Influence of media composition on the production of alkaline alfa-amylase from Bacillus subtilis CB-18
Background. Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving a-l,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior altemative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. Material and methods. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell-free culture supernatant after centrifugation at 2515 x g for 15 minutes. Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. Results. Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with α-amylase activity of 758 U/mL after 48 h. Peptone was the best nitrogen source for enzyme production with a-amylase activity of 680 U/mL after 48 h. Metal ions including Ca2+, Mn2+ and Mg2+ stimulated enzyme production while Hg2+ and Ag+ repressed enzyme production. The best enzyme yields were observed in basal media containing agro-based substrates. Conclusion. This work reports the production of alkaline α-amylase by Bacillus subtlis CB-18 in different media. Enzyme production was highest when agro-based media were used to formulate the media.
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