CONVERSION OF ALDOSE TO KETOSE IN THE PRESENCE OF A COMPLEXING REAGENT

THIS INVEHTION BELATES to a process for the conversion of an aldose or aldose derivative to a ketose or ketose derivative in the presence of an oxyanion and in particular to the conversion of glucose to fructose, marmose to fructose, glucose 6-phosphate to fructose 6-phosphate, maltose to maltulose, galactose to tagatose, and lactose to lactulose and to other analogous reactions such as the conversion of xylose to xylulose. The process can be carried out in the presence or absence of an enzyme catalysing the conversion.

When the conversion of glucose to fructose is conducted in the presence of an enzyme, glucose isomerase, equilibrium is very often reached when 50 to 55% of the glucose in the reaction medium has been converted to fructose. Hitherto it has not been possible to convert glucose to fructose by a nbn-enzymic reaction without the production of by-products. There are numerous publications, for example US Patents Hos 2,487,121, 3,452,545. 3,558,555 and 5,514,527 and German Patent No 1,163,307, relating to non-enzymic conversion of glucose to fructose all of which describe processes in which the fructose produced is accompanied by alkaline dégradation and other products arising from.the purely chemical and non-enzymic reaction. Because of this any large scale production of fructose up to the present has been based on an' enzyme catalysed reaction.

In the enzymic conversion process as the proportion of fructose in the enzymic reaction medium increases, the rate of the reaction whereby fructose is produced decreases. Therefore in a commercial process for converting glucose to fructose the fructose yield is optimised by balancing a loss in fructose production against the decreasing reaction rate whereby the proportion of fructose in the reaction medium can be increased. A commercially operated process B 27975 may be optimised to produce a syrup in which typically 409a of glucose has "been converted to fructose. In most chemical non-enaymic conversions of glucose to fructose, the amount of fructose present drops after reaching a maximum.

Clearly it is advantageous to increase the proportion of glucose which can economically be converted to fructose during the reaction.

This can be achieved by the effective removal of fructose from the reaction medium by the inclusion in the medium of a reagent which forms a stronger complex with fructose than with glucose although the complexing agent may have additional properties that increase or decrease the reaction rate. Eeagents which have been proposed for this purpose are borate compounds - see Y Takasaki, Agr Biol Chem 1971, 35(9), 1371-5 and USP 3.689,362 (enzymic reaction) and J F Mendicino, J Amer Chem Soc 82, I960, 4975 (chemical reaction).

Also arena boronates have been proposed by S A Barker, P J Somers and B W Hatt in UK Specification No 1,369,185 for both chemical and enzymic reactions. Disadvantages of these prior proposals are, in the case of borate compounds, that these are toxic and could constitute a health .hazard in a product intended for use as a sweetener in foodstuffs for human consumption, and, in the case of benzene boronate it has a limiting solubility, cannot form complexes with a 2;1 ratio of sugars boronate and high concentrations of fructose in the product cannot be attained at high sugar concentrations ( S A Barker, B W Hatt and P J Somers, Carbohydrates Res, 26 (1973) 41-53)- !Phus these complexing reactions do not lend themselves readily to use in commercially operated conversion processes. In ord,er to develop a commercial process in which the proportion of fructose in the syrup produced is increased it is necessary to find a complexing reagent which does not have such disadvantages associated with its use.

B 27975 According bo the present invention we provide a process for the an aldose or conversion/of an aldose derivative to a ketose or ketose derivative wherein the conversion takes place in the presence of a complexing reagent which is an oxyanion or mixed complex oxyanion of gexmanim or tin which forms a stronger complex with the ketose or ketose derivative than with the aldose or aldose derivative.

Also according to the present invention we provide a process for the conversion of an aldose or aldose derivative to a ketose or ketose derivative wherein the conversion takes place in the presence of a complexing agent which is an oxyanion or mixed complex oxyanion • of germani-um which forms a stronger complex with the ketose or ketose derivative than with the aldose or aldose derivative.

Very suitably the aldose derivative is an aldose phospftate oif'.a glycosyl deriva-tive of an aldose.

Whilst the invention is applicable to a wide range of conversions and in particular to those conversions specified hereinabove it is most usefully employed in the conversion of glucose to mannose to fructose.

During a conversion an enzyme catalyst may be present wheste this enables milder reaction conditions to be employed or has other advantages such as the selective action of the enzyme employed on only one isomer (3) or L) of the aldose or aldose derivative. When an enzyme-catalysed conversion is carried out the enzyme may be present in solution or in an immobilised form on a solid matrix that may be a living cell, an inactivated cell or any other suitable support. The enzyme may also be in soluble form. The isomerase-type enzyme suitable for conversions of the type (examples listed in Table A) to which the invention is applicable may constitute one or more of a series of enzymes engaged in sequential reactions.

B 27975 106743? TABLE A Conversion 1 IV-galactose = 3>-tasatose 2 L-arabinose z=5' L-ritxaose Enzyme L-arabinose isomerae (D-galactose isomerase) Reference JSTo.

J Biol Chem, 1971» 246, 5102-6 E.G.

5.3.1.4 As in 1 3 L-fucose Ir-fUCTllOSe As in 1 4 L-rhamnose L-rharaulose L-matnnose L-fructoae Ir-fucose isomerase (D-arabinose isomerase) L-rhamnose isomerase J Biol Chem, 1958, 250, 457 Methods Enzymol, £, 597-82, (1966) EC 5.3.1.3 B.C.

5.3.1,4 Ir-mannose isomerase 6 D-mannose I>-fractose 7 3)-glucose B-fructose B-lyxose isomerase (D-mannose isomerase) Carb Res, .1968, 8, J Biol C3iem, 218 (1956) 535 Not listed E.Ç.

5.3.1.7 B-glucose isomerase (B-xylose isomerase) 8 D-glycero- j=5 B-sedohept- As in 6 B-manno- ulose heptose LO D-xylose 11 L-xylose =£ L-xylose 12 I>-arabinose ==ï B-ribulose 9 B-lycose B-xylulose B-xyliilose Biochem Biophys Acta, (1969) 178, 376-9 E.G.

5.3.1.5 J Biol Chem, 218, (1956) 535 As in 6 As in 7 13 B-ribose =à B-rihulose 5-phosphate • 5-phosphate 14 D-arabinose ci. B-ribulose 5-phosphate 5-phosphate L-xylose isomerase As in 3 As in 6 As in 7 Btot Fed Proc, 19 (i960) 82 listed As in 3 B-araMnose 5- isomerase B-glyceralde =Bihydroxy ijyae-3- acetone phosphate 3-phosphate J Biol Chem, 1957, 226 - Methods Enzymol, 2, E.G.

585-8 (1966) 5.3.1.13 Biochem J, 1968, 107,. 775 B 27975 106743? TABLE A (CONT'D) Conversion Enzyme 16 D-galactose iD-tagotose 6-phosphate 6-pliosphate Yj Jj.-glxicose D-fructose 6-phosphate 6-phospliate 18 D-maimose D-fructose 6-phosphate 6-phosphate 19 D-glucosamine B-fructose 6-phosphate 6-phosphate Eeferenoe Biochem Biophys Rea Gomm, 1975» SE» 641-7 Glucose phosphate isomerase Mannose phosphate xsomerase Glucoseamine phosphate isomerase J Biol Chem, 1973» 248, 2219 J Biol Chem, 1968, 245, 5410-19 No.

K. C .

5.3.1.9 E.G.

5.3.1.8 Adv Eazymol, £L» 491* E.G.

(1975) 15.3.1.10 In this specification the term ketose is to be -understood to mean a ketulose, see the discussion of the nomenclature of ketosea in «The Editorial Report on Homehclatuxe", Journal of the Chemical Society P 5110, (1952).

The complexing reagent may he introduced into the conversion process in any suitable manner, eg as an aldose-oxyanion complex or a derivative of an aldose oxyanion complex, or as a salt or as a compound such as an oxide which forms oxyanions or mixed complex oxyanions under the conditions of the conversion process. «Che complexing reagent may also he introduced as an oxyanion which was previously held on a polyol or an ion exchange resin or other insoluble support that chelates with the complexing reagent or has the complexing reagent as a counter ion.

Suitably the mixed complex oxyanion is one formed by the interaction of an oxyanion of germanium or tin with an ion of another Group IV element or of an element from Group V or YI. Preferably the oxyanion or mixed complex oxyanion contains germanium.. Particularly suitable complexing reagents are genaanate or poly-germanate ions, included in the process as for example sodium germanate or germanium dioxide, and used in solution, as immobilised chelates or as B 27973 coxmter ions of ion exchaaag-e resins. Mixed ions such as £Ge 0 (SG.Jg/ /flGe 0 (POSj2"" or lactate-gexmanim species may be advantageously employed in some cases.

It is known from Lindberg and Swan, Acta Chem Scand, 14» 0-90), 1045-50, that when separated by electrophoresis at pH 107 fructose- germanate complexes axe very different from glucose-germanate complexes, and the former have more tnan twice the mobility of the. latter at 4© G. , V A Nazarenko and G V Plyantilcova (Zh Efeorgan Khim, 8 ($65) 22?!, 1370) cite ionization constants for glucose and fructose with germanate of 8.3 x 10 and. 1.04 x 10" respectively. Œîiey further cite instability -2 constants for glucose and fructose with germanate of 5•54 * 10 and —5 4.24 x 10 y respectively.

It is surprising that germanate ions should be useful as reagents in the conversion of glucose to fructose for the following reasons 1. It has been shown that germanate exists as a monogermanatë =5.

pentagermanate — heptagermanate equilibrium which is displaced to the right on increasing the germanium concentration and to the left by increasing the pH above pH 9 (l> -A. Everest and J C Harrison, J Ghem Soc, 1959, p 2178-2182). For an economic process it is preferred to have the minimum weight of germanate species for the maximum conversion rate as well as avoiding the production of alkaline degradation byproducts.

2. Germanium dioxide and sodium germanate have a very limited solubility in water - see P J Antikainen (Suomen Kenustilehti, JJB . (i960) 58-40). Gulzian and Mailer, J Amer Chem Soc* 1932, M> 3142 quote 31-33 mM for Ge02 in water. D A Everest and J 0 Harrison, J Chem Soc, 1959, 2178, quote 870 mM for sodium germanate.

3. Magnesium ions are generally present in the reaction media used in glucose to fructose enaymic conversions. Magnesium ortho germanate B 27973 (Mg GeO ) is extremely insoluble in water and is uaedin the analytical determination of germanium - see J H Muller, J Amer Chem Soc, 1923, p 2493-2498. Under the conditions cited below it did not precipitate out of solution.

4. Glucose isomerase has a steric reçtuirement for -D-glucose (K J Schray and I A Eose, Biochemistry 10 (1971) 1058-1062) and the glucose-germanate complex known to be formed could have . interfered with the enzyme reaction by inhibiting it partially or wholly. Indeed the 1,2 cia glycol of <£.~D-glucose is suitable for complexing with germanate rather than o-D-glucose.

5. Mannose complexes more strongly than glucose with germana-fce (P J Antikainen, Acta Chem Scajid., 13 (1959) 312).

The conversion of glucose to fructose may be performed by a purely chemical reaction using the germanate species or starmate complex to displace the pseudo equilibria described by S A Barker, B W Eatt and P J Somers (Garb Res, 26 (1973) 41-53)- Preferably however it is performed as an enzyme catalysed reaction in the presence of glucose isomerase. Any glucose isomerase enzyme may be used in the conversion but these enzymes vary as to their optimum pH and temperature. Suitable isomerases include those derived from bacteria of the genera Aerobactor, Pseudomonas, Lactobacillus (K Yamanaka, Agr Biol Chem, 22., 1963» 265-270) Streptomyces, Curtdba.cterium (as described in our co-pending Canadian Application Serial No. 223,169, filed on March 26, 1975), or, particularly, Arthrobacter (described in U.K.

Specification No 1,328,970). Glucose isomerases from the thermophilic microorganisms of the genera aaiermoactinomyces, Thermopolyspora, Ihermomonospora and Pseudonocardia such as are described in Japanese Patent Publication 74/30588 are also suitable.

Some of the above glucose isomerases have a requirement for cobalt ions for optimum activity.

B 27973 The conversion of glucose to fructose may be perfoimed continuously by passing a solution containing glucose through a column containing the immo'bilised enzyme or other catalyst.

Preferably the enzyme is immobilised by being contained in flocculated whole microbial cells in the manner described in JK Specification ¥o 1,368,650. The complexing reagent, eg germanate or stanate species, may be present in the solution passed into the column or with the immobilised enzyme or other catalyst in the column.

In the latter case the column may be packed with immobilised enzyme or other catalyst having the reagent admixed with it and homogeneously dispersed throughout the column or alternatively the column may contain alternate layers of immobilised enzyme or other.catalyst and reagent separated by meshes or grids. When the reagent is present in the column it is in an insoluble form, for example in gel form, as a zeolite or as an inorganic or organic polymeric derivative.

After a glucose to fructose conversion fructose can be separated from the mixture containing the complexing reagent and removed from the process either alone or in admixture with glucose. The product of the process is fructose, a glucose/fructose syrup or both fructose and a glucose/fructose syrup. The complexing agent alone, together with glucose or together with glucose and complexed glucose can be recycled. Separation and recycling may be performed by any suitable method. Two particularly suitable methods for separation and recycling are as follows:

a) A method wherein the initial product of the glucose/fructose conversion is passed through a column containing a cation exchange resin with cationic counterions of a metal selected from Group II of the Periodic Table and hydrogen ions. This divides the initial product into fructose which is removed as the final product of the JO process and glucose plus the complexing reagent which is recycled.

B 27975 Pxeferatly the Group II metal ions are calcium ions.

b) A method wherein the initial product of the g-lucose/fruotose conversion is passed first through a column containing; a cationic exchange resin with cationic counterions of a metal of Groups I or II of the Periodic Table, preferably sodium ions. This divides the initial product into two parts namely (i) a syrup containing glucose and fructose and (ii) fructose plus the complexing reagent. Part (i.); is removed whilst part (ii) is passed through a column as described under a) above to separate fructose from the complexing agent which latter is recycled.

In both methods a) and b) above the resin is preferably a nuclearly sulphonated polystyrene cation exchange resin containing & cross-linking agent.

Alternatively other methods may be used such as by breaking down the fructose-containing complex using "Borasorb" (Registered Trade Mark) sold by Calbiochem Ltd - a polymer comprising a long chain of cis hydroxy! groups linked through a tertiary N. atom to a polystyrene divinylbenzene grid and normally sold to absorb borate i to give fructose and the complexing reagent. If the reaction is conducted with the complexing reagent in solution in the reaction medium this reagent may be also recycled. Thus the germanate absorbed on the "Borasorb" can be eluted with alkali or acid. All these manipulations can be avoided if the complexing reagent (eg germanate).is used in an immobilised form.

The conversion of glucose to fructose is preferably conducted enzymatically and continuously using a column of flocculated whole cells containing the enzyme as described above. When the reagent is present in the reaction medium entering the column it is preferably present in concentrations between 200 nuM and 800 mM, especially 500 mM and 600 mM. The reaction medium entering the column preferably B 2797? contains 30% w/w to 50% w/w glucose in aqueous solution and the reaction is conducted in such a manner that the concentration of fructose in the medium leaving the column is between 40% and 85% especially 7% and 80%. The pH of the reaction medium preferably is in the range 6 to 10 with optimum activity occurring iii the region of pH 8 particularly at pH 7«8 hut varying somewhat with every species of enzyme, the preceding values relating to enzyme derived from Arthrobacter organisms. The operating temperature is preferably within the range 50 to 100 G, particularly within the range 45° to 80OG. The pH of the eluate is in general lower than that of the feed because of the product fructose conrplexing with the oxyanion.

Besides glucose and various species of germanate ions the reaction medium suitably contains the following constituents present in the following proportions:

Mg , ions at concentrations of about 4 mM - with chloride ions of equivalent concentration and HaOH for adjustment of pH For other enzyme reactions (eg phosphogluociosomerase which converts glucose 6-phosphate to fructose 6-phosphate and where increased yields of the product have been found at 25 C in the presence of species of germanate) the reaction conditions will be very different and must be optimised for each enzyme. Glucose isomerase can also be used to convert B-xylose to D-xylulose and is a suitable candidate for this technology and germanate displaces the equilibrium in favour of increased xylulose yields.

Use of germanate ions as the complexing reagent has advantages over the use of borate compounds suggested previously in that germanate species form strong complexes with fructose and are more selective than borate stereo chemically in sugar complexing. The toxicity pro¬ blems associated with the use of borate compounds are avoided.

Unlike areneboronic acids, germanate can form a 1:2 germanate- fructose chelate so economising on the use of complexing agent.

The normal optimum pH of the enzyme alone may not be the optimum pH for the combined enzyme/complexing reagent pro¬ cess. Thus any ability to lower the optimum pH of an enzyme, eg of glucose isomerase from pH 8.5 to pH 7, will be beneficial in reducing the costs of removing the colour produced during the enzyme process. This is unexpectedly true in the case of Arthrobacter glucose isomerase and may be true with other impor¬ tant enzymes. Equally the ability to reduce the operating tem¬ perature and yet attain the same percentage-conversion in the same operating time will also be beneficial. While this ijjif not the case with Arthrobacter glucose isomerase it is possible to save operating time because of another unexpected advantage. The addition of germanate markedly increases the initial reaction rate of conversion of glucose to fructose so that economic con¬ versions are obtainable with a shorter residence time. Further it is fortunate that germanate causes no destabilising effect on the Arthrobacter glucose isomerase over the time studied provid¬ ing the pH fall accompanying the production of fructose in the presence of germanate is ameliorated.

Figure 2 of the drawings shows the percentage change in optical rotation as a result of complex formation against pH for germanate complexes with glucose, fructose and mannose. As can be seen the fructose complex forms at a lower pH than those of glucose and mannose.

The invention is illustrated by the following examples:

Example 1 Enzymatic conversion of glucose to fructose A series of aqueous glucose solutions containing different B 27973 concentrations of glucose together with magnesium ions and germanate ions were passed throxigh a column 52 cms in length and 0.4 cms internal diameter packed with flocc-ulated whole cells containing glucose isomerase. Corresponding solutions containing no germanate were also passed through the column for the purpose of comparison. The percentage conversion of glucose to fructose in the eluate from the column was measured on the Autoanalyser using the resorcinol method.

The reactions were carried out at an initial pH of 8.5 and at a temperature of 60oC, there being a concentration of 0.004M magnesium chloride in the aqueous glucose solutions.

For the preparation of glucose solutions up to 100 mM, in order to prevent the germanate ions causing precipitation of magnesium from the solutions, solutions containing the germanate ions were always added to the glucose solutions entering the column before the magnesium chloride since magnesium is not precipitated by the glucose-germanate complex which forms in the solutions. The solutions containing germanate ions were prepared by suspending germanium dioxide in water, adding a concentrated alkaline solution until pH 10.5 is reached and thereafter adding glucose solution followed by magnesium chloride.

On final adjustment to pH 8.5 the solutions became clear.

For substrate solutions containing 200 mM germanate or more the geimanium dioxide was added to 1M glucose solution so that on addition of alkali intermittently to pH 8.5 the germanate went slowly into solution. A slightly cloudy solution was obtained after adding magnesium chloride but the eluate from the column was perfectly clear.

The results are set out in Table 1.

B 27973 TABLE 1 SlucoseConcentration Germanate ionsConcentration(nM) % Conversion ofglucose tofructose pH ofeluate Flow rate(ml/min) 0.528 0 59 n.a. 0.12 0.528 25 75 n,a. 0.12 4-65 0 57 •8.3 0.10 , 4-58 25 82 8.1 0.10 122 0 47.5 7.0 0.11 118 25 80.5 6.8 0.11 337 0 44.5 6.8 0.11 270 25 63 6,7 0.11 259 0 46+ 6.5 0.11 265 25 53 6.3 0.11 .

300 50 52 6.0 0.11 284 100 59-5 6.4 0.11 275 200 72.5* •7.2 0.11 +55.5% at equilibrium * 82% at equiiibritim (final pH 6.5) As can "be seen from fable 1 in each case where comparison solutions containing no germanate ions were tested the- fructose concentration was increased in the presence of germanate ions.

Example 2 Enzymatic conversion of glucose to fructose The procedure of Example 1 was repeated using a column of similar dimensions (ie 30 cms in length and 0.4 cms internal diameter) in order to study the effect of higher glucose concentrations. In this'case the flow rate was reduced to 0.03 ml/min in order to give a maximum 14' B 27973 residence time of 125 minutes to attain eduilibrium over several houxs and the equilibrium value for this set of conditions was recorded. The glucose concentration was always assayed after the mixture had been made to correct for dilution with alkali/masnesium salts.

The results are set out in Table 2.

TABLB 2 Initial glucoseconcentration(% w/v) Germanate ionsconcentration(mm) Yo Fructose(w/v) % Conversion 47-4 0 25.6 54.0 45-6 364 32.0 70.0 43.7 524 35.0 80.0 43-5 696 56.5 84.0 50.0 800 39.7 79.4 As can be seen from the results the presence of gexmanate ions greatly increased the % conversion to fructose.

Fructose was assayed by the Chaplin-Kennedy method (Carbohydrate Res., 1975, 40, 227-33). This methpd tends to give higher results than the resorcinol method which was used in all subsequent examples for fructose.

In a further experiment assayed by the resorcinol method (Carbohydrate Ees., 26, (1975) 41) ™*ing a, column of the same dimensions at 60° and pH-8.5 the following results were obtained using different flow rates on the column.

42.5 69 B 27975 Flow rate COLUMN FEEDS (ml/mln) (with added 4mM MgCl) 51.2% w/v 47*8% w/v glucose only glucos.e + 800mM genoanat® 0.015 0.050 0.050 1.

% Conversions to fmotose The product from the initial glucose concentration of 43.5% in Table 2 above was fractionated on the Jeol Ltd anion exchange resin in the borate form using a gradient elution with borate buffers (O.IJM borate pH7 to O.JÇM borate pH 9.8) to effect à separation between the glucose and fructose in the product. "Ehe same separation was also performed after removal of the germanate on a coluion of "BOEASOHB" (registered Trade Mark, see previously).. The ratio of fructose to glucose was 3.31 : 1 without prior removal of germanate.

Fructose and glucose were assayed using the cysteiner-sulphuric acid method.

Example 3 Chemical conversion of glucose to fraotosa The purely chemical conversion of glucose to fructose in the presence of germanate ions was assessed by heating a solution containing 50% w/v glucose, 4mM magnesium salt and 600 mM germanate ions to 60 C at pH 8.5. The fructose concentrations found after various times were; as follows.

Time (Min) % Fructose 90 3.3 135 4.6 180 5-7 B 27975 The experiment was repeated at 90 C with a glucoae concentration of 48.4-50% w/v and a gexmanate concentration of 600mW-582mM. Ifte % conversions to fructose obtained after various times are set out in Table 5. Fructose was assayed by the resorcinol method. Nitrogen was present in the heated syrup throughout the course of the experiment.

In all cases the initial pH which had been measured at 51 0 fell and was readjusted to the original pH at the time stated.

TABLE 48.4% w/v glucoseInitial pH 7.0No added MgOlg 50% w/v glucoseInitial pH 7.5No added MgClg 50% w/v glucoseTnitial pH-8.0 -,No added MgCl2 582mM Germanate 600mM Germanate. 600niM Germanate Time fo Conversion(hr) to fructose Time % Conversion(hr) to fructose Time % Conversion(hr) to fructose 0.5 2.0 0.5 4.5 0.5 9.1 1.0 5-8 1.0 8.2 1.0 12.5 1.5 4-7 1.5 9.7 1.5 14.4 2.5 6.8 2.0 15.2 5.0 7.4 5.0 12.0 5.0 15.45 5.5 7-84.0 8.5 5.5 12.6 5.5 15.4(pH 6.74)4.0 18.5 4.5 8.7 6.0 12.2(final pH 6.57) 4.5 18.5 Similar experiments were carried out in the presence of added MgClg.

Results obtained by the resorcinol method are given in Table 4.

The following results obtained with 1.245M glucose, ôOOmM germsmate and 4mM MgCl heated at pH 8.5 and 90° illustrate the importance of the ratio of glucose s germanate.

B 27975 2.0 5&.6 6.5 2.5 (pH 7-48) 38.6 Time (hr 0.5 l'O 1'5 % Conversion to 19.8 51.6 56.2 fructose Time (hr) 3.66 4.55 6 % Conversion to 46.1 48«5 48 fructose The product obtained a few hours subsequently was separated (with andwithout the prior removal of germanate) on a borate column.

Virtually identical values of 59-7% fructose were oTrfcained eluting at the calibrated position for fructose.

TABLE 4 50% w/v glucose Initial pH 8 0.004 M MgCl2 600mM Germanate #> w/v glucose initial pH .8.5 0.004 HMgClg 600mM Germanate Time % Conversion (hr) to fructose 2.17 16.9 2.85 19.0 5.55 18.8 4.0(pH 6.37) 18.0 4.5 22.1 4-85 • 23 5.33 22.7 Time (hr) 0.5 1.0 2.85 % Conversion to fructose 19-5 28.5 50% w/v glucose Initial pH 9*0 O.OO4 M MgClg 600hiM Germanate 3.33 (pH 6.42) 4-5 5.0 29.8 27.2 34.5 55.5 Time (hr) 0.5 1.0 2.0 2.33 % Conversion to fructose 30.3 35-2 35-7 36.7 2.67 (pH 6.98) 3.33 3-67 35.8 36.2 a 55% w/v glucose, 600mM germaxiate, .004 M MgClg solution heated at 90° and of initial pH 7-5 after 6 hours showed a 13.7% conversion to fructose as assayed by the resoxcinol method.

The chemical conversion of glucose to fructose proceeded very slowly at low germanate concentrations at 90° and pH 8.5 with.no added magnesium chloride.

B 27975 lOOmM glucoselOmM germanateTime % Conversion 20inM glucoselOmM germanateTime % Conversion 20MM glucoae20mM germanateTime % Conversion 0.5 5-5 0.5 4.3 0.5 4.3 1 7-6 1 8.5 1 8.5 1.5 10-7 1.5 11.7 2 15.5 2.67 18.2 2.5 18.7 3+ (pH 8.16) 18.8 3+ (pH 7-78) 19.8 5.5 21.5 3-5 25.3 3+ '--. •19.8(pH 8.22) 4 24.2 4 29.1 3.5 20.44 25.6 4-5 25.6 27-5 5+ 33.4(pH 7.8) 5 29.7 5.5 29.1 5.8+ 32.2ÇpH 8.0) 6 50.3 6 36.8 6 33.5 7 31.8 7 37.3 7.5+ 51.9(pH 7-82) 7.5 39-7 8.5 33.4 8 40.6 8 39-4 9 34.6 9 40.6 9 38.9 ''"pH adjusted to 8.5 Examole 4 Conversion of fflucose to ; fructose using soluble glx icose isomerase The initial reaction rates using glucose isamerase in a soluble form (200/1) were investigated at 60° and pH 8.5 with substrate D-glucose at 0.5 mM and added AM MgClg and 0-5 mM CoClg constant in a series of solutions (25 ml) containing different amounts of added germanate.

The solutions were assayed with the automated resorcinol method.

None, 6.25/g fructose/min/ml enzyme 0.5mM germanate, 8.75/<-g fructose/miiv'nil enzyme 25inM germanate, 15/*g fructose/min/ml enzyme B 27973 (Che % conversion to fructose after 21 hours was also assessed.

None, 4336 conversion; 0.5niM, 51% conversion; 25inM, 62% conversion.

ExampjLe Conversion of Mannose to IFructose A solution containing 50% w/v mannose, 4jriW KfeClg and 600 mM germanate was heated at 90OC and pH 8.5- The concentrations of fructose assayed "by the resorcinol method are set out in Tahle 5.

Table Time (min) % Conversion to jO fructose 3.44 60 6.42 120 -4 150 15.4 170 (pH 7.25 readjusted to pH 8.5) 15.9 260 20.4 280 20-6 Exam-pie 6 Conversion of Glucose-6-phosphate to ffructose-é-phosphate Using the technique of Example 1 a series of experiments was performed using a 0.5 niM solution of glucose-6-phosphate to which different amounts of germanate ions were added. The experiments were carried out at 250and at a series of different pH values. The enzyme used was Sigma Grade III from yeast (crystalline suspension) and was diluted 200 times and dialysed against distilled water to remove "buffer salts "before use.

Fructose-6-phosphate produced was assayed "by the resorcinol method.

The percentage conversions obtained are set out in Table 6.

B 27975 TABLE 6 Gennanateions con¬centration % Conversion pH 10.5 pH 10.0 pH 9-5 pH 9-0 pH 8.5 pH 8.0 pH 7-5 pH 7.p 100 81 81 • 77 68 48 40 36 32 12.5 76 69 64 56 40 29 25 22 6.25 62 55 48 40 32 25 23 22 0 - i i 23 22 As can be seen the % Conversion increased in the presence of gennanate ions.

Bxample 7 Enzymatic conversion of glucose to fructose -stability A feed solution containing "41.2%, glucose 600mM germanate and 4n®t magnesium chloride at an initial pH of 7.0 was passed continuously through an enzyme column similar to that of Example 1; The final pE and % conversion to fructose were measured after various intervals of time. The experiment was divided into three periods.

After a first period of 43 hours the feed solution was clarified "by sintered filtration to remove the slight precipitate which tends to form in glucose/germanate solutions after lengthy- periods. This led to a rise in activity which had been falling slightly. After a second period extending from 43 "to 102 hours the feed pH was increased to 7.8 with beneficial results. The pH of the eluate tends to fall markedly after prolonged passage through the column. It appears that conversions in excess of 70% can be maintained over a prolongea period if the pH of the column eluate is not allowed to fall significantly below 7.0. The results are set out in Table 7A.

B 27973 TABLE 7A Time Initial pH Final pH after. passage throughenzyme reactor <>/& conversionto fructose 2 hrs 7.0 6.6 67.2 17 hxs 7.0 5.6 61.5 43 hrs 7-0 5-5 60.8* 54 hrs 7.0 5-8 64.9 102~hrs 7.0 5-7 65.5** 126 hrs 7.8 6.9 76.5 150 hrs 7,8 6.9 78.3 174 lu?s 7-8 6.9 76.5 198 hrs 7-8 6.8 71.6 * End of first period - feed solution then clarified ** End of second period - initial pH then raised The effects of different temperatures on stability was meastired rising an enzyme column the same as that -used above. The results are set out in Tables 7B and JC. Eeaction conditions are given at the heads of the Tables. In Table TS> the conversion is that achieved at equilibrium whilst in Table 70 the conversion is that achieved initially. In both Tables 7B and 70 the results show the cumulative effect.

TABLE 7B 44% w/v Glucose + 4mM MgCl„ + 600nM germanate pH 7.1 at a flow-rate of 0.05 ml/min through the enzyme Temp 0C Date % Conversion to Fructose (enzyme washed 2 days ago) 26.11.75 69.2", 60 26.11.75 75-4 65 ' 26.11.75 75.1 70 27.11.75 71.5 75 27.11.75 72.1 80 27.11.75 . 75.0 85 27.11.75 6o«5 B 27973 TABLE 70 4496 w/v Glucose + 4mM NgCl + 600mM germanate pH- 7.1 at a flow-rate of 0.16 ml/min through the enzyme ► Temp 0C Date % Conversion Time through Cumulativeto Fructose column at a time solution(enzyme particular has passedwashed 14 temperature through columndays ago) (hrs) (hxs) 65 » 51.8 li 4i 70 h 34.1 li 6 75 » 35-8 2£ 8i 80 n 32.4 2 ioi 85 n 25.6 liExample 8 12 Chemical Conversions of Sugars a) Glucose to Fructose Glucose solutions (55-60 w/v) were prepared hoth with and without inclusion of 600mM germanate at pH 12 at 20 C. Immediately after preparation 50/A.l aliquots were taken, diluted to 5 ml and then stored at a temperature of -20 C until required for analysis. The syrups were then placed in stoppered flasks in a refrigerator at 4.5 0. The flasks were shaken daily and alictuots were removed at the time intervals shown in Table 8A.

All samples were analysed toy separation on an ion exchange resin with borate and the peaks were monitored by the automated cysteine/ sulphuric acid assay (Anal Biochem., 26, (1968) p219)v H* was noted that the germanate containing solution developed only a slight off white colour over the whole period covered by Table 8A B 27975 whilst the geimanate free isolution rapidly acquired a green colour which intensified throughout the incubation.

The results are given in Table SA.

TABLE 8A Time ofstorage(days) Solutions containing germanate Solutions without germanate Fructoseproduced w/v Glucoseremaining w/v Fructoseproduced w/v Glucoseremaining w/v * Equivalent to 5496 conversion. Only 3.1% maimose present.

** 5% mannose present b) The effect of incubation at pH 12 and 50C in the presence and absence of germanate ions was investigated with a number of sugars. The results obtained and basic conditions in respect of solutions of fructose, mannose, maltose and 3-0-Methyl D-glucose are set out in Tables 8B, 80, 8I> and 8E respectively.. In all cases the presence of germanate has two effects a) it delays destruction of sugars and b) it alters the constitution of the equilibrium mixture. In the Tables the figures do not add up to 100% because of decomposition.

TABLE 8B Initial D-Fructose concentration Germanate Components Found (%) r Glucose Mannose D-Fructose Unknown a Unknown b 52.1% w/v None 17J>t%B 22.8 27.8 30 24.3 21-..2 50.5% w/v 600 niM 17igT 26.7 51.2 TABLE 80 B 27973 Initial D-Mannose concentration Germanate Compounds found (%) 3>-Mannose B-Pructose TTnknovm 1 TTnfcnown 2 3)-Glticose 51.14% w/v Hone 17 days 50 days 79.3 53.4 13.65 18.3 7.2 18.8 53.3% w/v 600 mM 17 days 30 days 60.2 7.1 27.45 44.4 13.36.

TABLE 8D Initial Maltose concentrationGexmanate 59% w/vNone 58.7% w/v358mM Comppunds found (%)MaltoseMaltulose*FructoseGlucosei . , 15 days 27 days58.5 26.524.5 21.52.4 4-3922.1 22.8 15 days 27 days48.5 13.951.9 67.9- ++,trace 9.3 * calculated as M.Wt. with 0.5 molar glucose response + 0.5 molar fructose response.

TABLE 8E Initial 3-0-Methyl D-glucose* concentrationSermanate 41.8% w/v 58.7% w/vNone 464 vM.

Components found (%)3-0-methyl fructose**3-0-methyl glucose 15 days 15 days18.5 29.736.1 46 * known to be veiy alkali labile ** calculated with reference to fructose response Example 9 Enzymic Conversion of jçylose to xylulose A solution of xylose was made up containing 44% w/v xylose B 2797? 106737 and 4iaM MgCl at pH 7.0 and was passed through the gitioose isomerase enzyme coliamn used in the previois examples for the enzymic conversion of glucose to fructose (Column teinp = 60 C and flow ratte =; 0.05 ml/min). In a similar manner a solution .

containing 44% xylose, 4mM MgCl and 600 mM germanate ions was passed through the column at pE 7-0.

After two hours the fractions were collected and separated on an ion exchange resin in the borate form. A control xylose standard was also passed down the column as a comparison. An automate version of the cysteine/ï SO assay for pentoses was used to analyse the peaks in the order they camsoff the column. The assay also reacts with pentuloses and the product xylulose from the xylose solutions that had been passed through the enzyme column was clearly visible.

Because a standard xylulose for calibration purposes is not yet available the extent of the reaction was ascertained by comparing the areas, under the peaks. This ratio of xylulose formed from total xylose and xylulose was 41sl00 but with germanate ions present the ratio was 58 ' 100.

Example Conversion of glucose to fructose using stagnate ions Two feed solutions containing 44.&/o glucose and 4mM MgClg at pH 8.5 were passed through the glucose isomerase enzyme column used in.

previous examples. One solution contained no stannate ions whilst the other contained 600mW stannate ions obtained from, ciystalline Na-SnO-j.JH 0. The results are shown in Table 9- s/ C.

B 27975 TABLE 9 /. s % Conversion to Tune (hra) No Stannate 42.6 % Conversion to Pructose (600iriM Stannati»), 3sr 55-5 trr 59.. 4.

56.6 56.1 Samples from the reactor colximn èluate, when mixed with additional glucose did not produce farther fructose indicating that active glucose isomerase had not been leached from the reactor column.

Example 11 . Enaymic conversion of glucose to fructose : Effect of -pH and germanate levels .a) Two series of experiments were performed using a glucose isomerase column (length 51 cm, diameter 4 cm) similar to that used in earlier examples. Each series compared results obtained using feed solutions comprising glucose and 4mM MfeCl with and without ôOOmM germanate. The flow rate thsough the column was 0.05 ml/min and the temperature of the enzyme was 60 C. The initial pH of the feed solutions was varied between different experiments in each series and the final pH of eluate was measured.

The results are set out in Table 10.

TABIiE B 27975 Series InitialFeedpH Orderofanaly¬sis Tni tial%glucoseconcn Germanateconcn. % conversionto fructose Final pHof eluate 1 7 5 48 0 48.2 - 1 6.9 8 47 0 49.6 7.2 1 8.5 1 49.2 0 49.2 - 1 8.5 2 49-5 0 49-2 - 1 7 5 40.2 600 76.5 7.0 1 7 6 40.0 600 70.3 6.9 1 8.5 4 38.2 600 74.6 7.2 1 6.5 7 40.0 600 72.5 6.6 1 8.3 9 37.0 600 71.2 7.0 2 7.0 2 42.0 0 54.0 *•? 2 8*4 1 41.6 Q 49*3 7.1 2 8.5 4 44-6 0 54,9 7.0 2 7-1 5 53-4 600 .67.4 6.8 .2 8.6 5 50.0 600 66.7 6.8 b) Two series of experiments were performed using a glucose isomerase column similar to that used in earlier examples. ïfhe feed solutions comprised 50. w/v nominal glucose concentration, 4idM MgCl_ and 200 and 600 niM germanate respectively in the different series. The experiments were performed at a variety of pH values.

A comparative series of experiments was performed using solutions containing no germanate. In all the experiments'the solutions were pumped througb the column of immobilized enzyme at a flow rate of 0.05 ml/min (75 min nominal residence time). The results are shown in Table 11.

B 27975 TABLE 11 pH Eluate Composition {% Fructose) No germanate 200idM germanata 600 01 germanate 9.0 51 1 1 Mill 68 8.5 51 64 69 8.0 49. 59 70 7-5 50 60 70 7-0 50 60 70 6.5 59 59 69 6.0 5850 Example 12 Enzymic oonveraion of glucose to fructose - reversiMlity Solutions of glucose to fructose having the carbohydrate concentrations' shown in Table 12 were prepared. Those solutions to contain germanate were prepared by dissolving weighed amounts of Ge02 in aliquots of these solutions by stirring in small amounts of 50% NaOH solution. Aliquots of MgOlp solution were added to all these solutions to a final concentration of 4* and the pH of all solutions was adjusted to 8.5 at 25 C. ' The solutions were then pumped at the flow rates shown through the enzyme column (50 x 0.4 cm) at 60 C. The level of fructose in the column eluate was monitored, after dilution. For calibration syrups of 0.55% w/v were prepared and passed through the whole analytical system. To check the initial level of glucose in the feed syrups the samples and the calibration standard syrups were- diluted 2 x 104 manually and analysed by the cysteine - sulphuric acid method. The results are set out in Table 12 and are shown graphically in Figure 1 of the drawings which clearly shows that with no germanate present the equilibrium position is between 52 and 55% B 27975 fructose whether one starts with a glucose or a fructose feed syrup and is between 74 and 79% in the presence of 600mM germanate.

Again starting either from glucose or fructose as the feed syrup.

TABLE 12 mï Carbohydratefeed compan.& w/v •without ".~.i.germanate CarbohydratecoiQpsan»% w/v withgexmanatein feed NominalHesi-'.- denoetime Eluate coffiDOsition Feedtype ÎTogexmanate 600mMgexmanate 0.1 54.6 50.5 38 22.5 31.0 0.05 51.2 47.8 . 75 36.0 ',- 49-5 Glucose 0.03 51.2 47-8 125 42.5. 68.5 0.015 51.2 47.8 251 53.0 74. G 0.1 55.0 48.2 38 71.0 .79.0 0.05 55.0 48.2 75 55-5 77.5 0.05 55-0 ' 48.2 125 53.5 • 77-0 Fructose 0.015 55.0 48.2 251 52.0 77.0 Example 13 Enzymio conversion of glucose to fructose - concentration dependence A series of solutions containing 600mM gexmanate and 4mM MgClp but having differing concentrations of glucose were passed through a column of glucose isomerase similar to that used in previous examples at a temperature of 60 C. The results are shown in Table 13.

TABLE 13 Glucose feedconcentration(% w/v) % Conversion to Fructose Final pH 20.6 92.7 7-8 30.0 $0.0 7.8 40.0 72.5 7.5 53-4 67.4 6.8* 60.0 65.0 7.5.

* From a previous experiment.

106737 B 27973 Example 14 Chemical oonversion of melibiose The chemical conversion of melibiose (6 - 0 - tk -D-galactopyxanosyl- D-glucose) was studied in the presence and absence of 324mM gexmanate at pH 12.0 and at 4 C. The concentration of melibiose in the initial solution was 51.6% w/v. The results are shown in Table 14.

TABLE 14 Compound % of given sugar in total sugar after no. ofdays set out at heads of individual columns Germanate 0 15 29 42 Melibiose 92-r2 55.5 57-5 49.6 6-0--3)galacto-pyranosyl-D-fructose 3.2 9.8 12.8 11.0 Absent Galactose - 9.9 11.9 12.5 Recovery ofidentifiedproducts 95.-4 75.2 82.2 72.9 Melibiose - 42.7 35.0 25.2 6-0-qC-Dgalacto-pyranosyl-B-fructose - 37.8 42.5 31.1 Present Galactose - - . 7-9 12.8 Eecovery ofidentifiedproducts 80.5 85.4 69.I pa/jha/ab 28.5.76.

WHAT WE CLAIM IS:

1. A process fox the convexsion of an aldose ox aldose dexivative to a ketose ox ketose dexivative whexein the convexsion takes place in the presence of a complexing reagent which is an oxyanion or mixed complex oxyanion of germanium ox tin which forms a stxongex complex with the ketose or ketose derivative than with the aldose ox aldose derivative.

2. A process for the conversion of an aldose or aldose derivative to a ketose ox ketose dexivative whexein the conversion takes place in the presence of a complexing reagent which is an oxyanion ox mixed complex oxyanion of germanium which forms a stxonger complex with the ketose ox ketose dexivative than with the aldose ox aldose dexivative..

J. A pxocess according to Claim 1 wherein mannose ox glucose is converted to fructose.

4- A pxocess according to Claim 1 whexein during the conversion an enzyme catalyst is present.

5. A process according to Claim 1 wherein mannose ox glucose is converted to fructose in the presence of glucose isomerase.

6. A process accoxding to Claim 4 wherein the enzyme is present in an immohilised form on a solid matrix which is a living cell, an inactivated cell or any other suitable support.

7. A process according to Claim 1 wherein the complexing reagent is introduced into the process as an aldose-oxyanion complex or a complex dexivative of an aldose oxyanior/or as a salt ox an oxide or another compound which forms oxyanions or mixed complex oxyanions under the process conditions.

8. A process accoxding to Claim 1 wherein the mixed complex oxyanion is one formed by the interaction of an oxyanion or germanium or tin with an ion of anothex element fxom Group IV of the Periodic Table or of an element from Group V or Group VI, 9. A process according to Claim 5 wherein the glucose isomerase is dexived fxom a stxain of Arthxobacter.

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