Purification and Characterization of Alkaline Protease from Bacillus megaterium Isolated from Thai Fish Sauce Fermentation Process

An alkaline protease produced by Bacillus megaterium isolated from a fermented broth of Thai fish sauce was purified by hydrophobic interaction combined with gel filtration techniques. After final purification step, the enzyme was purified 148-fold with an increase in specific activity from 0.09 to 13.33 U/mg protein. The properties of the purified enzyme were then analyzed. The molecular weight of the enzyme under denaturing condition was estimated to be 27 kDa. The optimum pH and temperature for protease activity were 10 and 50 o C, respectively. This protease could retain the activity in the pH and temperature ranging from pH 7.5 to 9.5 and 30 to 45 o C, respectively, resulting in the relative activity of higher than 80%. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP) suggesting that it was a serine protease. Phenylmethylsulfonyl fluoride (PMSF), p-Chloromercuribenzoic acid (PCMB), ethylenediaminetetraacetic acid (EDTA), 1, 10-phenanthroline and Fe 3+ strongly inhibited the activity of this purified enzyme activity, decreasing relative activity to lower than 15%. Protease activity was enhanced by Mn 2+ , Ca 2+ and Mg 2+ . Cytochrome C, soybean protein isolate and casein were good substrates specific to the enzyme with the relative activity of more than 100%.