Determination of alkaline protease vitality (forin-phenol reagent method)

< p style" text-align: left;" > (2) to master the principles and methods of use of dicing photonometers.< p style is "text-align: left;" > (3) learn how to draw the curve.

II, experimental principle

forin-phenol "reagent is a mixture of phosphate and tungsten phosphate. It is unstable under alkaline conditions and can be reduced by a phenolic base in tyrosine to produce a mixture of platinum blue and tungsten blue. Tyrosine produced by casein after protease action can react with forin-phenol reagents, and the resulting bluecompounds can be measured by coloration.

III, experimental equipment

(1) split

< a href>"ringing thermostat

(2) condenser

four, materials and .

< p style" "text-align: left;" > (1) commercially available alkaline protease preparations.

(2)0.4mo/L sodium carbonate: 42.4gNa2CO3 dissolved with distilled water.

< p style s"text-align: left;"> (3)4mo1/L trichlorocetic acid: 65.6g triclosate dissolved in distilled water, fixed to 1000 mL.

(4) 2% casein: 2.0g casein plus 40mL distilled water, plus 3-5 drops of ammonia, dissolved in a boiling water bath.

< p style" "text-align: left;" > with pH11 sodium borate-sodium hydroxide buffer solution to 1000mL.

(5)pH11 sodium borate-sodium hydroxide buffer solution: 0.1mol/LNaOH mixed with volume 0.05mol/LNa2B4O7.

(6) forin-phenol reagents: 50g sodium tungstenate (Na2W04.2H20), 12.5g sodium platinumate (Na2Mo04.2H20),

2H20 350mL water, 25mL 85% phosphoric acid, 50mL thick hydrochloric acid into a 1000mL round bottom burner, wen fire reflow (keep slightly boiling) 10 h, remove the condenser, add 150g sulfate (Li2O4), 25mL water, mix and add bromine water about 5mL decolorization. Until golden yellow, then slightly boil 15min, remove residual bromine, cooling, with 4-5 acid-resistant bacteriafunnel filtration , fixed to 500mL, in a cleandry brown bottle, when used in 1:2 dilution.

5, how to do it

(1) sample processing

(2) color measurement

< text-align: left;"> takes 10mL centrifugal tube 4 to add diluted rib fluid 1.0mL.

parallel test 3 blank experiments 1

(1) warms up 2-3min in a 40c water bath. (1) Preheat 2-3min in a water bath at 40 degrees C.

(2) plus 1.0mL 40C preheated 2% cheese egg (2) plus 2.0mL 0.4mol/L tachlorocetic acid 40c protection

< "text-align left;" > white, at 40C reaction 10minmin. Temperature 10min.

(3) plus 2. OmL 0.4mol/L triclosan acetate anti (3) plus l.0mL 2% casein, reaction 15min

should be filtered by 15min. Filter.

< p style"text-align: left;"> (4) filter solution 1.0mL into the

(5) plus 1.0mL l:2 diluted forling-phenol reagent (which should be added last) at 40 OC water bath hair color 20 min.

(6)7220 ionometer at 680nm color, 1cm thicker than cuvette.

(3) color constant (K)'s method

number12. 345 tyrosine/ml00.2. 0.40.60.8< d style: 154.0px; text-align: left; ">H2 O/ml1.00.8. 0.60.4: left; ">0.2< td style: "width: 154.0px; text-align: left;">Na2 CO3 /ml5. 555< "width: 154.0px; text-align: left;">forin-phenol/ml11 1111px" OD680". <td style""width: 77.0px; text-align: le.