알룰로오스 함유 감미료 조성물의 제조 방법

The method of the present invention number tank, glucose, fructose and with the rule which it will know five [su including sweetener composition as number bath method, 1) immobilized with the rule which it will know five [su a number 1 enzyme activity ratio epitaxial [...][...] immobilized glucose isocyanate number. 49:1 - 5. 61:1 Is to be filled with one column A preparing process, 2) glucose solution through a column by said enzymatic reaction process embodiment B, and 3) from said column effluent liquor, glucose, fructose and with the rule which it will know five [su including a process including a C characterized harvested treated. Hereinafter, the method of the present invention number bath enhancer is disclosed therein.

Defined

In the present invention, 'glucose isocyanate (U) of enzyme activity [...] number' is, glucose as a substrate, reacting a reaction temperature 55 °C, 1 minutes to 1 μmol of fructose 1 enzyme force generating units are disclosed. Specifically, including 50 mm magnesium sulfate 2 mm phosphate buffer (pH8. 0) Cyanuric acid 0. 2M 2500 μl of glucose solution, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH8. 0) Is charged into a screw cap number 333 μl [...] 2167 μl and glucose in vitro with isocyanate, in 55 °C immersed in hot water bath to about 15 minutes reaction. 5 Mass % aqueous solution of hydrochloric acid by adding pH3. 0 - 2. 5 Enzyme is regulated so as to deactivated ([...]), ion exchange resin by desalination, filter after filtering, HPLC analysis as follows. Fructose generated using enzymatic activity to determine the peak area ratio.

In the present invention, 'number (U) of enzyme activity with the rule which it will know five [su epitaxial [...]' is, as a substrate with the rule which it will know five [su, reacting a reaction temperature 55 °C, 1 minutes to 1 μmol of fructose 1 enzyme force generating units are disclosed. In particular measurement sequence, they are five [su with the rule which it will know the substrate addition, enzyme activity is required when the number of said isocyanate [...] of glucose are disclosed.

In the present invention, '([...]) (U/ml) of active isocyanate [...] immobilized glucose number' is, 'Japanese industrial standard industrial glucose ISO [...] number K7002 provided 1988' thermal maximum activity measuring method a measured value. Specifically, by adding 2 mm magnesium sulfate and sodium carbonate, pH7. 8 - 8. 0 45 Mass % glucose solution regulated to 4700. A number taken in 30 ml glucose isocyanate 4 °C day attended swelling amount [...] immobilization organic material, said herein without further glucose solution 100 ml, 30 min or 50 °C immersed in hot water bath while maintaining the pressure of the degassing etched. An inside diameter of the jacket it oxidation is 20 mm, length 400 mm glass number column after filling, said glucose solution liquid transfer pump to down stream 55 °C jacket temperature in etched through a. Effluent sampling, and a part of the ion exchange resin by desalination, after filter, HPLC analysis, 2042 obtain isomerization rate generated from fructose peak area ratio. The, 0 having an elongation of isomerization. 39 - 0. 44 To enter flow rate range of switchboard. And, by immobilized glucose isocyanate of formula then [...] inactive 2042 obtain a number.

E=(FS/W) 0. 50Ln {0. 50/(0. 50 A-X)}

E: inactive (U/ml)

F: flow rate (ml/min)

S: temperature difference (μmol/ml) glucose solution

W: capacity of immobilized glucose [...] number (ml) isocyanate

X: isomerization rate

In the present invention, 'number of an inert (U/ml) epitaxial [...] immobilization with the rule which it will know five [su' is, instead of using glucose concentration in addition to the fructose, glucose measuring method of immobilizing said number of isocyanate [...] enzyme activity measured thermal value. Specifically, by adding 2 mm magnesium sulfate and sodium carbonate, pH7. 8 - 8. 0 Thereof adjusted to 45 mass % of fructose solution 4700. 4 °C followed by immobilization with the rule which it will know five [su a number taken in 30 ml day attended swelling epitaxial [...] amount, said herein without further fructose solution 100 ml, 30 min or 50 °C immersed in hot water bath while maintaining the pressure of the degassing etched. An inside diameter of the jacket it oxidation is 20 mm, length 400 mm glass number column after filling, fructose solution liquid transfer pump downward through said jacket in changing the etched through a temperature 55 °C. Effluent sampling, and a part of the ion exchange resin by desalination, after filter, HPLC analysis, with the rule which it will know five [su 2042 obtain isomerization rate generated from peak area ratio. The, 0 having an elongation of isomerization. 21 - 0. 24 To enter flow rate range of switchboard. And, with the rule which it will know five [su immobilization by epitaxial [...] inactive 2042 obtain a number of formula then.

E=(FS/W) 0. 27Ln {0. 27/(0. 27 A-X)}

E: inactive (U/ml)

F: flow rate (ml/min)

S: fructose concentration (μmol/ml) fructose in solution

W: epitaxial [...] number (ml) capacity of immobilized with the rule which it will know five [su

X: isomerization rate

In the present invention, the each saccharide (glucose, fructose, with the rule which it will know five [su) ratio of sweetener compositions, glucose, fructose, and total mass with the rule which it will know five [su (100%) sugar, mass (%) ratio of each saccharide occupies are disclosed.

In the present invention, 'space velocity (SV)' is, solution through a column which is a unit of speeds, 'space velocity (SV)=column volume (ml) (ml)// time (h) as a result of liquid measure' are calculated.

A process

In process A, number 1 and number as the activity of immobilized with the rule which it will know five [su epitaxial melter [...] immobilized glucose isocyanate ratio. 49:1 - 5. 61:1 Is to be filled with one column 4700.

(Isocyanate number [...] immobilized glucose)

Number [...] isocyanate is glucose, glucose fructose interaction with the enzyme able to catalyze the conversion D - D - are disclosed. For the number of isocyanate used in the present invention glucose [...] derived, not one specifically number, microorganism, such as derived from animals and plants either may be disclosed. Number of isocyanate [...] glucose derived from microorganisms include, for example, Bacillus coagulans, Streptomyces griseofuscus, Streptomyces rubinosus, such as Streptomyces murinus are exemplified. In addition, the isocyanate used in the present invention glucose [...] number, said least one isolated from organisms may be made, in addition gene recombinant prepared by the number by engineering method may be disclosed. Not immobilized glucose [...] isocyanate number is, for example, multi right, such as from a commercially available chemical yarn which, commercial products may be used in the present invention glucose [...] immobilized isocyanate number of 2000.

In addition, as in the present invention immobilized glucose ISO [...] number, product number (products) number of positive number article or regulating glucose immobilized isocyanate [...] installed in people, in addition to glucose [...] microorganism that produces is immobilized isocyanate number even when used now.

For the number of which is used for the support for immobilizing glucose [...] isocyanate, immobilization of the enzyme makes it possible to limit as is, but not particularly number one, for example, ion exchange resin, polyvinyl alcohol, polypropylene, acrylic, chitosan, hydrophobic adsorption resin, chelating resin, such as synthetic resin and organic polymer carrier; 2000 light, laying, the car it raises but this [thu, silica gel, molecular sieve (molecular sieve), porous glass, activated carbon, ceramic such as activated sludge or the like is cited. In addition, when a microorganism used as enzyme, 4 dialkylamide pyridine compounds (for example electrochemical industrial stock Company number except the DAC) can be used as a carrier for immobilizing or alginate salt. This for immobilizing carrier, installed in the 1 species used alone, at least one combination in addition 2 may be disclosed. Among other things this fixed support for immobilizing, by number of immobilized glucose ISO [...] active hold, inexpensive and easy to handle for example in terms of pressure loss, preferably ion exchange resin, more preferably weakly basic anion exchange resin, more preferably polystyrene weakly basic anion exchange resin is cited.

Isocyanate is immobilized glucose [...] number, number of publicly known method for immobilizing the glucose [...] isocyanate or process for microorganisms by immobilized on the carrier can be achieved. For example, the ion exchange resin is used as a carrier for immobilizing glucose [...] fixed isocyanate number is necessary, hereinafter-dependent sequence embodiment can. Ion exchange resin to fill the column, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH8. 0) After cleaning, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH8. 0) A glucose solution [...] number predetermined concentration dilution isocyanate, 1 - 16 temporal extent, rotationally adsorption reaction chamber. Then, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH8. 0) Cleaning solution, immobilized glucose [...] obtain isocyanate number. In addition, the glucose [...] immobilized isocyanate number after, if necessary, to the writing base tar it knows and it holds such as crosslinked polyethyleneimine, pulse number [...] immobilization of glucose isocyanate notification may be disclosed.

For the immobilized glucose ISO [...] inactive number, carry on with the rule which it will know five [su number ratio of enzyme activity with immobilized glucose ISO [...] number appropriately coupled to the epitaxial [...] immobilization meeting but good range, number in the bath with the rule which it will know five [su enhance the sweetener compositions containing more than one layer in terms of ratio, 100 U/ml or more, preferably 100 - 150 U/ml, more preferably 100 - 200 U/ml is cited. In particular, the above-mentioned isocyanate immobilized glucose [...] satisfied a range in which a large amount of an inert number, further process when a space velocity in the range of temperature difference as a result of misfortune it will do carry B solution by, the sweetener compositions containing 13% ratio in number equal to or more than the bath with the rule which it will know five [su, glucose, fructose and with the rule which it will know five [su 50:37 containing ratio: 13 - 43:42: 15 of sweetener composition bath capable of efficiently number via the network.

(Epitaxial [...] immobilization with the rule which it will know five [su number)

With the rule which it will know five [su epitaxial [...] number is, the interaction with the enzyme able to catalyze the conversion with the rule which it will know five [su fructose and D - are disclosed. With the rule which it will know five [su used in the present invention for the number of epitaxial [...] derived, not one specifically number, microorganism, such as derived from animals and plants either may be disclosed. For example, with the rule which it will know five [su number is epitaxial [...], Arthrobacter globiformis M30 produced by known main (strain NITE BP a-1111) which, in the present invention, with the rule which it will know five [su can be derived from microorganism using epitaxial [...] number. With the rule which it will know five [su number of epitaxial [...] derived from microorganisms include, for example, Pseudomonus cichorii, Agrobacterium tumefaciens, Clostridium sp, Clostridium scindens, Clostridium bolteae, Ruminococcus sp, like Clostridium cellulolyticum is cited. In addition, in the present invention is used with the rule which it will know five [su epitaxial [...] number, said least one isolated from organisms may be made, in addition gene recombinant prepared by the number by engineering method may be disclosed.

In addition, as in the present invention immobilized with the rule which it will know five [su epitaxial [...] number, positive number (products) with the rule which it will know five [su number of epitaxial [...] article or regulating number article immobilized installed in people, in addition a microorganism that produces is immobilized with the rule which it will know five [su epitaxial [...] number even when used now.

With the rule which it will know five [su number of which is used for the epitaxial [...] for immobilizing carrier, as is immobilization of the enzyme makes it possible to limit, but not one specifically number, for example, ion exchange resin, polyvinyl alcohol, polypropylene, acrylic, chitosan, hydrophobic adsorption resin, chelating resin, such as synthetic resin and organic polymer carrier; 2000 light, laying, the car it raises but this [thu, silica gel, molecular sieve (molecular sieve), porous glass, activated carbon, ceramic such as activated sludge or the like is cited. In addition, when a microorganism used as enzyme, 4 dialkylamide pyridine compounds (for example electrochemical industrial stock Company number except the DAC) can be used as a carrier for immobilizing or alginate salt. This for immobilizing carrier, installed in the 1 species used alone, at least one combination in addition 2 may be disclosed. Among other things this fixed support for immobilizing, by epitaxial [...] immobilized with the rule which it will know five [su number of active hold, inexpensive and easy to handle for example in terms of pressure loss, preferably ion exchange resin, more preferably polystyrene divinylbenzene systems weakly basic anion exchange resin is cited. Weakly basic anion exchange resin ion exchange include, for example 3 amine is cited.

With the rule which it will know five [su epitaxial [...] number is immobilized, with the rule which it will know five [su number of publicly known method or process for epitaxial [...] by immobilized on the carrier for immobilizing the microorganism can be achieved. For example, the ion exchange resin is used as a carrier for immobilizing, fixed with the rule which it will know five [su derived from Arthrobacter globiformis epitaxial [...] necessary number, hereinafter-dependent sequence embodiment can. First, 0. With the rule which it will know five [su to a minimum salt medium (MSM: Minimal Salts Medium) including 5% D - Arthrobacter globiformis by of various germs, culture embodiment as follows. By collecting a bacterial cells from a culture medium obtained by centrifuging, 50 mm phosphate buffer (pH8. 0) Easily. Then, 50 mm phosphate buffer cells of cleaning (pH8. 0) Suspended in solution, 10% by weight wet process thereto by 5% weight per weight per weight degree of lighting element and wet process by adding of sodium chloride degree, 120 minutes in 37 °C extraction of reaction in a next decomposing embodiment as follows. Then, in embodiment 15 minutes more 55 °C degree by heating, deactivated to non-thermoresistant enzymes, recovering supernatant by centrifuging, bath ([...]) doped with enzyme solution. Separate, ion exchange resin column filling solution, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH8. 0) After cleaning, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH8. 0) Said dilution to a coenzyme solution, time 1 - 16 temporal extent, rotationally to an ion exchange resin to adsorb the enzyme through a reaction chamber. Then, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH8. 0) Cleaning solution, obtain epitaxial [...] immobilization with the rule which it will know five [su number. In addition, the immobilized with the rule which it will know five [su epitaxial [...] number after, if necessary, to the writing base tar it knows and it holds such as crosslinked polyethyleneimine, pulse number with the rule which it will know five [su epitaxial [...] immobilization of notification may be disclosed.

For the immobilization with the rule which it will know five [su epitaxial [...] inactive number, carry on with the rule which it will know five [su isocyanate number number ratio of enzyme activity with immobilized glucose [...] meeting appropriately coupled to the epitaxial [...] immobilization but good range, number in the bath with the rule which it will know five [su enhance the sweetener compositions containing more than one layer in terms of ratio, 20 U/ml or more, preferably 20 - 150 U/ml, more preferably 25 - 80 U/ml is cited. In particular, the above-mentioned epitaxial [...] immobilization with the rule which it will know five [su satisfied a range in which a large amount of an inert number, further process by setting the temperature difference as a result of misfortune it will do carry B solution when a space velocity range, the sweetener compositions containing 13% ratio in number equal to or more than the bath with the rule which it will know five [su, glucose, fructose and with the rule which it will know five [su 50:37 containing ratio: 13 - 43:42: 15 of sweetener composition bath capable of efficiently number via the network.

(Isocyanate number column number with the rule which it will know five [su immobilization on immobilized glucose [...] epitaxial [...] filling)

In the present invention, glucose is epitaxial with the rule which it will know five [su isocyanate [...][...] number and number, different support for immobilizing to 10th, isocyanate is mixed in forward and reverse with the rule which it will know five [su number [...] immobilization on immobilized glucose epitaxial [...] number column filled with and may, in addition, both the number and the same number with the rule which it will know five [su isocyanate for immobilizing carrier [...] epitaxial [...] immobilization to glucose, to filled with this column may be disclosed.

In the present invention, immobilization glucose ISO [...] number: activity of immobilized with the rule which it will know five [su epitaxial [...] number ratio (isocyanate immobilized glucose number [...] inactive: epitaxial [...] immobilization with the rule which it will know five [su number of an inert) is 1. 49:1 - 5. 61:1 So that the, same column fills the enzyme amount. The isocyanate number combination with the rule which it will know five [su number by immobilization on epitaxial [...][...] glucose immobilization ratio, high sweetener composition bath capable of efficiently number ratio with the rule which it will know five [su via the network. With the rule which it will know five [su [...] immobilized on said epitaxial active ratio number out of the immobilized glucose isocyanate [...] number if, in the sweetener compositions with the rule which it will know five [su number bath ratio lowered, in order to reduce space velocity through a width and with the rule which it will know five [su containing an extremely needs, with the rule which it will know five [su cannot be efficiently number having a high percentage sweetener composition containing high pressure liquid coolant to be coated.

With the rule which it will know five [su high pressure liquid coolant containing more than one layer having a high percentage sweetener composition that efficiently in terms of number, number [...] immobilized glucose isocyanate: activity of immobilized with the rule which it will know five [su epitaxial [...] number rate, preferably 2. 94:1 - 3. 74:1 Is cited.

Process B

In process B, glucose solution through a column by said enzymatic reaction embodiment as follows. Said glucose solution through a column by, and subjected to the action of a portion of the isocyanate number [...] glucose fructose to glucose reversibly conversion, and subjected to the action of a portion of the epitaxial [...] fructose produced further with the rule which it will know five [su reversibly with the rule which it will know with five [su number from outside. Method of the present invention number tank it, isocyanate number with the rule which it will know five [su glucose in one column number detection reaction on epitaxial [...][...] reversible, for cassette is transferred, glucose, fructose and with the rule which it will know five [su sweetener can be efficiently number including high pressure liquid coolant.

(Glucose solution)

Glucose solution, the glucose solution is dissolved in water to which the substrate are disclosed. For the glucose solution used derived from glucose, but not one specifically number, for example, starch hydrolyzing enzyme, glucose obtained by positive number is cited.

Glucose for the glucose solution, the sweetener composition number bath to be appropriately set according to the concentration reducing saccharide in the form a space therebetween but, with the rule which it will know five [su containing a bath having a high percentage sweetener composition in terms of efficiently number, as Brix concentration, typically 30 - 50%, preferably 35 - 45% a is cited.

In addition, glucose solution are, water-soluble magnesium salt may be carried disclosed. Such water-soluble magnesium salt included body to, in accordance with the number of epitaxial [...] stable with the rule which it will know five [su. Glucose solution that is added to a water-soluble magnesium salt include, but are not limit in particular number as food is permitted is a number larger than the size, magnesium sulfate, magnesium chloride or the like is cited. This water soluble magnesium salt, 1 installed in the species used alone, at least one combination in addition 2 may be disclosed.

Content of the water-soluble magnesium salt of glucose in solution, but not one specifically number, e.g. 1 - 5 mm, preferably 1. 5 - 2. A 5 mm is cited.

For the glucose solution pH, the effect of immobilized with the rule which it will know five [su [...][...] immobilized glucose isocyanate number number appropriately coupled to the epitaxial but within an acceptable range, normal 7. 0 - 8. 5, Preferably 7. 8 - 8. 0 Is cited. The adjustment of the pH glucose solution, using pH adjusting number of publicly known embodiment can.

(Glucose solution through a conditions)

Said glucose solution subjected to conditions through a column, the column filled with inactive enzyme, the sweetener composition to be reducing the number bath ratio according to the appropriately adjusting a space therebetween but with the rule which it will know five [su, the number bath ratio with the rule which it will know five [su fructose sweetener compositions that effectively in terms of height and in more layer, through a space velocity (SV) as a glucose solution, 0. 2 - 1. 0, Preferably 0. 3 - 0. 5 A is cited.

In particular, the above-mentioned isocyanate number with the rule which it will know five [su number on each inactive immobilized glucose [...] epitaxial [...] immobilization satisfied a range in which, in addition a space velocity (SV) is a glucose solution through a range by said visitor, 37% or more sweetener compositions containing 13% ratio in addition with the rule which it will know five [su is produced in at least the ratio of fructose, glucose, fructose and with the rule which it will know five [su 50:37 containing ratio: 13 - 43:42: 15 efficiently over a number of sweetener composition in accordance with the bath. The sweetener composition is a ratio of the number tank it will be less than 37% fructose, alternatively a composition ratio of isomerization number per article, the second article per isomerization number used as free, processing can be required in addition. In addition, it will be less than the number the sweetener composition containing a bath ratio with the rule which it will know five [su 13%, glucose fructose composition ratio is lower value added to the sweetener composition number tank, separate, can be added with the rule which it will know five [su required.

In addition, the space velocity to the lower end of which is higher than the range above, throughput of but per unit time, space velocity is too high, the LAN containing fructose and with the rule which it will know five [su lower ratio as substrate. In addition, conversely, lower than the range to the lower end of said space velocity. Throughput of per unit time decreases, lowering the efficiency of number bath tend to appear other. I.e., in bath method of the present invention number, column number by a space velocity of said range to said through a glucose solution, it can be used as produced sweetener composition per isomerization number article, further additional it is with the rule which it will know five [su can contain a high content and efficiently, and in addition can be sheet using height per unit time.

In addition, as a result of misfortune it will do in respect of the temperature conditions in said column when glucose solution, the effect of the enzyme column filled with a space therebetween but appropriately set within an acceptable range, for example, column temperature as, 45 - 65 °C, preferably a 55 - 60 °C is cited. Column temperature adjustment is, for example, using column like heating jacket heating method is cited.

C process, and then process

In C process, said column effluent liquor taking other. The portion thus recovered effluent liquor, glucose, fructose and with the rule which it will know five [su insulates the, sweetener compositions can be used as it is. In addition, with the rule which it will know five [su 13 - 15% of the effluent liquor containing ratio is one case, and the effluent liquor, overweight (obesity) or hyperglycemia, such as metabolic syndrome resulting low-calorie sweetener composition can be used as low risk.

In addition, C process in retrieving the effluent, like embodiment needed to concentrated, raising the concentration containing saccharides may be disclosed.

In addition, in process C recovered effluent, simulated moving bed chromatography such as to the number of separately processing [...], including trace including a mixture of glucose fructose fraction can be fraction with with the rule which it will know five [su. With the rule which it will know five [su including fraction is obtained in such a manner, with the rule which it will know five [su purity of at least 90%, to calorie number, number billion blood glucose levels rise, anti obesity prevention material can be used as domestic habit. In addition, a fraction including a mixture of glucose fructose, existing isomerization number per second (glucose: fructose=58:42 - 50:50 weight ratio) can be used as a product.

In addition, with the rule which it will know five [su of said fraction comprising, an n C process recovered leakage ratio with the rule which it will know five [su centrifugal sweeteners composition can be achieved. Increasing the sweetener composition ratio with the rule which it will know five [su, low-calorie sweetener can be used as with the rule which it will know five [su to enhanced function.

{In the embodiment {

In the embodiment to the present invention but represented a further detailed hereinafter, the present invention refers to limited to are not correct. In addition, hereinafter, 'an inert liquid ratio (I: E)' was burnt into the, '[...] immobilized glucose isocyanate number of an inert: epitaxial [...] immobilization with the rule which it will know five [su number of an inert' means other. In addition, 'active ratio', " and 'enzyme activity ratio enzyme activity ratio' to laminate, meaning such as are disclosed.

Number attainments 1. Measuring enzyme activity of enzyme number tub

Immobilization of glucose [...] commercially available isocyanate [kheym company number (number width life: [su altruism number GN) was obtained from leaving the cell. The number article includes, microorganisms having isocyanate number activity is immobilized granular number number [...] glucose carriers which, swelling 110 of non-activity. 43 U/ml was.

When the isocyanate number [...] immobilized glucose directly tank number, order of his embodiment hereinafter. First, ion exchange resin (number [phyu roller [thu company, hanger: PUROLITE A103S) to fill a 50 ml column, 2 mm magnesium sulfate including 50 mm phosphate buffer (pH 8. 0) After washing, liquid glucose number [...] 4500U isocyanate (multi right chemical yarn number, width life number: Spezyme Gifp) including 500 ml of a 50 mm phosphate buffer containing 2 mm magnesium sulfate (pH 8. 0) A, 4 °C 16 in time, rotationally to an ion exchange resin to adsorb a number [...] been through a isocyanate by glucose. Then, 2 mm magnesium sulfate containing 50 mm phosphate buffer (pH 8. 0) And cleaning, immobilized glucose ISO [...] number are obtained. The handset [...] immobilized glucose obtained by isocyanate number 107 non-activity. 65 U/ml was.

With the rule which it will know five [su epitaxial [...] number is immobilized, the following (1) - (3) is represented by the method number was high pressure liquid coolant. (1) Process cultivation and process recovery: 0. With the rule which it will know five [su of Arthrobacter globiformis M30 to a minimum salt medium (MSM: Minimal Salts Medium) comprising 5% of various germs and 4L, water (jar fermentor) 30 °C 24 in using time, 400 rpm stirring speed, per minute ventilation amount 0. 10L/L was cultured in the medium. 100G bacterial cells from the culture is by centrifuging ([sup weight) recovery and, 50 mm phosphate buffer (pH 8. 0) Cleaning of children. (2) Extraction process of coenzyme: 100g a 50 mm phosphate buffer ([sup weight) obtained process (pH 8. 0) And suspended in 1000 ml, egg albumen 10g therein by lysine (food additive, a data coding yarn number) and 5g element by adding sodium chloride, 37 °C by heating in 120 minutes, conducting extraction of enzyme reaction. Then, in 15 minutes more 55 °C heating, centrifugation (12000 rpm, 30 minutes) coenzyme a supernatant liquid obtained by electrodes respectively. (3) Coenzyme of immobilizing: to fill a 50 ml column of wet ion exchange resin (number [phyu roller [thu company, hanger: PUROLITE A103S), 2 mm magnesium sulfate including 50 mm phosphate buffer (pH 8. 0) After cleaning, including 500 ml of a 50 mm phosphate buffer containing 2 mm - 3 - D - 4500 unit number with the rule which it will know five [su epitaxial [...] magnesium sulfate (pH 8. 0) 4 °C 16 in a time, rotationally through a enzyme is adsorbed onto the ion exchange resin, magnesium sulfate containing 2 mm 50 mm phosphate buffer (pH 8. 0) Epitaxial [...] immobilization by polishing with the rule which it will know five [su number are obtained. Immobilization of activity with the rule which it will know five [su epitaxial [...] number 29. 55 U/ml was.

In the embodiment 1. Immobilized glucose On [...] number isocyanate Immobilization With the rule which it will know five [suA number epitaxial [...]FillingA glucose solution column By through a Identifying obtained sweetener composition (space velocity SV=0. 2 Conditions)

Number 1 or number of commercially available isocyanate prepared on the attainments of immobilizing glucose [...] immobilized glucose on a number number number [...] immobilization with the rule which it will know five [su epitaxial [...] independently bath isocyanate, table 1 to locate the percentage shown positive and active mixing, oxidation is an inside diameter of the jacket 32 mm, length 300 mm glass number column filling of children.

2 Mm magnesium sulfate and sodium carbonate added to pH 7. 8 - 8. 0 Thereof adjusted to 35 mass % of glucose solution Royal. Said column jacket temperature 50 °C glucose solution, space velocity (SV) is 0. 2 The number so that the while, through a continuously upward flow fronts. Effluent 24 is sampled at known time intervals of 3 times, each time from collection space velocity SV with liquid measure collection from falling. Next, each taking a portion of the effluent liquor, ion exchange resin by desalination, after filter, HPLC analysis (analysis column: non MCIGEL CK08EC america [ssu of a note number) number pollution, glucose, fructose and with the rule which it will know five [su that the peak area, reaction product a composition ratio was calculated.

2 Exhibits a table to the result obtained. As shown in table 2, the effluent liquor, even other sampling time, and substantially uniform, glucose, fructose and with the rule which it will know five [su composition ratio (weight ratio) is 43. 6:41. 3:15. 1 - 43. 4:41. 5:15. 1 To the gas.

In the embodiment 2. Immobilized glucose On [...] number isocyanate Immobilization With the rule which it will know five [suNumber of epitaxial [...] Its amount is, glucose, fructose and With the rule which it will know five [su Influence of composition ratio (space velocity SV=0. 3 - 0. 4 Conditions A)

Number 1 or number of commercially available isocyanate prepared on the attainments of immobilizing glucose [...] immobilized glucose on a number number number [...] immobilization with the rule which it will know five [su epitaxial [...] independently bath isocyanate, table 3 to locate the percentage shown positive and active mixing, oxidation is an inside diameter of the jacket 32 mm, length 300 mm glass number column filling of children.

2 Mm magnesium sulfate and sodium carbonate added to pH 7. 8 - 8. 0 Thereof adjusted to 35 mass % of glucose solution Royal. Said column jacket temperature 50 °C glucose solution, space velocity (SV) is 0. 3 - 0. 4 While the number so, through a continuously upward flow fronts. Effluent 24 is sampled at time intervals 3 - 4 times, each time from collection space velocity SV with liquid measure collection from falling. Then, each taking a portion of the effluent liquor, ion exchange resin by desalination, after filter, HPLC analysis (analysis column: non MCIGEL CK08EC america [ssu of a note number) number pollution, glucose, fructose and with the rule which it will know five [su that the area, reaction product a composition ratio was calculated.

4 Exhibits a table to the result obtained. As a result in, one column number 1 on the activity of immobilized isocyanate number ratio with the rule which it will know five [su epitaxial [...][...] immobilized glucose filling. 25:1 - 5. 61:1 Is in a range of, in effluent liquor containing 13% is beyond the ratio with the rule which it will know five [su, with the rule which it will know five [su been containing pluralities. On the other hand, filling one column number with the rule which it will know five [su number 1 on the activity of immobilized glucose ISO [...] epitaxial [...] immobilization ratio. 25 Less than: 1 or 5. 61 Or more: 1 is filled in, 13% in effluent liquor ratio with the rule which it will know five [su will not reach it. As a result from, with the rule which it will know five [su of effluent liquor ratio from an evaporator, filling with the rule which it will know five [su number column number 1 on the activity of immobilized glucose ISO [...] epitaxial [...] immobilization ratio. 25:1 - 5. 61:1 Was found that needs to be set.

In the embodiment 3. Immobilized glucose On [...] number isocyanate Immobilization With the rule which it will know five [suNumber of epitaxial [...] Its amount is, glucose, fructose and With the rule which it will know five [su Influence of composition ratio (space velocity SV=0. 5 Conditions)

Space velocity SV ratio equal to or more than 13% with the rule which it will know five [su under conditions high to indicate that an immobilization enzyme activity ratio, hereinafter embodiment test was. I.e., in a test, high throughput per unit time efficient number bath method verifying his condition.

With the rule which it will know five [su immobilization on a number number number 1 immobilized glucose isocyanate prepared on the attainments [...] epitaxial [...], table 5 to locate the percentage shown positive and active mixing, oxidation is an inside diameter of the jacket 20 mm, length 400 mm glass number column filling of children.

2 Mm magnesium sulfate and sodium carbonate added to pH 7. 8 - 8. 0 Thereof adjusted to 35 mass % of glucose solution Royal. Said column jacket temperature 50 °C glucose solution, space velocity (SV) is 0. 5 While the number so, through a continuously upward flow fronts. Effluent 24 is sampled at time intervals 3 - 4 times, each time from collection space velocity SV with liquid measure collection from falling. Then, each taking a portion of the effluent liquor, ion exchange resin by desalination, after filter, HPLC analysis (analysis column: non MCIGEL CK08EC america [ssu of a note number) number pollution, glucose, fructose and with the rule which it will know five [su that the area, reaction product a composition ratio was calculated.

6 Exhibits a table to the result obtained. As a result in, one column number 2 on the activity of immobilized with the rule which it will know five [su epitaxial [...][...] immobilized glucose isocyanate number filling ratio. 94:1 - 3. 74:1 Is in a range of, space velocity is 0. 5 Even when high, containing 13% or more effluent liquor ratio with the rule which it will know five [su been found stably obtained.

In the embodiment 4. Per isomerization Product and number With the rule which it will know five [su Simultaneous number tank number article

Chromatographic separation for an inside diameter of the jacket a cation-exchange resin (hanger: CR1310, this year tugofwar number) 400 ml oxidation is 42 mm, length 500 mm column filled with glass to form a compacted column number 60 °C, No test in the embodiment 2. 7 Effluent liquor obtained in (glucose: fructose: composition ratio with the rule which it will know five [su=45. 2:40. 3:14. 5) 21. 86G pouring, a process of the eluent ([...]) SV=1. 5 Through a fronts. Eluent through a disclosure after 15 minutes from attain elution, using a fraction collector (fraction collector), 30 acquired a boiling point in the 88, was sampled. Weight (g) of the respective solution, after measuring Brix (%), and a part of the, after filter, HPLC analysis (analysis column: a note number of non MCIGEL CK08EC america [ssu), glucose, fructose and with the rule which it will know five [su that the peak area, is set to of the respective composition ratio.

This measurement result, the respective glucose in safety, solid dose (g) was calculated fructose and with the rule which it will know five [su respectively. The result 1 also exhibits. In Figure 1 you will, fraction 1 - 45 46 - 88 on fraction separated from front and rear semi ([...]), sampling the, fraction 1 - 45 side overall ([...]), isomerization sugar (glucose: fructose: with the rule which it will know five [su=53. 2:45. 6:1. 2) Can be obtained, 46 - 88 side half ([...]) fraction, purity 91. With the rule which it will know five [su of 5% (glucose: fructose: with the rule which it will know five [su=1. 5:7. 0:91. 5) Can be obtained as in the picomolar. I.e., the power unit, said in the embodiment 1 - 3 obtained in effluent, the ion exchange resin by seeds, isomerization product and with the rule which it will know five [su number article number can be simultaneously high pressure liquid coolant per number has been found.