Application and Principle
This alkaline protease is used to determine protease activity, expressed as PC units, of preparations derived from Bacillus subtilis var. and Bacillus licheniformis var. The assay is based on a 30-min proteolytic hydrolysis of casein at 37°and pH 7.0. Unhydrolyzed casein is removed by filtration, and the solubilized casein is determined spectrophotometrically.
Reagents and Solutions Of Alkaline Protease
Casein Use Hammarsten-grade casein (United States Biochemical Corp., Catalog No. 12840, or equivalent).
Tris Buffer (pH 7.0) Dissolve 12.1 g of enzyme-grade (or equivalent) tris(hydroxymethyl)aminomethane in 800 mL of water, and titrate with 1 N hydrochloric acid to pH 7.0. Transfer into a 1000-mL volumetric flask, dilute to volume with water, and mix.
TCA Solution Dissolve 18 g of trichloroacetic acid and 19 g of sodium acetate trihydrate in 800 mL of water in a 1000-mL volumetric flask, add 20 mL of glacial acetic acid, dilute to volume with water, and mix.
Substrate Solution Dissolve 6.05 g of enzyme-grade tris(hydroxymethyl)aminomethane in 500 mL of water, add 8 mL of 1 N hydrochloric acid, and mix. Dissolve 7 g of Casein in this solution, and heat for 30 min in a boiling water bath, stirring occasionally.
Cool to room temperature, and adjust to pH 7.0 with 0.2 N hydrochloric acid, adding the acid slowly, with vigorous stirring, to prevent precipitation of the casein. Transfer the mixture into a 1000-mL volumetric flask, dilute to volume with water, and mix.
Sample Preparation Using Tris Buffer, prepare a solution of the sample enzyme preparation so that 2 mL of the final dilution will contain between 10 and 44 bacterial protease units.
Procedure Of Alkaline Protease
Pipet 10.0 mL of the Substrate Solution into each of a series of 25- × 150-mm test tubes, allowing one tube for each enzyme test, one tube for each enzyme blank, and one tube for a substrate blank. Equilibrate the tubes for 15 min in a water bath maintained at 37° ± 0.1°.
Starting the stopwatch at zero time, rapidly pipet 2.0 mL of the Sample Preparation into the equilibrated substrate. Mix, and replace the tubes in the water bath. Add 2 mL of Tris Buffer (instead of the Sample Preparation) to the substrate blank. After exactly 30 min, add 10 mL of TCA Solution to each enzyme incubation and to the substrate blank to stop the reaction. Heat the tubes in the water bath for an additional 30 min to allow the protein to coagulate completely.
At the end of the second heating period, shake each tube vigorously, and filter through 11-cm Whatman No. 42, or equivalent, filter paper, discarding the first 3 mL of filtrate.
[Note: The filtrate must be perfectly clear.]
Determine the absorbance of each sample filtrate in a 1-cm cell, at 275 nm, with a suitable spectrophotometer, using the filtrate from the substrate blank to set the instrument at zero. Correct each reading by subtracting the appropriate enzyme blank reading, and record the value so obtained as AU.
Standard Curve Transfer 100.0 mg of L-tyrosine, chromatographic-grade or equivalent (Aldrich Chemical Co.), previously dried to constant weight, to a 1000-mL volumetric flask. Dissolve in 60 mL of 0.1 N hydrochloric acid. When completely dissolved, dilute the solution to volume with water, and mix thoroughly. This solution contains 100 µg of tyrosine in 1.0 mL. Prepare three more dilutions from this stock solution to contain 75.0, 50.0, and 25.0 µg of tyrosine per mL. Determine the absorbance of the four solutions at 275 nm in a 1-cm cell on a suitable spectrophotometer versus 0.006 N hydrochloric acid. Prepare a plot of absorbance versus tyrosine concentration.
Calculation Of Alkaline Protease
One bacterial protease unit (PC) is defined as that quantity of enzyme that produces the equivalent of 1.5 µg/mL of L-tyrosine per min under the conditions of the assay.
From the Standard Curve, and by interpolation, determine the absorbance of a solution having a tyrosine concentration of 60 µg/mL. A figure close to 0.0115 should be obtained. Divide the interpolated value by 40 to obtain the absorbance equivalent to that of a solution having a tyrosine concentration of 1.5 µg/mL, and record the value thus derived as AS.
Calculate the activity of the sample enzyme preparation by the equation
PC/g = (AU/AS) × (22/30W)
in which 22 is the final volume, in mL, of the reaction mixture; 30 is the time, in min, of the reaction; and W is the weight, in g, of the original sample taken.
Packaging and storage Of Alkaline Protease
Solid food packaging bag, 25 kg / barrel.
Alkaline Protease should be stored in dry, cool and well ventilated places away from the sun, heat.
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