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    Home > Biochemistry News > Biotechnology News > The determination principle and experimental steps of the protease vitality of the salil bacteria.

    The determination principle and experimental steps of the protease vitality of the salil bacteria.

    • Last Update: 2020-10-25
    • Source: Internet
    • Author: User
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    related topics . Analysis of protease activity determination Focus protease research new progress
    First, the experimental purpose 1. to deepen understanding of the concept of enzyme vitality..2. Learn to master the method of determining protease vitality., experimental principles,enzyme vitality refers to the ability of enzymes 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 s mu;g 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 the product tyrosine under certain conditions.III, instruments and reagentsinstruments:
    string
    water bath pot,
    hydrometer
    ,
    test tube
    and test tube rack,
    drying
    filter paper, glass
    funnel
    .raw materialswithcobacteria protease: said to take 1g of hashella protease powder, with a small amount of 0.02mol/L, pH7.5
    phosphoric acid buffer
    dissolved and fixed to 100mL, shock 15 minutes, so fully dissolved, dry gauze
    filtering
    , take the filter refrigerator backup. When using, the buffer is properly diluted with the energy of the enzyme.Reagents 1. Folin-phenol reagents:add sodium tungstenate (Na2WoO4 . 2H2O) to the 2L reflow bottle 100g, sodium tantalum (Na2WoO4 . 2H2O) 25g, distilled water 700mL, 85% phosphate 50mL and 100mL thick hydrochloric acid, 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..2. 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 6. 0.02mol/LpH7.5
    phosphoric acid buffer
    :called take phosphate dodium phosphate (Na2HPO4 . 12H2O) 7.16g, water capacity to 100mL (A liquid); After mixing A liquid 84mL and B liquid 16mL, 0.2mol/LpH7.5
    phosphoric acid buffer
    be stored for long periods of time. Dilute 10 times when you use it. . 7. Standard tyrosine solution (50 sm;g/mL): 12.5 mg is taken to dry tyrosine to constant weight, dissolved with 0.2mol/L hydrochloric acid of about 30mL, distilled water is determined 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 are used, and the absorbance value is the standard curve for the ordinates. (ii) Enzyme vitality measurement 1. Enzyme reaction: take a test tube, add 2.0mL0.5% casein solution, preheat in a 30c water bath for 5 minutes, and then add 1.0mL preheated Bacillus protease solution, immediate timing, water bath quasi-ensure the temperature of 10min, from the water bath, immediately add 2.0mL of 10% triclosal acetic acid solution, shake and rest for a few minutes, dry filter paper filtration, collection filter (sample fluid). Take another test tube, first add 1.0mL preheated herb protease solution and 2.0mL 10% triclosan acetic acid 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 fluid (control fluid). the above two processes, we should do a parallel experiment. . 2. Determination of tyrosine content in the filter take 3 test tubes, add 1.0mL water, A liquid, B solution, and then add 5.0 mL 0.55mol/L sodium carbonate solution and 1.00mLFolin-phenol reagent, shake and smooth according to the standard curve production method to keep warm and absorb the light value. 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. V. Calculating the absorbance value of A sample-sample fluid A-control-control fluid absorbance value K-standard curve on the A-1 corresponding volume of V-enzymatic reaction fluid (5mL in this experiment) T-enzymatic reaction time (this experiment is 10min) N-enzyme solution dilution
    times.
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