2019-05-14Author:adminpraise:1
1 Characteristics of catalysis
① High
efficiency
② Uniqueness
Unique bond, Unique radical group, Absolutely or almost
absolutely unique
③ Adjustable
2 Factors affecting enzyme activity
① Test method of enzyme activity.
To test enzyme activity, the whole way of enzyme catalytic reaction must be known. The disappearance or the generation of substrate should be tested by the analysis procedures. And the supporting factors, the proper pH value of enzyme and temperature should be taken into considerations.
② Enzyme link test method
The peroxidase and its co-substrate or supporting enzyme must be excessive, so that the enzyme-linked test does not belong to the rate-limiting step.
③ The reaction rate of enzyme reaction.
④ Concentration of substrate: Enzyme is saturated.
⑤ Enzyme concentration.
⑥ Temperature.
⑦ pH value
3. The extraction of enzyme
① Breakdown
method
Mechanical homogenization
method, ultrasonic method, freeze-thaw method, osmotic pressure method,
enzymatic digestion method.
② Freeze-thaw liquid
Protease
inhibitor PMSF, compounding agent EDTA, reducing agent DTT should be added to
avoid the target enzyme being damaged.
③ Enzyme
digestion method
4 The reason of losing enzyme activity
①Protease
hydrolysis and autolysis.
The reason that
enzyme loses its activity in utilizing and storage is because of the
Hydrolase action of microbe and exogenous protein.
②Polymerization.
③Extreme pH
value.
④Oxidation -
oxygen molecules, hydrogen peroxide, oxygen free radicals.
⑤Surfactants and
detergents.
⑥Denaturants -
urea and guanidine hydrochloride, high concentration salts, chelation, organic
solvents.
⑦Heavy metal ions
and sulfhydryl reagents
⑧Heat.
⑨Mechanical
force.
⑩Freezing and
dehydration.
5 Calculation of enzyme activity
① Definition of enzyme activity
Enzyme activity unit: One unit of enzyme activity is the amount of enzyme by which 1μmol of substrate is catalyzed to a product at 25℃ for 1 min under the optimum conditions of the enzyme.
② Timing calculation
The enzyme and the substrate are incubated under specific conditions, and the enzymatic reaction begins. After a certain period, the reaction is terminated with a stop solution, and the total amount of change of the substrate or product is measured by metabolism assay methods, divide the total amount of change by time. The rate of substrate consumption or the rate of product formation can be calculated, convert the speed to μmol/min, which is the enzyme activity expressed in international units.
For example:
Suppose the enzyme activity of a sample is XU/L, incubate the sample amount VS with the substrate buffer solution Vr (mL). After t min, add the stop solution Ve (mL), the detected net absorbance is increased to A. The molar extinction coefficient of the product is ε (generally determined by the band standard), and the cuvette has a light path of b cm.
③ Calculation of continuous supervision
method.
For example:
Suppose the enzyme activity of a sample is XU/L, and incubate the sample volume VS (mL) with the substrate buffer Vr (mL). The lag phase is t0 min, the measurement interval is t1 min, and the number of readings is n. The average change in absorbance at intervals is A1, the molar extinction coefficient of the product is ε (generally determined by the band standard), and the optical path of the cuvette is b cm. The reaction rate is expressed by the rate of product formation and the catalytic ability of the enzyme in the sample: