Orthogonal method to determine the influence of several factors on enzyme activity

, the purpose of the experiment
1. Preliminary understanding of the use of orthodectative method (orthosectative test design method).
2. Orthothotic method was used to determine the effect of substrate concentration, enzyme concentration, temperature and pH on enzyme vitality.
2, experimental principle
the catalytic action of enzymes is carried out under certain conditions, it is affected by a variety of factors, such as enzyme concentration, substrate concentration, temperature, inhibitors and activators can affect the speed of enzyme catalysis reaction. Usually under the condition that other factors are constant, the influence of a factor is determined by the enzyme vitality under a series of changing conditions, which is a single-factor experimental method.
for multi-factor tests can be done by orthosecting test design method (orthosectance method). Orthosecting method is to simplify table calculation and analyze the results correctly by using orthosecting table. Find the best conditions for the experiment, the main number of factors, so that we can achieve good experimental results by a relatively small number of experiments.
this experiment used orthophic method to determine the effect of the four factors of substrate concentration, enzyme concentration, temperature and pH on enzyme activity, and to find out what substrate concentration, enzyme concentration, temperature and pH value when the enzyme is most active. 3, instruments, raw materials and
reagements instruments
test tubes
and test tube racks,
ringing temperature
water bath, small
fleppers
and filter paper,
hydrometers
, suction tubes, small funnels and filter paper, pH meter.
raw
raw
serum
albumin, bovine tryplin.
reagents
. 1. Bovine serum albumin: 20mL distilled water added bovine serum albumin 2.2g, urea 36g, 1mol/LNaOH solution 8mL, room temperature placed 1h, so that
protein
denatured. If there is an insoluble substance,
can
the filter. Add 0.2 mol/LNaH2PO4 solution to 110mL and urea 4g, adjust the solution pH up to about 7.6.
2. Protease solution: 3 mg bovine trypsin frozen dry powder, dissolved in 10mL distilled water.
3. 15% tricloste solution: 15g tricloste is dissolved in distilled water. and diluted to 100mL.
4. lmol/LpH 7, 8, 9 barbito buffer
. 5. Fo1in-phenol
(1) 4% sodium carbonate solution; (2)0. 2% sodium hydroxide solution;
(3) 1% copper sulfate solution; (4) 2% potassium sodium stataid solution
(2) and (4) and other volumes are made into copper sulfate - potassium sodium sodium sulphate solution. The two reagents are then mixed in a 50:1 ratio. It is called Folin-Phenol meth reagent. This reagent is in use and is preparation and is valid for one day.
6. Folin-phenol ethyl reagent
100g tungsten acid (NaWO4.2H2O), 25g sodium tantalum (NaMoO4H2O) and 700mL steamed into a 2L volume grinding reflow bottle Distilled water, plus 85% phosphate 50mL and thick hydrochloric acid 100mL, fully mixed, followed by a return condensation tube, to a small fire back 10h (the bottle with a few small glass beads, to prevent the solution from boiling overflow).
150g lithium sulfate (Li2SO4), 50mL distilled water and drops of liquid bromine are added after the return flow, and then the opening continues to boil for 15min to remove excess bromine, and the cooled solution is bright yellow (If it is still green, repeat the step of dripping brominated water), after cooling and distilled water to 1000mL, filtration, that is, Folin-phenol b reagent, filter in a brown reagent bottle, can be stored in the refrigerator for a long time.
if this storage fluid is used for too long, the color changes from yellow to green, can add a few drops of liquid bromine, boil for a few minutes, restore the primary color can continue to use.
folin-phenol reagent storage solution makes the acidity eventually 1moL/L before use. A standard NaOH solution (around 1mol/L) can be used as an indicator of phenolic phenolic, which is the end point of titration when the color of the solution is purple and purple-grey ink green. The acidity of the reagent storage fluid should be 2moL/L right, diluted to the equivalent of 1moL/L, acidity can be used.
7. 0.1mol/LNaH2PO4 solution
. 8. Urea
9. 1mol/LNaOH solution
IV, operation step (i) Experimental design
1. Determining test factors and levels
Each factor selects three levels (levels are the "points" to be tested within the per allowable variation of the factors). The test factors and selection levels are shown in the table below.
.