Abstract
Aim: To produce the highest amount of cold-active alpha-amylase within a short time using mesophilic bacteria with optimized media to save the energy consumption cost and obtain higher enzyme production. Methodology: Amylase producing twenty-three strains were isolated on starch peptone agar plates. Among them, one strain, A5 was selected on the basis of highest clear (12 mm) zone on starch peptone agar plates. It was characterized and identified following Bergey's Manual of Systematic Bacteriology. Enzyme was characterized as Cold alpha amylase. All physico-chemical parameters (temperature, pH, Inoculum size) including carbon, nitrogen, metal ion and amino acid sources were optimized for maximum production of the enzyme. The optimized media was used for enhancing the cold active amylase production. Results: The strain, A5 was identified as Bacillus cereus RGUJS2023 by 16S rRNA sequencing analysis for further experiments. This strain showed the highest activity (9.922± 0.143 U ml-1) on the basal starch peptone media. Though, crude enzyme showed its activity at 4°C to 48°C temperature, but the temperature was 28°C. The highest cold active enzyme was produced (18.87±0.06 U ml-1) at 16 hr of bacterial growth at 35 °C with a pH 6.5 in the optimized media containing 0.5% starch, 0.1% peptone and 0.03% MgSO4.7H2O as a carbon, nitrogen and metal ion sources, respectively, with addition of 0.03% arginine. Interpretation: The cold active alpha amylase could be used commercially for the benefit of pharmaceutical and starch processing industries. Key words: Bacillus cereus RGUJS2023, Cold-active amylase, Starch hydrolysis