Attachment  
Psicose Epimerase  
プシコースエピメラーゼ  
 
Standard for U se 
 
Not established.  
 
 
Compositional S pecifications  
 
Substance  Name  Psicose Epimerase, Allulose Epimerase  
CAS Number  [1618683 -38-7] 
Definition   Psicose Epimerase is an enzyme that mutually isomerizes fructose and 
psicose  to each other . It is derived from the culture of Escherichia coli (limited to E. coli 
K12 W3110 strain) in which the psicose epimerase gene, intrinsically occurring in the 
bacterium (limited to Arthrobacter globiformis ), is introduced.  
It may contain foods used exclusively for bulking, powdering, diluting, stabilizing, or 
preserving it or f or adjusting its activity. It may also contain food additives used for 
bulking, powdering, diluting, stabilizing, or preserving it or for adjusting its pH or 
activity.  
Enzyme Activity  Psicose Epimerase has an enzyme activity of not less than 230 units 
per gram.  
Description   Psicose Epimerase  occurs as a light to dark br own liquid or as a gray 
powder.  
Identification  When tested by the enzyme activity determination specified below, 
Psicose Epimerase shows activity.  
Purity  
(1) Lead   Not more than 5 μg/g as Pb (0.80 g, Method 1, Control Solution: Lead 
Standard Solution 4.0 mL, Flame Method).  If the residue does not dissolve in 5 mL of 
diluted nitric acid (1 in 100) in the preparation of the test solution  specified in Method 1, 
procee d as directed in Method 3 . 
(2) Arsenic   Not more than 3 μg/g as As  (0.50 g, Method 5, Standard Color: Arsenic 
Standard Solution 3.0 mL, Apparatus B).   
 
Microbial Limits  Proceed as direct ed under Microbial Limit Tests.  
Total plate count:  Not more than 50,000 per gram.  
Escherichia coli : Negative per test.  
Salmonella : Negative per test.  
Sample Fluid  Prepare as directed in Method 3 for total  plate count.  
Pre-enrichment Culture  Prepare as directed in Method 3 for the Escherichia  coli 
test and Method 2 for the Salmonella test. 
Enzyme Activity Determination  
(i) Substrate Solution  Weigh 0.18 g of D(+)-psicose, dissolve it in water, and add 
water to make exactly 5 mL. Prepare fresh before use.  
(ii) Sample Solution  Weigh accurately about  1.0 g of Psicose Epimerase , and 
dissolve it in the diluent prepared as directed below to make a constant volume so that 
the resulting solution has 4−10 units per mL.  The diluent: Mix phosphate buffer (0.05 
mol/L) at pH 8.0 and magnesium chloride TS (1 mo l/L) at a rate of 199 : 1.  
(iii) D(−)-Fructose  Standard Solutions  Weigh accurately about 0.27 g of  D(−)-
fructose for enzyme activity determination,  dissolve it in water to make exactly 100 mL. 
Use this solution as the standard stock solution. Prepare four  standard solutions with 
different concentrations —10 mol, 5 mol, 3 mol, and 1 mol of D(−)-fructose (C 6H12O6 
= 180.16) per mL —by exactly diluting the standard stock solution  with water  to 1.5, 3, 
5, and 15 times, respectively.  
(iv) Procedure  
Test Solution   Place 0.100 mL of the sample solution into a test tube, mix it with 
0.400 mL of the diluent prepared in (ii), lid the test tube, and equilibrate at 50°C ± 0.5°C 
for 5 minutes. Add 0.500 mL of the substrate solution to the test tube, mix, incubate  at 
50°C ± 0.5°C for exactly  10 minutes, and then heat in a water bath for 2 minutes. After 
cooling, add about 100 mg of strongly acidic cation -exchange resin and about 100 mg of 
weakly basic anion -exchange resin (free -form), both whose surface water was p reviously 
removed with a filter paper, shake for 15 minutes, and filter through a membrane filter 
(pore size: 0.2 m). 
For strongly acidic cation -exchange resin, wash with water before use as directed in 
the Strongly Acidic Cation -exchange Resin section under C. REAGENTS, SOLUTIONS, 
AND OTHER REFERENCE MATERIALS,  beginning with “ Weigh about 50 g of strongly 
acidic cation -exchange resin,”  and confirm that the pH of the effluent is 5.0−6.5.  
Reference Solution  Place 0.100 mL of the diluent into a test tube instead of the 
sample solution, and  proceed as directed  for the test solution.  
Determination  Analyze 10 L portions of the test solution, the reference solution, 
and the four D(−)-fructose  standard solutions by liquid chromatography using the 
operating conditions given below. Prepare a calibration curve from the peak area and 
the concentration ( mol/mL) of each D(−)-fructose standard solution. Measure the peak 
areas of D(−)-fructose in the test solution and the reference solution, and then determine  
 
the concentration ( mol/mL) of D(−)-fructose in each solution from the calibration curve. 
Calculate the enzyme activity by the following formula. One unit of the enzyme activity 
is equivalent  to the amount of the enzyme required to liberate 1 mol of D(−)-fructose  
per minute when  determined as directed in the Procedure.  
Enzyme activity (unit g⁄)  = (CT − CB)× VT
 M  
CT = concentration ( mol/mL) of  D(−)-fructose  in the test solution,  
CB = concentration ( mol/mL) of D(−)-fructose  in the reference solution,  
VT = whole volume (mL) of the sample solution prepared,  
M = weight (g) of the sample.  
 
Operating conditions  
Detector: Differential refractometer.  
Column: A stainless steel tube (8 mm in ternal diameter and 30 cm length).  
Column packing material: About 9 -m cation -exchange resin for liquid 
chromatography (Ca -form).  
Column temperature: 80°C.  
Mobile phase: Water.  
Flow rate: 0.4 mL/min.  
 
 
Reagents, Solutions, and Other Reference Materials  
D(−)-Fructose for Enzyme Activity Determination   C6H12O6  [57-48-7]  Colorless to 
white crystals or powder.  
Specific rotation   [α]D20: −90 to −94°  Weigh accurately about 4 g of D(−)-fructose  
for enzyme activity determination , add 0.2 mL of ammonia TS and 80 mL of water to 
dissolve it, allow to stand for 30 minutes, and add water to make exactly 100 mL. 
Measure the optical rotation of the resulting solution.  
Purity (1) Clarity of solution   Clear (1.0 g, water 20 mL).  
(2) Loss of drying   Not more than 2.0% (reduced pressure, 18 hours).  
(3) Related substances  Prepare a test solution by dissolving 20 mg of D(−)-fructose  
for enzyme activity determination  in 2 mL of water. Prepare a control solution by 
diluting exactly measured 1 mL of the t est solution with water to exactly 50 mL. Analyze 
10 L portions of the test solution and the control solution by liquid chromatography 
using the operating conditions given below. Continue the chromatography for three 
times the retention time of the main p eak, and measure the peak areas. The sum of the 
areas of all peaks, other than the main peak and the solvent peak, from the test solution 
is not greater than the area of main peak from the control solution.     
 
Operating conditions  
Detector: Differential re fractometer.  
Column: A stainless steel tube (3−8 mm internal diameter and 15−30 cm length).  
Column packing material: 5−10 m aminopropyl -bonded silica gel for liquid 
chromatography.   
Column temperature: A constant temperature of 35−40°C.  
Mobile phase: A 7  : 3 mixture of acetonitrile/water.  
Flow rate: Adjust the retention time of D(−)-fructose  to 4−7 minutes.  
Magnesium Chloride TS (1 mol/L)   Dissolve 203 g of magnesium chloride hexahydrate 
in water to make 1000 mL.  
D(+)-Psicose  C6H12O6  [551-68-8]  A white to slightly pale yellow crystalized powder 
or powder.  
Specific rotation  [α]D20: +2.0 to +6.0° (0.1 g, water, 10 mL).  
Purity   Related substances   Prepare a test solution by dissolving 20 mg of D(+)-
psicose in 2 mL of water. Prepare a control solution by diluting exactly measured 1 mL 
of the test solution with water to exactly 50 mL. Analyze 10 L portions of the test 
solution and the control solution by liquid chromatography using the operating 
conditions given below. Continue the chromatography for three times the retention time 
of the main peak, and measure the peak areas. The sum of the areas of all peaks, other 
than the main peak and the solvent peak, from the test solution is not greater than  the 
area of main pe ak from the control solution.  
Operating conditions  
Detector: Differential refractometer.  
Column: A stainless steel tube (3−8 mm internal diameter and 15−30 cm length).  
Column packing material: 5−10 m aminopropyl -bonded silica gel for l iquid 
chromatography.   
Column temperature: A constant temperature of 35−40°C.  
Mobile phase: A 7 : 3 mixture of acetonitrile/water.  
Flow rate: Adjust the retention time of D(+)-psicose  to 6−9  minutes.