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Calculation
One D-amylase dextrinizing unit (DU) is defined as the quantity of D-amylase that will
dextrinize soluble starch in the presence of an excess of E-amylase at the rate of 1 g/h at 30°.
Calculate the D-amylase dextrinizing units in the sample as follows:
DU (solution) = 24/(W × T), and
DU (dry basis) = DU (solution) × 100/(100  M),
in which W is the weight, in grams, of the enzyme sample added to the incubation mixture in
the 5 ml aliquot of the Sample Preparation used; T is the elapsed dextrinizing time, in
minutes; 24 is the product of the weight of the starch substrate (0.4 g) and 60 min; and M is
the percent moisture in the sample, determined by suitable means.

Antibacterial Activity
Scope
This procedure is designed for the determination of antibacterial activity in enzyme
preparation derived from microbial sources.
Principle
The assay is based on the measurement of inhibition of bacterial growth under specific
circumstances.
Culture Plates
Six organisms are tested: Staphylococcus aureus (ATCC 6538); Escherichia coli (ATCC
11229); Bacillus cereus (ATCC 2); Bacillus circulans (ATCC 4516); Streptococcus
pyrogenes (ATCC 12344): and Serratia marcescens (ATCC 14041).
Make a test plate of each organism by preparing a 1:10 dilution of a 24 h Trypticase Soya
Broth culture in Trypticase Agar (TSA) (for Streptococcus pyrogenes a 1:20 dilution).
Pour 15 ml of plain TSA into a Petri dish and allow the medium to harden. Overlay with 10
ml of seeded TSA and allow to solidify. Place a paper disk prepared according to Disk
Preparation of the tested enzyme on each of the six inoculated plates.
Disk Preparation
Make a 10% solution of the enzyme by adding 1 g of enzyme to 9 ml of sterile, distilled
water.
Mix thoroughly with a Vortex mixer to obtain a homogeneous suspension. Autoclave suitable
paper disks (for instance, S & S Analytical Filter Papers No. 740-E, 12.7 mm in diameter),
then saturate them with the enzyme by application of 0.1 ml (about 3 drops) of a 10%
solution of the enzyme to the disk surface. Prepare six disks (one for each of the six
organisms) for each enzyme: place one disk on the surface of the six inoculated agar plates.
Incubation
Keep the six plates in the refrigerator overnight to obtain proper diffusion. Incubate the plates
at 37° for 24 h. Examine the plates for any inhibition zones that may have been caused by the
enzyme preparation.
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Interpretation
A visually clear zone around a disk (total diameter: 16 mm) indicates the presence of
antibacterial components in the enzyme preparation. If an enzyme preparation shows obvious
antibacterial activity against three (or more) organisms, it is concluded that antimicrobial
agents are present.

Catalase Activity
Scope
This procedure is designed for the determination of catalase activity, expressed as Baker
Units.
Principle
The assay is an exhaustion method based on the breakdown of hydrogen peroxide by
catalase, and the simultaneous breakdown of the catalase by the peroxide, under controlled
conditions.
Reagents and Solutions
0.250 N Sodium thiosulfate: Dissolve 62.5 g of sodium thiosulfate, Na2S2O3·5H2O in 750 ml
of recently boiled and cooled water, add 3.0 ml of 0.2 N sodium hydroxide as a stabilizer,
dilute to 1,000 ml with water, and mix. Standardize as directed for 0.1 N Sodium thiosulfate
(Volumetric Solutions), and adjust to exactly 0.250 N if necessary.
Peroxide substrate solution: Dissolve 25.0 g of anhydrous dibasic sodium phosphate
(Na2HPO4), or 70.8 g of Na2HPO4·12H2O, in about 1,500 ml of water, and adjust to pH 7.0 ±
0.1 with 85% phosphoric acid. Cautiously add 100 ml of 30% hydrogen peroxide, dilute to
2,000 ml, in a graduate, and mix. Store in a clean amber bottle, loosely stoppered. The
solution is stable for more than one week if kept at 5° in a full container.
Note: With freshly prepared substrate, the blank will require about 16 ml of 0.250 N sodium
thiosulfate. If the blank requires less than 14 ml, the substrate solution is unsuitable and
should be prepared fresh again. It is essential that the sample titration is between 50% and
80% of that required for the blank.
Procedure
Pipet an aliquot of not more than 1.0 ml of the sample, previously diluted to contain
approximately 3.5 Baker Units of catalase, into a 200-ml beaker. Rapidly add 100 ml of
Peroxide Substrate Solution, previously adjusted to 25°, and stir immediately for 5 to 10 sec.
Cover the beaker, and incubate at 25 ± 1° until the reaction is completed. Stir vigorously for 5
sec and then pipet 4.0 ml from the beaker into a 50-ml Erlenmeyer flask. Add 5 ml of 2 N
sulfuric acid to the flask, mix, then add 5.0 ml of 40% potassium iodide, freshly prepared,
and 1 drop of 1% ammonium molybdate and mix. While continuing to mix, titrate rapidly to
a colourless endpoint with 0.250 N Sodium thiosulfate, recording the required volume, in ml,
as S. Perform a blank determination with 4.0 ml of Peroxide Substrate Solution, and record
the required volume, in ml, as B.
Note: When preparations derived from beef liver are tested, the reaction is complete within
30 min. Preparations derived from Aspergillus and other sources may require up to 1 h. In
assaying an enzyme of unknown origin, a titration should be run after 30 min and then at 10
min intervals thereafter. The reaction is complete when two consecutive titrations are the
same.