Description
ApplicationIt is used for the development and mass formulation of β-hydroxybutyrate reagents.Enzymatic propertiesSource: MicroorganismEnzymology Committee Number: EC1.1.1.30Molecular weight: 27 kDa (SDS-PAGE)Isoelectric point: 6.1Km value: 1.46×10-2M (D-3-Hydroxybutyrate), 9.5×10-5M (NAD+)Inhibitor: Hg²⁺，Ag⁺，SDS Optimal pH: 8.5 Figure 1Optimum temperature: 60℃ Figure 2pH stability: 5.0-10.0 (25℃,16h) Figure 3Thermal stability: Stable below 50℃ (pH8.5, 30min) Figure 4Stability: -25 ~ -15℃ standing store for 12 months. More than 90% activity Figure 5 Assay method for activity1. PrincipleThe amount of NADH produced by the reaction can be measured by spectrophotometer at 340nm.2. Definition of enzyme activityUnit enzyme activity is defined as the amount of enzyme required to produce 1µmol of NADH per minute under the following reaction conditions.3. Reagent preparationReagent I: 100mM pH8.5 Tris-HCl buffer.Reagent II: 158mM sodium 3-hydroxybutyrate solution (dissolved with reagent I).Reagent III: 27.9mM NAD+ (dissolved with reagent I).Reagent IV: 100mM Tris-HCl, pH8.5, containing 0.1%BSA.Sample to be tested: Dilute the enzyme solution with reagent IV to 0.1-0.5 U/mL.4. Operation procedure4.1 Add 3mL reagent I, 0.5mL reagent II and 0.2mL reagent III into a 3mL colorimetric dish and preheat it at 37°C for 5min.4.2 Add 100µL sample to be tested and mix well.4.3 Reaction at 340nm at 37°C, record absorbance change within 1min (∆As)* 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 computingThe formula can be captured or edited in this positionVt: total volume of reaction liquid (3.1mL);Vs: enzyme liquid volume (0.1mL);1.0: optical path length (cm);df: dilution ratio;C: Enzyme concentration (mg/mL);6.22: Under standard reaction conditions, the millimolar absorption coefficient (cm2/µmol) of the color-producing group at 340nm