Purification And Characterization Of Alkaline Protease

The alkaline proteases are kinds of hydrolytic enzymes which the optimum pH value is alkaline. They play a very important role in medicine, feed, food and other fields. The conditions of enzyme production, purification and characterization of the alkaline protease from Cellulomonas bogoriensis sp. nov were studied in this paper, and concluded as follows:Using the single factor analysis and the Box-Behnken experimental response surface methodology, we got the optimum fermentation conditions for the production of the alkaline protease: sucrose 1.5%(v/v), beef extract and yeast powder(1:1) 1.5%(v/v), the pH of culture medium 10.5, Volume of medium 75/250 mL, NaCl 2.67%, cultivate time 120 h, and the culture temperature 30 ℃.The alkaline protease was purified by ammonium sulfate precipitation and the CM Sepharose Fast Flow column chromatography. Its molecular mass was measured to be 18.3 kDa by SDS-PAGE. The alkaline protease purified up to 10.83 times with a recovery of 25.79%.The optimum reaction temperature and pH of the enzyme were 70℃ and 11, respectively; The alkaline protease is stabile within temperature 4-60 ℃, and pH 3-12;1mM Mg2+and Fe2+ with 1mM will enhance the activity of the protease, However, Ba2+, Mn2+and Ca2+ with 1mM have no effect on protease activity almost, while at 10 mM concentration, they will have an obvious inhibition on protease activity.EDTA as the metal ion chelating agent has no effect on the enzyme activity at 1mM concentration, and when the concentration increased to 10 mM, the residual enzyme activity was 77%. The enzymatic activity was completely inhibited by serine inhibitor PMSF and the chymotrypsin inhibitors TPCK, which suggested that this enzyme is a kind of chymotrypsin along to serine protease family. The alkaline enzyme we got was very stabile in organic solvents, but cannot resistant to Triton X-100. The best substrate for the alkaline enzyme is casein, and it can also hydrolysis hemoglobin and bovine. The Michael’s constant Km is 19.2 μg/mL, and the Vmax is 2000 μ g/min. The alkaline protease has a good compatibility with detergents, good hair removal effect, salt resistance and the ability to deal with bloodstains. These excellent properties make this alkaline protease a promising candidate for industrial application.