Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PROCESS FOR ~ KING GLUCOSONE
This invention is concerned with a new and useful
process for the prttl~ t;on of ~lucosone and m^re particulall
for the production glucosone from which food-grade fructose
5 can be obtained.
Colr~nercial 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
10 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-consuming.
15 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
20 glucosone (D-arabino-2-hexosulose) which in turn can be re-
duced to fructose with zinc and acetic acid ~Folia Micriobiol.
23, 292-298 (1978) and Czechoslovakian Patent No. 175897 to
Volc et al.].
The reaction of glucose-2-oxidase with glucose to
25 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 7176. In
the published application, the in situ formation of hydrogen
30 peroxide is proposed by inclusion of glucose-2-oxidase and
glucose in the reaction mixture which includes a halogenating
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enzyme and a source of inorganic halide into which the selected alkene
is to be introduced. me 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 ar.d the alkene conversion reaction. In particular, the
latter reaction produces halohydrins and alkylene oxides, e.g. ethylene
oxide, which are highly toxic material even at levels in the region of
parts per million. mus, fructose produced by such a process 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 initial processing state 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.
SUWMARY QF 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
m~mbrane 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. mis reaction
is catalyzed by os~ium, 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),
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1 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 ~ri gi nAl ~l ken~.
reactant by reaction with hydrogen peroxide, a halogenating
5enzyme 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 app:Lication 7176.
The membranes employed in the present process are
for the prupose of establishing two separate zones and per-
lOmitting migration of hydrogen peroxide from the first to thesecond 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
15can 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 accomplished through establishment
of an equilibrium predicated on the relative concentrations
of H202 on each side of the membrane. As the concentration
of hydrogen peroxide in the first zone increases, the H202
25tends to migrate to the secon zone until equilibriu~ 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,
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1 Employing the present process results in considerable
advantage particularly in the further processing of glucosone
to fructose. The migration of hydr~g~n p~roxlde from the flrst
reaction zone of course affects the rate of the enzymatic oxi-
sdation 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 con-
taminants 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
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 trimer, and whatever contaminants
that may have been introduced in the original glucose charge.
Vsually, the glucose charge will be a hydrolysate of a natural
product containing glucose units, most commonly starch, which
will contain soluble contaminants such as other carbohydrates,
e.g. 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 effect food grade
status for the product, the contaminants being derived only
from the glucose natural sources, e.g. starches such as corn
25starch.
The alkene reaction zone also is cleaner than attain-
able when both reactions are conducted in the same reactor.
6SS
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 protectors for the enzyme
can also be present. For example, as described in the aforesaid
Folia Microbiol. 23, 292-298 tl978), 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.
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1 The enzymatic oxidation reaction is carried out until
substantially complete as can be determined by monitoring the
mixture using aliquots to test for glucose c~ntent~ or hy
colorimetric determination of glucosone or by determination of
5 of hydrogen peroxide. Usually, reaction periods of about 24-
48 hours are sufficent, de~ending on enzyme potency or activity.
A wide variety of micxoorganisms can be used to pro-
duce the glucose-2-oxidase employed in the present process.
For example, the following organisms are described in the
10 literature for this purpose:
I Aspergillus parasiticus [siochem. 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
20 is not critical. The reaction can be conducted at room tempera-
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 50C. ~nd above.
with heat stable enzyme systems in which range bacterial infection
25 of the reaction mixture is minimized. Alternatively, the enzy-
matic reaction mixture can contain antibacterial agents to pre-
clude extensive bacterial growth.
The first reaction zone of course should contain no
significant amounts of a reducing agent for hydrogen peroxide
30 so that the beneficial results of the present process can be
realised. Thus~ the system should be substantially free of
~ reducing agents for n202 t i e. d ~IOn reducing system.
5~
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 procuedures employed for the conversion of alkenes to
oxygenated products are those described in the hereinbefore
described references.
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
atmoshpere and even higher while the temperature can range up to
about 200C. Preferred is 100 to 150C. and a pressure of about
500 atmospheres.
The following example illustrates the invention.
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1 EXAMPLE
Mycelium of o. mucida are grown in accordance with
Example 1 of Czechoslovakian patent 175897 and the equivalent
of 15 g. (dry weight) of the mycelium is susPended in 3 L.
5o~ 2.5% glucose solution o~o5rl 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 hubbled
through an aqueous solution of chloroperoxidase and halide ion
buffered with a phosphate buffer (O.lM potassium phosphate) as
described in European patent application 7176 (Examples 1-18).
The suspension in the first zone is mixed at 25C.
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 Ni at 500 atmos-
15pheres hydrogen gas and 100C. The aqueous mixture is filteredclear 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 oktained 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 flanus
Polyporus versicolor
Pellic~laria ~ilamentosa
Armillaria mellea
Schizophyleum com~une
Corticium caeruleum
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