GB1596662A

GB1596662A - Glucose isomerase compositions comprising iron salts

Info

Publication number: GB1596662A
Application number: GB35368/77A
Authority: GB
Original assignee: Novo Industri AS
Current assignee: Novo Nordisk AS
Priority date: 1966-09-15
Filing date: 1977-08-23
Publication date: 1981-08-26
Also published as: GB1148412A

Description

(54) GLUCOSE ISOMERASE COMPOSITIONS COMPRISING IRON SALTS (71) We, NOVO INDUSTRI A/S, a Danish Company, of Novo Allé, DK-2880 Bagsvaerd, Denmark, do hereby declare the invention, for which we pray that apatent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to an iron containing glucose isomerase composition and more particularly to a glucose isomerase particle form composition containing at least 0.050 wt% of iron, incorporated as an iron salt therein.

A basic difficulty facing this art is that glucose isomerase enzyme seemed to require cobalt ions in the feed syrup to be isomerised, yet cobalt is widely considered to be a toxic substance and, therefore, the cobalt level present in the iso-syrup product produced must be reduced to the parts per billion level, for example by ion exchange of the iso-syrup product. Heretofore, the approach employed by the applicants herein and their co-workers has been to adjust processing conditions so that cobalt ions need not be present in the feed-syrup for enzyme activation purposes. As an example to this approach, reference is made to U.S. Patent 4,025,389.

Recently, it has been noted that iron can activate glucose isomerase enzymes. It has been suggested to include small quantities of an iron salt in the feed syrup for enzyme activation purposes. However, it should be noted that standard feed syrups often contain small amounts of iron in soluble form.

Nonetheless, introduction of soluble iron salts into the glucose syrup feed-stream is easier to suggest than to practice. For one thing, the operator of the glucose isomerization system must have a reasonable degree of chemical sophistication, and the system itself should be sophisticated. The iron salt must be metered into the glucose syrup. Chemical analysis of the glucose syrup entering the isomerization reactor for its iron content must be made periodically, if only as a cross check against satisfactory operation of the metering equipment. Secondly, since the iron binding capacity of the enzyme is either negligible or at the most limited, the point of saturation is likely to be reached during a long run isomerization process. In either case leakage of iron into the product stream will commence at some point during the process. The presence of iron in the product may induce color formation to an extent which would necessitate its removal. for example by ion exchange.

and thus add to the costs of purification. In total. addition of iron salts to the glucose syrup is a nuisance.

It is an object of the present invention to provide an enzyme product having iron incorporated therein, since this is generally more advantageous, particularly if the iron were retained for the useful life of the enzyme product and remained so firmly bound that practically no iron leakage occurs in use of the enzyme.

According to the first aspect of the present invention there is provided a method of activating a cell mass form as hereinafter defined of glucose isomerase, which process comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a non-toxic water-soluble iron salt. The cell mass form may then be converted into a particulate form and dried to obtain an enzyme product suitable for marketting in a pre-soaking state (as hereinafter defined).

According to the second aspect of the present invention there is provided a method of activating a cell mass form (as hereinafter defined) of glucose isomerase, which process comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a solid non-toxic water-soluble iron salt. The cell mass form may then be converted into a particulate form and dried to obtain an enzyme product suitable for marketting in a pre-soaking state (as hereinafter defined).

According to the third aspect of the present invention there is provided a method of activating a cell mass form (as hereinafter defined) of glucose isomerase in accordance with the disclosure and/or claims of Specification No. 1,516,704 and/or United States Patent Specification No. 3,980,521, which process comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a non-toxic water-soluble iron salt. The cell mass form may then be converted into a particulate form and dried to obtain an enzyme product suitable for marketting in a pre-soaking state (as hereinafter defined).

According to the fourth aspect of the present invention there is provided a method of activating a cell mass form (as hereinafter defined) of glucose isomerase in accordance with the disclosure and/or claims of Specification No. 1,516,704 and/or United States Patent Specification No. 3,980,521, which process comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a solid non-toxic water-soluble iron salt. The cell mass form may then be converted into a particulate form and dried to obtain an enzyme product suitable for marketting in a pre-soaking state (as hereinafter defined).

The invention also provides an iron activated cell mass form (as hereinafter defined) of glucose isomerase in dried particulate form, having incorporated therein at least 0.05% w/w of iron as a non-toxic water-soluble iron salt. Preferably. the cell mass form is in accordance with the disclosure and/or claims of Specification No. 1,516,704 and/or United States Patent No. 3,980,521.

Thus, the invention enables the provision of a particulate product ready for soaking in sugar solution prior to use in isomerisation.

Herein, by the term "pre-soaking state" is meant suitable for glucose isomerisation following a soaking process in glucose syrup.

As a practical matter, using iron as the activating metal in glucose isomerase preparations represents a significant advance in the art, because iron in small quantities is recognized as a non-toxic material. The iron salt added can, of course, be of food grade quality.

Accordingly, the fear of leaving toxic substances in the product syrup disappears. A few parts per million of iron salt in the product is permissible.

Glucose isomerase is an intracellular enzyme which need not be isolated from the microorganism cells to produce an active enzyme product (see for example United States Patent Specification Nos. 3,821,086. 3,779,869 and 3.980,521). All such preparations use the microorganism cell, whole or disrupted, as basis for the glucose isomerase product.

Herein the terms "cell mass form", "cell mass preparation". and "cell mass particulate form" are employed to define forms, preparations and particles obtained, formed or otherwise fabricated from the substance of the microorganism cells along with organic reactants, for example glutaraldehyde, proteins or agglomerating agents, for example polyelectrolytes. On a weight basis the glucose isomerase content of a cell mass preparation is normally a very small fraction of the preparation as a whole.

It has now been discovered that cell mass glucose isomerase preparations can bind therein substantial proportions of iron, and, moreover, relatively little of the iron is lost through extended contact with glucose and glucose-fructose syrups. The quantity of iron incorporable into cell mass preparations far exceeds the activation requiremets of the glucose isomerase.

In particular, non-toxic water soluble salts of iron in solid form may be mixed incorporated into the cell mass preparation during forming thereof, for example just prior to extrusion of a particulate form. The salts may also be introduced, in appropriate circumstances, as a concentrated aqueous solution.

This invention encompasses as a product a dry cell mass enzyme preparation with a non-toxic water-soluble iron salt incorporated in such an amount as to provide iron in an amount of at least 0.05% w/w, preferably from 0.05 to 2.0% w/w of the cell mass form of glucose isomerase. More iron than 2.0% w/w could, of course, be incorporated but no useful purpose would be served thereby. The preferred iron content is in the range of from 0.2 to 0.5cue w/w, especially from 0.2 to 0.25cm w/w.

In all instances, once the iron is incorporated within the cell mass preparation in from 0.05cue to 2.08/o wt/wt dry weight basis, little if any of the iron is ldst to syrup over the useful life of the preparation for glucose isomerisation purposes. Indeed, the iron containing enzyme preparation can strip iron from the syrup. For example, a syrup entering an isomerisation reactor with 4 ppm of iron might well leave the isomerisation reactor with an iron content of less than 1 ppm of iron.

In practice, it has been found that improvement in productivity and/or stability of the glucose isomerase can occur when other solid ingredients are also admixed into the enzyme preparation. In particular, the initial pH drop which occurs during a period of 1-2 days after loading a column with fresh enzyme has caused some problems. A decrease in pH of the column is undesirable because it induces shrinkage of the enzyme bed which in turn may lead to bed channeling. In addition, a decrease in activity and, in severe cases, a lower stability of the enzyme product may ensue. Incorporation of from 0.5 to 3.0% by weight of magnesium oxide, based on the dry weight of the cell mass form of glucose isomerase, into the cell mass preparation has been found to overcome the initial pH drop to a substantial degree, thus affording relatively stable syrup outlet PH values. In addition, the admixture of solid glucose (for example glucose monohydrate), serving principally as a mixing aid diluent, to the cell mass in amounts of from 2 to 15% by weight, based on the dry weight of the cell mass form of glucose isomerase, has often been found to be desirable.

The preferred glucose isomerase particles contemplated herein are the glutaraldehyde reacted homogenized cells preparations disclosed and/or claimed in Specification No.

1,516,704 and/or United States Patent Specification No. 3,980,521.

In a preferred mode, the water-soluble iron salt is admixed with the magnesium oxide and the glucose and then added to the cell mass before extrusion that forms the final granulate.

Although practice of this invention contemplates incorporation of any non-toxic water-soluble iron salt into the cell mass enzyme preparation, certain iron salts are preferred, namely: Ferric sulphate Ferrous sulphate Ferric chloride Ferrous lactate Ferric citrate Ferrous citrate Ferric ammonium citrate Ferrous acetate Ferric nitrate Ferric pyrophosphate The present invention also provides a method of isomerasing glucose, which comprises isomerasing glucose utilising a all mass form of glucose isomerase in accordance with the invention.

The following Examples illustrate the present invention. In the Examples, all parts and percentages are by weight, unless otherwise specified.

In the Examples, the following terminology is used: Defittinoti of Activity The unit of activity is defined as the amount of enzyme which forms fructose at an initial rate of 1 Fmol of fructose per min. at a given set of isomerisation conditions. Assay of Activity The activity is determined under the following conditions: Syrup 40% w/w dissolved dextrose pH inlet 8.5 Mg++ 0.004 M Temperature 65"C Column diameter 2.5 cm - height 35 cm Flow direction downflow The activity is expressed in IGIC units per g.

In long run isomerisations, the activity decay curves are fitted to exponential decay models of the form: Act = Ac, X e -b where t is No. of hours after start of isomerization Act is the activity at t = t Ao is the activity at t = 0 and b is the decay constant in hrs-' from this equation half life is defined as in In 2 Tl'2= b and is given in hours.

Productivity Productivity is defined as kg of glucose d.s. converted to a mixture of 45% fructose and 55% glucose per kg of enzyme after a given time of isomerization.

In the examples the productivity is calculated according to an equation of the above given form after an isomerization time of 2 x To,2.

Iron The iron content is determined according to the o-phenanthroline method (Nordisk Metodik Komite for Levnedsmidler Nr. 22. 1955 U.D.C. 664.7: 546.72).

Color The color is determined according to the CIRF method.

Color Stability Color stability is determined after 1 hour heating at 100"C at pH 4.2 (CIRF).

Magnesium oxide employed was heavy type ER/B from Pharmelko, Milan, Italy.

Example 1: Addition of ferric citrate, ferrous lactate and ferric sulphate together with magnesium oxide and dextrose. Addition of ferric oxide.

A filter cake was produced according to example V in U.S. Patent No. 3,980,521.

The cake was granulated by means of an oscillating granulator equipped with a screen with 1 cm holes.

The coarse granulate contained about 76% of water (measured by drying at 1OS"C). It was divided into 6 lots.

+) A. 8.5 kg of the coarse granulated filter cake was extruded by means of an axial extruder equipped with a screen with holes of a diameter of 0.8 mm. The extrudate was dried in a fluid bed with 60"-65"C air to a water content of about 10%.

B. To 8.5 kg of the coarse granulated filter cake was added a mixture of 20 g magnesium oxide, 85 g dextrose monohydrate and 40 g ferric citrate with an iron content of 16%.

After thorough mixing the mixture was extruded and dried as in A.

C. 8.5 kg of the coarse granulate was mixed with a mixture of 20 g magnesium oxide, 85 g dextrose monohydrate and 40 g ferrous lactate with an iron content of about 19%.

After thorough mixing, the mixture was extruded and dried as in A.

D. 8.5 kg of the coarse granulate was mixed with a mixture of 20 g magnesium oxide, 85 g dextrose monohydrate and 30 g ferric sulphate with an iron content of about 20%.

After thorough mixing, the mixture was extruded and dried as in A.

+J E. 8.5 kg of the coarse granulate was mixed thoroughly with a mixture of 20 g magnesium oxide and 85 g dextrose monohydrate. The mixture was extruded and dried as described under A.

+J F. 8.5 kg of the coarse granulate was mixed throughly with 25 g ferric oxide containing about 58% of iron. The mixture was extruded and dried as in A.

+) comparative example The preparations were sieved to between 0.35 mm and 1.0 mm and the products analysed.

The pH was measured in the syrup outlet stream in samples taken after 20 hours and 43 hours, respectively. Before the pH determination the samples were cooled to 25"C.

TABLE I Activity pH in outlet syrup after Found Corrected Preparation IGIC/g IGIC/g gain 20 hrs. 43 hrs.

+)A 246 246 0 6.68 7.62 B 307 326 33 7.99 8.20 C 296 315 28 7.60 7.98 D 308 328 33 7.90 8.14 +'E 254 267 8 7.99 8.20 +)F 257 260 6 6.86 7.65 As can be seen from Table I. only addition of soluble iron components gives activity gain of any importance. Addition of ferric oxide gave only about 6% compared to about 30coo for the soluble salts.

Example II: Addition of magnesium oxide + glucose and magnesium oxide + glucose + iron salt.

A filter cake was produced according to example V in US patent No. 3,980,521. The cake was granulated by means of an oscillating granulator equipped with a screen with 1 cm holes.

The coarse granulate contained about 79% of water. It was divided into 5 lots of 8.5 kg.

+)A. 8.5 kg was extruded and dried as in example 1A without addition of additives.

+)B. To 8.5 kg granulated filter cake was added 25 g magnesium oxide. After thorough mixing it was extruded and dried as in A.

+)C. To 8.5 kg granulated filter cake was added 25 g magnesium oxide and 200 g glucose monohydrate. After mixing it was extruded and dried as in A.

+)D. To 8.5 kg granulated filter cake was added a mixture of 25 g magnesium oxide and 300 g glucose. After mixing it was extruded and dried.

E. 8.5 kg of the filter cake was mixed with a mixture of 25 g magnesium oxide, 200 g glucose monohydrate and 40 g ferric sulphate containing about 20% iron. Thereafter it was extruded and dried.

+) Comparative example The dried preparations were sieved to between 0.35 mm and 1.0 mm and the products analysed.

The pH in the outlet syrup was measured in samples taken after 20 hours and 43 hours, respectively. and cooled to 25"C.

TABLE II Activity IGIC/g pH in outlet syrup after %added Found Corrected for % Preparation material added inact. gain 20 hrs. 43 hrs.

mat.

+'A () 220 220 0 6.85 7.40 1 1 222 224 2 8.18 8.23 10 10 216 240 9 8.14 8.22 14 14 916 251 14 8.15 1.21 E 12 272 309 40 8.15 8.27 As can be seen from Table II. only addition of an iron salt affords a significant increase in activity.

Example Ill: Addition of ferric citrate, ferric pyrophosphate. ferric ammonium citrate and ferrous sulphate.

A coarse granulated filter cake with about 76% of water as in example I was divided into 6 lots of each 8.5 kg.

A. 8.5 kg granulated filter cake was extruded and dried as in example 1 to give a reference composition.

B. To 8.5 kg of the coarse granulate was added a mixture of 25 g magnesium oxide, 25 g ferric citrate with about 16cos of iron and 250 g glucose monohydrate. After thorough mixing the granulate was extruded and dried as in A.

+' Comparative example C. 8.5 kg of the coarse granulate was extruded and dried as in A after addition of 25 g magnesium oxide, 50 g ferric citrate and 250 g glucose monohydrate.

D. 8.5 kg of the coarse granulate was treated as C except that the 50 g ferric citrate was replaced by 30 g ferric pyrophosphate with an iron content of about 12%.

E. To 8.5 kg of the coarse granulate was added 25 g magnesium oxide, 250 g glucose monohydrate and 30 g ferric ammonium citrate with an iron content of about 15%.

After thorough mixing the granulate was extruded and dried as in A.

F. To 8.5 kg of the coarse granulate was added 25 g magnesium oxide, 250 g glucose monohydrate, and 30 g ferrous sulphate with an iron content of about 30%. After thorough mixing the granulate was extruded and dried as in A.

The dried preparations were sieved to between 0.35 and 1.0 mm and the products obtained were analysed. The pH of the outlet syrup was measured after 20 and 43 hours.

TABLE III Activity IGIC/g % pH in outlet syrup after Found Corrected gain 20 hrs. 43 hrs.

+'A 229 229 0 6.64 7.25 B 261 293 28 7.90 8.24 C 273 306 34 7.84 8.22 D 266 299 31 7.79 8.19 E 268 301 31 7.70 8.14 F 263 295 29 7.87 8.03 +)Comparative example No significant difference in activating effect of the applied iron salts is observed, +' Example IV: Effect of magnesium oxide incorporation on pH drop, activity and stability.

a. Three enzyme preparations were produced according to the same procedure as described in Example I. To the coarse granulated filter cake was added magnesium oxide in sufficient amounts to give preparations with the following magnesium oxide content in the final'dried preparations.

Prep. B1 no additive - B2 2% magnesium oxide - B3 5% magnesium oxide Isomerizations were performed in 6() ml jacketed glass columns (h x d = 35 x 1.5 cm) using 15 grams of each of the three preparations. The parameters for isomerization were: Syrup 45% w/w redissolved dextrose pH inlet 8.0 + 0.1 Mg add. to syrup 0.0008 M Temperature 65"C An inlet pH of 8.0 is lower than the one normally used and regarded as optimum, but here it was applied to screen the effect of magnesium oxide addition.

The isomerizations were continued until the preparations had decreased in activity to an arbitrarily chosen activity of 20-25 umol/min/g.

The following results were obtained: TABLE IV (a) i Preparation Max.measured Running Half life Productivity activity/after time,hrs. hours after 2 x T112 hours B1 88/72 665 257 369 B2 143/16 665 238 436 B3 124/16 378 161 253 Outlet pH's of syrups from the column were found as tabulated below: TABLE IV (a) ii Preparation Hours after start 0 17 42 70 140 230 350 665 (soaking) B1 - 6.2 6.2 6.1 6.0 6.0 5.9 6.3 B2 8.4 7.4 7.0 6.7 6.4 6.2 6.2 6.9 B3 9.3 8.6 7.7 7.2 6.7 6,4 6.3 The results demonstrate that addition of 5% magnesium oxide gives rise to high initial outlet pH's. This appears to influence the maximum observed activity as well as the stability and productivity in descending direction.

In this test, isomerizing with an inlet pH of 8.0, a higher maximum activity and productivity resulted from presence of 2% added magnesium oxide as compared to no additives.

b. To optimize the addition of magnesium oxide four additional preparations were produced according to the procedure described in Example I. The additive content of the dried preparations were: A no magnesium oxide B 1/2% magnesium oxide + 9% glucose C 1% magnesium oxide + 9% glucose D 2% magnesium oxide + 9% glucose Isomerizations were performed in 60 ml jacketed glass columns (h x d = 35 x 1.5 cm) using the following parameters: Syrup 45ago w/w redissolved glucose pH inlet 8.4 + 0.1 Mg add. to syrup 0.0016 M Temperature 65"C The isomerizations were continued for 351 hours.

The following results were obtained: TABLE IV (b) i Preparation Max. measured ac- Activity after Productivity af tivity/after hours 351 hours ter 351 hours A 103/67 79 397 B 105/18 75 384 C 103/18 72 371 D 98/18 65 354 Outlet pH's of syrup from the column were measured as seen from the table: TABLE IV (b) ii Preparation Hours after start 19 43 67 140 210 303 A 6.6 6.7 7.0 7.3 7.4 7.2 B 7.1 7.0 7.3 7.5 7.5 7.1 C 7.4 7.6 7.7 7.6 7.6 7.2 D 8.4 8.0 7.8 7.6 7.5 7.2 The results indicate no great differences in activity, stability, or productivity between the four preparations. Outlet pH's are influenced. Addition of 1% magnesium oxide gives almost constant outlet pH during the run and is therefore the preferred level of addition.

Both 1/2 and 2% magnesium oxide addition have effect on the outlet pH compared to the control, but in both cases some pH variation during the first 150 hours was found.

Example V: Isomerization experiments.

A coarse granulated filter cake prepared according to U.S. Patent No. 3,980,521 Example V was used for the following preparations. The filter cake contained about 77% water.

410/A. No addition.

+)410/B. About 10 parts by weight of mix 1 were added to about 90 parts by weight on a dry basis, of the filter cake. Mix 1 consisted of glucose (100 parts) and magnesium oxide (8 parts).

410/C. About 2 parts by weight of mix 2 were added to about 98 parts by weight of the filter cake dry basis. Mix 2 consisted of glucose (100 parts), magnesium oxide (10 parts) and ferric sulphate (12 parts).

410/D. About 7 parts by weight of mix 2 were added to about 93 parts by weight of the filter cake, dry basis.

+)410/E No addition.

+'Comparative example The mixtures 410/A to 410/E were then extruded through a screen with 0.8 mm holes, and then dried in a fluid bed to a water content of about 10%.

The iron contents of the five final preparations were determined.

410/A 0.04% 410/B 0.03% 410/C 0.08% 410/D 0.18% 410/E 0.04% Isomerizations were performed with material from preparations 410/A, 410/B, 410/D and 410/E, using the following conditions.

Syrup 45% w/w redissolved glucose pH inlet 8.4 + 0.1 Mg2+ 0.0016 M Temperature 62"C Column dimensions height 40 cm diameter 5.8 cm volume 1 litre Weight of enzyme 260 g The enzyme was soaked for 2 hours at room temperature in the above described syrup, but at pH 8.0 and then packed into the column.

The following results were obtained: TABLE V (a) Prepara- Max. measured Total run pH outlet Half life Produc tion activity time after Tl, tivity hours 21h 48h 92h hours after 2xT1,2h.

410/A 158 1293 6.9 6.8 7.2 842 1880 410/B 155 936 7.4 7.7 8.0 818 179 410/D 202 1316 7.3 7.5 7.7 843 2295 410/E 151 1147 6.9 6.9 7.7 828 1755 The concentration of iron in the outlet syrup from these columns was determined.

TABLE V (b) Fe (ppm) in outlet syrup Preparation 2 1/2 hours 21 hours 27 hours after start after start after start 410/A < 1 < 1 < 1 410/B < 1 < 1 < 1 410/D approx. < 1 < 1 < 1 410/E < 1 < 1 < 1 A second set of isomerization experiments was performed with materials from preparations 410/C. 410/D and 410/E, using the following conditions: Syrup 45% w/w redissolved glucose pH inlet 8.4 I 0.1 Mg t 0.0016 M Temperature 650C Column dimensions height 20 cm diameter 2.5 cm volume 100 ml Weight of enzyme 20 g The enzyme was soaked for one hour at room temperature in the above described syrup, and then packed into the column.

The following results were obtained: TABLE V (c) Prepara- Max. measured Total run pH outlet Half life Producti tion activity time, after T 1/2, vity af hours 17h 45h 200h hours ter 2xTX 410/C 210 900 7.0 7.8 8.1 512 1510 410/D 250 900 7.5 8.0 8.2 484 1725 410/E 190 900 6.9 7.4 8.2 485 1340 The concentration of iron in the outlet syrup from these columns was determined.

TABLE V (d) Fe (ppm) in outlet syrup Preparation 0 hrs 24 hrs 72 hrs 140 hrs 850 hrs (soaking) after after after after start start start start 410/C 0.8 < 0.5 < 0.5 < 0.5 < 0.5 410/D 3.6 < 0.5 < 0.5 < 0.5 < 0.5 410/E < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 The CIRF color of the outlet syrup from these columns was determined.

TABLE V (e) CIRF color in syrup Preparation 0 hrs. 24 hrs. 72 hrs.

(soaking) after start after start 410/C 0.266 0.030 0.019 410/D 0.247 0.036 0.020 410/E 0.232 0.036 0.022 For comparison, the CIRF color of three samples of the inlet syrup used during this period were measured to 0.019, 0.012 and 0.014.

The color stability of the outlet syrup from these columns was determined.

TABLE V (f) Color stability of syrup Preparation 0 hrs. 24 hrs. 72 hrs.

(soaking) after start after start 410/C 0.21 0.040 0.014 410/D 0.21 0.050 0.017 410/E 0.22 0.044 0.017 For comparison, the color stability of three samples of the inlet syrup used during this period was measured. The results were 0.004, 0.002 and 0.004.

They iron content of the enzyme preparations was determined before and after use.

TABLE V (g) mg iron in column packed with 20 g enzyme Preparation At start After 900 hrs.

410/C 16 24 410/D 36 42 410/E 8 14 It will be noted that the iron content after 900 hours was greater than at the start of the experiment. Thus the enzyme adsorbed iron from the input syrup. Since, no iron was added to the input syrup used in these experiments, the iron adsorbed by the enzyme originated from the traces of iron naturally present in the solutions of crystalline glucose. Analysis of the iron content of the 45% w/w redissolved glucose syrup showed < 0.5 ppm, and approximately 0.1 ppm iron. In the course of the 900 hours that these columns ran, approximately 75000 g of syrup were passed through each column containing 20 g enzyme.

If the average iron concentration of this inlet syrup was 0.1 ppm, then the total iron content of the inlet syrup was 75000 x 10-7 g = 7.5 mg.

This corresponds well to the amount picked up by the enzyme preparations during the course of the test.

Conclusions Addition of magnesium oxide has a significant influence on the outlet pH in the period between 0 and 100 hours after start up. With magnesium oxide, as in 410/B and 410/us the outlet pH was 0.5-1.0 unit higher than without magnesium oxide, as in 410/A and 410/E.

Addition of iron salt, as in 410/D, increased the activity

Example VI: Comparison of Ferrous and Ferric salts.

A mixture of iron salt, dextrose, and magnesium oxide was added to samples of a coarse granulated filtercake made according to U.S. patent 3,980,521 Example V. The mixture was then further processed by extrusion through a screen with 0.8 mm holes and finally by drying in a fluid bed to a water content of about 10%. The composition and amount of the mix consisting of iron salt, glucose and magnesium oxide was such as to give final preparations with the following compositions: TABLE VI (a) Preparation Iron salt Dextrose Magnesium oxide IG 403 II C 1.2% Ferric 8% 1% sulphate IG 403 II D 1.2% Ferrous 8% 1% sulphate IG 403 II E- None 8% 1% Analysis of the preparations gave the following results for the actual Fe content: IG 403 II C 0.22% IG 403 II D 0.27% IG 403 II ER 0.05coo * Comparative Example Isomerizations were performed under the following conditions: Syrup 45% redissolved glucose pH inlet 8.4 + 0.1 Mug++ 0.0016 M Temperature 65"C Column dimensions height 20 cm diameter 2.5 cm volume 100 ml Weight of enzyme 20 g The enzyme was soaked for one hour at room temperature in the above described syrup and then packed into the column. The following results were obtained: TABLE VI (b) Max. measured Total run Half life Productivity Preparation activity time, To,2, hours after 2xTI,2 hours IG 403 II C 290 755 482 2093 IG 403 II D 274 755 467 1914 IG 403 II E* 254 755 431 1635 The iron contents of the enzyme preparations were determined before and after use.

Preparation mg Fe in column packed with 20 g enzyme at start after 755 h IG 403 II C 44 52 IG 403 II D 54 68 IG 403 II E* 10 16 Again the iron content increased slightly during the course of the test, indicating that the preparations adsorbed iron from the traces of iron present in the redissolved dextrose syrup.

Conclusion Addition of either ferrous or ferric sulphate increased the activity and productivity of the enzyme preparation.

Example VII: Demonstration of iron saturation.

A coarse granulated filter cake according to U.S. Patent No. 3,980,521 Example V was used for the following preparations: 415/A No addition 415/B About 10 parts by weight of mix 2 were added to about 90 parts by weight of the filter cake on a dry weight basis. The filter cake contained approximately 77% water. Mix 2 consisted of glucose (100 parts), magnesium oxide (10 parts) and ferric sulphate (12 parts).

The mixtures 415/A and 415/B were then extruded through a screen with 0.8 mm holes and then dried in a fluid bed. to a water content of about 10%.

The iron contents of the two final preparations were determined: 415/A 0.03% 415/B 0.26% Isomerizations were performed with preparations 415/A and 415/B using the following conditions: Syrup 45% w/w redissolved glucose pH inlet 8.3 + 0.1 Mg 0.0016 M Fe 0.00007 M (4 ppm) Temperature 65"C Column dimensions height 20 cm diameter 2.5 cm volume 100 ml Weight of enzyme 20 g The enzyme was soaked in the syrup for one hour at room temperature and then packed into the column.

The following results were obtained: TABLE VII (a) Max. measured Time to Total Half life Producti activity reach run, T112,hours vity after Prepara- max. ac- time, 2 x T1,2 tion tivity, hours hours 415/A 272 160 906 611 2560 415/B 275 20 906 547 2260 The concentration of iron in the outlet syrup from these columns was determined.

TABLE VII (b) Fe (ppm) in outlet syrup at Preparation 0 hours (soaking) 20 hrs. 70 hrs. 350 hrs. 900 hrs.

415/A < 0.5 < 0.5 < 0.5 < 0.5 0.5 415/B 7 < 0.5 < 0.5 < 0.5 0.6 The iron contents of the enzyme preparations were determined before and after use.

Preparation mg iron in column packed with 20 g enzyme at start after 900 hours 415/A 6 320 415/B 52 380 Coilclusions 415/A gave 13% higher productivity than 415/B. However, it should be noted that 415/B contains approximately 10% by weight of non-enzyme material. Thus, calculated on the basis of the original enzyme containing filter cake, both preparations gave approximately the same productivity.

The activity of 415/A increased during the first 160 hours of the run. This is in contrast to 415/B which gave maximum activity after 20 hours. This indicates that 415/A was slowly adsorbing iron from the input syrup with a resulting slow activation. This slow activation is also the reason for the longer exponential decay half life observed for 415/A, i.e. activation and exponential decay occurred simultaneously.

During the 900 hours of the experiment about 90000 g of syrup were passed through each column containing 20 g enzyme preparation. The iron content of this syrup was 4 ppm. Thus 90000 g syrup contained 360 mg iron. The iron content of the two columns increased by 314 and 328 mg. Thus the greater part of the iron in the input syrup was removed by the enzyme preparations. The results show that after 900 hours the level of iron in the output syrup had started to increase. This suggests that the enzyme preparations were approaching the limit of their abilitv to absorb iron.

WHAT Wif CLAIM IS: 1. A method of activating a cell mass form (as hereinbefore defined) of glucose isomerase, which method comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a non-toxic water-soluble iron salt.

2. A method of activating a cell mass form of glucose isomerase, which method comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a solid non-toxic water-soluble iron salt.

3. A method according to Claim 1 or 2. wherein, after incorporation of the iron, the glucose isomerase is converted into a particulate form and dried to obtain an enzyme product in a pre-soaking state (as hereinbefore defined).

4. A method according to Claim 1. 2 or 3, wherein the cell mass form of glucose isomerase is in accordance with the disclosure and/or claims of Specification No. 1,516,704 and/or United States Patent Specification No. 3,980,521.

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Claims

**WARNING** start of CLMS field may overlap end of DESC **.

TABLE VII (a) Max. measured Time to Total Half life Producti activity reach run, T112,hours vity after Prepara- max. ac- time, 2 x T1,2 tion tivity, hours hours

415/A 272 160 906 611 2560

415/B 275 20 906 547 2260 The concentration of iron in the outlet syrup from these columns was determined.

TABLE VII (b) Fe (ppm) in outlet syrup at Preparation 0 hours (soaking) 20 hrs. 70 hrs. 350 hrs. 900 hrs.

415/A < 0.5 < 0.5 < 0.5 < 0.5 0.5

415/B 7 < 0.5 < 0.5 < 0.5 0.6 The iron contents of the enzyme preparations were determined before and after use.

Preparation mg iron in column packed with 20 g enzyme at start after 900 hours

415/A 6 320

415/B 52 380 Coilclusions

415/A gave 13% higher productivity than 415/B. However, it should be noted that 415/B contains approximately 10% by weight of non-enzyme material. Thus, calculated on the basis of the original enzyme containing filter cake, both preparations gave approximately the same productivity.

The activity of 415/A increased during the first 160 hours of the run. This is in contrast to 415/B which gave maximum activity after 20 hours. This indicates that 415/A was slowly adsorbing iron from the input syrup with a resulting slow activation. This slow activation is also the reason for the longer exponential decay half life observed for 415/A, i.e. activation and exponential decay occurred simultaneously.

During the 900 hours of the experiment about 90000 g of syrup were passed through each column containing 20 g enzyme preparation. The iron content of this syrup was 4 ppm. Thus 90000 g syrup contained 360 mg iron. The iron content of the two columns increased by 314 and 328 mg. Thus the greater part of the iron in the input syrup was removed by the enzyme preparations. The results show that after 900 hours the level of iron in the output syrup had started to increase. This suggests that the enzyme preparations were approaching the limit of their abilitv to absorb iron.

WHAT Wif CLAIM IS: 1. A method of activating a cell mass form (as hereinbefore defined) of glucose isomerase, which method comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a non-toxic water-soluble iron salt.
2. A method of activating a cell mass form of glucose isomerase, which method comprises incorporating therein at least 0.05% w/w (dry basis) of iron as a solid non-toxic water-soluble iron salt.
3. A method according to Claim 1 or 2. wherein, after incorporation of the iron, the glucose isomerase is converted into a particulate form and dried to obtain an enzyme product in a pre-soaking state (as hereinbefore defined).
4. A method according to Claim 1. 2 or 3, wherein the cell mass form of glucose isomerase is in accordance with the disclosure and/or claims of Specification No. 1,516,704 and/or United States Patent Specification No. 3,980,521.
5. A method according to any one of the preceding claims, wherein the amount of iron

is in the range of from 0.05 to 2.0% w/w.
6. A method according to Claim 5, wherein the amount of iron is in the range of from 0.2 to 0.25% w/w.
7. A method according to any one of the preceding claims, wherein from 0.5 to 3.0% by weight of magnesium oxide, based on the dry weight of the cell mass form of glucose isomerase, is incorporated into the cell mass form.
8. A method according to any one of the preceding claims, wherein from 2 to 15% by weight of solid glucose, based on the dry weight of the cell mass form of glucose isomerase, is incorporated into the cell mass form.
9. A method according to Claims 7 and 8, wherein the magnesium oxide and glucose are admixed with the water-soluble iron salt and then added to the cell mass form of glucose isomerase, whereafter the mass is extruded, thereby to form granules.
10. A method according to any one of the preceding claims, wherein the water-soluble iron salt is selected from ferric sulphate, ferrous sulphate, ferric chloride, ferric citrate, and ferrous citrate, ferric ammonium citrate, ferric nitrate, ferric pyrophosphate, ferrous lactate and ferrous acetate.
11. An iron activated cell mass form of glucose isomerase in dried particulate form which comprises a cell mass form (as hereinbefore defined) of glucose isomerase having incorporated therein at least 0.05% w/w of iron as a non-toxic water-soluble iron salt.
12. An iron activated cell mass form of glucose isomerase in dried particulate form, which comprises a cell mass form (as herein before defined) of glucose isomerase having incorporated therein at least 0.05% w/w of iron as a non-toxic water-soluble iron salt, incorporated as a solid iron salt.
13. A cell mass according to Claim 11 or 12, which is in accordance with the disclosure and/or claims of Specification No. 1,516,704 and/or United States Patent Specification No.

3,980,521.
14. A cell mass according to Claim 11, 12 or 13, wherein the amount of iron is in the range of from 0.05 to 2.0% w/w.
15. A cell mass according to Claim 14, wherein the amount of iron is in the range of from 0.2 to 0.25% w/w.
16. A cell mass according to any one of Claims 11 to 15. which has from 0.5 to 3.0% by weight of magnesium oxide, based on the dry weight thereof, incorporated therein.
17. A cell mass according to any one of Claims 11 to 16, which has from 2 to 15auk by weight of solid glucose, based on the dry weight of the cell mass form of glucose isomerase, incorporated therein.
18. A cell mass according to any one of Claims 11 to 17, wherein the water-soluble iron salt is selected from ferric sulphate, ferrous sulphate. ferric chloride, ferric citrate, ferrous citrate, ferric ammonium citrate, ferric nitrate, ferric pyrophosphate, ferrous lactate and ferrous acetate.
19. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example 1, B.
20. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example 1. C.
21. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example 1, D.
22. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example II. E.
23. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example III, B.
24. A method of activating a cell mass form of glucose isomerase. substantially as described in foregoing Example III. C.
25. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example III. D.
26. A method of activating a cell mass form of glucose isomerase. substantially as described in foregoing Example III, E.
27. A method of activating a cell mass form of glucose isomerase. substantially as described in foregoing Example III, F.
28. A method of activating a cell mass form of glucose isomerase. substantially as described in foregoing Example V, 410/C.
29. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example V, 410/D.
30. A method of activating a cell mass form of glucose isomerase, substantially as described in foregoing Example VI, IG 403 II C.
31. A method of activating a cell mass form of glucose isomerase. substantially as described in foregoing Example VI, IG 403 II D.
32. A cell mass form of glucose isomerase, substantially as described in foregoing Example 1, B.
33. A cell mass form of glucose isomerase, substantially as described in foregoing Example 1, C.
34. A cell mass form of glucose isomerase, substantially as described in foregoing Example 1, D.
35. A cell mass form of glucose isomerase, substantially as described in foregoing Example II, E.
36. A cell mass form of glucose isomerase, substantially as described in foregoing Exampl III, B.
37. A cell mass form of glucose isomerase, substantially as described in foregoing Example III, C.
38. A cell mass form of glucose isomerase, substantially as described in foregoing Example III, D.
39. A cell mass form of glucose isomerase, substantially as described in foregoing Example III, E.
40. A cell mass form of glucose isomerase, substantially as described in foregoing Example III, F.
41. A cell mass form of glucose isomerase, substantially as described in foregoing Example V, 410/C.
42. A cell mass form of glucose isomerase, substantially as described in foregoing Example V, 410/D.
43. A cell mass form of glucose isomerase, substantially as described in foregoing Example VI, IG 403 II C.
44. A cell mass form of glucose isomerase, substantially as described in foregoing Example VI, IG 403 II D.
45. A cell mass form of glucose isomerase whenever activated by the method of any one of Claims 1 to 10 and 19 to 31.
46. A method of isomerasing glucose. which comprises isomerasing glucose utilising a cell mass form of glucose isomerase in accordance with any one of Claims 11 to 18 and 32 to 45.