TENTATIVE
Prepared at the 49th JECFA (1997)
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These specifications are designated tentative because Appendix B "General Considerations and Specifications for Enzymes from Genetically Manipulated Microorganisms" of Annex 1 of the Compendium of Food Additive Specifications, FNP 52, Rome 1992, is tentative. |
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SYNONYMS |
ALDC |
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SOURCE |
Produced extracellularly by submerged fermentation of Bacillus subtilis UW193 (a dal-transformant strain of ToC46) which through rDNA techniques contains the gene for a -decarboxylase from Bacillus brevis ATCC 11031 in plasmid pUW235. When fermentation is complete, the broth is filtered then stabilised with glutaraldehyde before further filtration. |
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ACTIVE PRINCIPLE |
a -Acetolactate decarboxylase |
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SYSTEMATIC NAME AND NUMBER |
(S)-2-Hydroxy-2-methyl-oxobutanoate carboxy-lyase |
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REACTIONS CATALYSED |
Decarboxylation of a -acetolactate to acetoin |
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DESCRIPTION |
Commercial preparations are brown liquids containing the active enzyme. These preparations are standardised with glycerol and lactic acid and preserved with 0.2% potassium sorbate |
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FUNCTIONAL USES |
Enzyme preparation |
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GENERAL SPECIFICATIONS |
Must conform to the "General Specifications for Enzyme Preparations Used in Food Processing" in Annex 1 of the Compendium of Food Additive Specifications, FAO Food and Nutrition Paper 52, Rome 1992. |
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CHARACTERISTICS |
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IDENTIFICATION |
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a -Acetolactate decarboxylase activity |
The sample shows a -acetolactate decarboxylase activity |
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TESTS |
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a -Acetolactate decarboxylase activity |
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PRINCIPLE
The enzyme a -acetolactate decarboxylase (ALDC) decarboxylates a -acetolactate to produce acetoin. The resultant acetoin can be reacted with a mixture of naphthol and creatine, resulting in quantitative formation of a characteristic red colour.
APPARATUS
1. Spectrophotometer or equivalent capable of measuring optical density (absorbance) at 522 nm
2. Water bath, held at 30±1 °, containing test tube rack
3. Volumetric glassware
REAGENTS
1. MES buffer, 0.05M (pH = 6.0).
Dissolve 9.76 g of MES (2-(N-morpholino) ethanesulphonic acid) in approximately 900 ml of deionised water. Adjust pH to 6.0 ± 0.5 with 1 N NaOH. Transfer to a 1-litre volumetric flask and make to volume with deionised water. This solution may be kept for two weeks at room temperature.
2. Brij 35 solution, 15% w/v.
Dissolve 15.0 g of Brij 35 (polyoxyethylene lauryl ether, Atlas Chemie, BDH, or equivalent) in approximately 70 ml of deionised water, heating to 60° to aid dissolution. After cooling, transfer to a 100-ml volumetric flask and make to volume with deionised water. This solution should be stored in a refrigerator, and can be kept for up to two months.
3. MES/Brij 35/NaCl solution.
Dissolve 48.8 g of MES and 175.32 g of NaCl (AR grade) in approximately 4.5 litres of deionised water. Add 17 ml of 15% Brij 35 solution (see 2. above). Adjust pH to 6.0 ± 0.5 with 1 N NaOH. Transfer to a 5-litre volumetric flask and make to volume with deionised water. This solution may be kept for two weeks at room temperature.
4. a -Acetolactate substrate, 0.2% v/v.
Pipette 100 m l of ethyl-2-acetoxy-2-methylacetolactate into a 50 ml volumetric flask. Add 6.0 ml of 0.5 N NaOH to the flask and stir for 20 minutes. Add MES buffer (see 1. above) to bring the volume to approximately 40 ml. Adjust pH to 6.0 ± 0.5 with 0.5 N HCl. Make to volume with MES buffer (see 1. above). This substrate should be made just before use.
5. Naphthol/Creatine colour reagent.
Dissolve 5.00 g of 1-naphthol and 0.50 g of creatine in 1 N NaOH, make to volume with 1 N NaOH in a 500-ml volumetric flask. This colour reagent should be made fresh just before use and shielded from light as much as possible.
6. Acetoin stock solution, 1000 mg/l.
Dissolve 0.100 g of acetoin (3-hydroxy-2-butanone) in deionised water in a 100-ml volumetric flask. Make to volume with deionised water.
7. Acetoin standards.
Dilute 0, 1.0, 2.0, 4.0, 6.0, and 8.0 ml of stock acetoin solution (see 6. above) to volume with deionised water in 100-ml volumetric flasks to give standard solutions of 0, 10, 20, 40, 60, and 80 mg/l of acetoin. These solution may be kept for two weeks stored in a refrigerator.
8. Enzyme solutions.
Test samples of the enzyme a -acetolactate decarboxylase (ALDC) are diluted with MES/Brij 35/NaCl solution (see 3. above). The exact dilution will need to be determined by experience and experimentation to give an enzyme concentration which produces a result within the range of the acetoin standards (see 7. above) under the conditions of the test.
PROCEDURE
Preparation of standard curve
Prepare a standard curve by treating the acetoin standards (7.) in the following manner:
Pipette 400 m l of the acetoin standard solutions into 10-ml plastic test tubes. Add 4.6 ml of colour reagent (5.) to each test tube, mix, and let the test tube stand at room temperature for exactly 40 minutes. At the end of this 40 minute period, place 0 mg/l acetoin standard in the spectrophotometer set to read absorbance at 522 nm and adjust absorbance reading to zero. Measure the absorbance of the remaining acetoin standards at 522 nm.
Determination of enzyme activity
Preheat enzyme solutions (see 8. above), MES buffer (see 1. above), and the substrate (see 4. above) in a water bath at 30± 1°C for approximately 10 minutes.
Four solutions must be prepared for each analysis:
1. Enzyme blank (B1). Pipette 200 m l of enzyme solution (8.) and 200 m l of MES buffer (1.) into a 10 ml plastic test tube. Mix, and immediately place the test tube back into the water bath.
2. Sample value (HI). Pipette 200 m l of enzyme solution (8.) and 200 m l of substrate (4.) into a 10 ml plastic test tube. Mix, and immediately place the test tube into the water bath.
3. Buffer blank (B2). Pipette 200 m l of MES buffer (1.) and 200 m l of MES/Brij 35/NaCl solution (3.) into a 10 ml plastic test tube. Mix, and immediately place the test tube into the water bath.
4. Buffer value (H2). Pipette 200 m l of MES/Brij 35/NaCl solution (3.) and 200 m l of substrate (4.) into a 10 ml plastic test tube. Mix, and immediately place the test tube into the water bath.
For each enzyme determination the test tubes, are prepared in the order: B1, H1, B2, H2.
Exactly 20 minutes after mixing of each of solutions B1, H1, B2, and H2, remove from water bath, add 4.6 ml of colour reagent (5.), mix and leave at room temperature for exactly 40 minutes. At the end of this 40 minute period measure the absorbance of the solutions at 522 nm on a spectrophotometer or equivalent previously adjusted so that the 0 mg/l acetoin standard gives an absorbance reading of zero.
CALCULATION
The activity of the ALDC enzyme is defined in terms of activity units where:
1 ADU (a -Acetolactate Decarboxylase Unit) = the amount of enzyme which, by decarboxylation of a -acetolactate produces 1 m mol of acetoin per minute under the test reaction conditions.
Plot optical density values at 522 nm for the acetoin standards against acetoin concentration (in mg acetoin/l) of the standard and generate a standard curve. Use the standard curve to convert the optical density measurements for solutions H1, B1, H2, and B2 to mg acetoin/l.
Activity of enzyme (in ADU/g)
Where:
[H1], [B1], [H2], [B2] are the concentrations of acetoin in the respective solutions in mg/l
F is the final volume of enzyme solution, after dilutions, in millilitres
W is the weight of enzyme in volume F, in grams
The factor 0.0011351 contains:
· the conversion from mg acetoin/l to m mol acetoin/min
· the conversion for the actual volumes of substrate and enzyme solution used in the analysis.
