Description
ApplicationIt is used for the development and mass preparation of total bile acid (TBA) reagents.Enzymatic properties (note that 2+, +, 3+ are superscripts)Source: MicroorganismEnzymology Committee Number: EC1.1.1.50Molecular weight: 30 kDa (SDS-PAGE)Isoelectric point: 5.2Km value: 3.6×10-5M (androsterone), 4.7×10-5M (NAD+)Inhibitors: Cu²⁺，Ag⁺，Hg²⁺，Zn²⁺，Fe³⁺ Optimum pH: 8.5-9.5 Figure 1Optimum temperature: 55℃ Figure 2pH stability: 6.0-9.0 (25℃,16h) Figure 3Thermal stability: Stable below 37℃ (pH9.0, 30min) Figure 4Stability: -25 ~ -15℃ standing store for 12 monthsMore than 90% activity Figure 5Protective agent: BSA Assay method for activity1. PrincipleThe NADH produced by the reaction can be detected with a spectrophotometer at 340nm.2. Definition of enzyme activityUnit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1µmolNADH per minute under the following conditions.3. Reagent preparationReagent I: 0.1M sodium pyrophosphate (adjust pH to 8.9 with HCl).Reagent II: Dissolve 319mg NAD+ into 25mL double steaming water, adjust the pH to 7.0-7.5 with solid NaHCO3, and adjust the volume to 30mL with double steaming water.Reagent III: Dissolve 30mg into 100mL methanol.Enzyme diluent: 10mMTris-HCl, pH 9.0.4. Operation procedure1. Add 2.6mL reagent I, 0.2mL reagent II and 0.1mL reagent III into a 3mL colorimetric dish and mix well.2. Preheat the reaction mixture at 25°C for 5min.3. Add 0.1mL enzyme liquid to the reaction mixture, mix it well, react at 25°C, and record the absorbance change within 1min at 340nm with a spectrophotometer (∆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 computing3.00: total volume of reaction liquid (mL);0.10: enzyme liquid volume (mL);1.0: optical path length (cm);df: dilution ratio;C: Enzyme concentration (mg/mL);6.22: Nanomolar absorption coefficient of NADH at 340nm (cm2/µmol)