AFU (α-L-Fucosidase)
This enzyme is used in the reagent calibration of α-L-fucosidase and research of quality control products.
Preparation and specification
|
Appearance |
Colorless transparent liquid |
|
Protein purity |
≥90% (from SDS-PAGE) |
|
Activity |
≥5 U/ml |
|
Β-N-acetylglucosaminidase |
≤ 0.2% |
|
a-mannosidase |
≤ 0.05% |
|
β-galactosidase |
≤ 0. 1% |
Properties
|
EC number |
3.2.1.51 (Recombinant from microorganism) |
|
Molecular weight |
55 kDa (SDS-PAGE) |
|
Isoelectric point |
6.2 |
|
Michaelis constants |
7.5 Fig. 1 |
|
Optimum pH |
80 ℃ Fig. 2 |
|
Optimum temperature |
pH 7.0-10.0 (25 ℃, 16 h) Fig. 3 |
|
pH stability |
Below 90 ℃ (pH 8.0, 30 min) Fig. 4 |
|
Thermal stability |
At least one year at -25 ~ -15 ℃ Fig. 5 |
Storage condition
Store at -20°C and protected from light,valid for 2 year.
Figures
Assay principle
Unit definition
One unit (U) is defined as the amount of enzyme which produces 1 μmol of CNP per min under the conditions described below.
Reagents preparation
Reagent I: 20 mM pH 8.0 Tris-HCl.
Reagent II: 10 mM CNP-AFU, dissolved by DMSO.
Enzyme diluent: 20mM pH 8.0 Tris-HCl.
Procedure
1. Add 0.94 mL Reagent I and 0.05 mL Reagent II to 1 mL cuvette.
2. Preincubate the reaction mixture at 37 ℃ for 5 min.
3. Add 0.01 mL the enzyme solution in the reaction mixture and mix to start the reaction, record the ΔAs at 405 nm in 1 minute in a spectrophotometer thermostated at 37 ℃.
4. At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab.
Calculation
Vt: Total volume (1mL)
Vs: Enzyme volume (0.01mL)
1.0: Light path length (cm)
df: dilution factor C: Enzyme concentration (mg/mL)
18.4: Millimolar extinction coefficient of CNP under 405 nm (cm2/μmol)
