Process for making glucosone

Disclosed are 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.

BACKGROUND OF THE INVENTION 
This invention is concerned with a new and useful process for the 
production of glucosone and more particularly for the production of 
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 and/or 
fractionation techniques which are costly and time-consuming. More 
detailed description of the various methods of isomerizing glucose can be 
found in the literature, e.g. U.S. Pat. Nos. 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-arabino-2-hexosulose) 
which in turn can be reduced to fructose with zinc and acetic acid [Folia 
Micriobiol. 23, 292-298 (1978) and Czechoslovakian Pat. No. 175897 to Volc 
et al.]. 
The reaction of glucose-2-oxidase with glucose to produce glucosone also 
yields hydrogen peroxide in equimolar 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 
No. 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 enzyme and a source of 
inorganic halide into which the selected alkene is to be introduced. The 
disclosure of the European patent application further indicates that the 
glucosone product of the enzymatic oxidation of glucose can be converted 
to fructose by simple chemical hydrogenation. 
However, fructose produced by the said process can be contaminated with 
significant amounts of by-products from both the enzymatic conversion of 
glucose and the alkene conversion reaction. In particular, the latter 
reaction produces halohydrins and alkylene oxides, e.g. ethylene oxide, 
which are highly toxic materials even at levels in the region of parts per 
million. Thus, fructose produced by such a process will require careful 
and costly purification to attain foodgrade purity. Further, the potential 
for contamination of fructose by virtue of secondary reactions during the 
initial processing stage is quite high due to the highly reactive 
products, halohydrins and alkyleneoxides, and substantial purification 
procedures are required to assure the high level purity required for food 
grade fructose. 
SUMMARY OF THE INVENTION 
This invention provides a method for the production of glucosone by 
enzymatic oxidation of glucose to glucosone in a reaction zone from which 
hydrogen peroxide is removed by use of a hydrogen peroxide-permeable 
membrane into a second 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 invention, the alkene is converted 
to a glycol by reaction with hydrogen peroxide. This reaction is catalyzed 
by osmium, vanadium or chromium oxide or by ultraviolet light in 
accordance with the procedure described J.A.C.S. 58, 1302 (1936); 59, 543, 
2342, 2345 (1937), incorporated herein by reference. 
In accordance with a second embodiment of the invention, 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 No. 7176. 
The membranes employed in the present process are for the purpose of 
establishing two separate zones and permitting 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 invention, 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 accomplished through establishment of an equilibrium 
predicated on the relative concentrations of H.sub.2 O.sub.2 on each side 
of the membrane. As the concentration of hydrogen peroxide in the first 
zone increases, the H.sub.2 O.sub.2 tends to migrate to the second zone 
until equilibrium is reestablished. 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. 
Employing the present process results in considerable advantage 
particularly in the further processing of glucosone to fructose. The 
migration of hydrogen peroxide from the first reaction zone of course 
affects the rate of the enzymatic oxidation of glucose so that the 
reaction tends to be more complete and the reaction times can be shorter 
than normally required. Further, the first reaction zone is essentially 
free of contaminants that will accumulate primarily in the second reaction 
zone where the so produced hydrogen peroxide is reacted. The glucosone 
solution produced in the first reaction zone can be filtered or 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 timer, 
and whatever other saccharides that may have been introduced in the 
original glucose charge. Usually, the glucose charge will be a hydrolysate 
of a natural product containing glucose units, most commonly starch, which 
will contain soluble saccharides such as maltose, formed in the starch 
hydrolysis. 
Accordingly, the reduction of the reaction product of the first zone will 
provide a product, fructose, which will be comparatively free of 
contaminants that affect 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 attainable when both 
reactions are conducted in the same reactor.

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 mycelia of the selected microorganism are used 
by merely suspending them in the reaction solution. Promoters and 
protectors 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 O. mucida. 
Protectors for enzymes can also be used, e.g. Co, Mn and Mg salts. 
The enzymatic oxidation reaction is carried out until substantially 
complete as can be determined by monitoring the mixture using aliquots to 
test for glucose content, or by colorimetric determination of glucosone or 
by determination of hydrogen peroxide. Usually, reaction periods of about 
24-48 hours are sufficient, depending on enzyme potency or activity. 
A wide variety of microorganisms can be used to produce 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 [Biochem J. 31, 1033 (1937)] 
II. Iridophycus flaccidum [Science 124, 171 (1956)] 
III. Oudemansiella mucida [Folia Microbiol, 13, 334 (1968) ibid. 23, 
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. 1895-7] 
The temperature for the enzymatic oxidation reaction is not critical. The 
reaction can be conducted at room temperature, 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.degree. 
C. and above with heat stable enzyme systems in which range bacterial 
infection of the reaction mixture is minimized. Alternatively, the 
enzymatic reaction mixture can contain antibacterial agents to preclude 
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 H.sub.2 O.sub.2, i.e. a non 
reducing system. 
During the course of the present process, it is possible for some diffusion 
of material from the second reaction 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 listed 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 usual 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 atmosphere and even higher while the temperature can range 
up to about 200.degree. C. Preferred is 100.degree. to 150.degree. C. and 
a pressure of about 500 atmospheres. 
The following example further illustrates the invention. 
EXAMPLE 
Mycelia of O. mucida are grown in accordance with Example 1 of 
Czechoslovakian Pat. No. 175897 and the equivalent of 15 g. (dry weight) 
of the mycelia are suspended in 3 L. of 2.5% glucose solution 0.05 M NaF 
in one zone of a 10 L. reactor fitted with a hydrogen peroxide-permeable 
membrane to form two zones. In the second zone, ethylene gas is bubbled 
through an aqueous solution of chloroperoxidase and halide ion buffered 
with a phosphate buffer (0.1 M potassium phospate) as described in 
European patent application No. 7176 (Examples 1-18). 
The suspension in the first zone is mixed at 25.degree. C. and aerated with 
oxygen. After 24 hours the mycelia are then separated from the solution in 
the first zone and resulting clear solution is then hydrogenated over 
Raney Ni at 500 atmospheres hydrogen gas and 100.degree. C. The aqueous 
mixture is filtered clear of the catalyst, decolorized with carbon, 
deionized with ion-exchange (anionic and cationic) and concentrated to a 
fructose syrup at reduced pressure. Alternatively, the aqueous mixture is 
concentrated and fructose allowed to crystallize. 
The fructose obtained as either syrup or crystalline product is of food 
grade quality. 
The halohydrins obtained by the reaction in the second zone are converted 
to the corresponding epoxides by treatment with sodium hydroxide. 
Essentially the same results are obtained when O. mucida is replaced with 
the following organisms: 
Polyporus obtusus 
Radulum casearium 
Lenzites trabea 
Irpex flavus 
Polyporus versicolor 
Pellicularia filamentosa 
Armillaria mellea 
Schizophyleum commune 
Corticium caeruleum