FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules  2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. IMMOBILIZATION OF PSICOSE-EPIMERASE AND A METHOD OF PRODUCING D-PSICOSE USING THE SAME

2.

1. (A) CJ CHEILJEDANG CORPORATION
(B) Republic of Korea
(C) 500  Namdaemunro 5-ga  Jung-gu  Seoul  100-749  Republic of Korea

centrifugation, filtration, etc.; or supernatant obtained by homogenizing and centrifuging the separated cells; or the psicose-epimerase which is separated and purified from the supernatant by fractionation, chromatography, etc. may be used as an enzyme for converting D-fructose to D-psicose.
In one embodiment of the present invention, the psicose-epimerase may be in the form of the cell mass in the culture or the psicose-epimerase separated from the cell mass.
In one embodiment of the present invention, the step of preparing D-psicose may further comprise the step of immobilizing the psicose-epimerase or the cell mass expressing the same onto a carrier.
Since in case of immobilizing the enzyme or the cell mass onto a carrier, there may be provided environment for maintaining the activity for a long time, the immobilization has been used in the industrial production method using an enzyme or an microorganism. The carrier which may be used for the immobilization may include alginates such as sodium alginate, but is not limited thereto.
In one embodiment of the present invention, the immobilization may be carried out by using sodium alginate as the carrier. Sodium alginate is a natural colloidal polysaccharide which is present abundantly in cell wall of algae, consists of ß-D-mannuronic acid and a-L- guluronic acid, and is formed by random ß-1,4 links in content, and thus, it is advantageous that the cell mass or the enzymes can be stably immobilized onto it.
In one embodiment of the present invention, the immobilization may be carried out by using 1.5% to 4.0% of sodium alginate as the carrier.
In one embodiment of the present invention, the immobilization may be carried out by using 2% of sodium alginate as the carrier.
In one embodiment of the present invention, by using sodium alginate as the carrier for immobilization, the immobilization may be carried out by adding 1 - to 2-fold volume of an aqueous sodium alginate solution to a sample containing the enzyme or the cell mass, dropping the resulting mixture to a 0.1 M calcium ion solution to create the enzyme or the cell mass-alginate complex in beads by using a syringe pump and a vacuum pump.
In one embodiment of the present invention, the step of immobilizing the psicose-epimerase onto the carrier may further comprise the steps of packing a column with the immobilized enzyme and supplying the packed column with a D-fructose solution.
The column to be packed with the carrier onto which the enzyme or the cell mass is immobilized and the method of packing the column with the same may be suitably and easily selected by those skilled in the art to which the present invention pertains depending on the

enzyme or the cell mass, or the carrier for immobilization used.
In one embodiment of the present invention, it is possible to prepare a packed-bed column by packing the column with the immobilized enzyme. Enzymatic reaction, i.e., conversion of D-fructose to D-psicose may be carried out by supplying the packed-bed column with a D-fructose solution that is a substrate thereof.
In one embodiment of the present invention, when the packed-bed column which is packed with the cell mass including the psicose-epimerase is supplied with the D-fructose solution at a constant concentration, the epimerization reaction proceeds by the immobilized cell mass, resulting in the conversion of D-fructose to D-psicose. The converted D-psicose is subject to separation and purification by using separation columns, and then, is available as pure D-psicose.
The term "immobilization reactor" as used herein means a reactor wherein the reaction for the production of D-psicose occurs by the cell mass or the enzyme immobilized onto the carrier, or the column packed with the cell mass or the enzyme immobilized onto the carrier. Namely, the immobilization means that a substance providing biological activities, in this case, a psicose-epimerase or a D-glucose epimerase or a cell mass including the same is immobilized onto the carrier.
The term "operational stability" as used herein means that a biological reactor for the successive production of a target product such as D-psicose may be operated while maintaining a suitable level of productivity as compared with initial activity thereof, and generally, is represented by an operation period.
In one embodiment of the present invention, the column packed with the carrier onto which the cells expressing the psicose-epimerase are immobilized is supplied with 300 g/L of
D-fructose at an inflow rate of 0.1 ml/min at 50°C, which makes it possible to guarantee 25
days or longer of operational stability.
The present invention further provides a method of preparing D-psicose from D-glucose, comprising the step of immobilizing a psicose-epimerase and a D-glucose isomerase onto carriers.
As described above, since D-fructose used as a substrate for the psicose-epimerase is expensive, it is required to produce D-psicose from a cheap substrate such as D-glucose for the industrial mass production of D-psicose. The D-glucose isomerase is an enzyme to convert D-glucose to D-fructose, and thus, may provide a substrate for a psicose-epimerase by converting D-glucose to D-fructose. Therefore, it is possible to produce D-psicose from

enzyme. Measuring the activity of the psicose-epimerase as described above was used in the epimerization reaction. Fig. 2b shows the HPLC analysis results of epimerization reaction.
Example 2: Immobilization of a recombinant strain and preparation of D-psicose
(1) Immobilization of a recombinant strain
For the mass production of D-psicose, Corynebacterium glutamicum ATPE (KCCM 11046) expressing the psicose-epimerase derived from Agrobacterium tumefaciens and prepared in the above Example 1 was immobilized onto the carrier in this example. For the immobilization of the recombinant strain, the modification medium for Corynebacterium used in the Example 1 was first inoculated with the recombinant strain at an initial
concentration of OD600 = 0.6 followed by the incubation at 30°C for 20 hours. After the
completion of the incubation, the resulting culture was subject to the centrifugation to recover the cells, which were resuspended in the 50 mM EPPS buffer (pH 8.0) to be 20%. The resuspended cells of the recombinant strain were added to 2% (v/v) of an aqueous sodium alginate solution. The resulting mixture was dropped to a 100 mM CaCl2 solution by using the syringe pump and the vacuum pump to create a cell-alginate complex in which the cells were trapped in sodium alginate beads.
(2) Preparation of D-psicose by using the immobilized recombinant strain
XK16 packed-bed column (16 mm X 20 mm, Amersham pharmacia) was packed with the recombinant strain immobilized onto sodium alginate in the above (1) followed by measurement of % conversion of D-fructose to D-psicose. In order to determine an optimal reaction temperature of an immobilization reactor and a substrate concentration of D-fructose, the conversion reaction of D-fructose to D-psicose was carried out under combined
conditions of 40°C, 45°C and 50oC of reaction temperatures, and 100 g/L, 300 g/L and 500
g/L of D-fructose concentrations. According to Figs. 3a to 3c, It was found that the best
conversion is achieved at a temperature of 50°C and 100 g/L of D-fructose concentration,
and 300 g/L and 500 g/L of D-fructose concentration also give high conversion. Further, different from other D-glucose conversion reactions, the epimerization reaction for D-psicose showed low dependency on temperature.
(2-1) Productivity depending on an inflow rate of D-fructose

followed by the measurement of % conversion of D-glucose to D-psicose. In order to determine the optimal reaction condition for the two mixed enzymes, the conversion reaction
of D-glucose to D-psicose was conducted under combined conditions of 40°C, 45°C, or 50°C
of a reaction temperature, and 100 g/L, 300 g/L, or 500 g/L of D-glucose concentration. Fig. 6 shows % conversion of D-glucose to D-psicose depending on changes in the reaction temperature and the substrate concentration. According to Figs. 6a to 6c, different from the epimerization reaction for D-psicose, the isomerization reaction for D-glucose was a temperature-dependent reaction. That is, the higher the reaction temperature the faster the conversion rate of D-glucose to D-psicose. It was found that this had effect on the productivity of D-psicose finally. The best conversion of D-glucose to D-fructose was
achieved at a temperature of 50°C, and there was no significant difference in the reaction rate
as the substrate concentration increased higher. These results demonstrate that it is possible to operate the immobilization reactor under the condition of a higher concentration of a substrate and to increase the productivity of D-psicose from D-glucose.
(2) Productivity depending on an inflow rate of D-glucose
The two packed-bed columns were packed with the immobilized recombinant strain including a psicose-epimerase and immobilized D-glucose isomerase, respectively, and the productivity of D-psicose was measured depending on an inflow rate of D-glucose. Based on the results of the above examples, the concentration of D-glucose and the reaction
temperature were set at 300 g/L and 50°C, respectively. The productivity was measured by
adjusting the inflow rate of D-fructose, a substrate to be 0.4, 1, 2, 4, and 8 h'1 of space velocity (1/h). The results are shown below in Table 2 and Fig. 7.
Table 2. Productivity and % conversion depending on an inflow rate of D-glucose

Inflow rate (ml/min) D-glucose (g/1) D-fructose (g/1) Productivity (g/l/hr) Conversion (%)
4 281.465 31.136 374 9.96
3 278.849 40.042 360 12.56
2 267.061 50.196 301 15.82
1 238.615 78.854 237 24.84
0.5 207.168 113.081 170 35.31
0.1 172.826 155.850 47 47.42

29

We Claim:-
?Claim 1?
A method of preparing D-psicose from D-fructose by using a psicose-epimerase derived from Agrobacterium tumefaciens  and expressed in a GRAS (generally accepted as safe) strain comprising steps of:
expressing the psicose-epimerase derived from Agrobacterium tumefaciens in the GRAS strain; and
preparing D-psicose from D-fructose by using the expressed psicose-epimerase.
?Claim 2?
The method as claimed in claim 1  wherein the psicose-epimerase used in the preparation of D-psicose is contained in a culture of the GRAS strain or separated from the culture.
?Claim 3?
The method as claimed in claim 1  wherein the GRAS strain is Corynebacterium sp.
?Claim 4?
The method as claimed in claim 3  wherein the GRAS strain is Corynebacterium glutamicum KCCM 11046.
?Claim 5?
The method as claimed in one of claims 1 to 4  wherein the psicose-epimerase derived from Agrobacterium tumefaciens has the amino acid sequence of SEQ ID NO: 1.
?Claim 6?
The method as claimed in one of claims 1 to 4  wherein the step of preparing D-psicose comprises the step of immobilizing the psicose-epimerase onto a carrier.
?Claim 7?
The method as claimed in claim 6  wherein the carrier is sodium alginate.
?Claim 8?
The method as claimed in claim 6  wherein the method further comprises the steps of packing a column with the carrier onto which the psicose-epimerase is immobilized and supplying the packed column with a D-fructose solution.
?Claim 9?
A method of preparing D-psicose from D-glucose  comprising the step of immobilizing a psicose-epimerase and a D-glucose isomerase onto carriers.
?Claim 10?
The method as claimed in claim 9  wherein the psicose-epimerase is contained in a culture of the GRAS strain expressing a psicose-epimerase derived from Agrobacterium tumefaciens or separated from the culture.
?Claim 11?
The method as claimed in claim 10  wherein the GRAS strain is a strain transformed with a recombinant vector including a gene encoding a psicose-epimerase.
?Claim 12?
The method as claimed in claim 10  wherein the GRAS strain is Corynebacterium sp.
?Claim 13?
The method as claimed in claim 12  wherein the GRAS strain is Corynebacterium glutamicum KCCM 11046.
?Claim 14?
The method as claimed in one of claims 10 to 13  wherein the psicose-epimerase derived from Agrobacterium tumefaciens has the amino acid sequence of SEQ ID NO: 1.
?Claim 15?
The method as claimed in claim 9  wherein the carrier is sodium alginate.
?Claim 16?
The method as claimed in one of claims 9 to 13  wherein the method further comprises the steps of packing a column with the carriers onto which the psicose-epimerase and the D-glucose isomerase are immobilized and supplying the carrier-packed column with a D-fructose solution.
?Claim 17?
The method as claimed in claim 16  wherein each of the carriers onto which the psicose-epimerase and the D-glucose isomerase are immobilized  respectively  is packed into a separate column  and the two columns are connected in communication with each other.
?Claim 18?
Corynebacterium glutamicum KCCM 11046 strain useful for the preparation of D-psicose from D-fructose or D-glucose.

Dated this 7th day of March  2012.