganisms from honey as a low water activity medium, a suitable source for selecting stable and cost-beneficial bacterial enzyme production system. Materials and method: Plackett-Burman method was used to select twelve effective factors including pH, inoculum size, temperature, time, corn starch, KH2PO4, peptone, MgSO4, CaCl2, NaCl, glycerin and yeast extract concentrations on bacterial alpha-amylases production and pH, inoculum size, temperature, time, soybean protein, KH2PO4, MgSO4, CaCl2, NaCl, Glycerin and glucose concentration for protease production yield. The stability of the bacterial enzymes was also determined in low pH and high-temperature conditions. Results: Among all isolated and characterized microorganisms, Bacillus megaterium and Bacillus safensis produced the highest amount of alpha-amylase and protease, respectively. Based on Box–Behnken results, the temperature 35 ̊C, pH 5 and starch 40 g/l for alpha-amylase and temperature 35 °C, Glucose 0.5 g/l, and soybean protein 38.32 g/l for protease were determined as the optimum level of significant factors to achieve the highest enzymes unit. These bacterial enzymes showed the stability at pH 5 and a range of temperature from 40 ̊C to 60 ̊C for alpha-amylase and pH at 9 and temperature at 50 °C for protease. Conclusion: The achieved data indicate that the enzyme may possess the potential for using industrial processes.