CA1100065A - Iron containing cell mass glucose isomerase preparation - Google Patents
Iron containing cell mass glucose isomerase preparationInfo
- Publication number
- CA1100065A CA1100065A CA309,622A CA309622A CA1100065A CA 1100065 A CA1100065 A CA 1100065A CA 309622 A CA309622 A CA 309622A CA 1100065 A CA1100065 A CA 1100065A
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- Prior art keywords
- iron
- cell mass
- syrup
- enzyme
- glucose isomerase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
- C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
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- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Jellies, Jams, And Syrups (AREA)
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Abstract
ABSTRACT OF THE DISCLOSURE
The invention relates to a method of activating a cell mass of glucose isomerase. This method comprises incor-porating in the said cell mass at least 0.05% w/w (dry basis) of iron as a non-toxic water soluble iron salt. The iron is re-tained for the useful life of the enzyme product and remains so firmly bound that practically no iron leakage occurs in the use of the enzyme. The invention also relates to an iron activated cell mass form of glucose isomerase in dried parti-culate form, having incorporated therein at least 0.05% w/w of iron as a non-toxic water soluble iron salt, and the invention furthermore relates to a method of isomerase as defined above.
The invention relates to a method of activating a cell mass of glucose isomerase. This method comprises incor-porating in the said cell mass at least 0.05% w/w (dry basis) of iron as a non-toxic water soluble iron salt. The iron is re-tained for the useful life of the enzyme product and remains so firmly bound that practically no iron leakage occurs in the use of the enzyme. The invention also relates to an iron activated cell mass form of glucose isomerase in dried parti-culate form, having incorporated therein at least 0.05% w/w of iron as a non-toxic water soluble iron salt, and the invention furthermore relates to a method of isomerase as defined above.
Description
This invention relates to an iron containing glucose isomerase composition and more particularly to a glucose iso-merase 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 syrup, yet cobalt is widely considered to be a toxic substance and, therefore, the cobalt level present in the product iso-syrup 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 ~,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 acti~ation purposes. Howe~er, it should be noted that standard 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 isomeriza-tion system must have a reasonable degree of chemical sophis-tication, and the system itself should be sophisticated. The iron salt must be me~ered into the glucose syrup. Chemical analysis of the glucose syrup entering the isomerization reactor for its iron content must be made periodically, i 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 isomer-llOO~S
ization process. In either case leakage of iron into the pro-duct 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 bit of 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 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 sal-t. The cell mass form will then be converted into a particulate form and dried to obtain an enzyme product suitable for marketing in a pre-soaking state.
According to the second aspect of the present invention there is provided a method of activating a cell mass form of glucose isomerase, which process comprises incorporating therein at least 0.05~0 w/w (dry basis) of iron as a solid non-toxic water soluble iron salt. The cell mass form will then be con-verted into a particulate form and dried to obtain an enzyme product suitable for marketing in a pre-soaking state.
According to the third aspect of the present invention there is provided a method of activating a cell mass form of glucose isomerase in accordance with the disclosure and/Gr claims of British Patent 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 will then be converted into a particulate form and dried to obtain an enzyme product suitable for marke-ting in a pre-soaking state.
According to the fourth aspect of the present invention there is provided a method of activating a ce]l mass form of glucose isomerase in accordance with the disclosure and/or claims of British Patent Specification No. 1,516,704 and/or United States Patent Specification No. 3,980,~21, which process comprises incorporating therein at least 0. o5~0 w/w ( dry basis) of iron as a solid non-toxic water soluble iron salt. The cell mass form will -then be converted into a particulate form and dried to obtain an enzyme product suitable for marketing in a pre-soaking state.
The invention also provides an iron activated cell mass form of glucose isomerase in dried particulate form, having incorporated therein at least 0.05~0 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 British Patent 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 isomerization.
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 recog-nized to be 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, prepara-~ions 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, ~or example polyelectrolytes. On a weight basis the glucose isomerase content of a cell mass preparation is normally a very small fraction o~ 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 pre-parations far exceeds the activation requirements oP the glucoseisomerase.
In particular, non-toxic water soluble salts of iron in solid form can be mixed incorporated into -the cell mass pre-paration during forming thereof, for example just prior to ex-trusion of a particulate form. The salts could also be intro-duced, in appropria-te circumstances, as a concentrated aqueous solution This invention encompasses as a product a dry cell mass enzyme preparation with a non-toxic water soluble iror salt incorporated in iron amounts of at least 0.05% w/w, generally from 0.05-2.0% w/w of the cell mass preparation. r~ore iron than 2 0% w/w cou]d, of course, be incorporated but no ~A 4 _ llOW65`
useful purpose would be served the~eby. The preferred iron content is in the range of from 0.2 to 0.5% w/w, especially from about 0,2 to 0.25% w/w.
In all instances, once the iron is incorporated within the cell mass preparation in from 0,05%-2 0% wt/wt dry weight basis, little if any of the iron is lost to syrup over the use-ful 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 char.neling. ln addition, a decrease in activity and, in seYere 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 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 preparation in amounts of from 2 to 15~o by weight (dry basis) has often been found to be desirable.
The preferred glucose isomerase particles contempla-te~d herein are the glutaraldehyde reacted homogenized cells prepara--tions disclosed and/or claimed in U.K~ Patent Specification 5 ~
No 1,516,704 and/or United States Patent Specification No.
3,980,521.
In the preferred mode, the water soluble iron salt is admixed with the magnesium oxide and the glucose, and then added to the cell mass before the ex-trusion step 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 ~errous lactate Ferric citrate Ferrous citrate Ferric ammonium citrate Ferrous acetate Ferric nitrate Ferric pyrophosphate The following Examples i.llustrate the present invention~
In the Examples, the following terminology is used:-Definition of Activit~
The unit of activity is defined as the arnount of enzyme which forms fructose at an initial rate of 1 ~ mol of fructose per min. at a given set of isomerisaticn conditions.
Assa~ of Activit~
The activity is determined under the following conditions:
Syrup 40% W/W dissolved dextrOS~
pH inlet 8.5 Mg 0 o0L~ M
Temperature 65C
Column diameter 2.5 cm - - height 35 cm ~10~6~
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:-Ac~ = Ao x e b x t where t is No. of hours after start of isomerizationAct 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 Tl/2 = lnb2 and is given in hours.
Productivit~
Productivity is defined as kg of dextrose d.s. con-verted to a mixture of 45~0 fructose and 55% glucose per kg of enzyme after a given time of isomerization.
In the examples the productivity is calculated accord-ing to an equation of the above given form after an isomeriza-tion time of 2 x Tl/2, I _ The iron content is determined according to the o-phenanthroline method (Nordisk Metodik Komite for ~evnedsmidler Nr. 22, 1955 U.D.C. 664.7: 546.72).
Color The color is determined according to the CIRF method.
Color stabilit~
Color stability is determined after 1 hour heating at 100C 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 in connection with magnesium oxide and dextrose.
Addition o~ 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 e~uipped with a screen with 1 cm holes.
The coarse granulate contained about 76~o of water (measured by drying at 105C). lt 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 o.8 mm.
Th8 ex~rudate was dried in a fluid bed with 60 -65~Cair 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 dextros,e monohydrate and 30 g ferric sulphate with an iron content of about 20~o, After thorough mixing, the mixture was extruded and dried as in A
) 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.
) F. 8.5 kg of the coarse granulate was mixed thoroughly with 25 g of ferric oxide containing about 58% of iron The mixture was extruded and dried as in A
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 25C.
~) Compara-tive example .~
~100~5 TABLE I
Activity pH in outlet syrup after . ... _ _ Found Corrected %
Preparation IGIC/g IGIC/g grain 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. Additio~
of ferric oxide gave only about 6~o compared to about 30% for the soluble sal-ts.
Example II: Addition of magnesium oxide + dextrose and magnesium oxide + dextrose + iron salt.
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 granula-tor equipped with a screen with 1 cm holes.
The coarse granulate contained about 79~0 of water.
It was divided into 5 lots of 8.5 kg.
)A, 8~ 5 kg was extruded and dried as in example lA
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.
)~, To 8.5 kg granula-ted filter cake was added 25 g magnesium oxide and 200 g dextrose 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 o~.ide and 300 g dex-trose.
After mixing it was extruded and dried.
) Comparative example llOO.Q~$..
E. 8.5 of the filter cake was mixed with a mixture of 25 g magnesium oxide, 200 g dextrose monohydrate and 40 g ferric sulphate containing about 20~o iron. Thereafter it was extruded and dried.
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 25C, Table II
Activity IGIC/g pH in outlet syrup after % added Found Corrected for¦ ~0 Preparation material mat gain 20 hrs 43 hrs _ )A 0 220 220 0 6.85 7.40 )B 1 222 224 2 8.18 8.23 C 10 216 240 9 8.14 8,22 )D 14 216 251 14 8.15 1.21 E 12 272 309 40 8.15 8.27 As can be seen from Table II, only addition o~ an iron salt affords a significant increase in activity, Example III: Addition of ferric citrate, ferric pyrophosphate, ferric ammonium citrate and ferrous sulphate.
A coarse granulatèd filter cake with about 76~o of water as in example 1 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 ref'erence 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 16~ of iron and 250 g dextrose monohydrate, After thorough mixing the granulate was extruded and dried as in A, C, 8.5 kg of the coarse granulate was extruded and dried as in A after additîon of 25 g magnesium oxide, 50 g ferric citrate and 250 g dextrose monohydrate.
) Comparative example 1~00065 D. 8.5 kg of the coarse granulate was treated as C
except that the 50 g fe~ric citrate was replaced by 30 g ferric pyrophosp~ate with an iron content of about 12~o.
E, To 8. 5 kg of the coarse granulate was added 25 g magnesium oxide, 250 g dextrose monohydrate and 30 g ferric ammonium citrate with an iron content of about 15%. A~ter thorough mixing the granulate was extruded and dried as in A.
F. To the last lot of 8. 5 kg was added 25 g magnesium oxide, 250 g dextrose 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 svrup after ~ v Found Correctedgain 20 hrs. 43 hrs.
)A 229 1 229 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, o3 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 ~ilter cake was added magnesium o~.ide in sufficient amounts to give preparations with the following magnesium oxide content in the final dried preparations.
) Compara-tive exarnple ,,~OOQ6~
Prep. Bl No additive - B2 2% magnesium oxide - B3 5% magnesium oxide Isomerizations were performed in 60 ml jacketed glass columns (h x d = 35 x 1.5 cm) using 15 grams of each o~ the three preparations, The parameters for isomerization were:
Syrup 45% w/w redissolved dextrose pH inlet 8.0t`0,1 Mg add. to syrup 0.0008 M
Temperature 65C
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 prepara-tions had decreased in activity to an arbitrarily chosen activity of 20-25~cmol/min/g.
The following results were obtained:
?able IV (a) Preparation Max. measured Running Half life Producitivity activity/after time,hrs. hours after 2xTl/2 hours Bl 88/72 665 257 369 Outlet pH's of syrups from the column were found as tabulated below:
Table I~ (a) ii Preparation Hours a~ter start _ (soaking) 1742 7 140 230 350 665 Bl _ 6.2 6.26.1 6.o 6.o 5 9 6.3 3G B2 8.4 7 4 7~o6.7 6.4 6.2 6.2 6.9 B3 9.3 8.6 7'77 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 in~luence the max. observed activity as well as the stability - 12 _ ,~., llOd0~i5 and productivity in descending direction.
In this test, isomerizing with an inlet pH of 8.0, a higher max. activity and productivity resulted from presence Of 2~o added magnesium oxide as compared to no additives.
b, To optimize the addition Of magnesium oxide four additional preparations were produced according to the proce-dure described in Example I. The additive content of the dried preparations were:
A no magnesium oxide B 2% magnesium oxide + 9% dextrose C 1% magnesium oxide + 9% dextrose D 2% magnesium oxide + 9~ dextrose Isomerizations were performed in 60 ml jacketed glass columns (h x d ~ 35 x 1.5 cm) using the following parameters;
Syrup 45% w/w redissolved dextrose pH inlet 8.4 + 0.1 Mg add. to syrup 0.0016 M
Temperature 65C
The isomerizations were continued for 351 hours. The follow-ing results were obtained:
Table IV (b) i Preparation Max. measured ac- Activity after Productivity af-ti~ity/after hours 351 hours ter 351 hours ~105/18 75 384 Outlet pH's of syrup from the column were measured as seenfrom the table:
Table IV (b) ii Preparation Hours after start A 6.6 6.7 7.07.3 7.4 7.2 B 7.1 7.0 7.37.5 7.5 7.1 C 7,4 7.6 7.77.6 7.6 7.2 D 8,4 8.0 7.87.6 7~5 7.2 ;~ - 13 -The results indicate no great differences in activity, stability, or productivity between the four preparations. Out-let 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 ~% 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 follow-ing 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 dextrose (100 parts) and magnesium oxide (8 parts).
410/C. About 2 parts by weight of mix Z were added to about 98 parts by weight of the filter cake dry basis. Mix 2 consis-ted of dextrose (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, 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 fi~re final preparations were determined.
410/A o.o4~0 410/B o,o3%
410/~ 0.08%
410/D 0.18%
410/E 0.04%
+) Comparative example 1l~
~l~O~iS
Isomerizations were performed with material from preparations 410/A, 410/B, 410/D and 410/E, using the follow-ing conditions.
Syrup 45% w/w redissolved dextrose pH2~ inlet 8.4 ~ 0,1 Mg 0.0016 M
Temperature 62C
Column dimensions h 40 cm d 5.8 cm v 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 Prod~c-tion activitytime 21h 48h 92h T1/2 tivity hours ho~rs after 20410/A 158 1293 6.~ 6.8 7.2 842 188 410/B 155 936 7'4 7'7 8.0 818 1790 410/D 202 1316 7.3 7.~ 7'7 843 229S
410/E 1~1 1147 6.9 6,9 7.7 828 1755 The concentration of iron in the outlet syrup fromthese columns was determined.
Table V (b):
Fe (pp~ in outlet syrup Preparation2 l/2hours 21 hours 27 hours after start after start after start 30410/A < 1 c 1 ~ 1 410/B < 1 < 1 ~ 1 410/Dapprox ~ 1 < 1 ~ 1 410/~ < 1 < 1 < 1 A second set of isomerization experiments was per-formed with material from preparations 410/C, 410/D and 410/E, using the following conditions:
~=~
1100~6S
Syrup 45% w/w redissolved dextrose pH inlet 8,4 + 0.1 Mg2* 0,0016 M
Temperature 65C
Column dimensions h 20 cm d 2,5 cm v 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 obtaineds Table V (c) Prepara- Max. measured Total run pH outlet Half life Produc-tion activity time after tivity hours 17h 45h 200h hours after 2xT2 410/C 210 900 7,0 7.8 8.1 512 1510 410/D 250 900 7.5 8.o 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 those columns was determined.
Table V (d) Fe (ppm) in ou-tlet syrup Preparation 0 hrs 24 hrs 72 hrs 140 hrs 850hrs (soaking)after after af-ter after start s-tart start start 410/~0.8 cO.5 < 5 < 5 ~'5 410/D3.6 <0.5 ~0.5 <0.5 cO.5 410/E< o,5 cO.5 <0,5 cO.5 <o,5 The CIRF color of the outlet syrup from these columns was determined.
Table V (e) CIRF color in syrup Preparation0 hrs. 24 hrs 72 hrs.
(soaking) after start after start 410/~ 0.266 0.030 0,019 410/~ 0.247 o.o36 0.020 410/E 0,232 o.o36 0,022 1~00066 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.
Ta~le V (f) Color sta~ility of syrup Preparation 0 hrs 24 hrs 75 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.
The iron content of the enzyme preparations was deter-mined before and after use.
Table V (~) mg iron in column packed with 20 g enzyme PreparationAt start After 900 hours 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 hy the enzyme originated from the traces of iron natur-ally present in the solutions of crystalline dextrose. Analysis of the iron content of the 45% w/w redissolved dextrose syrup showed ~ 0.5 ppm, and approximately 0~1 ppm iron. In the course of the ~00 hours that these columns ran, approximately 75000 q of syrup were passed through each column containing 20 g enzyme.
.. . _, .. .. .. .... .
llQ~6S
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 g = 7.5 mg.
This corresponds well to the amount picked up by the enzyme preparations during the course of the test.
_onclusions 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/D, the out-let pH was 0.5-1.0 unit higher than without magnesium oxide, as in 410/A and 410JE.
Addition of iron salt, as in 410/D, increased the act-ivity without impairing the stability, giving rise to an overall increase in productivity of between 20 and 30%.
Addition of smaller amounts of magnesium oxide and iron salt, as in 410/C, gave a smaller increase in outlet pH and a smaller increase in productivity, but these increases were still significant.
Example VI: Comparison of Ferrous and Ferric salts.
A mixture of iron salt, dextros~, and magnesium oxide was added to samp es 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 approximately l~/o. The composition and amount of the mix con-sisting of iron salt, dextrose and magnesium oxide was such as to give final preparations with the following compositions:
Table VI (a) Preparation Iron saltDextrose Magnesium oxide IG 403 II C 1.2% Ferric 8% 1%
sulphate IG 403 II D 1.2% Ferrous8% 1%
sulphate IG 403 II E None 8% 1%
Analysis of the preparations gave the following results llOOQ65 for the actual Fe content:
IG 403 II C 0.22%
IG 403 II D 0.27%
IG 403 II E 0.05%
Isomerizations were performed under the following conditions:
Syrup 45% redissolved dextrose pH inlet 8.4 + 0.1 Mg + 0.0016 M
Temperature 65C
Column dimensions h 20 cm d 2.5 cm v 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 ~
Max. measured Total run Half life Productivity Preparation activity time, Tl/2, hours after 2xT1/2 The iron contents of the enzyme preparations were de-termined before and after use.
Preparationmg Fe in column packed with 20 g enzyme at start after 755 h 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 ~Q
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 prep-arations:
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 dextrose (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 dextrose pH inlet 8.3 ~ 0.1 Mg 0.0016 M
Fe 0.00007 M (4 ppm) Temperature 65~C
Column dimensions h 20 cm d 2.5 cm v 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 o~tained:
! - 20 -11~Q6S
Table VII (a) Max. measured Time to Total Half life Product-Preparationactivity reach run, T 2' ivity max. ac- time, hl/rs after 2 x tivity, hours Tl/2 hours The concentration of iron in the outlet syrup from these columns was determined.
Table VII (b) Preparation Fe (ppm) in outlet syrup at 0 hours (soaking) 20 hrs. 70 hrs. 350 hrs. 900 hrs, 415/A ~ 0.5 < 0.5 c 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.
Preparationmg iron in column packed with 20 g enzyme at start after 900 hours Conclusions 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 fir~t 160 hours of the run. This is in contrast to 415/B which gave maxi-mum activity after 20 hours. m is indicates that 415~A was slowly adsorbing iron from the input syrup with a resulting slow activa-tion. I'his 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.
, . " ...i, llQ~
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 prepar~-tions. 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 ability to absorb iron.
A basic difficulty facing this art is that glucose isomerase enzyme seemed to require cobalt ions in the syrup, yet cobalt is widely considered to be a toxic substance and, therefore, the cobalt level present in the product iso-syrup 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 ~,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 acti~ation purposes. Howe~er, it should be noted that standard 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 isomeriza-tion system must have a reasonable degree of chemical sophis-tication, and the system itself should be sophisticated. The iron salt must be me~ered into the glucose syrup. Chemical analysis of the glucose syrup entering the isomerization reactor for its iron content must be made periodically, i 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 isomer-llOO~S
ization process. In either case leakage of iron into the pro-duct 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 bit of 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 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 sal-t. The cell mass form will then be converted into a particulate form and dried to obtain an enzyme product suitable for marketing in a pre-soaking state.
According to the second aspect of the present invention there is provided a method of activating a cell mass form of glucose isomerase, which process comprises incorporating therein at least 0.05~0 w/w (dry basis) of iron as a solid non-toxic water soluble iron salt. The cell mass form will then be con-verted into a particulate form and dried to obtain an enzyme product suitable for marketing in a pre-soaking state.
According to the third aspect of the present invention there is provided a method of activating a cell mass form of glucose isomerase in accordance with the disclosure and/Gr claims of British Patent 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 will then be converted into a particulate form and dried to obtain an enzyme product suitable for marke-ting in a pre-soaking state.
According to the fourth aspect of the present invention there is provided a method of activating a ce]l mass form of glucose isomerase in accordance with the disclosure and/or claims of British Patent Specification No. 1,516,704 and/or United States Patent Specification No. 3,980,~21, which process comprises incorporating therein at least 0. o5~0 w/w ( dry basis) of iron as a solid non-toxic water soluble iron salt. The cell mass form will -then be converted into a particulate form and dried to obtain an enzyme product suitable for marketing in a pre-soaking state.
The invention also provides an iron activated cell mass form of glucose isomerase in dried particulate form, having incorporated therein at least 0.05~0 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 British Patent 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 isomerization.
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 recog-nized to be 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, prepara-~ions 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, ~or example polyelectrolytes. On a weight basis the glucose isomerase content of a cell mass preparation is normally a very small fraction o~ 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 pre-parations far exceeds the activation requirements oP the glucoseisomerase.
In particular, non-toxic water soluble salts of iron in solid form can be mixed incorporated into -the cell mass pre-paration during forming thereof, for example just prior to ex-trusion of a particulate form. The salts could also be intro-duced, in appropria-te circumstances, as a concentrated aqueous solution This invention encompasses as a product a dry cell mass enzyme preparation with a non-toxic water soluble iror salt incorporated in iron amounts of at least 0.05% w/w, generally from 0.05-2.0% w/w of the cell mass preparation. r~ore iron than 2 0% w/w cou]d, of course, be incorporated but no ~A 4 _ llOW65`
useful purpose would be served the~eby. The preferred iron content is in the range of from 0.2 to 0.5% w/w, especially from about 0,2 to 0.25% w/w.
In all instances, once the iron is incorporated within the cell mass preparation in from 0,05%-2 0% wt/wt dry weight basis, little if any of the iron is lost to syrup over the use-ful 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 char.neling. ln addition, a decrease in activity and, in seYere 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 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 preparation in amounts of from 2 to 15~o by weight (dry basis) has often been found to be desirable.
The preferred glucose isomerase particles contempla-te~d herein are the glutaraldehyde reacted homogenized cells prepara--tions disclosed and/or claimed in U.K~ Patent Specification 5 ~
No 1,516,704 and/or United States Patent Specification No.
3,980,521.
In the preferred mode, the water soluble iron salt is admixed with the magnesium oxide and the glucose, and then added to the cell mass before the ex-trusion step 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 ~errous lactate Ferric citrate Ferrous citrate Ferric ammonium citrate Ferrous acetate Ferric nitrate Ferric pyrophosphate The following Examples i.llustrate the present invention~
In the Examples, the following terminology is used:-Definition of Activit~
The unit of activity is defined as the arnount of enzyme which forms fructose at an initial rate of 1 ~ mol of fructose per min. at a given set of isomerisaticn conditions.
Assa~ of Activit~
The activity is determined under the following conditions:
Syrup 40% W/W dissolved dextrOS~
pH inlet 8.5 Mg 0 o0L~ M
Temperature 65C
Column diameter 2.5 cm - - height 35 cm ~10~6~
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:-Ac~ = Ao x e b x t where t is No. of hours after start of isomerizationAct 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 Tl/2 = lnb2 and is given in hours.
Productivit~
Productivity is defined as kg of dextrose d.s. con-verted to a mixture of 45~0 fructose and 55% glucose per kg of enzyme after a given time of isomerization.
In the examples the productivity is calculated accord-ing to an equation of the above given form after an isomeriza-tion time of 2 x Tl/2, I _ The iron content is determined according to the o-phenanthroline method (Nordisk Metodik Komite for ~evnedsmidler Nr. 22, 1955 U.D.C. 664.7: 546.72).
Color The color is determined according to the CIRF method.
Color stabilit~
Color stability is determined after 1 hour heating at 100C 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 in connection with magnesium oxide and dextrose.
Addition o~ 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 e~uipped with a screen with 1 cm holes.
The coarse granulate contained about 76~o of water (measured by drying at 105C). lt 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 o.8 mm.
Th8 ex~rudate was dried in a fluid bed with 60 -65~Cair 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 dextros,e monohydrate and 30 g ferric sulphate with an iron content of about 20~o, After thorough mixing, the mixture was extruded and dried as in A
) 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.
) F. 8.5 kg of the coarse granulate was mixed thoroughly with 25 g of ferric oxide containing about 58% of iron The mixture was extruded and dried as in A
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 25C.
~) Compara-tive example .~
~100~5 TABLE I
Activity pH in outlet syrup after . ... _ _ Found Corrected %
Preparation IGIC/g IGIC/g grain 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. Additio~
of ferric oxide gave only about 6~o compared to about 30% for the soluble sal-ts.
Example II: Addition of magnesium oxide + dextrose and magnesium oxide + dextrose + iron salt.
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 granula-tor equipped with a screen with 1 cm holes.
The coarse granulate contained about 79~0 of water.
It was divided into 5 lots of 8.5 kg.
)A, 8~ 5 kg was extruded and dried as in example lA
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.
)~, To 8.5 kg granula-ted filter cake was added 25 g magnesium oxide and 200 g dextrose 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 o~.ide and 300 g dex-trose.
After mixing it was extruded and dried.
) Comparative example llOO.Q~$..
E. 8.5 of the filter cake was mixed with a mixture of 25 g magnesium oxide, 200 g dextrose monohydrate and 40 g ferric sulphate containing about 20~o iron. Thereafter it was extruded and dried.
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 25C, Table II
Activity IGIC/g pH in outlet syrup after % added Found Corrected for¦ ~0 Preparation material mat gain 20 hrs 43 hrs _ )A 0 220 220 0 6.85 7.40 )B 1 222 224 2 8.18 8.23 C 10 216 240 9 8.14 8,22 )D 14 216 251 14 8.15 1.21 E 12 272 309 40 8.15 8.27 As can be seen from Table II, only addition o~ an iron salt affords a significant increase in activity, Example III: Addition of ferric citrate, ferric pyrophosphate, ferric ammonium citrate and ferrous sulphate.
A coarse granulatèd filter cake with about 76~o of water as in example 1 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 ref'erence 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 16~ of iron and 250 g dextrose monohydrate, After thorough mixing the granulate was extruded and dried as in A, C, 8.5 kg of the coarse granulate was extruded and dried as in A after additîon of 25 g magnesium oxide, 50 g ferric citrate and 250 g dextrose monohydrate.
) Comparative example 1~00065 D. 8.5 kg of the coarse granulate was treated as C
except that the 50 g fe~ric citrate was replaced by 30 g ferric pyrophosp~ate with an iron content of about 12~o.
E, To 8. 5 kg of the coarse granulate was added 25 g magnesium oxide, 250 g dextrose monohydrate and 30 g ferric ammonium citrate with an iron content of about 15%. A~ter thorough mixing the granulate was extruded and dried as in A.
F. To the last lot of 8. 5 kg was added 25 g magnesium oxide, 250 g dextrose 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 svrup after ~ v Found Correctedgain 20 hrs. 43 hrs.
)A 229 1 229 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, o3 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 ~ilter cake was added magnesium o~.ide in sufficient amounts to give preparations with the following magnesium oxide content in the final dried preparations.
) Compara-tive exarnple ,,~OOQ6~
Prep. Bl No additive - B2 2% magnesium oxide - B3 5% magnesium oxide Isomerizations were performed in 60 ml jacketed glass columns (h x d = 35 x 1.5 cm) using 15 grams of each o~ the three preparations, The parameters for isomerization were:
Syrup 45% w/w redissolved dextrose pH inlet 8.0t`0,1 Mg add. to syrup 0.0008 M
Temperature 65C
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 prepara-tions had decreased in activity to an arbitrarily chosen activity of 20-25~cmol/min/g.
The following results were obtained:
?able IV (a) Preparation Max. measured Running Half life Producitivity activity/after time,hrs. hours after 2xTl/2 hours Bl 88/72 665 257 369 Outlet pH's of syrups from the column were found as tabulated below:
Table I~ (a) ii Preparation Hours a~ter start _ (soaking) 1742 7 140 230 350 665 Bl _ 6.2 6.26.1 6.o 6.o 5 9 6.3 3G B2 8.4 7 4 7~o6.7 6.4 6.2 6.2 6.9 B3 9.3 8.6 7'77 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 in~luence the max. observed activity as well as the stability - 12 _ ,~., llOd0~i5 and productivity in descending direction.
In this test, isomerizing with an inlet pH of 8.0, a higher max. activity and productivity resulted from presence Of 2~o added magnesium oxide as compared to no additives.
b, To optimize the addition Of magnesium oxide four additional preparations were produced according to the proce-dure described in Example I. The additive content of the dried preparations were:
A no magnesium oxide B 2% magnesium oxide + 9% dextrose C 1% magnesium oxide + 9% dextrose D 2% magnesium oxide + 9~ dextrose Isomerizations were performed in 60 ml jacketed glass columns (h x d ~ 35 x 1.5 cm) using the following parameters;
Syrup 45% w/w redissolved dextrose pH inlet 8.4 + 0.1 Mg add. to syrup 0.0016 M
Temperature 65C
The isomerizations were continued for 351 hours. The follow-ing results were obtained:
Table IV (b) i Preparation Max. measured ac- Activity after Productivity af-ti~ity/after hours 351 hours ter 351 hours ~105/18 75 384 Outlet pH's of syrup from the column were measured as seenfrom the table:
Table IV (b) ii Preparation Hours after start A 6.6 6.7 7.07.3 7.4 7.2 B 7.1 7.0 7.37.5 7.5 7.1 C 7,4 7.6 7.77.6 7.6 7.2 D 8,4 8.0 7.87.6 7~5 7.2 ;~ - 13 -The results indicate no great differences in activity, stability, or productivity between the four preparations. Out-let 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 ~% 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 follow-ing 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 dextrose (100 parts) and magnesium oxide (8 parts).
410/C. About 2 parts by weight of mix Z were added to about 98 parts by weight of the filter cake dry basis. Mix 2 consis-ted of dextrose (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, 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 fi~re final preparations were determined.
410/A o.o4~0 410/B o,o3%
410/~ 0.08%
410/D 0.18%
410/E 0.04%
+) Comparative example 1l~
~l~O~iS
Isomerizations were performed with material from preparations 410/A, 410/B, 410/D and 410/E, using the follow-ing conditions.
Syrup 45% w/w redissolved dextrose pH2~ inlet 8.4 ~ 0,1 Mg 0.0016 M
Temperature 62C
Column dimensions h 40 cm d 5.8 cm v 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 Prod~c-tion activitytime 21h 48h 92h T1/2 tivity hours ho~rs after 20410/A 158 1293 6.~ 6.8 7.2 842 188 410/B 155 936 7'4 7'7 8.0 818 1790 410/D 202 1316 7.3 7.~ 7'7 843 229S
410/E 1~1 1147 6.9 6,9 7.7 828 1755 The concentration of iron in the outlet syrup fromthese columns was determined.
Table V (b):
Fe (pp~ in outlet syrup Preparation2 l/2hours 21 hours 27 hours after start after start after start 30410/A < 1 c 1 ~ 1 410/B < 1 < 1 ~ 1 410/Dapprox ~ 1 < 1 ~ 1 410/~ < 1 < 1 < 1 A second set of isomerization experiments was per-formed with material from preparations 410/C, 410/D and 410/E, using the following conditions:
~=~
1100~6S
Syrup 45% w/w redissolved dextrose pH inlet 8,4 + 0.1 Mg2* 0,0016 M
Temperature 65C
Column dimensions h 20 cm d 2,5 cm v 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 obtaineds Table V (c) Prepara- Max. measured Total run pH outlet Half life Produc-tion activity time after tivity hours 17h 45h 200h hours after 2xT2 410/C 210 900 7,0 7.8 8.1 512 1510 410/D 250 900 7.5 8.o 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 those columns was determined.
Table V (d) Fe (ppm) in ou-tlet syrup Preparation 0 hrs 24 hrs 72 hrs 140 hrs 850hrs (soaking)after after af-ter after start s-tart start start 410/~0.8 cO.5 < 5 < 5 ~'5 410/D3.6 <0.5 ~0.5 <0.5 cO.5 410/E< o,5 cO.5 <0,5 cO.5 <o,5 The CIRF color of the outlet syrup from these columns was determined.
Table V (e) CIRF color in syrup Preparation0 hrs. 24 hrs 72 hrs.
(soaking) after start after start 410/~ 0.266 0.030 0,019 410/~ 0.247 o.o36 0.020 410/E 0,232 o.o36 0,022 1~00066 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.
Ta~le V (f) Color sta~ility of syrup Preparation 0 hrs 24 hrs 75 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.
The iron content of the enzyme preparations was deter-mined before and after use.
Table V (~) mg iron in column packed with 20 g enzyme PreparationAt start After 900 hours 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 hy the enzyme originated from the traces of iron natur-ally present in the solutions of crystalline dextrose. Analysis of the iron content of the 45% w/w redissolved dextrose syrup showed ~ 0.5 ppm, and approximately 0~1 ppm iron. In the course of the ~00 hours that these columns ran, approximately 75000 q of syrup were passed through each column containing 20 g enzyme.
.. . _, .. .. .. .... .
llQ~6S
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 g = 7.5 mg.
This corresponds well to the amount picked up by the enzyme preparations during the course of the test.
_onclusions 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/D, the out-let pH was 0.5-1.0 unit higher than without magnesium oxide, as in 410/A and 410JE.
Addition of iron salt, as in 410/D, increased the act-ivity without impairing the stability, giving rise to an overall increase in productivity of between 20 and 30%.
Addition of smaller amounts of magnesium oxide and iron salt, as in 410/C, gave a smaller increase in outlet pH and a smaller increase in productivity, but these increases were still significant.
Example VI: Comparison of Ferrous and Ferric salts.
A mixture of iron salt, dextros~, and magnesium oxide was added to samp es 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 approximately l~/o. The composition and amount of the mix con-sisting of iron salt, dextrose and magnesium oxide was such as to give final preparations with the following compositions:
Table VI (a) Preparation Iron saltDextrose Magnesium oxide IG 403 II C 1.2% Ferric 8% 1%
sulphate IG 403 II D 1.2% Ferrous8% 1%
sulphate IG 403 II E None 8% 1%
Analysis of the preparations gave the following results llOOQ65 for the actual Fe content:
IG 403 II C 0.22%
IG 403 II D 0.27%
IG 403 II E 0.05%
Isomerizations were performed under the following conditions:
Syrup 45% redissolved dextrose pH inlet 8.4 + 0.1 Mg + 0.0016 M
Temperature 65C
Column dimensions h 20 cm d 2.5 cm v 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 ~
Max. measured Total run Half life Productivity Preparation activity time, Tl/2, hours after 2xT1/2 The iron contents of the enzyme preparations were de-termined before and after use.
Preparationmg Fe in column packed with 20 g enzyme at start after 755 h 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 ~Q
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 prep-arations:
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 dextrose (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 dextrose pH inlet 8.3 ~ 0.1 Mg 0.0016 M
Fe 0.00007 M (4 ppm) Temperature 65~C
Column dimensions h 20 cm d 2.5 cm v 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 o~tained:
! - 20 -11~Q6S
Table VII (a) Max. measured Time to Total Half life Product-Preparationactivity reach run, T 2' ivity max. ac- time, hl/rs after 2 x tivity, hours Tl/2 hours The concentration of iron in the outlet syrup from these columns was determined.
Table VII (b) Preparation Fe (ppm) in outlet syrup at 0 hours (soaking) 20 hrs. 70 hrs. 350 hrs. 900 hrs, 415/A ~ 0.5 < 0.5 c 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.
Preparationmg iron in column packed with 20 g enzyme at start after 900 hours Conclusions 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 fir~t 160 hours of the run. This is in contrast to 415/B which gave maxi-mum activity after 20 hours. m is indicates that 415~A was slowly adsorbing iron from the input syrup with a resulting slow activa-tion. I'his 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.
, . " ...i, llQ~
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 prepar~-tions. 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 ability to absorb iron.
Claims (6)
1. A method of activating a cell mass form 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.
2. A method of activating a cell mass form 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.
3. A method according to claim 1 wherein, after incorporation of the iron, the glucose isomerase is converted into a particulate form and dried to obtain an enzyme product in a presoaking state.
4. A method according to any one of claims 1 to 3, wherein at least 0.5% by weight of magnesium oxide, based on the dry weight of the cell mass form of glucose isomerase and at least 2% by weight (dry basis) of solid glucose are admixed with the iron and then added to the cell mass, whereafter the mass is extruded and granules formed.
5. An iron activated cell mass form 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.
6. A method of isomerasing glucose, which comprises utilizing a cell mass form of glucose isomerase in accordance with claim 5.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/827,030 US4152211A (en) | 1977-08-23 | 1977-08-23 | Iron containing cell mass glucose isomerase preparation |
| GB35368/77A GB1596662A (en) | 1966-09-15 | 1977-08-23 | Glucose isomerase compositions comprising iron salts |
| GB35368/77 | 1977-08-23 | ||
| US827,030 | 1977-08-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1100065A true CA1100065A (en) | 1981-04-28 |
Family
ID=26262696
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA309,622A Expired CA1100065A (en) | 1977-08-23 | 1978-08-18 | Iron containing cell mass glucose isomerase preparation |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS5856639B2 (en) |
| AT (1) | AT364879B (en) |
| AU (1) | AU515471B2 (en) |
| CA (1) | CA1100065A (en) |
| DE (1) | DE2833644C2 (en) |
| DK (1) | DK147236C (en) |
| ES (1) | ES472735A1 (en) |
| FR (1) | FR2401170A1 (en) |
| IT (1) | IT1174334B (en) |
| NL (1) | NL187584C (en) |
| SE (1) | SE465369B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6049479B2 (en) * | 1981-03-19 | 1985-11-01 | 東レ株式会社 | Glucose isomerase deactivation prevention material and glucose isomerization reaction method |
| JPS60168644U (en) * | 1984-04-18 | 1985-11-08 | 株式会社日立製作所 | Lighting circuits for car radios, etc. |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3779869A (en) * | 1971-05-13 | 1973-12-18 | Miles Lab | Enzyme stabilization |
| JPS5439472B2 (en) * | 1974-06-26 | 1979-11-28 | ||
| US3980521A (en) * | 1974-08-28 | 1976-09-14 | Novo Industri A/S | Immobilization of glucose isomerase |
| US3935069A (en) * | 1974-12-23 | 1976-01-27 | R. J. Reynolds Tobacco Company | Enzymatic process using immobilized microbial cells |
-
1978
- 1978-07-25 AU AU38338/78A patent/AU515471B2/en not_active Expired
- 1978-08-01 DE DE2833644A patent/DE2833644C2/en not_active Expired
- 1978-08-16 NL NLAANVRAGE7808495,A patent/NL187584C/en not_active IP Right Cessation
- 1978-08-18 CA CA309,622A patent/CA1100065A/en not_active Expired
- 1978-08-21 DK DK368278A patent/DK147236C/en not_active IP Right Cessation
- 1978-08-22 IT IT50815/78A patent/IT1174334B/en active
- 1978-08-22 AT AT0611178A patent/AT364879B/en active
- 1978-08-22 SE SE7808865A patent/SE465369B/en not_active IP Right Cessation
- 1978-08-22 ES ES472735A patent/ES472735A1/en not_active Expired
- 1978-08-23 FR FR7824511A patent/FR2401170A1/en active Granted
- 1978-08-23 JP JP53101910A patent/JPS5856639B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| AU3833878A (en) | 1980-01-31 |
| NL187584C (en) | 1991-11-18 |
| JPS5444092A (en) | 1979-04-07 |
| DK368278A (en) | 1979-02-24 |
| DK147236C (en) | 1985-01-14 |
| FR2401170A1 (en) | 1979-03-23 |
| NL7808495A (en) | 1979-02-27 |
| AU515471B2 (en) | 1981-04-02 |
| SE7808865L (en) | 1979-02-24 |
| FR2401170B1 (en) | 1983-08-26 |
| DE2833644A1 (en) | 1979-03-01 |
| ES472735A1 (en) | 1979-02-16 |
| IT1174334B (en) | 1987-07-01 |
| ATA611178A (en) | 1981-04-15 |
| SE465369B (en) | 1991-09-02 |
| NL187584B (en) | 1991-06-17 |
| JPS5856639B2 (en) | 1983-12-15 |
| DE2833644C2 (en) | 1985-07-11 |
| IT7850815A0 (en) | 1978-08-22 |
| AT364879B (en) | 1981-11-25 |
| DK147236B (en) | 1984-05-21 |
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