Peptidase

Definition of Peptidase

1g enzyme powder (or 1mL liquid enzyme), at 40 ℃, pH6.0, 1min hydrolyzes casein to produce 1ug tyrosine as an enzyme activity unit in u / g (u / mL).

Forint method

 

Principle Of Peptidase
Peptidase hydrolyzes casein substrates at 40 ° C and pH 6.0 to produce amino acids containing phenolic groups. Under alkaline conditions, Folin reagent is reduced to form molybdenum blue and tungsten blue. Measure with spectrophotometer and calculate. Its enzyme activity.

Reagents and solutions
1 Preparation of Peptidase
In a 2000 mL mill-mouth reflow device, add 100 g of sodium tungstate (Na2WO4 · 2H2O), 25 g of sodium molybdate (Na2MOO4 · 2H2O), 700 mL of water, 50 mL of 85% phosphoric acid, 100 mL of concentrated hydrochloric acid, boil over low heat for 10 h, remove the reflux cooler In a fume hood, add 50 g of lithium sulfate (LiSO4), 50 mL of water and a few drops of concentrated bromine water (99%), and boil for 15 minutes to remove excess bromine (add green bromine water after cooling and boil slightly Remove excess bromine), cool, add water to volume to 1000 mL, mix well and filter. The reagents should be golden yellow and stored in brown bottles.
Use solution: Mix one part of Fulin reagent with two parts of water and shake well.
2 Sodium carbonate solution c (Na2CO3) = 0.4mol / L
Take 42.4 g of anhydrous sodium carbonate, dissolve in water and make up to 1000 mL.
3 Trichloroacetic acid c (CCL3 · COOH) = 0.4mol / L
Take 65.4 g of trichloroacetic acid, dissolve in water and make up to 1000 mL.
4 Sodium hydroxide solution c = 0.5mol / L
Prepared according to GB 601.
5 Hydrochloric acid solution c = 1mol / L and 0.1mil / L
GB 601: Measure 90mL of hydrochloric acid, pour into 1000mL of water, and shake to obtain 1mol / L hydrochloric acid.
Measure 9mL of hydrochloric acid, pour it into 1000mL of water, and shake to obtain 0.1mol / L hydrochloric acid.
6 Buffer solution
Phosphate buffer (pH = 6.0) for flavor proteases
Weigh 45.23 g of disodium hydrogen phosphate (Na2HPO4 · 12H2O) and 8.07 g of citric acid (C6H6O7 · H2O), dissolve in water and make up to 1000 mL. After the preparation, use a pH meter to correct it.
7 10g / L casein (casein) solution
Weigh 1.000g of casein, accurate to 0.001g, wet with a small amount of 0.5mol / L sodium hydroxide solution, add about 80mL of pH 6.0 buffer, and stir while heating in a boiling water bath until completely dissolved. , Transfer to a 100 mL volumetric flask, and dilute to the mark with pH 6.0 buffer. This solution is stored in the refrigerator for a period of 3 days.
8 100ug / mLL-tyrosine standard solution
a. Weigh 0.1000 g of L-tyrosine dried to constant weight at 105 ° C, accurate to 0.0002 g, and dissolve with 60 mL of 1 mol / L hydrochloric acid to make up to 100 mL.
b. Pipette 10.00 mL of a 1 mg / mL tyrosine standard solution and make up to 100 mL with 0.1 mol / L hydrochloric acid to obtain a 100 ug / mLL-tyrosine standard solution.

Instruments and equipment Of Peptidase
1 Constant temperature water bath: 40 ± 0.2 ℃
2 Spectrophotometer: should meet the requirements of GB 9721

Analysis steps Of Peptidase
1 Drawing of standard curve
a. L-tyrosine standard solution.Prepared according to Table A3

Table A3

 

 

b. Take 1.00mL of each of the above personnel (parallel test is required), add 5.00mL of 0.4mol / L sodium carbonate solution, 1.00mL of Folin reagent use solution, place in a 40 ± 0.2 ℃ water bath for 20min, and take it out. Use a spectrophotometer at a wavelength of 680nm and 10mm cuvette. Take the "0" tube containing no tyrosine as blanks, and measure the absorbance respectively. Take the absorbance A as the ordinate and the tyrosine concentration c as the abscissa. , Draw a standard curve (this curve should pass the zero point).

According to the plot or the regression equation, the amount of tyrosine (ug) when the absorbance is 1 is calculated, which is the absorbance constant K value. Its K value should be in the range of 95-100.

 

2 Determination
(1) Preparation of Peptidase
a. Weigh 1~2g of enzyme powder, accurate to 0.0002g (or absorb 1.00mL of liquid enzyme), dissolve with a small amount of pH6.0 buffer solution, and grind it with a glass rod, then carefully pour the supernatant into a volumetric flask. Add a small amount of the above-mentioned buffer solution to the sediment to dissolve it, and grind it for 3 to 4 times, and finally transfer it all into a volumetric flask, make up to volume with the buffer solution, and shake well. Filter through four layers of gauze, and the filtrate is once again diluted to the appropriate concentration with a buffer according to the enzyme activity for measurement (diluted to the measured absorbance within the range of 0.25 to 0.40).
b. For the determination of enzyme powder, refer to Table A4 for the dilution factor (liquid enzyme can also refer to Table A4 for dilution).

 

(2) Measurement

a. First put the casein solution into a 40 ± 0.2 ℃ constant temperature water bath and preheat for 5min.
b. Follow the procedure below:
Test tube A (blank)
→ Add 1.00mL of enzyme solution (40 ± 0.2 ℃)
→ Add 2.00mL of trichloroacetic acid (shake well) (40 ± 0.2 ℃)
→ Add casein 1.00mL (shake well)
→ Take it out for 10 minutes and filter
→ take 1.00mL filtrate
→ Add sodium carbonate solution 5.00mL
→ 1.00 mL of Jiafulin reagent solution (40 ± 0.2 ℃, color development 20min)
→ At a wavelength of 680nm, measure its absorbance with a 10mm cuvette

Test tube B (enzyme sample, three parallel samples are required)
→ Add 1.00mL of enzyme solution (40 ± 0.2 ℃)
→ Add casein 1.00mL (shake well, react at 40 ± 0.2 ℃ for 10min)
→ Add 2.00mL of trichloroacetic acid (shake well)
→ Take it out for 10 minutes and filter
→ take 1.00mL filtrate
→ Add sodium carbonate solution 5.00mL
→ 1.00 mL of Jiafulin reagent solution (40 ± 0.2 ℃, color development 20min)
→ At a wavelength of 680nm, measure its absorbance with a 10mm cuvette

Calculation
X = A × K × 4/10 × n = 2/5 × A × K × n
In the formula:
X ---- enzyme activity of the sample, u / g (u / mL);
A ---- The average absorbance of the sample in parallel test;
K ---- absorption constant;
4 ---- the total volume of the reaction;
10 --- reaction time 10min, calculated as 1min;
n ---- Dilution multiple.

The result obtained is expressed as an integer.

The allowable difference between the results of parallel tests shall not exceed 3%.

 

CERTIFICATE OF ANALYSIS 

 

 

 

 

Packaging and storage

Solid food packaging bag, 25 kg / barrel.

 

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

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