Acid Protease

Application and Principle

This acid protease is used to determine the proteolytic activity, expressed as hemoglobin units on the tyrosine basis (HUT), of preparations derived from Aspergillus oryzae var and Aspergillus niger var, and it may be used to determine the activity of other proteases at pH 4.7. The test is based on the 30-min enzymatic hydrolysis of a hemoglobin substrate at pH 4.7 and 40°. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration. The quantity of solubilized hemoglobin in the filtrate is determined spectrophotometrically.

Reagents and Solutions Of Acid Protease
Hemoglobin Use Hemoglobin Substrate Powder (Sigma Chemical Co., Catalog No. H2625) or a similar high-grade material that is completely soluble in water.
Acetate Buffer Solution Dissolve 136 g of sodium acetate (NaC2H3O2·3H2O) in sufficient water to make 500 mL. Mix 25.0 mL of this solution with 50.0 mL of 1 M acetic acid, dilute to 1000 mL with water, and mix. The pH of this solution should be 4.7 ± 0.02.
Substrate Solution Transfer 4.0 g of the Hemoglobin into a 250-mL beaker, add 100 mL of water, and stir for 10 min to dissolve. Immerse the electrodes of a pH meter in the solution, and while stirring continuously, adjust the pH to 1.7 by adding 0.3 N hydrochloric acid. After 10 min, adjust the pH to 4.7 by adding 0.5 M sodium acetate. Transfer the solution into a 200-mL volumetric flask, dilute to volume with water, and mix. This solution is stable for about 5 days when refrigerated.
Trichloroacetic Acid Solution Dissolve 140 g of trichloroacetic acid in about 750 mL of water. Transfer the solution to a 1000-mL volumetric flask, dilute to volume with water, and mix thoroughly.

Sample Preparation Dissolve an amount of the sample in the Acetate Buffer Solution to produce a solution containing, in each mL, between 9 and 22 HUT. (Such a concentration will produce an absorbance reading, in the procedure below, within the preferred range of 0.2 to 0.5.)

Procedure Of Acid Protease
Pipet 10.0 mL of the Substrate Solution into each of a series of 25- × 150-mm test tubes: one for each enzyme test and one for the substrate blank. Heat the tubes in a water bath at 40° for about 5 min. To each tube, except the substrate blank, add 2.0 mL of the Sample Preparation, and begin timing the reaction at the moment the solution is added; add 2.0 mL of the Acetate Buffer Solution to the substrate blank tube. Close the tubes with No. 4 rubber stoppers, and tap each tube gently for 30 s against the palm of the hand to mix. Heat each tube in a water bath at 40° for exactly 30 min, and then rapidly pipet 10.0 mL of the Trichloroacetic Acid Solution into each tube. Shake each tube vigorously against the stopper for about 40 s, and then allow to cool to room temperature for 1 h, shaking each tube against the stopper at 10- to 12-min intervals during this period.   Prepare enzyme blanks as follows: Heat, in separate tubes, 10.0 mL of the Substrate Solution and about 5 mL of the Sample Preparation in the water bath for 30 min, then add 10.0 mL of the Trichloroacetic Acid Solution to the Substrate Solution, shake well for 40 s, and to this mixture add 2.0 mL of the preheated Sample Preparation. Shake again, and cool at room temperature for 1 h, shaking at 10- to 12-min intervals.
At the end of 1 h, shake each tube vigorously, and filter through 11-cm Whatman No. 42, or equivalent, filter paper, refiltering the first half of the filtrate through the same paper. Determine the absorbance of each filtrate in a 1-cm cell, at 275 nm, with a suitable spectrophotometer, using the filtrate from the substrate blank to set the instrument to zero. Correct each reading by subtracting the appropriate enzyme blank reading, and record the value so obtained as AU.
[Note: If a corrected absorbance reading between 0.2 and 0.5 is not obtained, repeat the test using more or less of the enzyme preparation as necessary.]

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 the L-tyrosene is 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. Determine the slope of the curve in terms of absorbance per µg of tyrosine. Multiply this value by 1.10, and record it as AS. A value of approximately 0.0084 should be obtained.

Calculation Of Acid Protease
One HUT unit of proteolytic (acid protease) activity is defined as that amount of enzyme that produces, in 1 min under the specified conditions, a hydrolysate whose absorbance at 275 nm is the same as that of a solution containing 1.10 µg/mL of tyrosine in 0.006 N hydrochloric acid.
Calculate the HUT per g of the original enzyme preparation by the equation

HUT/g = (AU/AS) × (22/30W)

in which 22 is the final volume of the test solution; 30 is the reaction time, in min; and W is the weight, in g, of the original sample taken.

[Note: The value for AS, under carefully controlled and standardized conditions, is 0.0084; this value may be used for routine work in lieu of the value obtained from the standard curve, but the exact value calculated from the standard curve should be used for more accurate results and in cases of doubt.]

CERTIFICATE OF ANALYSIS

Product Name	Acid Protease
Other Name	N/A	Country of Origin	China
Strain	Aspergillus oryzae	Manufacture Date	MAY 13, 2019
Batch Number	SP19050903	Expiration Date	MAY 12, 2021
Package	25 kg/bucket	Quantity	100 kg
PROTOCOL	SPECIFICATIONS	RESULTS	METHOD
PHYSICAL & CHEMICAL ANALYSIS
Description	brown powder	Complies	Visual
Odor & Taste	Characteristic odor and taste	Complies	Taste
Moisture	NMT 7%	5.1%	Moisture Analyzer
IDENTIFICATION
Identifiable Activity	Positive for Bromelain Activity	Complies	In-house
ACTIVITY
Protease activity	NLT 500,000 HUT /g	508,270 HUT /g	FCC VII
MICROBIOLOGICAL
Total bacteria count Coliform bacteria (CFU/g) Molds and Yeasts E.Coli Salmonella Staphylococcus aureus Pseudomonas aeruginosa	NMT 3,000 CFU/g NMT 30 CFU/g NMT 100 CFU/g Absent Absent Absent Absent	500 CFU/g ＜10 CFU/g ＜10 CFU/g Not Detected Not Detected Not Detected Not Detected	FDA BAM Online Ch.3 FDA BAM Online Ch.4 FDA BAM Online Ch. 2 FDA BAM Online Ch.4 FDA BAM Online Ch.5 FDA BAM Online Ch.12 AOAC
HEAVY METALS
Lead Mercury Cadmium Arsenic	NMT 3 ppm NMT 0.1 ppm NMT 1 ppm NMT 1 ppm	0.21 ppm ＜0.1 ppm ＜1 ppm ＜1ppm	USP <231> USP <231> USP <231> USP <231>
Storage: Store in cool and dry place protected from light, keep drum close when not in use. Conclusion: Conforms to IU Standards. Shelf Life: Shelf life of Acid Protease under prescribed storage conditions and air-tight packing will be 2 years. The product COMPLIES with the above said specifications Approved by RICKY H. ZHU Authorized Signatory

*The above information is a direct translation of the information provided to Newgen Biotech from the manufacturer of acid protease. This Certificate of Analysis should be used for informational purposes and is not intended as a substitute for strict quality control analysis by the purchaser of this product.

Packaging and storage

Solid food packaging bag, 25 kg / barrel.

Acid Protease should be stored in dry, cool and well ventilated places away from the sun, heat.

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