PROCESS FOR MAKING GLUCOSONE

Abstract: Methods of producing glucosone which comprises enzymatically oxidizing glucose with glucose-2-oxidase in a first zone and separating the concomitantly produced hydrogen peroxide from said first zone through a semi-permeable membrane into a second zone wherein an alkene is reacted with said hydrogen peroxide to form oxygenated products of said alkene, said membrane being permeable only to compounds of a molecular weight of less than about 100.

Description: PROCESS FOR MAKING GLUCOSONE This invention is concerned with a new and useful process for the production of glucosone and more particularly for the production glucosone from which food-grade fructose can be obtained. Commercial methods for the production of fructose, a commercially important sweetner, primarily involve a two-step • process, the first, hydrolysis of a polysaccharide such as starch to produce glucose and the second, isomerization of the so-produced glucose to form fructose. The latter step, as is well-known, produces a mixture ' of glucose and fructose from which it is difficult to separate the desired product, fructose. The commercial separation method involves the use of crystallization techniques which are costly and time-consumin More detailed description of the various methods of isomerizing glucose can .be found in the literature, e.g. U.S. Patent 3,788,945, and 3,616,221. Glucose can also be converted to fructose by the action of an enzyme, designated glucose-2-oxidase, to form glucosone (D-arabin©-2-hexosulose) which in turn can be re¬ duced to fructose with zinc and acetic acid [Folia Micriobiol. 23, 292-298 (1978) and Chechoslovakian Patent No. 175897 to * Vole et al. ] . The reaction of glucose-2-oxidase with glucose to produce glucosone also yields hydrogen peroxide in eguimolar amount. The use of the so-produced hydrogen peroxide in the conversion of alkenes to corresponding halohydrins and epoxides has been proposed in European Patent Application 7176. In the published application, the in situ formation of hydrogen peroxide is proposed by inclusion of glucose-2-oxidase and glucose in the reaction mixture which includes a halogenating 1 enzyme and a source of inorganic halide into which the sele alkene is to be introduced. The disclosure of the European patent application further indicates that the glucosone pro duct of the enzymatic oxidation of glucose can be converted 5 to fructose by simple chemical hydrogenation. However, fructose produced by the said process ca be contaiminated with significant amounts of by-products fr both the enzymatic conversion of glucose and the alkene con version reaction. In particular, the latter reaction produ -'- 0 halohydrins and alk lene oxides, e.g. ethylene oxide, which are highly toxic material even at levels in the region of parts per million. Thus, fructose produced by such a proce will require, careful and costly purification to attain food grade purity. Further, the potential for contamination of -^ fructose by virtue of secondary reactions during the initia processing state is quite high due to the highly reactive products, halohydrins and alkyleneoxides, and substantial purification procedures are required to assure the high lev purity required for food grade fructose. 20 SUMMARY OF THE INVENTION This invention provides a method for the producti of glucosone by enzymatic oxidation of glucose to glucosone a reaction zone from which hydrogen peroxide is removed by use of a hydrogen peroxide-permeable membrane into a second - 3 reaction zone where the .hydrogen peroxide is reacted with an alkene to convert the alkene to an oxidation product. In accordance with one embodiment of the inventio the alkene is converted to a glycol by reaction with hydrog peroxide. This reaction is catalyzed by osmium, vanadium o 3° chromium oxide or by ultraviolet light in accordance with procedure described J.A.C.S. 5_8, 1302 (1936); 59., 543, 2342, 2345 (1937) , incorporated herein by reference. In accordance with a second embodiment of the inven¬ tion, the alkene is converted to a halohydrin and then to an alkylene oxide or glycol corresponding to the original alkene reactant by reaction with hydrogen peroxide, a halogenating enzyme and a halide ion source, to form the halohydrin which is then converted to an epoxide or glycol, by the methods described in European patent application 7176. The membranes employed in the present process are for the prupose of establishing two separate zones and per- mitting migration of hydrogen peroxide from the first to the second zone. The membranes therefore should be of suitable pore size to selectively permit hydrogen peroxide migration, but to preclude passage of larger molecules in the first reaction zone. Such membranes are readily available commercially and can be defined in terms of the molecular weight of solute particles to pass through the membrane. In the present inven¬ tion, membranes which permit substances of a molecular weight of less than about 100 are to be used, and preferably less than 50. The migration or passage of hydrogen peroxide through the aforesaid membrane is accomplis-hed through establishment of an equilibrium predicated on the relative concentrations of H_0 2 on each side of the membrane. As the concentration of hydrogen peroxide in the first zone increases , the H-O- tends to migrate to the secon zone until equilibrium is rees- stablished. The reaction with an alkene in the second zone increases the rate of flow of hydrogen peroxide through the membrane by offsetting the equilibrium in the direction of the second zone. Ut'- . : ι 2 Employing the present process results, in considerab advantage particularly in the further processing of glucosone to fructose. The migration of hydrogen peroxide from the fir reaction zone of course affects the rate of the enzymatic oxi cjdation of glucose so that the reaction tends to be more compl and the reaction times can be shorter than normally required. Further, the first reaction zone is essentially free of con¬ taminants that will accumulate primarily in the second reacti zone where the so. produced hydrogen peroxide is reacted. The 0glucosone solution produced in the first reaction zone can be used as such in the hydrogenation step or can be concentrated or otherwise processed as desired. The glucosone solution is substantially free of contaminants other than some unreacted glucose, or glucose dimer or tri er, and whatever contaminant 5that may have been introduced in the original glucose charge. Usually, the glucose charge will be a hydrolysate of a natura product containing glucose units, most commonly starch, which will contain soluble contaminants 'such as other carbohydrates, e.g. maltose, formed in the starch hydrolysis. 0 Accordingly, the reduction of the reaction product of the first zone will provide a product, fructose, which wil be comparatively free of contaminants that effect food grade status for the product, the contaminants being derived only from the glucose natural sources, e.g. starches such as corn starch. The alkene reaction zone also is cleaner than attai able when both reactions are conducted in the same reactor. tøOflJβtNAL PREFERRED EMBODIMENTS The membranes to be used in the present process are any of those commonly employed in aqueous systems and include .a wide variety. Most commonly, the membranes will be comprised of nylon, a styrene polymer, usually polystyrene teflon, or a cellulose ester such as cellulose acetate or propionate. In a first embodiment, the membrane is fitted into a reactor to provide two zones in a manner to preclude unintended mixing of the contents of the two zones . In a second embodiment, separate reactors can be coupled with the selected membrane providing the requisite interface in the coupling. For maximum migration of hydrogen peroxide from the first zone to the second zone, membranes of significant exposed surface area are of course preferred for which reason the first embodiment is more preferable. The glucose-2-oxidase enzyme can be provided in the form of the enzyme solution in water, immobilized enzyme or immobilized cells or mycelium or the free cells or mycelium. Most commonly since the enzyme is intracellular, the cells or mycelium of the selected microorganism are used by merely suspending them in the reaction solution. Promoters and pro- _ tectors for the enzyme can also be present. For example, as described in the aforesaid Folia Microbiol. 23, 292-298 (1978) , the presence of fluoride ion promotes the enzymatic oxidation of glucose with 0. mucida. Protectors for enzymes can also be used, e.g. Co, Mn and Mg salts. " B JR£4^ The enzymatic oxidation reaction is carried out un substantially complete as can be determined by monitoring th mixture using aliquots to test for glucose content,- or by colori etric determination of glucosone or by determination of hydrogen peroxide. Usually, reaction periods of about 2 48 hours are sufficent, depending on enzyme potency or activ A wide variety of microorganisms can be used to pr duce the glucose-2-oxidase employed in the present process. For example, the following organisms are described in the literature for this purpose: I Aspergillus parasiticus [Bioche . J. _31, 1033 (1937)] II Iridophycus flaccidu [Science 124, 171 (1956) ] III Oude ansiella mucida [Folia Microbiol. JL3, 334 (1968) ibid. 2 , 292-298 (1978)] IV Gluconobacter roseus [J. Gen Appl. Microbiol. _1,152 (1955 V Polyporus obtusus [Biochem. Biophys. Acta 167, 501 (1968) VI Corticium caeruleum [Phytochemistry 1977 Vol. 16, p 1895- The temperature for the enzymatic oxidation reactio is not critical. The reaction can be conducted at room tempe ture, or. even somewhat higher than room temperature where the enzyme system employed is of reasonable heat stability.. In particular, it is preferable to operate at 50°C. and above, with heat stable enzyme systems in which range bacterial infe of the reaction mixture is minimized. Alternatively, the enz matic reaction mixture can contain antibacterial agents to pr clude extensive bacterial growth. The first reaction zone of course should contain no significant amounts of a reducing agent for hydrogen peroxide so that the beneficial results of the present process can be realised. Thus, the system should be substantially free of reducing agents for SoC ? ' -i- -6 -* a non -reducing system. - 7- During the course of the present process, it is possible for some diffusion of material from the second reation zone into the first zone, especially where anions, cations or low molecular weight compounds are present in the second zone, but such diffusion is not significant under the present conditions The procedures employed for the conversion of alkenes to oxygenated products are those described in the hereinbefore described references which are incorporated herein by reference for the said disclosure. The reduction of glucosone to fructose is accomplished by known procedures including chemical reduction as with zinc and acetic acid as well as catalytic hydrogenation, with the usual metal catalysts. Of these, the preferred metal catalyst is Raney Ni since its use is compatible with the desired food grade of fructose, i.e. no residues or contaminants are left by this catalyst. In the ususal procedure employed, the glucosone is hydrogenated at elevated pressure and temperature over the selected metal catalyst until the desired degree of hydrogenation has been achieved. Pressures can range from 100 to 700 atmos¬ phere and even higher while the temperature can range up to about 200°C. Preferred is 100° to 150°C. and a pressure of about 500 atmospheres. The following example further illustrates the invention. EXAMPLE Mycelium of 0. ucida are grown in accordance with - ■*■ - Example 1 of Czechos'lova ian patent 175897 and the equivalent ' " : of 15 g. (dry weight) of the mycelium is suspended in j L. * c -o " 2.5% "glucosό"solution 0.05M NaF in one zone of a 10 L. reactor fitted with a hydrogen perioxide-permeable membrane to form two zones. In the second zone, ethylene gas is bubble through an aqueous solution of chloroperoxidase and halide ion buffered with a phosphate buffer (0.1M potassium phosphate) as - |Q described in European patent application 7176 (Examples 1-18) . The suspension in the first zone is mixed at 25°C. and aerated with oxygen. After 24 hours the mycelium is then separated from the solution in the first zone and resulting clear solution is then hydrogenated over Raney Mi at 500 at os c pheres hydrogen gas and 100°C. The aqueous mixture is filtere clear of the catalyst, decolorized with carbon, deionized with ion-exchange (anionic and catiόnic) and concentrated to a fructose syrup at reduced pressure. Alternatively, the aqueou mixture is concentrated and fructose allowed to crystallize. 2o The fructose obtained as either syrup or crystalline product is of food grade quality. The halohydrins obtained by the reaction in the seco zone are converted to the corresponding epoxides by treatment with sodium hydroxide. 25 Essentially the same results are obtained when 0. mucida is replaced with the following organisms: Polyporus obtusus Radulum casearium Lenzites Trabea _ . 30 Irpex flanus Polyporus versicolor i. τ f_ _c__ i.x ι l -5 v -...u-._ * !.ax x : " a_- - x_*x- x *ι aiuc —j -_i. -i_-.u —. a — a _- Armillaria mellea Schizophyleum commune 35 Corticium caeruleum

Claims: 1 WHAT IS CLAIMED IS: ,, ._ „ , 0 . 1. A method of prα αpiqg..glucosone which..comprises 5 brane into a second zone where.in.an alkene.. . is reacted with said hydrogen peroxide to .form oxygenated product * * 3 9$_. said alkene, said membrane being.permeable only,.to compounds of a molecular weight of less than about 100. 0 2. The method according to Claim 1 wherein the pro¬ duct of said reaction of an alkene with hydrogen peroxide is a glycol. 3. The method according to Claim 1 wherein the pro- ** duct of said reaction of an alkene with hydrogen peroxide is 5 the corresponding alkylene oxide. 4. .The method according to ' Claim 1 where said mem¬ brane is permeable only to compounds of a molecular weight of less than about 50. 5. A method of producing fructose ' which comprises the 0 steps of a) producing glucosone which comprises enzymatically oxidizing glucose with glucose-2-oxidase in a first zone and separating the concomitanly produced hydrogen peroxide from said first zone through a semi-permeable membrane into a second 5 zone wherein an alkene is reacted with said hydrogen peroxide to form oxygenated products of said alkene, said membrane being permeable only to compounds of a molecular weight of less than about 100, and b) ' reducing the so-produced glucosone to obtain 0 fructose. 6. The method according to Claim 5 wherein the - reduction is effected by catalytic hydrogenation. _ 0ΛPI ****-*•* * •.-;:.. --.*;*- ■ -.nit--. "-,<_. . -_>,_. nc:;* ,.-i;..i * i. * s-i 5- •-. **■ . ** * * 'C ** . * ,.. .< * .. '* '•$._ ■' _ TVNIΘIUO αvs

Inventors: HORWATH R, MASELLI J
Applicants: STANDARD BRANDS INC
Application year: 1981
Main classification: C12P7
IPC codes: C07H1, C07H3, C12P7, C12P17, C12P19