Alkaline protease vitality determination.

enzyme vitality refers to the enzyme's ability to catalyz certain chemical reactions. The size of the enzyme's vitality can be expressed by the speed of a chemical reaction that it catalyzes under certain conditions. Determining enzyme vitality is actually measuring the speed of chemical reactions catalyzed by enzymes.

enzymatic reaction can be expressed in the amount of reduction in the reaction substrate or an increase in the product per unit of time, usually to determine the production of the product per unit of time for the sake of sensitivity. Since the speed of enzymatic reaction can gradually decrease its value-added over time, in order to measure enzyme vitality correctly, it is necessary to determine the initial speed of enzymatic reaction.

< p style is "text-align: left;" > alkaline protease, under alkaline conditions, can catalytic casein hydrolysis to produce tyrosine. Tyrosine is aamino acid containing phenolic hydroxyl, which can react with forin (a mixture of tungsten phosphorus and phosphate).

(forinol reaction: forin reagents are extremely unstable under alkaline conditions, easily reduced quantitatively by phenolic compounds, resulting in a mixture of tungsten blue and tantalum blue, showing different shades of blue. The amount of tyrosine produced by alkaline protease in hydrolyzed casein can be determined by coloring method to indicate enzyme vitality."experimental materials"

1. experimental equipment>

"< electric heatef " thermostat" < analysis> Balance ; capacity bottle; pipe pipe; 721 dicing photonometer

2. Experimental reagents

(1) Forrest Reagent: Add 50g sodium tungstenate (Na2WO4.2H2O) and 125g sodium tantalum in a 1L volume reflow bottle ( Na2MoO4.2H2O), 350 ml distilled water, 25 ml 85% phosphoric acid and 50 ml of hydrochloric acid, fully mixed and then reflow 10h.

reflow, plus 25g lithium sulfate, 25ml distilled water and several drops of liquid bromine, the opening continues to boil for 15 minutes, in order to remove excess bromine, cooled to 500ml. < the filter > andin a dark place in a brown bottle. Dilute with 4 times distilled water before use.

(2) 1% casein solution: 1 g of casein is said to be in the study, first moistened with a small amount of distilled water, slowly add 0.2 Mol/L NaOH 4ml, fully ground, washed in a 100ml bottle with distilled water, boiled in a water bath for 15 minutes, dissolved and cooled to 100ml, stored in the fridge.

(3) pH10 buffer solution:

A solution (0.05mol/L borax solution): take borax (Na2B4O7,10H2O) 19 grams, dissolved with distilled water and fixed to 1000 ml.

(4) standard tyrosine solution: precisely called tyrosine 50 mg, add 1 ml 1mol/L hydrochloric acid dissolved with distilled water to 50 ml, that is, 1mg/ml tyrosine standard solution.

(5) 0.4mol/L sodium carbonate solution, 0.4mol/L triclosate solution. "

1. Prepare tyrosine standard curve

(1) Take 7test tube, numbered, according to the table below the preparation of different content of tyrosine solution.

(2) In each of the above 7 test tubes, add 1% casein solution 1 ml, keep warm in a 40 oC water bath for 15 minutes, remove, add 0.4mol/L triclosan acetic acid 3 ml, shake well, each tube is filtered with filter paper.

(3) absorbs 1 ml of the filter into the other 7 test tubes, adds 0.4mol/L sodium carbonate solution 5 ml, forin reagent 1 ml, shakes well, keeps warm for 15 minutes in a 40c water bath, then adds 3 ml of distilled water to each tube, shakes well.

(4) uses a 721 terocometer to measure the density of light at 680nm against tube 0.

(5) draws a standard curve with light density as the ordinate and tyrosine content (micrograms) as the horizontal coordinates.

2.sample determination

(1) precisely called take dry enzyme powder 2 grams, add 10 ml pH10 buffer solution, in small < a href" "> beaver dissolved, and stirred with a glass rod, after a moment of rest, carefully pour the upper liquid into the capacity bottle, the slag part added a small amount of buffer, so repeatedly stirred to dissolve 4 times, and finally all into the 200ml capacity bottle.

with buffer solution to the scale, fully shake, with two layers of gauze or four layers of gauze filtration, absorb the filter 5 ml, into a 100 ml capacity bottle, diluted with distilled water to the scale, the resulting liquid is diluted 2000 times the enzyme solution.

(2) take 3 test tubes dhealing and press the table number, and add reagents and operations in strict order in the table.

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shakes well, each tube is filtered separately, absorbing 1 ml of filter fluid, adding 0.4mol/L sodium carbonate solution 5 ml, forin reagent 1 ml, fully shaken,