FI69638B - FOER FARING FOR FRAMSTAELLNING AV D-GLUCOSON - Google Patents

Description

1 69638

Method for preparing D-glucosone

This invention relates to a process for the preparation of D-glucosone from glucose, which process is characterized by forming an aqueous solution of glucose, converting at least about 95% of the solution from glucose to D-glucosone in solution by enzymatic oxidation in the presence or absence of oxygen.

The parent application (F1 patent application 802967) deals with the preparation of fructose from glucose, whereby glucose is converted to D-glucosone by enzymatic oxidation and then D-glucosone is hydrogenated to D-fructose.

It has been reported in the literature that small amounts of glucosones can be prepared from glucose. Several methods have been proposed for the oxidation of glucose, in which the oxidation is carried out by means of H_0 »or by means of copper acetate 5. In all of these reactions, the conversion yields are low (<30%) and several by-products are formed. Glucose is converted to D-glucosone by first preparing a glucose derivative (e.g., by reacting it with phenylhydrazine to produce glucosazone D or by reaction with p-toluidine) and then chemically treating this derivative to give D-glucosone. In these reactions, the yields were at most 50% and the unrecovered reactants 30 are too expensive for commercial use. In addition, the use of flavored reagents can cause difficulties in obtaining restaurant grade sugars.

The conversion of glucose to D-glucosone is also a known enzyme reaction. As early as 1932, glucose was reported to be oxidizable to D-glucosone by crystal formation of 2,69638 mollusca (Saxidomus giganteous).

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la. In 1937, D-glucosone was reported to be prepared by oxidizing glucose, starch, maltose or sucrose with a plasmolyzed preparation of two mold species, Aspergillus parasiticus and 5 Aspergillus flavus-oryzae. In 1956, the enzymatic oxidation of glucose to glucosone in red alga, Iridophycus flaccidum1, was reported. Carbohydrate oxidase was isolated from the mycelium of Polyporus obtusus of Basidiomycete, which oxidized glucose to 10 D-glucosone. There is no mention of yield. Finally, in 1978, glucose-2-oxidase activity was observed in basidiomycete, Oudemansiel1 a mucida, as well as in 12 other wood-decaying fungi. The best reported yields were at most 50%. In all these cases, the formation of D-glucosone is assumed to be related to the formation of hydrogen peroxide.

In contrast to the above-mentioned known methods, in which the conversion of glucose to D-glucosone is at most 50%, the process according to the invention achieves almost complete conversion of glucose to D-gluconone; with the process of the invention, the conversion is at least about 95%. This large conversion is achieved when the hydrogen peroxide formed as a by-product of the reaction is either removed or used in some parallel process.

If hydrogen peroxide is not removed, it will adversely affect the ability of microbial enzymes to catalyze the process of the invention.

The invention will be described with reference to the accompanying drawings, of which Figure 1 is a schematic representation of a preferred embodiment of the method according to the invention? Figure 2 is a representation of the molecular structure of D-glucose; and Figure 3 is a representation of the molecular structure of D-glucosone.

With respect to the molecular structure of D-glucosone, those skilled in the art know that several other molecular forms have been assumed to be present in aqueous solution 3,696,338. Many of these are cyclic.3'3 As used herein, the terms "glucose", "D-glucose" and "dextrose" are used interchangeably and refer to this monosaccharide in all forms as a solution or dry. Figure 2 shows glucose.

As used herein, the terms "D-glucosone" and "D-ara-bino-2-hexosulose" are used interchangeably.

Figure 3 shows D-glucosone.

According to the invention, glucose is easily and substantially completely converted to D-glucosone using a purified oxidoreductase enzyme such as carbohydrate oxidase or glucose-2-oxidase. The yields obtained by the present invention exceed those mentioned in the literature for this enzyme. This efficient conversion of D-glucose eliminates the need to physically separate the unchanged glucose residue.

Certain oxidoreductases catalytically act on the hydroxyl group of the second carbon of glucose, promoting the oxidation of this hydroxyl group to the keto group, for example by converting the structure of Figure 2 to the structure of Figure 3. The oxidoreductase enzymes exemplified are "glucose-2-oxidase" having glucose-2-oxidase activity and "carbohydrate oxidase" having carbohydrate oxidase activity, but the invention is not necessarily limited to the enzymes so defined. Glucose-2-oxidase has high specificity for glucose as a substrate, while carbohydrate oxidase, for which glucose is the preferred substrate, has a broader specificity for substrate. Any enzyme that is capable of converting a hydroxyl group on glucose at another carbon atom to a keto group and that does not otherwise substantially affect the remainder of the glucose molecule is within the scope of our invention. Such an enzyme 35 can be defined as having its own glucose-2-oxidase activity.

4,69638

The preferred carbohydrate oxidase enzyme is derived from the microorganism Polyporus obtusus. Sources of glucose-2-oxidase include several other microorganisms, mollusca and red algae, as mentioned above, for example Aspergillus oryzae and Oudemansiella mucida.

For ease of review, this invention is presented in connection with the use of the preferred carbohydrate oxidase Polyporus obtusus and the glucose-2-oxidase Aspergillus oryzae. The microorganisms can be grown in a mixed, immersed culture at room temperature using conventional methods. The enzyme is prepared from the mycelium of the microorganism under mixed, subsurface culture conditions.

The enzyme is preferably used in immobilized form, although free enzyme may also be used. Methods for immobilizing enzymes are known to those skilled in the art. The methods comprise the reaction of an enzyme solution with a surface-treated or uncoated organic or inorganic support. These include polyacrylamide, ethylene maleic acid copolymers, methacrylic acid-based polymers, polypeptides, styrene-based polymers, agarose, cellulose, dextran, silica, porous glass spheres, charcoal or carbon black, alumina and hydrotreated flour, wood and sawdust. In this form, the enzymes have greater stability, extended service life, and better utility and art acceptability than before. Reactions using immobilized enzymes can be performed in columns or reaction tanks or other suitable reactors.

In addition to the carbohydrate oxidase or glucose-2-oxidase enzyme, an oxygen source is required. There is still a need for a process for removing or utilizing hydrogen peroxide in the reaction for the most efficient conversion of glucose to D-glucosone. This is due to the fact that 5,69638 f 2 O 2 oxidizes certain critical sites in the enzyme molecule, impairing its function. Methods for removing hydrogen peroxide include: 1) decomposition by a catalase enzyme, 5 2) decomposition by known chemical methods, and 3) decomposition using degradable matrices 16 17 such as manganese oxides or carbon black as an immobilizing medium for the oxidoreductase enzyme. In a preferred alternative method, the hydrogen peroxide formed, instead of decomposing it, can be consumed to produce valuable by-products. Combining the preparation of D-glucosone with the preparation of propylene halohydrin or propylene oxide is a preferred example, as shown in Figure 1.

The enzymatic conversion of glucose to D-glucosone is preferably performed in water at approximately neutral pH, but may be performed in the pH range of about three to about eight using appropriate buffers. This conversion is preferably performed at room temperature, but may be performed at a temperature in the range of about 15-65 ° C. The pressure is preferably normal atmospheric pressure, but may be below or above it. Any carbohydrate material that yields glucose upon chemical or enzymatic treatment is a suitable source of glucose for conversion to D-glucosone. These substances may include, for example, cellulose, starch, sucrose and corn syrup.

The methods used in the analysis of the sugars obtained according to the present invention are as follows.

1. Thin layer chromatography (TLC). Avicel coated glass plates are developed in an 8: 8: 2: 4 (v / v) isopropanol: pyridine: acetic acid: 35 water-solvent system. The R 2 value of glucose is 0.5-0.6; 6 69638 D-glucosone has an Rf value of 0.4-0.5 (line). (Rf = distance the substance moves from the starting point / distance of the front edge of the solvent from the starting point). If plates are sprayed with triphenyltetrazolium chloride (2% TTC in 0.5 N NaOH 5 D-glucosone immediately forms a red spot; Glucose forms a red spot only when heated for 10 minutes at 100 ° C. If plates are sprayed with diphenylamine / aniline / phosphoric acid / ethyl acetate reagent, (0.15 g / 0.8 ml / 11 ml / 100 ml) gives a brown spot of Glu-10 cose, D-glucosone gives a purple band when heated for 10 minutes at 95 ° C.

2. High performance liquid chromatography (HPLC). A 15% aqueous acetonitrile solution containing 0.001 M potassium phosphate buffer, pH 7, is applied to a μ-Bondapak carbohydrate column supplied by Waters Associates at a rate of 2 ml / min. The Rt value of glucose is 11.5 and the Rf value of D-glucosone is 14.0 (Rt value = retention time). Analyzes were performed on a Spectra Physics SP8000 instrument using both a Waters Associates1 refractive index detector 20 and a Schoeffel variable wavelength UV detector set at 192 nm.

3. Mass spectrometry (MS). The following processing instructions were used to solubilize the chemical ingredients: a) To about 100 milligrams of lyophilized sample is added 110 milligrams of N, N-diphenylhydrazine (t ^ NNg ^) and 1 ml of 75% aqueous ethanol. The reaction mixture is stirred vigorously and allowed to stand overnight at room temperature.

B) 3 ml of water are added to the reaction mixture and the precipitate formed is separated from the supernatant by centrifugation and decantation. To this precipitate is added 1 ml of 1: 1 pyridine-acetic anhydride. The reaction mixture is placed in a water bath at 35-40 ° C for 15 minutes with occasional shaking.

7 69638 c) 2 ml of water are added to stop the reaction and the mixture is then extracted twice with 3 ml portions of ethyl ether. The ether is dried over a small amount of sodium sulfate anhydride and the ether is then removed by gentle heating (^ 40 ° C) and blowing nitrogen through the mixture.

(d) The resulting solid or syrup is ready for mass spectrometry analysis.

Expected reactions: 10 -CH0 -CH = NNO2> C = 0 (1) H2NNO2> C = 0 (no effect) -> -OH (2) Ac2O, pyridine -OAc 15 Glucose gives a molecular ion with a mass of 556 (C2gH32N2O) and a base ion with a mass of 168 (NO2-ionic part), · fructose gives a molecular ion with a mass of 390 (C and a powerful diagnostic ionic portion 20 having a mass of 223 (OC-CH = N-NO 2 ionic portion). The analyzes were performed on a Finninga GC / MS / DS Model 4023 mounted on an electron impact ionization of 70 eV and a sample temperature of 220 °.

4. Colorimetric test tube analysis. Analysis of D-glucosone 25 in the presence of excess glucose is readily performed by two colorimetric methods. When triphenyltetrazolium chloride (TTC) is used, the detection method is based on the difference in the rate of reduction of the two sugars. To a 20 ml test tube add 0.5 ml of a 30 ml sample and 0.1 ml of 1% aqueous TTC solution and 0.4 ml of 6 N NaOH solution. After exactly 5 minutes, add 15 ml of acetic acid / ethanol (1: 9) and shake the contents of the test tube. Using water as a reference, measure the absorbance at 480 nm 35 using a Varian 635 UV / VIS spectrometer. Glucose reduces TTC to a red pigment - triphenylformazane - about 100 times slower than the corresponding amount of D-glucosone. When a diphenylamine / aniline / phosphoric acid reagent is used, the detection method is based on different colors of sugars with different structures. To a 20 ml test tube add a 0,2 ml sample and 5 ml of the following reagent mixture: Diphenylamine 0,15 g

Aniline 0.80 ml 10 Isopropanol 100 ml

Phosphoric acid 11 ml

The test tubes are placed in a water bath at 37 ° C for 60 minutes. Glucose forms a yellow colored solution, D-glucosone gives a purple-15 red solution.

The sources of pure sugars used in the various analytical studies of the invention are as follows: 1. D-glucose was supplied by Applied Science Laboratories, 20 purity 99%.

2. D-glucosone was synthesized by the following method: 20 g of glucose are mixed with 1 liter of distilled water containing 27 ml of glacial acetic acid. 4 4 g of phenylhydrazine are added. The reaction is allowed to proceed for 3 hours at 80 ° C with vigorous stirring using a mechanical stirrer and then cooled to room temperature overnight. The solid is filtered off and washed with 10% acetic acid, water and then ethyl ether. The solid is dried well in a vacuum oven at 50 ° C. The experimental yield is 16.1 g of glucosone. Glucosazone is placed in a 3-necked 3-liter flask and 450 ml of ethanol, 750 ml of distilled water and 9 ml of glacial acetic acid are added. 27.8 g of fresh benzaldehyde are added and the mixture is refluxed with vigorous stirring with a mechanical stirrer. The reaction mixture is refluxed for 5 hours. The condenser is inverted and removed by distilling 450 ml by adding 750 ml of distilled water (via an addition tube) to the flask. The reaction mixture is allowed to cool overnight to give a benzaldehyde phenylhydrazone precipitate. The solution is filtered and the residue is washed with distilled water (about 1 L) until the water is clear. The filtrate and washings are concentrated to a volume of 500 ml and then extracted 10 times with 300 ml portions of ethyl ether. To remove residual ethyl ether from the aqueous solution, it is placed in a vortex evaporator for 30 minutes. The aqueous solution is passed through a 4 x 100 cm column containing Amberlite XAD-2 resin thoroughly washed with acetone. The column is washed with 200 ml of additional water to remove residual glucosone, the combined aqueous portions are lyophilized. The experimental yield of glucosone is 3.4 g (total yield 16%).

The following examples illustrate the invention. Unless otherwise noted, all temperatures are room temperatures (about 25 ° C) and all pressures are normal atmospheric pressures (about 1 atm).

Example 1 This example shows a substantially complete conversion of glucose to D-glucosone using immobilized carbohydrate oxidase.

Glucose (2 g) is added to 20 ml of distilled water in a 100 ml ml Pyrex flask and the sugar solution is stirred. Oxygen gas is bubbled into the flask and 3 mg of catalase (Sigma Chemical Co., C-40, bovine liver) is added. The immobilized carbohydrate oxidase prepared from 200 ml of culture is also added to the flask as follows.

To prepare the enzyme, mycelium of the species Polyporus obtusus ATCC ft. 26733 was grown on agar media containing yeast / malt extract as follows: Yeast extract (3 g), malt extract (3 g), agar (20 g), peptone 35 (5 g) and glucose (10 g) is added to distilled water 10 69638 (1 L) and the pH is adjusted to 6.7. The medium is sterilized at 121 ° C for 15 minutes. The pH is then adjusted to 6.4. The organism is inoculated onto agar media and grown for 7 days at 25 ° C. The medium grown in the medium is then used to inoculate the yeast / malt extract medium (20 ml of medium in a 125 ml Erlenmeyer flask) prepared as described above (except that no agar is added). The organism is allowed to grow for 9 days on a rotary shaker at 25 ° C.

10 The culture is vacuum filtered through No 541 Whatman paper through it in a Buchner funnel. The mycelium left on the filter paper contains the enzyme.

Obtained from mycelium, 400 ml of culture is washed twice with 0.05 M potassium phosphate buffer at pH 7.0.

The mycelium is then placed in a Waring mixer containing 70 ml of 0.05 M potassium phosphate buffer at pH 7.0 and homogenized for 3 minutes. The mixture is then centrifuged at 6000 rpm for 20 minutes and the supernatant is removed by decantation from the solids. The supernatant is placed in 500 ml

To an Erlenmeyer flask, add 19 g of polyethylene glycol (weight 4000) and stir the solution for 30 minutes. The suspension is then centrifuged at 7,000 rpm for 20 minutes. The supernatant is removed by decane-25 and discarded. 15 ml of 0.2 M sodium chloride solution and 15 ml of 0.05 M potassium phosphate buffer at pH 7.0 are then added to the precipitate and mixed vigorously. The solution is allowed to stand for 30 minutes, during which time a precipitate forms. The mixture is centrifuged at 14,000 rpm for 30 minutes to 20 minutes. The cloudy, white supernatant containing the cell-free, purified enzyme is removed by decantation.

Immobilization of the enzyme on agarose can be performed as follows: Cell-free, purified enzyme is dialyzed overnight in 500 ml of freshly distilled water. 5 ml of 0.1 M sodium bicarbonate at pH 8.0 are then added. To this solution is added 5 g of activated CH-Sepharose 4B (washed and swollen with a sintered glass filter using 500 ml of 1 mM HCl-5 solution). Using an upright drum mixer, stir the gel suspension for 1 hour at 25 ° C. The gel suspension is then washed first with 40 ml of 0.1 M sodium bicarbonate solution at pH 8.0, then with 40 ml of 0.05 M Tris buffer at pH 8.0 containing 0.5 M sodium chloro-10. ride solution and then 0.5 M sodium formate buffer at pH 4.0, which also contains 0.5 M sodium chloride.

The reaction mixture is sampled at different times and analyzed for glucose and D-glucosone. Using HPLC analysis, peak areas at 11.5 min (Glu-15 cose) and R 14.0 min (D-glucosone) are quantitatively determined and sugar levels are calculated. The following results are obtained:

Reaction- Glucose D-glucosone% conversion of glucose time gg to D-glucosone 20 0 2.0 0.0 0 1 1.2 0.8 40 2 0.5 1.5 75 3 0.2 1.8 90 4 < 1.9> 1.9 99+ 25 Substantially complete conversion of glucose to D-glucosone is also indicated by the disappearance of the spot at 0.58 (glucose) and the presence of a band at R ^ 0.43-0.49 (D-glucosone) during the reaction and the absorbance. increase at 480 nm during the reaction using TTC-30 colorimetric tube assay.

The fact that the product is indeed D-glucosone is confirmed on the basis of the above and by mass spectrometry, as described above. The product gives diagnostic D-glucosone mass ions with a mass value of 512 (molecular-35 ion) and a mass value of 223 (OC-CH = N-NO2 ion residue) · 12 6 9 6 3 8

Example 2

Glucose (1 g) is added to 50 ml of distilled water in a 250 ml Pyrex flask and the sugar solution is stirred. Agarose-immobilized carbohydrate oxidase, prepared according to Example 1 from 50 ml of cell-free purified enzyme, is then added to the flask together with 1 mg of catalase (as in Example 1).

After 18 hours, the aqueous solution is separated from the solids by decantation. Analysis of this solution shows that more than 99% of the glucose is converted to D-glucosone.

Example 3 This example demonstrates the substantially complete conversion of glucose to D-glucosone using glucose-2-oxidase.

The reaction of Example 1 and the conditions used therein (using agarose as the immobilizing medium) are repeated replacing glucose-2-oxidase-20a carbohydrate oxidase. After a reaction time of 5 hours, more than 99% of the glucose had been converted to D-glucosone.

To prepare the enzyme glucose-2-oxidase, mycelium cultures of Aspergillus oryzae ATTC 1 7252 were grown in broth / yeast extract / tryptone medium as follows: broth extract (5 g), yeast extract (5 g) tryptone (3 g), dextrose and Difco starch (24 g) is added to distilled water (1 L) and the pH is adjusted to 7.3. The medium is sterilized at 121 ° C for 35 minutes. Using spores obtained in a generally known manner, the medium is inoculated to give about 3 x 4 10 spores per ml and the culture is carried out in a rotary shaker (180 rpm) at 30 ° C for 2 days. The culture is filtered under vacuum through Whatman No 541 filter-35 tin paper in a Blichner funnel and washed several times with water. The mycelium left on the filter paper contains the enzyme.

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Purification and immobilization of the enzyme can then be performed by the procedure of Example 1.

Example 4 In Example 1, the level of free hydrogen peroxide evolved during the reaction of glucose with carbohydrate oxidase and the associated formation of D-glucosone is minimized using catalase to decompose hydrogen peroxide. An alternative method of minimizing the amount of free hydrogen peroxide is to combine its formation with a hydrogen peroxide consuming reaction to form the desired (valuable) by-product.

In this example, the formation of hydrogen peroxide is combined with the preparation of propylene bromohydrin, which is an intermediate in the synthesis of propylene oxide disclosed in our U.S. Patent 4,247,641. for the reaction of seagrass peroxidase with bromide and propylene to form propylene bromohydrin. The end product of this combined reaction is then the simultaneous formation of glucosone and propylene bromohydrin required for the subsequent production of fructose, the latter of which can be readily converted to propylene oxide, as disclosed in our U.S. Patent 4,247,641. Any enzyme capable of oxidizing a hydroxyl group on the 2-carbon atom of glucose and involving the formation of hydrogen peroxide can be combined with each halogenating peroxidase and the combination can be used to produce alkene halohydrin according to the principles of the aforementioned U.S. patent.

Cell-free, purified seagrass peroxidase-35 enzyme is prepared as follows.

14 69638

Obtained from Coralina sp. Off the coast of La Jolla, California, it is ground in a Virtis 45 homogenizer for 5 minutes in distilled water. The homogenized product is centrifuged at 20,000 rpm for 5 to 20 minutes. The supernatant is removed by decantation and recovered. The residue is resuspended in distilled water and centrifuged. This supernatant and the previous supernatant are combined. The solution is first brought to a saturation of 33% then 55% in ammonium sulfate. Centrifugation and separation of the residue are performed at each stage. The residual fraction of 33-55% is passed through a DEAE column using a 0.3 M to 1 M phosphate buffer gradient (pH 6.0). The fraction eluting with 1 M is dialyzed against 20 mM phosphate buffer (pH 6) overnight.

Immobilized seagrass peroxidase is prepared as follows.

Glass beads (sold by Sigma Chemical Company, PG-700-200) are activated by immersing 1 gram of glass beads in 18 ml of 20 deionized water. 2 ml of 10% (v / v) o-aminopropyltriethoxysilane are added and the pH of the mixture is adjusted to 3-5 with HCl solution (6 M). The mixture is shaken at 75 ° C for two hours. The glass beads are then dried under vacuum during the reaction at 80 ° C. To glass beads 25 are added 3.2 ml of purified Coralina sp. Enzyme prepared as described above and 50 mg of water-soluble carbodiimide. The pH of the mixture is adjusted to 4.5 and shaken at 4 ° C overnight. The product (enzyme-coated beads) is washed with water. Activity mi-30 is measured in 2 monochlorodimedone units g per bead.

Carbohydrate oxidase immobilized on agarose is prepared as in Example 1 from 10 ml of cell-free, purified enzyme.

The reaction mixture containing the following ingredients is placed in a 100 ml Pyrex flask: 15 69638 a) 1 g of glass beads coated with seagrass peroxidase, b) immobilized carbohydrate oxidase prepared as described above, 5 c) 800 mg of potassium bromide and d) 20 ml of 0.01 M potassium phosphate buffer, pH 7.0.

Both propylene and oxygen are bubbled continuously into the flask. The reaction is started with 1 g of glucose. After 10 hours, the reaction mixture is sampled and analyzed for residual glucose, D-glucosone and propylene bromohydrin. The propylene bromohydrin formed is analyzed as follows: 5 microliters of the reaction mixture is injected into a Hewlett-15 Packard Model 402 gas chromatograph equipped with a 1.8 m x 0.32 cm glass column packed with Porapak R material (80/100 mesh). The flow rate is 30 ml / minute for helium and the column temperature is adjusted to 200 ° C. The residence time of propylene bro-20 myhydrin is 9 minutes for 1-bromo-2-propanol and 10 minutes for 2-bromo-1-propanol.

The product is identified by comparison with authentic samples of propylene bromohydrin; 1-Bromo-2-propanol is sold by Pfaltz and Bauer, Inc .; 2-Bromo-1-propanol is synthesized by reduction of 1-bromopropionyl chloride with lithium aluminum hydride. The residence times of the reaction products and the original samples are the same and their mass spectra are identical; bromine is identified by the presence of M and M + 2 isotope joints of the same strength, 301. the molecular ion of both isomers is identified by chemical ionization using isobutane reagent gas (M +; m / e 138 + 140); For 1-bromo-2-propanol, the main cleavage is the expected CH2Br loss, while for 2-bromo-1-propanol, the main cleavage is the expected CH2Brr loss.

35 Analysis of the sample shows> 99% glucose to the 16,69638 version of D-glucosone and a propylene bromohydrin yield of 20 g / l.

Example 5 This example further illustrates the concepts mentioned and illustrated in Example 5. In this case, immobilized glucose-2-oxidase replaces immobilized carbohydrate oxidase.

Immobilized seagrass peroxidase enzyme is prepared as in Example 4. Immobilized glucose-2-oxidase is prepared as in Example 3.

The reaction mixture is prepared as described in Example 4, replacing immobilized glucose-2-oxidase with immobilized carbohydrate oxidase. The reaction mixture is sampled after 20 hours and analyzed for residual glucose, D-glucosone and propylene bromohydrin. The results show a> 99% conversion of glucose to D-glucosone and a propylene bromohydrin production of 19.5 g / l.

Example 6 This example demonstrates the efficient conversion of glucose to D-glucosone over a prolonged period of time using carbohydrate oxidase immobilized in a column reactor.

The carbohydrate-25 oxidase (cell-free, purified enzyme, 10 ml) prepared according to Example 1 is immobilized on hydroxyapatite (calcium phosphate hydroxide) as follows.

To 100 ml of cell-free, purified enzyme is added 20 g of hydroxyapatite in 100 ml of 1 mM potassium-30 phosphate buffer at pH 7.0. The mixture is stirred for 30 minutes, the solids are then separated from the liquid by decantation and the solids are washed first with 200 ml of 10 mM potassium phosphate buffer at pH 7.0 and then with 200 ml of distilled water.

35 This material is then packed in a glass column

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17 69638 (0/5 cm x 4.5 cm). A 1% Gluose solution is passed through the column at a flow rate of 1.5 ml per hour. The eluate is periodically analyzed for residual glucose and the D-glucosone formed.

5 The eluate, prepared continuously for 5 days, showed that more than 95% of the glucose had been converted to D-glucosone. No hydrogen peroxide was detected. The study was terminated before determining the true half-life of the enzyme. At the low flow rate used in this experiment, both oxygen availability and the absence of accumulated hydrogen peroxide contributed to substantially complete conversion of glucose to D-glucosone. In this case, the support, hydroxyapatite, caused the decomposition of hydrogen peroxide.

Thus, it can be seen that the process can be easily performed at room temperature and normal pressure, whereby the energy requirement can be minimized if desired. The by-products of the process are not difficult to dispose of or recover.

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References; 1. Specialized Sugars for the Food Industry, edited by

Jeanne C. Johnson, Noyes Data Corporation, 1976, pp. 130- c 201.

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31 (1937) 1033.

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Claims (5)

19 69638 A process for the preparation of D-glucosone from glucose, characterized in that an aqueous solution of glucose 5 is formed, at least about 95% of the glucose in solution is converted to D-glucosone by enzymatic oxidation in the presence of oxygen using an oxidoreductase enzyme and the hydrogen peroxide For the preparation of propylene halohydrin or propylene oxide. Process according to Claim 1, characterized in that the enzyme is an oxidoreductase having glucose-2-oxidase activity. Process according to Claim 2, characterized in that the enzyme is glucose-2-oxidase from Aspergillus oryzae or carbohydrate oxidase from Polyporus obtusus. A method according to claim 2, characterized in that the enzyme is immobilized. Process according to Claim 1, characterized in that the hydrogen peroxide is removed by enzymatic decomposition using catalase.