Technical indicators:
Appearance: White amorphous freeze-dried powder; Protein purity: ≥ 90%; Specific activity: ≥ 200 U/mg enzyme powder; NADH/NADPH oxidase: ≤ 0.01%; Malic acid dehydrogenase: ≤ 0.005%; Glutamate dehydrogenase: ≤ 0.003%; Pyruvate kinase: ≤ 0.03%
Enzymatic properties
Source: Microorganisms; Classification: EC 1.1.1.28; Molecular weight: 45 kDa (SDS-PAGE); Isoelectric point: 4.5; Km value: 2.2 × 10-6 M (pyruvic acid); Inhibitors: Ag+, Hg2+; Optimal pH: 6.5-7.0; Optimal temperature: 45 ℃; PH stability 5.5-10.0 (25 ℃, 16 h); Thermal stability is stable below 50 ℃ (pH 7.0,30 min); Stability: Keep at -25~-15 ℃ for 12 months and maintain over 90% activity; Protector: BSA.
Usage: Used for the development and large-scale preparation of reagents such as glutamic oxaloacetic transaminase and glutamic oxaloacetic transaminase.
Definition of enzyme activity
Unit enzyme activity is defined as oxidation of 1% per minute under the following conditions μ The amount of enzyme required for mol NADH.
Reagent preparation
Reagent I: Weigh 0.04 g of sodium pyruvate, dissolve in 0.1 M, pH7.7 Tris HCl buffer, and bring to volume to 100 mL.
Reagent II: Weigh 2.11 g of CAPS, dissolve it in 80 mL of water, adjust the pH to 9.7 with NaOH, add 120 mg of NADH, and dilute to 100 mL with double distilled water.
Enzyme diluent: 10 mM, pH 7.0 potassium phosphate buffer, and add BSA to the final concentration of 0.1%.
Sample: Dilute the enzyme to 1-2U/mL using pre cooled enzyme diluent.
Operating Steps
1. Add 1 mL of reagent I to a 1 mL cuvette, 20 μ L sample to be tested, mix well.
2. Preheat the reaction mixture at 37 ℃ for 5 minutes.
3. Add 0.2 mL of reagent II to the reaction mixture, mix well, react at 37 ℃, 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
