Technical indicators
Appearance: White amorphous freeze-dried powder; Protein purity: ≥ 80%; Specific activity: ≥ 10 U/mg enzyme powder; α- Amylase: ≤ 0.00001%
Source: Microorganisms, Classification: EC 3.2.1.20; Molecular weight: 65 kDa (SDS-PAGE); Isoelectric point: 5.2; Km value: 5.8 × 10-4 M; (p-Nitrophenyl- α- D-glucopyranoside); Inhibitors: SDS, Ag+, Hg2+, Cu2+; Optimal pH: 7.1; Optimal temperature: 50 ℃; PH stability: 6.0-8.0 (25 ℃, 16h); Thermal stability: Stable below 50 ℃ (pH 7.0,10 min); Stability: Keep at -25~-15 ℃ for 12 months; More than 90% activity; Protector: BSA
Purpose: For α- Development and large-scale preparation of amylase reagents.
Definition of enzyme activity
Unit enzyme activity is defined as the production of 1 enzyme per minute under the following reaction conditions μ The amount of enzyme required for mol PNP.
Reagent preparation
Reagent I: 100 mM phosphate buffer, pH 7.0.
Reagent II: 20 mM p-nitrobenzene- α- D-glucopyranoside (dissolved in water and can be stored at 4 ℃ for 2 weeks). Reagent III: 200 mM Na2CO3 (21.2mg/mL dissolved in water).
Reagent IV: 200 mM, pH 7.0 potassium phosphate buffer, containing 0.05% Tween 20 and 1 mM EDTA. Sample to be tested: Dilute the enzyme solution to 0.006-0.022 U/mL using reagent IV.
Operating Steps
1. Add 1.0 mL of reagent I and 0.5 mL of reagent II to a 5 mL centrifuge tube, and preheat at 37 ℃ for 5 minutes.
2. Add 0.5 mL of the sample to be tested, mix well, and react at 37 ℃ for 15 minutes. Then, add 2.0 mL of reagent III to terminate the reaction.
3. Measure the absorbance of the sample at 400 nm.
4. Exchange the addition sequence of reagent III and the sample to be tested, and measure the blank absorbance.
