Note: Descriptions are shown in the official language in which they were submitted.
1100Q65
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
, ~
1100065
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
- 6
~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
8 -
.~
~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
-- 10 --
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
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 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
A103/67 79 397
~105/18 75 384
C103/18 72 371
D98/18 65 354
Outlet pH's of syrup from the column were measured as seenfrom the table:
Table IV (b) ii
Preparation Hours after start
19 43 67140 210 303
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:
- 15 -
~=~
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
- 16 -
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
410/C 16 2
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 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.
- 17 -
.. . _, .. .. .. .... .
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
- 18 -
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
IG 403 II C290 755 482 2093
IG 403 II D274 755 467 1914
IG 403 II E254 755 431 1635
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
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.
-- 19 --
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
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)
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
415/A 6 320
415/B 52 380
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.
,
1 21
. " ...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.
