Isolation, Optimization, Partial Purification and Characterization of Amylase Produced by Indigenously Isolated Bacillus AS2 Strain

Abstract

Amylases are highly valuable starch degrading enzymes that catalyzes hydrolysis of starch into economically important low products such as maltose, glucose, oligosaccharides and maltodextrins. Amylases have about 25-30% of total enzyme production value across world and have wide spread applications in different sectors such as paper and pulp, textile, food, fermentation, pharmaceutical, biopharmaceutical, detergents and biofuel production. Microbial sources, most importantly members of genus Bacillus are used significantly for amylase production at commercial scale. The present study reports the production, optimization, partial purification, characterization and industrial applications of amylase produced by an indigenous strain. Out of 89 isolated strains from different soil samples, 39 were identified as members of genus Bacillus. Isolated Bacillus strains were subjected to the qualitative and quantitative amylase screening. Best amylase producer “Bacillus AS2” was selected for further studies. Molecular identification of selected isolate showed 87% similarity to the standard specie of Bacillus cereus. In the current investigations, amylase and biomass productions were optimized via one variable at a time (OVAT) technique and response surface methodology (RSM). Central composite design (CCD) with 6 independent variables (temperature, pH, incubation period, concentration of starch, glycine and CaCl2) was constructed for statistical optimization of amylase. Various parameters for OVAT were studied and it was noticed that maximum growth as well as amylase production was obtained when culture was incubated at 40°C and pH 7.0 for 76 hrs. Amylase production was enhanced when 4% starch and 0.5% glycine were supplemented as carbon and nitrogen source in the growth medium. Production of amylase was significantly increased in presence of CaCl2 (0.05%). A marked enhancement in amylase yield was also observed when 0.02% SDS was used as a surfactant. However, optimum parameters after RSM were found slightly different i.e., temperature (40°C), pH (7.0), starch (5.3%), glycine (0.1%), CaCl2 (0.068%) and incubation period (76 hrs). Significant differences were found among optimum parameters for maximum biomass production (35°C, pH 5.0, starch 2%, glycine 0.9%, xxxiii CaCl2 0.0237% and 24 hrs). Both models were found highly significant (on the basis of R2 values, lack of fit and actual vs. residuals probability plots). Amylase was subjected to the purification by using ammonium sulphate as precipitating agent but no significant results were obtained. A band of 66 kDa was observed for amylase from Bacillus cereus AS2 which was further confirmed by zymography. Amylase from Bacillus cereus AS2 was found optimally active at wide range of temperature (10°C-70°C) and pH (3.0-11.0) with optimum activity at 60°C and pH 6.0. It was also found stable at a wide range of temperature and pH with optimum stability at 50°C and pH 6.0. Significant increase in amylase activity was found in the presence of Ca+2, Na+ and Cu+2. Similarly, Ca+2, Na+, K+ and Cu+2 were also found the active stabilizing metal ions. Activity of amylase enzyme was boosted in presence of detergents and organic solvents such as SDS, EDTA, toluene and acetone. Substrate hydrolysis profile showed high affinity of amylase towards starch, dextrin and dextrose. Kinetic characterization revealed the optimum reaction after 15 min with 1% starch as substrate. Km, Vmax and Ea values were found to be 0.75 mg/ml, 125 IU/ml/min and 40.16 KJ/mole respectively. Storage stability of enzyme was showed that amylase was maximally active when stored at 4°C as compare to -18°C and 37°C. Amylase production under solid-state fermentation and submerged fermentation was also checked using a variety of agroindustrial wastes including potato peels, sugarcane baggas, rice bran, wheat bran, rice flour and maize flour. Enhanced Amylase production was observed under SmF than SSF. Potato peels were found most suitable substrate for enzyme production by both of the methods. Scanning electron micrographs to study surface modifications showed significant hydrolysis of different natural substrates by the action of amylase produced by Bacillus cereus AS2 Higher thermal stability and lower Ea values of amylase (from Bacillus cereus AS2) makes it suitable for different industrial applications. Amylase was found stable in various locally available laundry detergents. Furthermore, stain removal efficacy of detergents were boosted in the presence of amylase. Enzyme was found efficient in de-sizing and fabric was de-sized to TEGEWA scale 9 at 60°C in 15 hrs. Scanning electron xxxiv micrographs didn’t show any starch contents on amylase treated fabric fibers. Amylase was found efficient to be used in paper and pulp industry due to its significant deinking ability. Maximum deinking potential was observed in 1200 ppm of red ink for 72 hrs of incubation. Similarly all tested paper pulps were decolorized to a noticeable extent by fermentative decolorization. These results indicate that amylase from Bacillus cereus AS2 can be used in various commercial sectors.