Isolation and Purification of -amylase from a local bacteria Bacillus subtilis ITBCCB148 using carboxymethylcellulose (CMC)

Amylases are one of the main enzymes used in industry. Such enzymes hydrolyze the starch molecule into polymers composed of glucose units. Amylases have potential application in a wide number of industrial processes such as food, fermentation, and pharmaceutical industries. The aims of this research are to isolate and purify -amylase from a local bacteria Bacillus subtilis ITBCCB148. The purification of enzyme was conducted by few steps such as fractionation with ammonium sulphate, dialysis and carboxymethyl cellulose (CMC) cation exchange column chromatography. -amylase crude extract was produced by Bacillus subtilis ITBCCB148 at the fermentation temperature 32oC, the fermentation media at pH 6.0 and the duration time of fermentation was 72 hours with specific activity of 925.75 U/mg. The specific activity of purified enzyme by fractionation with ammonium sulphate was 4315.31 U/mg, increasing 4.67 times than the crude enzyme extract. The specific activity of purified enzyme by dialysis was 5586.77 U/mg, increasing 6.04 times than the crude enzyme extract. The specific activity of purified enzyme cation exchange column chromatography using carboxymethyl cellulose (CMC) was 10387.11 U/mg, increasing 11.22 times than the crude enzyme extract. The optimum pH of purified enzyme was 6.0 and the optimum temperature was 60oC, while the Km and Vmax value approximately were 6.18 mg mL-1 substrate and 909.09 mol mL-1 min-1. Keyword : -amylase, Bacillus subtilis ITBCCB148, isolation and purification, CMC