-
Categories
-
Pharmaceutical Intermediates
-
Active Pharmaceutical Ingredients
-
Food Additives
- Industrial Coatings
- Agrochemicals
- Dyes and Pigments
- Surfactant
- Flavors and Fragrances
- Chemical Reagents
- Catalyst and Auxiliary
- Natural Products
- Inorganic Chemistry
-
Organic Chemistry
-
Biochemical Engineering
- Analytical Chemistry
- Cosmetic Ingredient
-
Pharmaceutical Intermediates
Promotion
ECHEMI Mall
Wholesale
Weekly Price
Exhibition
News
-
Trade Service
. First, the purpose of the experiment1. To deepen understanding of the concept of enzyme vitality.
2. Learn how to determine the vitality of proteases., the experimental principle isthe ability of the enzyme to catalyz a particular reaction. Its size can be expressed by the reduction of substrates or the production of products in the reaction system after a certain amount of time under certain conditions.
unit of enzyme vitality is an important indicator of enzyme vitality size. This experiment specifies that the enzyme vitality unit (U) is the amount of enzyme required to break down 1 sg tyrosine per minute under certain conditions.
the reaction system of tyrosine produced by hydrolyzed casein, which is used in the experiment. The product tyrosine reacts with Folin-phenol
reagent
under alkaline conditions to produce a blue
compound
, which has maximum light absorption at 680nm and has a light absorption value that is directly related to the tyrosine content.
, the vitality of protease can be calculated by measuring the change of the content of tyrosine under certain conditions.
III, instruments and reagents
instruments:
stration
water bath pot, hydrometer,
test tube
and test tube rack,
drying
filter paper, glass
flepper
.
Raw material
withcobacterium protease: said to take 1g of bacillus protease powder, with a small amount of 0.02mol/L, pH7.5 phosphoric acid buffer dissolved and fixed to 100mL, shock 15 minutes, so that full solution, dry gauze
filtration
, take the filter refrigerator backup. When using, the buffer is properly diluted with the energy of the enzyme.
reagents
. 1. Folin-phenol reagents:
sodium tungstenate (Na2WoO4. 2H2O) 100g, sodium tantalum (Na2WoO4. 2H2O) 25g, distilled water 700mL, 85% phosphate 50mL and strong hydrochloric acid 100mL, fully mixed, micro-fire reflow
heating
10 hours. Then add lithium sulfate 150g, distilled water 50mL and several drops of liquid bromine, shake well after the opening continues to boil 15min, in order to remove excess bromine. After cooling, the distilled water is fixed to 1000mL, filtered, the solution is yellow-green and stored in a dark place in a brown reagent bottle. The acidity (approximately 2mol/L) can be measured with standard NaOH solution and phenolic phenolic as an indicator before use, and then diluted to 1mol/L with water.
0.2mol/L hydrochloric acid solution
. 3. 0.04mol/L Sodium Hydroxide Solution
4. 0.55mol/L Sodium Carbonate Solution
5. 10% Tetrachloroacetic Acid Solution
0.02mol/LpH7.5 Phosphate Buffer:
Called Na2HPO4. 12H2O) 7.16g, water capacity to 100mL (A liquid), called sodium phosphate (Na2HPO4.12H2O) 3.12g, water capacity to 100mL (B liquid). After mixing A liquid 84mL and B liquid 16mL, a 0.2mol/LpH7.5 phosphate buffer is obtained for long-term storage. Dilute 10 times when you use it.
7. Standard tyrosine solution (50 μg/mL): 12.5 mg to dry to constant tyrosine, dissolved with 0.2mol/L hydrochloric acid about 30mL, distilled water to 250mL.
8. Casein solution (0.5%): 1.25g casein, dissolved with 0.04mol/L sodium hydroxide solution (20mL), then 0.02mol/LpH7.5 phosphoric acid buffer to 250mL., operation step(i) Tyrosine standard curve production
take 6 test tubes (label 0, 1 to 5), in order to add 0.00, 0.20, 0.40, 0.60, 0.80 and 1.00mL standard tyrosine solution, and then filled with water to 1.00mL, shake well after each add 0.55mol/L sodium carbonate 5.0mL, shake well. Add Folin-phenol reagent 1.00mL in turn, shake well and time it, keep warm for 15min in a water bath pan at 30C. The absorbent value (controlled by tube No. 0) is then measured at 680nm. The horizontal coordinates of the tyrosine content (g) are used, and the absorbance value is the standard curve for the ordinates.
(ii) enzyme vitality determination
. 1. Enzyme reaction: Take a test tube, add 2.0mL0.5% casein solution, preheat in a 30c water bath for 5 minutes, then add 1.0mL preheated herb protease solution Immediate timing, water bath quasi-ensure the temperature of 10min, after removal from the water bath, immediately add 2.0mL 10% triclosal acetic acid solution, shake and leave for a few minutes, dry filter paper filtration, collection of filter (sample fluid).
Take another test tube, first add 1.0mL preheated herb protease solution and 2.0mL 10% triclosan acetate solution, shake well, place for a few minutes, then add 2.0mL0.5% casein solution, and then in 30 degrees C water bath insulation 10min, the same dry filter paper filtration, collection filter (control fluid).
the above two processes, we should do a parallel experiment.
2. Determination of tyrosine content in the filter fluid
Take 3 test tubes, add 1.0mL water, A liquid, B liquid, and then add 5.0 mL 0.55mol/L sodium carbonate solution and 1.00mLFolin-phenol reagents, shake and measure the photometric value according to the standard curve production method. According to the absorbance value, the difference of tyrosine content in A and B liquid can be determined by the standard curve, and the dynamic unit of enzyme can be calculated.
, the calculation
. A Sample - sample fluid absorbance value
A control - control fluid absorbance value
K - standard curve A - 1 corresponding to the volume of tyrosine sg
V - enzymatic reaction fluid volume (5mL in this experiment)
T - enzymatic reaction time (this experiment is 10min)
N - enzyme solution dilution multiplication .