ScienceAsia 32 (2006): 377-383 |doi: 10.2306/scienceasia1513-1874.2006.32.377

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

Siriporn Yossan^a, Alissara Reungsang^b* and Masaaki Yasuda^c

 
ABSTRACT: 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 oC, respectively. This protease could retain the activity in the pH and temperature ranging from pH 7.5 to 9.5 and 30 to 45 oC, 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 Fe3+ strongly inhibited the activity of this purified enzyme activity, decreasing relative activity to lower than 15%. Protease activity was enhanced by Mn2+, Ca2+and Mg2+. Cytochrome C, soybean protein isolate and casein were good substrates specific to the enzyme with the relative activity of more than 100%.

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^a Department of Biotechnology, Graduate School, Khon Kaen University, Khon Kaen, 40002, Thailand.
^b Fermentation Research Centre for Value Added Agriculture Products, Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand.
^c Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa, 9030129, Japan.

* Corresponding author, E-mail: alissara@kku.ac.th

Received 6 Feb 2006, Accepted 31 May 2006