COEI-1-ACTGLU Determination of beta-glucanase (β 1-3, β 1-6) Activity in enzyme preparations

General specifications

These enzymatic activities are usually present within a complex enzymatic preparation. In the degradation of ß-glucans from Botrytis cinerea, endo- β-glucanase activities of the type endo- β-1,3 and of the type exo- β-1,6 glucosidase, as well as exo- β-1,3 type activities are involved. They are summarized here under the commonly used term, “β-glucanases”. These enzymatic preparations are also capable of degrading β-glucans in the cell walls of dying Saccharomyces yeast cells which supports the process called “élevage de vin sur lie“ (aging of wine laying on lees). Endo- β-1,3 activities, endo- β-1,6 activities as well as exo- β-1,3 and exo- β-1,6 activities are involved in this process. Unless otherwise stipulated, the specifications must comply with resolution OIV/OENO 365/2009 concerning the general specifications for enzymatic preparations included in the International Oenological Codex.

1. Origin

Reference should be made to paragraph 5, “Sources of enzymes and fermentation environment”, of the general monograph on Enzymatic preparations.

The enzyme preparations containing β-glucanase activities are produced by direct fermentations, for example, of Trichoderma harzianum, Trichoderma longibrachiatum (T. reesei) and Penicillium funiculosum.

2. Scope of application

Reference should be made to the International Code of Oenological Practices, OENO 11/2004; OENO 12/2004; OENO 15/20 04 and AG 3/85- OEN.

The enzymatic preparations containing β 1-3 and β 1-6 glucanase activities are able to hydrolyse the glucan produced by Botrytis cinerea (noble rot and gray rot). This polysaccharide causes great difficulties during wine clarification and filtration. Such β-glucanases are therefore specifically used for clarification and filtration of wines made from botrytised grapes.

The glucans contained in the yeast cell walls are also hydrolysed by these β-glucanases. They may be used to improve the process of maturing on lees as well as the filterability.

3. Principle

The method of analysis is based on measuring the glucose released by the enzyme, using a standardised solution of Schizophyllum sp. glucan as substrate.

3.1.         Definition of units

A unit of β-glucanase (β-Glu-U) is defined as the quantity of reducing sugars, expressed as glucose, released in test conditions by 1 g (or 1 mL) of enzyme per minute.

3.2.         Role of the enzyme

As it grows on infected grapes (as noble or grey rot), Botrytis cinerea excretes a β-1,3-glucan which, at every third unit of glucose, possesses a β-1,6 glycosylated residue of glucose (Fig. 1). This glucan is very similar to glucan synthetised by Schizophyllum sp.

3.3.         Principle of measurement

The enzymatic activity releases glucose which, in an alkaline salt solution, reduces 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid. The addition of phenol increases the sensitivity of the reaction. Sodium bisulphite serves to stabilise colour.

4. Apparatus

4.1.         Spectrophotometer and cuvettes with an optical path length of 1 cm

4.2.         40°C, 100°C water bath

4.3.         Standard magnetic stirrer

4.4.         Submersible multi-point magnetic stirrer set at 300 rpm

4.5.         Measuring containers (volumetric flasks, beakers, conical flasks, etc.)

4.6.         Beaker

4.7.         Micro-pipettes

4.8.         Timer

4.9.         Ultrasonic bath

4.10.     pH meter

5. Reagents and products

5.1.         Substrate

Glucan stock solution supplied by the University of Braunschweig^[1]; the glucan content of which has been determined by the University of Braunschweig.

5.2.         Pure products

5.2.1.  Citric acid monohydrate (CAS No.  5949-29-1)

5.2.2.  Sodium hydroxide (CAS No.  1310-73-2)

5.2.3.  Potassium sodium tartrate (CAS No.  304-59-6)

5.2.4.  Sodium metabisulphite Na2S2O5 (CAS No.  7681-57-4)

5.2.5.  Phenol (CAS No. 108-95-2)

5.2.6.  Anhydrous glucose

5.2.7.  3,5-dinitro-2-hydroxybenzoic (3,5-dinitrosalicylic) acid (CAS No. 609-99-4)

5.2.8.  Distilled water

5.3.         Solutions

5.3.1.  1M sodium hydroxide solution

In a 100-mL volumetric flask, dissolve 4.0 g of sodium hydroxide (5.2.2) in distilled water (5.2.8) and make up to the required volume.

5.3.2.  Citrate buffer solution (pH 4.0) - 0.2 mol/L

In a 500-mL volumetric flask, dissolve 21.0 g of citric acid monohydrate (5.2.1) in 400 mL of distilled water, then adjust the pH to 4.0 with a molar solution of sodium hydroxide (5.3.1) and make up to the required volume with distilled water (5.2.8).

5.3.3.  Citrate buffer solution (pH 4.0) - 0.1 mol/L

In a 1,000-mL volumetric flask, dissolve 21.0 g of citric acid monohydrate (5.2.1) in 900 mL of distilled water (5.2.8), then adjust the pH to 4.0 with a molar solution of sodium hydroxide (5.3.1) and make up to the required volume with distilled water (5.2.8).

5.3.4.  Titrating solution: DNS (dinitrosalicylic) acid colour reagent with phenol

This is prepared from solutions A, B and C below:

5.3.4.1.                  Solution A:

Weigh out 154.2 g of potassium sodium tartrate (5.2.3) in an 800-mL beaker and dissolve completely in 500 mL of distilled water (5.2.8). Add 9.7 g of sodium hydroxide (5.2.2).

5.3.4.2.                  Solution B:

In a 2,000-mL beaker, completely dissolve 5.3 g of 3,5-dinitrosalicylic acid (5.2.7) in 500 mL of distilled water (5.2.8). The best results are obtained using an ultrasonic bath.

5.3.4.3.                  Solution C:

In a 100-mL beaker, dissolve 4.2 g of phenol (5.2.5) in 50 mL of distilled water (5.2.8). Then add 1g of sodium hydroxide (5.2.2) and, when completely dissolved, 4.2 g of sodium metabisulphite (5.2.4) and dissolve again.

5.3.4.4.                  0.3% glucose solution

In a 100-mL volumetric flask, put exactly 300 mg of glucose (5.2.6), dissolve in distilled water (5.2.8) and make up to the required volume with distilled water.

5.3.4.5.                  DNS acid colour reagent with phenol

Solutions A and C are mixed with solution B in a 2,000-mL beaker, which is then covered with aluminium foil.

Before using, keep in the dark for at least 3 days.

Transfer the reagent to a brown glass container.

If stored in a dark place at 15-20° C, this solution can be kept for a month.

For each newly-prepared reagent and before each measurement, a new calibration is carried out prior to each enzyme analysis.

Before each use, 3 mL of 0.3% glucose solution (5.3.3.4) should be added to 200 mL of the DNS acid colour reagent with phenol.

5.3.5.  Glucan in solution at 0.1%, pH 4.0

Weigh out the exact quantity of glucan stock solution (5.1) to obtain a final concentration of 1 g /L.

The final substrate solution should contain 50% of the citrate buffer solution (pH 4.0) - 0.2 mol/L (5.3.2).

To obtain 100 mL of substrate solution from the glucan stock solution (5.1) (actually containing 5.2 g/L), weigh out 19.2 g in a 100-mL beaker. Add 50 mL of the citrate buffer solution (pH 4.0) - 0.2 mol/L (5.3.2). Homogenize the glucan mixture by stirring for at least 15 minutes. When well-mixed, adjust the pH to 4.0 with a sodium hydroxide molar solution (5.3.1). Then transfer the solution to a 100- mL volumetric flask and make up to the required volume later with distilled water (5.2.8).

Store all glucan stock solutions at ambient temperature. If a new glucan stock solution is used, a glucan substrate factor (Gf = glucan factor) should be determined by means of the standard enzyme. The "Gf" is essential for comparing the results from previous glucan stock solutions with the new ones. The “Gf” is calculated with the values measured considering that standard enzymatic activity is 10,000 β-Glu U/g in the formula (See: Calculation of enzymatic activity).

5.4.         Enzyme preparations

5.4.1.  Glucanase standard enzyme solution:

Dissolve 0.5 g of glucanase standard enzyme preparation in 25 mL of the citrate buffer solution (pH 4.0; 0.1 mol/L) (5.3.3) and make up to 100 mL with distilled water (5.2.8).

5.4.2.  For all other enzyme preparations: Dissolve 1 mL of enzyme preparation or 0.5 g of solid powdered or granulated enzyme preparation in 25 mL of the citrate buffer solution (pH 4.0; 0.1 mol/L) (5.3.3) and make up to 100 mL with distilled water (5.2.8). If the absorption values are too high or too low (absorbance range 0.1-0.6), appropriate dilution is necessary. The enzyme dilution should contain 25% of citrate buffer solution (5.3.3).

6. Procedure

6.1.         Reagent “blank” test

Add 7 mL of DNS acid colour reagent with phenol (5.3.4) to 3 mL of distilled water (5.2.8) in a 50-mL volumetric flask and heat for exactly 10 minutes over a bath of boiling water. Cool for 5 minutes in an ice bath, then transfer the flask into a water bath at 20° C and add distilled water (5.2.8) to just below the mark. After 10 minutes at 20°C, make up to the required volume.

6.2.         Glucose calibration curve with DNS acid colour reagent with phenol

Dissolve 2.00 g of glucose (5.2.6) in a 200-mL volumetric flask and make up to volume with distilled water (5.2.8). Using this solution, prepare the following dilutions:

No.	V standard solution / 100 mL	glucose/100 mL	glucose (μg) in the trial (= 0.5 mL)
1	2 mL	20 mg	100 μg
2	5 mL	50 mg	250 μg
3	10 mL	100 mg	500 μg
4	15 mL	150 mg	750 μg
5	20 mL	200 mg	1,000 μg
6	30 mL	300 mg	1,500 μg
7	40 mL	400 mg	2,000 μg

Use a pipette to put 0.5 mL of each glucose dilution into a 50-mL volumetric flask and add 7 mL of DNS colour reagent with phenol (5.3.4) and 2.5 mL of distilled water (5.2.8). Heat the measuring containers for exactly 10 minutes in a bath of boiling water. Cool for 5 minutes in a bath of ice, then transfer the flask to a water bath at 20°C and add distilled water (5.2.8) to just below the mark. After 10 minutes at 20°C, make up to volume. Measure the absorbance of the solutions within the next 15 minutes, using a spectrophotometer with a wavelength of 515 nm against the “blank” (reagent alone).

On a diagram, plot the quantity of glucose released in the test against the absorbance at 515 nm (Fig. 2).

The calibration curve is produced the same day before every enzyme analysis.

6.3.         “Blank” testing of enzymes

Use a pipette to put 0.5 mL of each enzyme solution (5.4.1 or 5.4.2) into a 50-mL volumetric flask and add 7 mL of DNS acid colour reagent with phenol (5.3.4). Mix carefully and add 2.5 mL of substrate solution (5.3.5). Stir well by hand. Then heat all samples over a bath of boiling water for exactly 10 minutes, cool for 5 minutes in a bath of ice and transfer the flask to a water bath at 20° C, adding distilled water (5.2.8) to just below the mark. After 10 minutes at 20° C, make up to volume. Measure the absorbance of the solutions within the next 15 minutes, using a spectrophotometer with a wavelength of 515 nm against the “blank” (reagent alone).

6.4.         Measuring the activity of enzyme preparations

For each sample of enzymes, put 10 mL of substrate (5.3.5) into a conical flask in a water bath at 40° C for 5 minutes. Samples should be homogenized using a submersible multi-point magnetic stirrer set at 300 rpm. After 5 minutes, 2 mL of the enzyme solution (5.4.1 or 5.4.2) are added to the first sample and a timer started just after adding the first enzyme solution.

Then add the following enzyme solutions to all the other samples with an interval of 30 seconds between samples.

Samples should then be stirred at 300 rpm throughout the entire reaction time.

After exactly 15 minutes, remove 3 mL of the first mixture, followed by all the other samples, at intervals of 30 seconds.

Using a pipette, put each 3-mL mixture into as many 50-mL volumetric flasks as required, each of which contains 7 mL of DNS acid colour reagent with phenol (5.3.4).

Then heat all the samples, at 30-second intervals, for exactly 10 minutes over a bath of boiling water.

Cool for 5 minutes in a bath of ice, transfer the flask to a water bath at 20° C and add distilled water (5.2.8) to just below the mark.

After 10 minutes at 20° C, make up to volume. Measure the absorbance of the solutions within the next 15 minutes, using a spectrophotometer with a wavelength of 515 nm against the “blank” (reagent alone).

The difference in the absorbance between the “blank” reading of enzymes and the value after reaction should be between 0.1 and 0.6 absorbance units.

If the values are over the measuring range of the calibration curve, repeat the experiment with dilutions adapted to the enzymes.

For all enzymes, always prepare 1 “blank” enzyme reading and 2 values after reaction. The two values after reaction should be similar.

7. Calculations

To calculate the enzyme activity, use the mean value of the two readings.

The enzymatic activity of an enzyme preparation is calculated according to the following formula:

β-Glu-Unit activity/ g or mL=

Nkat/g or mL = (Actiβ-Glu-Unit/g or mL)

Where:

• G = Quantity of reducing sugars released during the test (reducing sugars released by Δ = the mean of 2 repetitions of the absorbance after reaction minus the absorbance of the “blank” enzyme, calculated in glucose from the glucose calibration curve in µg).
• E = Quantity of enzyme diluted to 100 mL in g or mL
• 200 = Dilution factor
• 15 = Reaction time in min
• Gf = Glucan factor (to be calculated)

Example of a calculation:

Enzyme	Measured value 1 2		“Blank” enzyme	E	μg glucose	β-Glu units /g or mL
Enzyme used	0.621	0.618	0.415	0.503	662	10325
Penicillium funiculosum ß-Glucanase	0.417	0.416	0.023	1	1249	9799

Gf calculation:

• 1 Measure using old substrate and standard enzyme (Value 1)
• 2 Measure using new substrate and standard enzyme (Value 2)

Calculation: Value 1 / Value 2

8. Bibliography

• Bertrand A. Détermination de l'activité β-glucanase de Botrytis des préparations enzymatiques, OIV FV 1263

^[1] Prof. Dr Udo Rau, Technical University Braunschweig, Department of Biochemistry and Biotechnology Spielmannstr. 7, 38106 Braunschweig - Germany