Invertase from Candida sp. is responsible for catalyzing the hydrolysis of sucrose into glucose and fructose and is widely used in the field of carbohydrate processing. Invertase from Candida sp. can be used for enzymatic determination of sucrose concentration as well as for structural analysis of carbohydrates containing β-D-fructofuranosyl residue.
PREPARATION and SPECIFICATION
Appearance | : White amorphous powder, lyophilized |
|---|
Activity: | ≥100U/mg enzyme powder (containing approx. 70 % of stabilizer) |
|---|
Stabilizer: | KH₂PO₄ |
|---|
PROPERTIES
| Stability | Stable at −20 ℃ for at least one year | (Fig.1) | | Molecular weight | approx. 260,000 | - | | Michaelis constant | 1.5 × 10⁻² M (Saccharose) | - | | Structure | Glycoprotein containing approx. 50% of carbohydrates | - | | Optimum pH | 3.5 − 4.0 | (Fig.3) | | Optimum temperature | 60 − 70 ℃ | (Fig.4) | | pH Stability | pH 4.0 − 6.0 (50 ℃, 10min) | (Fig.5) | | Thermal stability | below 60 ℃ (pH 4.5, 10min) | (Fig.6) | | Substrate specificity | This enzyme hydrolyzes saccharose and raffinose, but does not hydrolyze inulin and melezitose. | - |
|
APPLICATIONS
This enzyme is useful for enzymatic measurement of saccharose, and structural analysis of carbohydrates containing β-D-fructofuranoside residue.
ASSAY
Principle
The formation of reducing sugars is measured by the modified Fehling-Lehmann-Schoorl method.
Unit definition
One unit catalyzes formation of one milligram of glucose-equivalent reducing sugars in 3 minutes, under the conditions detailed below. This activity is equivalent to the international unit, i.e. hydrolysis of one micromole of saccharose per minute at the same temperature.
Method
Reagents
A. Saccharose solution 5.0 %: 5.0 g of saccharose / 100 mL of 80 mM acetate buffer, pH 4.5, with 2−3 drops of toluene added for preservation.
B. Alkaline solution 103 g of NaOH + 346 g of potassium sodium tartrate tetrahydrate / 1,000 mL of H₂O
C. CuSO₄ solution: 6.93% (69.3g CuSO₄·5H₂O/1,000ml of H₂O)
D. KI solution: 30% (300g KI/1,000ml of H₂O) (Store in a brown bottle)
E. H₂SO₄ solution: 25%
F. Na₂S₂O₃ solution: 50mM (49.638g Na₂S₂O₃·5H₂O, 4.0g Na₂CO₃ (stabilizer)/4,000ml of H₂O) (Store in a brown bottle and keep for 3−4 days before use)
G. Soluble starch solution: 2.0% (Dissolve by boiling) (Should be prepared fresh)
H. Enzyme diluent: 50mM acetate buffer, pH 4.5
Procedure
1.Pipette 1.0 mL of substrate solution (A) into a test tube and equilibrate at 20 ℃ for approximately 5 minutes.
2.Add 1.0 mL of the enzyme solution (pre-incubated at 20 ℃) and mix.
Concentration in assay mixture
| Acetate buffer | 65mM | | Saccharose | 73mM |
|
3.After exactly 3 minutes at 20 ℃, add 2.0 mL of alkaline solution (B) to stop the reaction.
At the same time, prepare the blank by first mixing the substrate solution with 2.0 mL of alkaline solution after incubation for 3 minutes at 20 ℃, then add the enzyme solution, and carry out the same procedure as with the test (Procedure 4−8).
4.Transfer the stopped reaction mixture from the test tube to a 100-mL Erlenmeyer flask. Rinse the inside of the test tube with approximately 3 mL of distilled water and transfer the rinsings to the flask. Repeat this procedure three times.
5.Add 2.0 mL of CuSO₄ solution (C) and place the flask directly on an electrothermic heater (1.2 KWH) in the presence of a glass bead (5 mmφ) to prevent bumping.
6.Keep for exactly 2 minutes in a boiling state and cool to room temperature under running water.
7.Add 2.0 mL each of KI solution (D) and H₂SO₄ solution (E) in this order.
8. Shake the flask and determine the amount of residual Cu²⁺ by titration with Na₂S₂O₃ solution (F) in the presence of a few drops of soluble starch (G) as an indicator.
9.Record the titers (ml) of the test (t) and the blank (b), and calculate the titration difference (Δtiter, mL).
*Dissolve the enzyme preparation in ice-cold distilled water and dilute to 2.0−9.0 U/mL with the enzyme dilute (H), immediately before the assay.
Calculation
Activity can be calculated by using the following formulas:
| Vs | : Sample volume (1.0mL) |
| 0.600 | : Titration difference (mL) of 50 mM Na₂S₂O₃ solution (F＝1.00) for 1.0 mg of reducing sugar (glucose) |
| F | : Concentration factor of 50 mM Na₂S₂O₃ (F should be determined by the iodotimetry method in each time of the preparation). |
| C | : Enzyme concentration in dissolution (c mg/mL) |
ZJ26F0434265