Technical indicators
Appearance: White amorphous freeze-dried powder; Protein purity: ≥ 90%; Specific activity: ≥ 25 U/mg enzyme powder; Glucose 6 phosphate dehydrogenase: ≤ 0.01%; Lactate dehydrogenase: ≤ 0.005%; Glutamate dehydrogenase: ≤ 0.001%; Malic acid dehydrogenase: ≤ 0.01%; Hexokinase: ≤ 0.01%
Enzymatic properties
Source: Microorganisms; Classification: EC 1.1.1.50; Molecular weight: 30 kDa (SDS-PAGE); Isoelectric point: 5.2; Km value: 3.6 × 10-5 M (androgen), 4.7 × 10-5 M (NAD+); Inhibitors: Cu2+, Ag+, Hg2+, Zn2+, Fe3+; Optimal pH: 8.5-9.5;
Optimal temperature: 55 ℃; PH stability: 6.0-9.0 (25 ℃, 16 hours); Thermal stability: Stable below 37 ℃ (pH 9.0,30 min); Stability: Keep at -25~-15 ℃ for 12 months and maintain over 90% activity; Protector: BSA
Purpose: Used for the development and large-scale preparation of total bile acid (TBA) reagents.
Definition of enzyme activity: Unit enzyme activity is defined as the catalytic production of 1 per minute under the following conditions μ The amount of enzyme required for mol NADH.
Reagent preparation
Reagent I: 0.1 M sodium pyrophosphate (adjust pH to 8.9 with HCl).
Reagent II: Dissolve 319 mg of NAD+in 25 mL of double distilled water, adjust the pH to 7.0-7.5 with solid NaHCO3, and dilute to 30 mL with double distilled water.
Reagent III: Dissolve 30mg into 100mL of methanol.
Enzyme diluent: 10mM Tris HCl, pH 9.0.
Operating Steps
1. Add 2.6 mL of Reagent I, 0.2 mL of Reagent II, and 0.1 mL of Reagent III to a 3 mL cuvette, and mix well.
2. Preheat the reaction mixture at 25 ℃ for 5 minutes.
3. Add 0.1 mL of enzyme solution to the reaction mixture and mix well,
React at 25 ℃ and record the absorbance change within 1 minute using a spectrophotometer at 340 nm（ Δ As)
*Replace the enzyme solution with enzyme diluent, and the other steps are the same. The absorbance of the obtained solution is blank absorbance（ Δ Ab) Δ A= Δ As- Δ Ab
