Description
ApplicationIt is used in the research and development of enzymatic glycosylated albumin reagents and mass formulation.Enzymatic propertiesSource: MicroorganismEnzymology Committee Number: EC1.5.3Molecular weight: 55kDa (SDS-PAGE)Isoelectric point: 6Km value: 5.0×10 ⁻⁴ M (Fructosyl-Ala)Inhibitors: Hg ²⁺, Pb ²⁺ Optimal pH: 7.7 Figure 1Optimum temperature: 42℃ Figure 2pH stability: 5.0-9.5 (25℃, 16h) Figure 3Thermal stability: Stable below 40℃ (pH8.0, 30min) Figure 4Stability: -25 ~ -15℃ standing store for 12 monthsMore than 90% activity Figure 5Protective agent: glycerin, trehaloseAssay method for activity1. Principle 2. Definition of enzyme activityUnit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1µmol H2O2 per minute under the following conditions.3. Reagent preparationReagent I: 1M potassium phosphate buffer, pH8.0Reagent II: 1kU/mL PODReagent III: 50mM TOOS solutionReagent IV: 50mM 4-AA solutionReagent V: 200mM Glycated alanineEnzyme diluent: 20mM Tris-HCl, pH8.0Prepare the reaction mixture as follows:Reagent I: 10mLReagent II: 0.1mLReagent III: 1mLReagent IV: 1mLReagent V: 10mLDouble steam water set volume to 100mL4. Operation procedure4.1 Add 980µL reaction mixture into 1mL colorimetric dish.4.2 Incubate at 37°C for 5 minutes.4.3 Add 20 µL of enzyme solution to be tested to the reaction mixture.4.4 Reaction at 37°C, the absorbance change (∆As) of the sample within 1min is detected by spectrophotometer at 555nm.* Blank control measurement method: Use 20 µL of enzyme dilution solution instead of the enzyme solution to be tested, and measure the absorbance change (ΔAb) of the sample within 1 minute.∆A=∆As-∆Ab5. Vitality computingVt: Total volume of reaction liquid (1.0 mL);Vs: Enzyme liquid volume (0.02 mL);t: Reaction time (1 min);df: Dilution ratio;C: Enzyme concentration (mg/mL);1.0: Optical path length (cm);1/2: 1 mole hydrogen peroxide to generate 1/2 mole quinone imide dye;39.2: Under standard reaction conditions, the millimolar absorption coefficient of the color group at 555nm (cm² /µmol)