Partially purified enzyme preparation with specific activities of 153.7 U/mg for α-amylase and 0.15 U/mg for protease was produced by selective adsorption on starch. Enzymes were purified until homogeneous electrophoretically by gel-filtration over HW-55 TSK-gel with specific activities of 245 U/mg for α-amylase and 1.44 U/mg for protease. The optimum temperature and pH for purified α-amylase activity are 40-50°C and pH 6.0. The effects of various metal ions on the activity and stability of the enzyme were studied.We have studied the local bacterial strain Bacillus subtilis-150, which produces active α-amylase and neutral protease and the conditions for active fermentation of the strain cultivated on the optimal nutrient medium [1, 2]. The strain B. subtilis-150 differed from the previously studied strain B. subtilis-7A in certain cultural signatures and α-amylase activity [3].Various methods for isolating highly purified and homogeneous forms of bacterial amylase have been reported [4,5]. Fractional precipitation by (NH 4 ) 2 SO 4 for which 60% saturation by (NH 4 ) 2 SO 4 produced an enzyme preparation with specific activity 31 U/mg, where the yield by activity was 66.5% [3], was used earlier to prepare α-amylase preparation from B. subtilis-7A.Herein we report the isolation and investigation of the physical chemical and catalytic properties of purified α-amylase from B. subtilis-150. We precipitated the protein fraction from culture liquid (CL) using acetone, ethanol, and isopropanol. A high yield by activity (82%) was obtained for precipitation of proteins by isopropanol in a 1:1 ratio. α-Amylase was less sensitive to the action of isopropanol and precipitated almost completely whereas the less stable protease was partially precipitated. The resulting enzyme preparation had specific activity 285.3 U/mg (Table 1).Successful use of various natural polysaccharides for purification of amylolytic enzymes has been reported [6,7]. The most suitable sorbent for specific binding of α-amylase was soluble starch, which is very cheap and available. α-Amylase was sorbed on soluble potato starch at -20°C in the presence of ethanol and calcium acetate for 30 min. After the sorption of enzymes on starch was complete, the enzyme-substrate complex was split by increasing the temperature to 40°C and incubating for 60 min to hydrolyze the starch. The α-amylase was released and transferred into buffer with a certain amount of associated protease. We showed previously that α-amylase and protease of this strain have different thermal stabilities [1]. Therefore, the α-amylase activity was retained during incubation for 30 min at 60°C whereas that of protease under the same conditions decreased by more than 80%. Thus, biospecific chromatography over starch was able to produce partially purified α-amylase preparation with a specific activity of 153.7 U/mg.Gel-filtration of partially purified α-amylase preparation was performed over a column (20 × 600 mm) packed with HW-55 TSK-gel (Toyopearl, Japan). The results showed that the eluted ...
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