Amendment to the Enforcement Ordinance of the Food Sanitation Law and the 
Standards and Specifications for Foods and Food Additives 
 
The government of Japan will designate Asparaginase from Aspergillus oryzae  
expressed in Aspergillus oryzae  as an authorized food additive. 
 Summary 
Under Article 10 of the Food Sanitation Law (hereinafter referred to as the “Law”), 
food additives shall not be used or marketed  without authorization by the Minister of 
Health, Labour and Welfare (hereinafter referred to as “the Minister”). In addition, when 
specifications or standards are established for f ood additives based on Article 11 of the Law and 
stipulated in the Ministry of Health, Labour a nd Welfare Notification (Ministry of Health and 
Welfare Notification No. 370, 1959), those additiv es shall not be used or marketed unless they 
meet the standards or specifications. 
 
In response to a request from the Minister, the Committee on Food Additives of the 
Food Sanitation Council that is establishe d under the Pharmaceutical Affairs and Food 
Sanitation Council has discussed the adequacy of the designation of Asparaginase from 
Aspergillus oryzae  expressed in Aspergillus oryzae  as a food additive. The conclusion of 
the committee is outlined below.  Outline of conclusion  
The Minister, based on Article 10 of the Law, should designate Asparaginase from 
Aspergillus oryzae  expressed in Aspergillus oryzae , as a food additive unlikely to harm 
human health, and establish standards for use and compositional specifications, based 
on Article 11 of the Law (see Attachment).     Attachment 
Asparaginase 
アスパラギナーゼ 
 
Revision of regulations 
Part of the definition and the specifications for Asparginase ( A. oryzae 
NZYM-SP-derived) will be added by revision th is time. The reagents and test solutions 
(TS) newly added for testing of A. oryzae NZYM-SP-derived asparaginase are given in 
the last part of this document.     Standards for Use 
Not established.   Compositional specifications 
Definition  Asparaginase is derived from th e filamentous fungi (limited to Aspergillus 
niger  ASP-72 and Aspergillus oryzae  NZYM-SP), in which asparaginase productivity is 
improved by amplifying the asparagina se gene intrinsica lly occurring in A. niger and A. 
oryzae. It is an enzyme that hydrolyzes aspara gine into aspartic acid and ammonia. 
There are two types of Asparaginase: A. niger  ASP-72-derived and A. oryzae  
NZYM-SP-derived products. It may contain gl ycerine, dextrin, maltodextrin, salt, or 
wheat flour.  
 
Asparaginase ( A. niger  ASP-72-derived) 
 
Enzyme Activity  Asparaginase has an enzyme activity of not less than 2375 units per 
gram or milliliter.    
Description   Asparaginase occurs as a clear, yell ow to brown liquid or as pale gray or 
slightly yellowish white granules.   
Identification  When tested by the enzyme activity  determination, Asparaginase shows 
activity.   
Purity  (1) Lead  Not more than 5.0 μg/g as Pb. 
Test Solution  Weigh 0.8 g of Asparaginase into a platinum, quartz, or porcelain 
crucible or a quartz beaker, and moisten it wit h a small amount of diluted sulfuric acid 
(1 in 4). Heat it by increasing the temper ature gradually until the sample is carbonized 
and the white fumes of sulfuric acid are no longer evolved. If necessary, add diluted sulfuric acid (1 in 4) again, and heat until the sample is almost carbonized. For a liquid 
s a m p l e  o r  a  s a m p l e  t h a t  i s  h a r d  t o  b e  c a rbonized, concentrated sulfuric acid may be 
used, instead of diluted sulfuric acid (1 in 4).  After the sample is carbonized, lid the 
crucible or beaker loosely if necessary, heat in an electric furnace by increasing the 
temperature gradually, and ignite at 450–600 C to incinerate. If any carbonized residue 
is present, crush the residue with a glass ro d if necessary, moisten with 1 ml of diluted 
sulfuric acid (1 in 4) and 1 ml of nitric ac id, heat until the white fumes of sulfuric acid 
are no longer evolved, and ignite in the elec tric furnace to completely incinerate it. To 
residue, add 10 ml of diluted hydrochloric acid (1 in 4), heat on a water bath, and 
evaporate to dryness. To the residue, add a sm all amount of diluted nitric acid (1 in 100), 
and warm to dissolve it.  After cooling, add d iluted nitric acid (1 in 100) to make exactly 
10 ml.  
When incineration is done at 500 C or below, a heat-resistant glass beaker can be 
used.     
Control Solution   To 1 ml of Lead Standard Stock Solution, exactly measured, add 
water to make exactly 100 ml. To exactly me asured 4 ml of this solution, add diluted 
nitric acid (1 in 100) to make 10 ml.  
Procedure   Proceed as directed in Method 1 of the Lead Limit Test.  
(2) Arsenic  Not more than 4.0 μg/g as As 2O3 (0.50 g, Method 3, Apparatus B). 
Microbial Limits  Proceed as directed under Microbial  Limit Tests. The total bacterial 
count is not more than 50,000/g. Escerichia coli and Salmonella are negative. For the 
Salmonella  test, proceed as directed in the mi crobial limit tests for Nisin.     
Enzyme Activity Determination  
(i) Substrate Solution  Weigh 1.50 g of L- asparagine monohydrate, add citric acid–
sodium hydroxide buffer (pH 5.0), and dissolve it  completely by stirring. Add citric acid–
sodium hydroxide buffer (pH 5.0) again to make exactly 100 ml. Prepare fresh before 
use.   
(ii) Sample Solution  Weigh accurately about 2.5 g of Asparaginase, dissolve in 20 
ml of citric acid–sodium hydroxide buffer (pH 5.0), and then add citric acid–sodium 
hydroxide buffer (pH 5.0) to make exactly 25 ml. Dilute this solution with citric acid–
sodium hydroxide buffer (pH 5.0) to prep are a solution containing 6 units/ml.  (iii) Control Stock Solution  Weigh an amount of A. niger -derived asparaginase for 
enzyme activity determination equivalent to  4000 units, dissolve in 20 ml of citric acid–
sodium hydroxide buffer (pH 5.0) , and then  add citric acid–sodium hydroxide buffer 
(pH 5.0) to make exactly 25 ml. Dilute this solution with citric acid–sodium hydroxide 
buffer (pH 5.0) to prepare a so lution containing 6 units/ml. 
(iv) Ammonium Sulfate Standard Soluti ons  Weigh accurately about 3.9 g of 
ammonium sulfate, add 40 ml of citric acid–sodium hydroxide buffer (pH 5.0), and 
shake for 15 minutes. Again add citric acid–s odium hydroxide buffer (pH 5.0) to make 
50 ml. Dilute this solution with citric acid–sod ium hydroxide buffer (pH 5.0) 4, 6, 10, 30, 
and 60 times, respectively, to prep are five standard solutions.   
(v) Procedure   
Test Solution and Control Solution   Place 2.0 ml portions of the substrate solution 
into two separate test tu bes, and warm them at 37 C for 10 minutes. Add 0.100 ml of 
the sample solution to one test tube and 0. 100 ml of the control st ock solution to the 
other, and stir. Warm them at 37 C exactly for 30 minutes, add 0.400 ml portions of 
trichloroacetic acid solution (1  in 4) to them, stir, and add 2.5 ml of water to each, and 
again stir. Take 0.100 ml from each test tube , and add 4.0 ml of water and 0.850 ml of 
basic phenol −nitroprusside TS, and stir. Add 0.850 ml of sodium hypochlorite −sodium 
hydroxide TS for A. niger  -derived asparaginase activity determination to each, and 
allow them to stand at 37 C for 10 minutes. Use them as the test solution and the 
control solution, respectively.        
Reference Solutions  Place 2.0 ml portions of the substrate solution into two 
separate test tubes, an d add 0.400 ml portions of trichloroa cetic acid solution (1 in 4) to 
them, and stir. Add 0.100 ml of the sample solu tion to one test tube and 0.100 ml of the 
control stock solution to the other, stir, and warm them at 37 C for 30 minutes. Add 2.5 
ml of water to each, and stir again. Take 0. 100 ml from each test tube, and add 4.00 ml 
of water and 0.850 ml of phenol-nitroprusside (basic), and stir. Add 0.850 ml of sodium 
hypochlorite −sodium hydroxide TS for A. niger -derived asparaginase activity 
determination to each, allow them stand at 37 C for 10 minutes. Use them as the 
reference solutions for the test solution  and the control solution, respectively.  
Calibration Curve   Place 2.0 ml portions of the subs trate solution into five separate 
test tubes, and warm them at 37 C for 10 minutes. To each test tube, add 0.100 ml 
portion of each of the ammonium sulfate standard solutions with different 
concentrations, instead of the sample solution, and proceed as directed for the test solution.  Measure the absorbance of th em against water at 600 nm. Prepare a 
calibration curve using the absorbance valu es obtained and the ammonium sulfate 
concentrations in the ammonium sulfate standard solutions.   Determination  Measure  the absorbance (A T and  AC) of the test solution and the 
control solution against wa ter at 600 nm. Also measure  the absorbance (A BT and  ABC) of 
the reference solutions for the test solution  and the control solution against water at 
600 nm. Measure the slope, a (ml/mg),  of the calibration curve. Calculate the enzyme 
activity of A. niger  -derived asparaginase for enzyme activity determination used for the 
preparation of the control solution by the formula given below. When the obtained activity is in the range of 91 to 109% of the labeled value, determin e the enzyme activity 
of the sample also by the formula. One unit of enzyme activity is equivalent to the 
amount of the enzyme required to liberate 1 μmol of ammonia per minute from 
L-asparagine when the enzyme activity is determined as directed in the Procedure.            
Enzyme	activity	 ሺunit/gሻൌ	A	ൈD
୤	ൈ25	ൈ2	ൈ	10ଷ
a	ൈW	ൈ132.14	ൈ30 
A = the value obtained by deducting the absorbance (A BT or ABC) of the 
corresponding reference solution from the absorbance (A T or AC) of the 
test solution or the control solu tion, whichever is appropriate, 
Df = the dilution factor of the sample solution or the control stock solution,  
W = the weight (g) of the sample or A. niger  -derived asparaginase for enzyme 
activity determination. 
 
Asparginase ( A. oryzae  NZYM-SP-derived) 
 
Enzyme Activity  Asparaginase has an enzyme activi ty of not less than 3,500 units per 
gram or milliliter.    
Description   Asparaginase occurs as a light brow n liquid or as white to grayish white 
granules.   
Identification  When tested by the enzyme activity  determination, Asparaginase shows 
activity.   
Purity  
(1)  Lead  Not more than 5.0 μg/g as Pb. 
Weigh 0.8 g of Asparaginase, and proceed as directed in Purity (1) for Asparaginase 
(A. niger ASP-72-derived).  
(2)  Arsenic  No t more than 4.0 μg/g as As 2O3 (0.50 g, Method 3, Apparatus B). 
Microbial Limits  Proceed as directed under Microbial  Limit Tests. The total bacterial 
count is not more than 50,000/g. Escerichia coli and Salmonella are negative. For the Salmonella  test, proceed as directed in the mi crobial limit test for Nisin.     
Enzyme Activity Determination  
(i) Substrate Solution  Weigh 0.25 g of L-asparagine monohydrate, add 15 ml of 
MOPS buffer (0.1 mol/L, pH7.0), and dissolv e it completely by stirring. Cover the 
container to block out light. Refe r the resulting solution to as Solution A. To Solution A, 
add 0.011 g of -nicotinamide adenine dinucleotide disodium salt hydrate (reduced 
form), 0.063 g of disodium 2- ketoglutarate, and an appropr iate amount of L-glutamic 
acid dehydrogenase (bovine liver-derived) equivalent to not less than 1680 units, and 
stir well to dissolve them. Add MOPS buffer (0 .1 mol/L, pH7.0) to make exactly 25 ml. 
Prepare fresh before use. 
(ii) Sample Solution  Weigh accurately abo ut 1.0 g of Asparaginase, and dissolve it 
in acetate buffer (0.1 mol/L, pH5.0, containing polyoxyethylene(23) lauryl ether) to 
make exactly 100 ml. Dilute this solution with acetate buffer (0 .1 mol/L, pH5.0, 
containing polyoxyethylene(23) lauryl ethe r) to prepare a solution containing 0.6 
units/ml.  
(iii) Standard Stock Solutions  Weigh an amount of A. oryzae -derived aparaginase 
for enzyme activity determinat ion equivalent to 775 units, dissolve in acetate buffer (0.1 
mol/L, pH5.0, containing po lyoxyethylene(23) lauryl ether) to make exactly 100 ml. 
Dilute this solution with acetate buffe r (0.1 mol/L, pH5.0, containing 
polyoxyethylene(23) lauryl ether) 8, 10, 15,  20, and 30 time to prepare five solutions 
containing 0.9688, 0.7750, 0.5167, 0.3875,  and 0.2583 units/ml, respectively.  
(iv) Procedure  
Test Solution  Transfer 4.6 ml of the substrate solu tion into a test tube, and warm at 
37.0 ± 0.5 C for 8 minutes.  Add  0.400 ml the sa mple solution, shake, and warm at 
37.0 ± 0.5 C for 90 seconds.   
Standard Solutions  Transfer 4.6 ml portions of the substrate solution into five 
separate test tubes, and warm at 37.0 ± 0.5 C for 8 minutes.  To each test tube, add 
0.400 ml of  each of the standard stock so lutions with different concentrations, instead 
of the sample solution, and proceed as directed for the test solution. 
Calibration Curve  Measure  the absorbance of the standard solutions against water 
at 340 nm  and prepare the calibration curve from the absorbance values obtained and 
the enzyme activity in 1 ml (unit/ml ) of each standard stock solution.  
Determination  Measure  the absorbance, A, of the test solution against water at 340 
nm.  Determine the enzyme activity, U (unit/ml) , of the sample solution from 
absorbance A and the calibration curve. Then  calculate the enzyme activity of the 
sample by the following formula.  One unit of enzyme activity is equivalent to the amount of the enzyme required to liberate 1 μmol of ammonia per minute from 
L-asparagine when the enzyme activity is determined as directed in the procedure.            
Enzyme	activity	 ሺunit/gሻൌ	U	ൈDൈ100
Weight	ሺgሻ	of	the	sample	 
U = the enzyme activity of the sample solution, 
D = the dilution factor of  the sample solution.  
 
Reagents and Test Solutions (TS)   
Acetate Buffer (0.1 mol/L, pH5.0, containi ng polyoxyethylene(23) lauryl ether)  
To 500 ml of acetate buffer (1 mol/L, pH 5.0), add 3500 ml of water and 7.5 ml of 
polyoxyethylene(23) lauryl ether TS. Adjust the pH to 5.0 with sodium hydroxide solution of an appropriate concentration,  and dilute with water exactly to 5000 ml.  
Acetate buffer (1 mol/L, pH 5.0)  Dissolve 88.8 g of sodium acetate trihydrate in 1800 
ml of water. Adjust the pH to 5.0 with acet ic acid and dilute with water to exactly 2000 
ml. 
Asparaginase (
A. niger -derived) for Enzyme Activity Determination   
 Obtained from the filamentous fungi,  A. niger  ASP-72, in which asparaginase 
productivity is improved by amplifying the asparaginase gene intrinsically occurring in 
Aspergillus niger.  Occurs as a clear, yellow to brown liquid or as pale gray or slightly 
yellowish white granules. Has an enzyme activity whose number of units is known. One unit of Asparaginase for Enzyme Activity De termination corresponds to the amount of 
the enzyme required to liberate 1 μmol of ammonia in one minute at pH 5.0 and at 37 C 
when 
L-asparagine as the substrate is used.  
Asparaginase ( A. oryzae -derived) for Enzyme Activity Determination   Obtained from 
the filamentous fungi,  A. oryzae NZYM-SP, in which asparaginase productivity is 
improved by amplifying the asparaginase gene intrinsically occurring in Aspergillus 
oryzae.  Occurs as a light brown liquid or as white to grayish white granules. It has an 
enzyme activity whose number of units is known.  One unit of this substance is equivalent to the amount of the enzyme required to liberate 1 μmol of ammonia in one 
minute at pH 5.0 and at 37 C when L-asparagine as the substrate is used.   
Disodium 2-ketoglutarate  C
5H4Na2O5  A white powder. Soluble in water. 
L-Glutamic Acid Dehydrogenase (bovine liver-derived)  Obtained from bovine liver. It 
has an enzyme activity whose number of units is known.  One unit of enzyme activity is equivalent to the amount of the enzyme required to liberate 1 μmol of L-glutamic acid 
per minute at pH 7.3 and at 25 C when 2-ketoglutaric acid as the substrate is used.  
3-(N-Morpholino)propanesulfonic Acid  C7H15NO 4S  A white crystalline powder.  
Freely soluble in water and practi cally insoluble in ethanol (99.5). 
Melting point  275–280 C.  
M O P S  B u f f e r  ( 0 . 1  m o l / L ,  p H 7 . 0 )   Dissolve 21 g of 3-( N-morpholino)propanesulfonic 
acid in 900 ml of water. Adjust the pH to  7.0 with sodium hydroxide solution of an 
appropriate concentration, and dilu te with water to exactly 1000 ml. 
-Nicotinamide Adenine Dinucleotide Dis odium Salt Hydrate (reduced form) 
C21H27N7Na2O14P2   A white to pale yellow powd er.  Soluble in water.  
Polyoxyethylene(23) Lauryl Ether  (C2H4O)nC12H26O  Use lauromacrogol specified in 
the Japanese Pharmacopoeia.  
Polyoxyethylene(23) Lauryl Ether TS   Add water to 15 g of polyoxyethylene(23) lauryl 
ether to make 100 ml. 
Sodium Hypochlorite −Sodium Hydroxide TS for Asparaginase ( A. niger -derived) 
Activity Determination   To 2.5 ml of sodium hypochlori te TS, add water to make 10 ml. 
Standardize the resulting solution using 3 ml of it, as directed in Assay for “Sodium Hypochlorite” in the Monographs, a
nd adjust it to make a soluti on of 0.32 to 0.38 mol/L 
sodium hypochlorite. Adjust its pH to 12. 5 with sodium hydroxide solution with an 
appropriate concentration. To 3 ml of this solu tion, add 85 ml of water, and adjust its pH 
to 12.5 with sodium hypochlorite solution with an appropriate concentration.  Add 
water to make 100 ml. Store in a cool, dark place.