Maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis

Prepared at the 49th JECFA (1997)

TENTATIVE

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.

SYNONYMS

Maltogenase

SOURCES

Prepared by submerged fermentation of Bacillus subtilis, strain DN 252 (a derivative of Bacillus subtilis 168, strain QB 1133, BGSC1A289) which, through recombinant DNA techniques contains the gene coding for maltogenic amylase in the plasmid pDN1413. The gene is imported from Bacillus stearothermophilus (NCIB 11837).

ACTIVE PRINCIPLES

Exo-acting a -amylase

SYSTEMATIC NAMES AND NUMBERS

Glucan 1,4-a -maltohydrolase, E.C. 3.2.1.133

REACTION CATALYZED

Exohydrolysis of 1,4-a -glucosidic linkages in amylose, amylopectin and related glucose polymers. Maltose units are successively removed from the non-reducing end of the polymer chain until the molecule is completely degraded or, in the case of amylopectin, a branch point is reached.

DESCRIPTION

Commercial preparations can be liquid, granulated or powdered form. The powdered form contains sodium chloride as a stabilizer, the granulated form contains sodium chloride and wheat grits, and the liquid form contains sodium chloride and sucrose.

FUNCTIONAL USES

Enzyme preparation Retardation of staling in baked goods, preparation of high maltose glucose syrup

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.

CHARACTERISTICS

IDENTIFICATION

Maltogenic amylase activity

The sample shows maltogenic amylase activity
See description under TESTS

TESTS

Maltogenic amylase activity

The method is based on the ability of the enzyme to hydrolyze maltotriose to maltose and glucose. The reaction is stopped by raising the pH of the reaction medium to about 11. The formed glucose is converted to gluconolactone by glucose dehydrogenase in the presence of NAD⁺ which is converted to NADH⁺H⁺. The change in the concentration of NAD⁺ is measured photometrically at 340 nm. One enzyme unit (Maltogenic Amylase Unit, MAU) is defined as the amount of enzyme which under standard assay conditions cleaves 1 m mol of maltotriose per minute. Assay conditions being as follows: Substrate concentration 10 mg/ml; temperature 37°; pH 5.0; incubation time 30 min.
Preparation of 20 mg/ml-maltotriose substrate:
Dissolve 1000 mg maltotriose (e.g. Sigma M8378) in 0.1M citrate buffer, pH 5.0, making a total volume of 50 ml (to be prepared each day of analysis).
Preparation of glucose dehydrogenase (GluDH) reagent:
Add buffer solution (Merck No. 14051 or equivalent) to the enzyme mixture (Merck No. 14055, bottles "1" and "2", or equivalent). Allow the mixture to stand for 15 min. Transfer the solution to a 500 ml volumetric flask, containing 200 ml of 0.1 M citrate buffer pH 5.0. Fill the flask to the mark with the same buffer. This solution can be stored for two weeks at room temperature (not in a refrigerator).
Preparation of standard enzyme solutions:
Dilute a maltogenic amylase standard with known activity in 10 mM NaCl to give the following enzyme units per ml: 0.015, 0.030, 0.045, 0.060 and 0.075.
Measurement of enzyme activity:
Add 500 m l of substrate solution (20 mg/ml maltotriose, prepared in 0.1 M citrate buffer, pH 5.0, pre-warmed to 37°) to an equal volume of enzyme solution also pre-warmed to 37°. Mix the resulting solution thoroughly and transfer to a water bath maintained at 37°. After 30 minutes remove the test tube from the water bath, and add 1000 m l of the stop reagent (0.06N NaOH) and shake vigorously. Add 3 ml of GluDH reagent and mix again. Leave the test tube at room temperature for exactly 30 min and measure optical density (OD) at 340 nm. Prepare a blank in a similar manner except that the stop reagent is added before the substrate.
Calculation of enzyme activity:
Draw the enzyme standard curve in a coordinate system using enzyme activity (MAU/ml) as the abscissa and D OD (sample - blank) as the ordinate. Calculate the activity of the sample by reading D OD of sample dilutions on the standard curve and then multiplying by the corresponding dilution factors. The standard curve is a straight line passing through the origin and linear regression can therefore be applied.
Basic standardisation:
Glucose solutions:
a) Stock solution: Accurately weigh 1.60 g of glucose, analytical grade, in a 1000-ml volumetric flask and make up to volume with demineralized water.
b) standard solutions: Dilute 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 ml of stock solution to volume with deionized water in 100-ml volumetric flask to give standard solutions of 88.8, 178, 266, 355, 444 and 533 m mol/l.
Standard curve:
Prepare the glucose standard curve by mixing 2.0 ml glucose solution with 3.0 ml GluDH reagent and incubating at room temperature for 30 min. Read OD at 340 nm. Prepare a common blank using demineralized water instead of glucose solution. Plot glucose concentration (m mol/l) versus D OD.
Calculation:
Calculate the enzyme activity by reading the equivalent glucose concentration on the standard curve and inserting it in the following formula:

where:
A = reading on the standard curve, m mol/l
F = dilution factor of the enzyme solution, ml/g
4 = the ratio between amount of enzyme dilution in sample and amount of glucose solution in standard
30 = incubation time, 30 min
1000 = conversion from litre to ml

TENTATIVE	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.
SYNONYMS	Maltogenase
SOURCES	Prepared by submerged fermentation of Bacillus subtilis, strain DN 252 (a derivative of Bacillus subtilis 168, strain QB 1133, BGSC1A289) which, through recombinant DNA techniques contains the gene coding for maltogenic amylase in the plasmid pDN1413. The gene is imported from Bacillus stearothermophilus (NCIB 11837).
ACTIVE PRINCIPLES	Exo-acting a -amylase
SYSTEMATIC NAMES AND NUMBERS	Glucan 1,4-a -maltohydrolase, E.C. 3.2.1.133
REACTION CATALYZED	Exohydrolysis of 1,4-a -glucosidic linkages in amylose, amylopectin and related glucose polymers. Maltose units are successively removed from the non-reducing end of the polymer chain until the molecule is completely degraded or, in the case of amylopectin, a branch point is reached.
DESCRIPTION	Commercial preparations can be liquid, granulated or powdered form. The powdered form contains sodium chloride as a stabilizer, the granulated form contains sodium chloride and wheat grits, and the liquid form contains sodium chloride and sucrose.
FUNCTIONAL USES	Enzyme preparation Retardation of staling in baked goods, preparation of high maltose glucose syrup
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.
CHARACTERISTICS
IDENTIFICATION
Maltogenic amylase activity	The sample shows maltogenic amylase activity See description under TESTS
TESTS
Maltogenic amylase activity	The method is based on the ability of the enzyme to hydrolyze maltotriose to maltose and glucose. The reaction is stopped by raising the pH of the reaction medium to about 11. The formed glucose is converted to gluconolactone by glucose dehydrogenase in the presence of NAD⁺ which is converted to NADH⁺H⁺. The change in the concentration of NAD⁺ is measured photometrically at 340 nm. One enzyme unit (Maltogenic Amylase Unit, MAU) is defined as the amount of enzyme which under standard assay conditions cleaves 1 m mol of maltotriose per minute. Assay conditions being as follows: Substrate concentration 10 mg/ml; temperature 37°; pH 5.0; incubation time 30 min. Preparation of 20 mg/ml-maltotriose substrate: Dissolve 1000 mg maltotriose (e.g. Sigma M8378) in 0.1M citrate buffer, pH 5.0, making a total volume of 50 ml (to be prepared each day of analysis). Preparation of glucose dehydrogenase (GluDH) reagent: Add buffer solution (Merck No. 14051 or equivalent) to the enzyme mixture (Merck No. 14055, bottles "1" and "2", or equivalent). Allow the mixture to stand for 15 min. Transfer the solution to a 500 ml volumetric flask, containing 200 ml of 0.1 M citrate buffer pH 5.0. Fill the flask to the mark with the same buffer. This solution can be stored for two weeks at room temperature (not in a refrigerator). Preparation of standard enzyme solutions: Dilute a maltogenic amylase standard with known activity in 10 mM NaCl to give the following enzyme units per ml: 0.015, 0.030, 0.045, 0.060 and 0.075. Measurement of enzyme activity: Add 500 m l of substrate solution (20 mg/ml maltotriose, prepared in 0.1 M citrate buffer, pH 5.0, pre-warmed to 37°) to an equal volume of enzyme solution also pre-warmed to 37°. Mix the resulting solution thoroughly and transfer to a water bath maintained at 37°. After 30 minutes remove the test tube from the water bath, and add 1000 m l of the stop reagent (0.06N NaOH) and shake vigorously. Add 3 ml of GluDH reagent and mix again. Leave the test tube at room temperature for exactly 30 min and measure optical density (OD) at 340 nm. Prepare a blank in a similar manner except that the stop reagent is added before the substrate. Calculation of enzyme activity: Draw the enzyme standard curve in a coordinate system using enzyme activity (MAU/ml) as the abscissa and D OD (sample - blank) as the ordinate. Calculate the activity of the sample by reading D OD of sample dilutions on the standard curve and then multiplying by the corresponding dilution factors. The standard curve is a straight line passing through the origin and linear regression can therefore be applied. Basic standardisation: Glucose solutions: a) Stock solution: Accurately weigh 1.60 g of glucose, analytical grade, in a 1000-ml volumetric flask and make up to volume with demineralized water. b) standard solutions: Dilute 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 ml of stock solution to volume with deionized water in 100-ml volumetric flask to give standard solutions of 88.8, 178, 266, 355, 444 and 533 m mol/l. Standard curve: Prepare the glucose standard curve by mixing 2.0 ml glucose solution with 3.0 ml GluDH reagent and incubating at room temperature for 30 min. Read OD at 340 nm. Prepare a common blank using demineralized water instead of glucose solution. Plot glucose concentration (m mol/l) versus D OD. Calculation: Calculate the enzyme activity by reading the equivalent glucose concentration on the standard curve and inserting it in the following formula: where: A = reading on the standard curve, m mol/l F = dilution factor of the enzyme solution, ml/g 4 = the ratio between amount of enzyme dilution in sample and amount of glucose solution in standard 30 = incubation time, 30 min 1000 = conversion from litre to ml