Description
ApplicationFor the detection of lactic acid content.Enzymatic propertiesSource: MicroorganismEnzymology Committee Number: EC 1.1.3.2Molecular weight: 42 kDa (SDS-PAGE)Isoelectric point: pH 4.6Km value: 7.5 × 10-4 M (L-Lactate)Inhibitors: Hg²⁺, Pb²⁺ Optimum pH: 6.0-7.0 Figure 1 Optimum temperature: 50℃ Figure 2pH stability: 6.0-8.5 (25℃,16h) Figure 3 Thermal stability: Stable below 50℃ (pH7.0, 30min) Figure 4Stability: -25 ~ -15℃ standing store for 12 monthsMore than 90% activity Figure 5 Enzyme activity definitionUnit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1µmol H2O2 per minute under the following conditions.Assay method for activity1. PrincipleThe amount of Quinoneimine dye produced by the reaction can be measured by spectrophotometer at 555 nm.2. Definition of enzyme activityUnit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 µmol H2O2 per minute under the following conditions.3. Reagent preparationReagent I: 0.2 MpH 6.5 potassium phosphate buffer.Reagent II: 1kU/mL peroxidase (POD) solution.Reagent III: 50 mM4-AA solution.Reagent IV: 0.5 MDL- lactic acid solution, pH6.5.Reagent V: 50 mMTOOS solution.Enzyme diluent: 10 mMpH7.0 potassium phosphate buffer containing 10 µM FAD.Sample: Dilute the enzyme with enzyme diluent to 0.05-0.2U/mL.Prepare the reaction mixture as follows:Reagent I is 10 mlReagent II 0.25 mLReagent III 1.5 mLReagent IV is 5 mLReagent V 1.5 mLDouble steam water to 50 ml4. Operation procedure4.1 Add 1mL reaction mixture into 1mL colorimetric dish.4.2 Preheat the reaction mixture at 37 °C for 5min.4.3 Add 20µL of enzyme liquid to be measured and mix well.4.4 The reaction is measured at 37 °C at 555 nm and the absorbance change (∆As) within 1min is recorded.* Replace enzyme liquid with enzyme diluent, other steps are the same, the absorbance of the resulting solution is blank absorbance (∆Ab)∆A=∆As-∆Ab5. Vitality computing1.020: total volume of reaction liquid (mL);0.020: enzyme liquid volume (mL);1: optical path length (cm);1/2:1 mole of hydrogen peroxide to generate 1/2 mole of quinone imide dye;df: dilution ratio;C: Enzyme concentration (mg/mL);39.2: Under standard reaction conditions, the millimolar absorption coefficient of the color group at 555 nm (cm2/µmol)