Notices on enzymes biochemical reagent kit
1 Characteristics of catalysis
① High efficiency
Unique bond，Unique radical group, Absolutely or almost absolutely unique
Enzyme concentration adjustment, Hormone adjustment, covalent modification adjustment, Hydrolysis of restriction proteases and enzyme activity adjustment, Inhibitor adjustment, Feedback adjustment, Metal ions and other small molecule compounds adjustment.
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 co-substrate: Enzyme is saturated.
⑤ Enzyme concentration.
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.
Protease inhibitor PMSF, compounding agent EDTA, reducing agent DTT should be added to avoid the target enzyme being damaged.
③ Enzyme digestion method
To break down the cells by enzymatic hydrolysis of cell membrane or cell wall by lysozyme, proteolytic enzyme and glycosidase.
4 T he 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.
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
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 chemical or biochemical 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.
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.
The enzyme is incubated with the substrate under specific conditions, and the change in the characteristic signal of a certain substrate or product during the enzymatic reaction is continuously measured at regular intervals (2-60 s) to calculate the rate of change of signal per minute. The continuous supervision method is to continuously measure the amount of product produced or the amount of substrate consumed at multiple time points, and select the rate of the linear phase to calculate the enzyme activity, also known as the rate method.
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: