Method of purifying glucose isomerase and a composition for storing same

A process for purifying glucose isomerase comprises the steps of acid treatment and salt fractionation. An enzyme solution is treated with an acid, such as acetic acid, to a pH from about 3.5 to about 5.0. The proteinaceous solids are collected and extracted with a buffer, such as imidazole, whose solution has a pH of about 6 to about 8. The solution is then collected and a salt, such as ammonium sulfate, is dissolved therein from about 40% to about 50% of its saturation point. The proteinaceous solids which form are removed and additional ammonium sulfate is dissolved to attain from about 41% to about 60% of its saturation point, followed by collection of the solids containing purified enzyme. A composition which preserves enzyme activity upon storage of glucose isomerase and which imparts resistance to thermal deactivation of said enzyme comprises an aqueous solution of glycerol, a buffer whose solution is at a pH of about 6 to about 8, divalent cobalt ions and magnesium ions.

BACKGROUND OF THE INVENTION 
It is known that fructose is substantially sweeter than glucose. Because 
the latter is relatively inexpensive and readily available, it is 
desirable to have an efficient and economical means of converting glucose 
to fructose. The alkali isomerization of glucose yields fructose, but the 
production of undesirable side products and the necessity of removing 
caustic and other materials from a food ingredient make this route 
unattractive. A preferred method of isomerization utilizing enzymes has 
the advantages of specificity of reaction and lesser likelihood of 
producing undesirable side products which must be removed before the 
fructose-containing material can be used in foods. The enzymes which 
effect the conversion of glucose to fructose are called glucose isomerases 
and are formed from such bacteria belonging, inter alia, to the genus 
Arthrobacter and the genus Actinoplanes. These enzymes are water soluble, 
and if they are merely added to aqueous solutions of glucose, recovery of 
enzyme for reuse is difficult and expensive. Using the enzyme only once 
also affords a process which is relatively expensive. Consequently, many 
techniques have been developed for immobilizing the enzyme in such a way 
that substantial enzymatic activity in isomerizing glucose to fructose is 
displayed while the enzyme itself remains rigidly attached to some 
water-insoluble support, thereby permitting reuse of the enzyme over 
substantial periods of time and for substantial amounts of 
glucose-containing solutions. One illustration of a method for 
immobilizing an enzyme is found in Levy and Fusee, U.S. Pat. No. 
4,141,857, where a polyamine is adsorbed on a metal oxide such as alumina, 
the resulting composite is treated with an excess of bifunctional reagent, 
such as glutaraldehyde, so as to crosslink the amine, thereby entrapping 
the resulting polymer in the pores of the metal oxide, and thereafter 
contacting the mass with an enzyme to form covalent bonds between the 
pendant aldehyde groups and a amino groups on the enzyme. It is highly 
desirable that the material used in making immobilized enzyme contain the 
desired enzyme, here glucose isomerase, in as chemically pure a state as 
possible, both to assure maximum loading on the support, and to assure 
that the immobilized enzyme product will be homogeneous in the kind of 
enzyme bound to the support, thereby insuring maximum specificity in the 
conversion of glucose to fructose. 
SUMMARY OF THE INVENTION 
Accordingly, a principal object of this invention is to provide a method of 
purifying glucose isomerase. One embodiment of the invention comprises 
treating an enzyme solution with acid to pH from about 3.5 to about 5.0 
and collecting solids, extracting these solids with a buffer whose 
solution is at pH 6-8 and collecting the solution, dissolving additional 
salt to 41-60% of its saturation point and collecting the solids 
containing purified enzyme. A more specific embodiment comprises 
application of the process wherein said glucose isomerase is produced 
during the growth of a microorganism of the genus Actinoplanes on a 
culture. A still more specific embodiment of our invention utilizes 
imidazole as the buffer, acetic acid as the acid precipitating agent, and 
ammonium sulfate as the salt wherein the temperature is maintained from 
about 0.degree. C. to about 20.degree. C. throughout the process. 
Another object of this invention is to provide a composition which imparts 
storage stability and thermal resistance to deactivation of the enzyme. A 
specific embodiment comprises an aqueous solution of glycerol containing a 
buffer whose solution is at a pH of about 6 to about 8 and containing 
divalent cobalt ions. 
Other objects and embodiments will be apparent from the description within. 
DESCRIPTION OF THE INVENTION 
Enzymes with glucose isomerase activity are produced by many 
microorganisms, including those of the genus Streptomyces, Lactobacillus, 
Curtobacterium, and Actinoplanes. Examples of particular species of 
glucose isomerase producers from the above genera include: A. 
missouriensis, A. philippenensis, A. armeniacus, L. pentosus, L. breves, 
C. citreum, C. luteum, C. helvolum, etc. The Streptomyces are particularly 
rich in glucose isomerase producers, and examples of such species include 
olivochromogenes, venezuelae, coelicolor, aureus, griseolus, and 
virginiae. By way of illustration only, A. missouriensis may be cultured 
on a medium containing a suitable carbon source and other appropriate 
nutrients for a time sufficient to give maximum, or near maximum, glucose 
isomerase activity. The whole cells containing the enzymes are collected 
by suitable means, such as filtration, washed, then freeze dried and 
resuspended in a buffer at a pH of about 6 to about 8. In a preferred 
embodiment the buffer is selected from the group consisting of imidazole, 
phosphate, and tris(hydroxymethylamino)methane, and any combination 
thereof. It is contemplated within the scope of this invention that other 
buffers may be used, but not necessarily with equivalent results. In 
addition, if so desired, the buffered solution may also contain divalent 
cobalt ions in the range from about 10.sup.-4 to about 10.sup.-2 molar, 
and magnesium ions in the range from about 10.sup.-3 to about 10.sup.-1 
molar. 
To release the enzyme from the cells the cell walls must be ruptured. 
Examples of suitable means include chemical rupture as by digestion with a 
lysozyme enzyme preparation, or physical rupture as by rending the walls 
with sound waves (sonication) or mechanical grinding. In one embodiment 
the enzyme may be released by sonication at a temperature between about 
0.degree. and about 15.degree. C. The cell debris which is formed is then 
removed by any means known to those skilled in art, as for example, by 
centrifugation, to afford a solution containing glucose isomerase. In 
order to denature undesired proteinaceous material, this solution may be 
heated to a temperature from about 40.degree. C. to about 80.degree. C. 
for an interval from about 5 to about 60 minutes. However, such heat 
treatment may be omitted, although not necessarily with equivalent 
results. In one embodiment the solution is heated from about 55.degree. C. 
to about 65.degree. C. for a period from about 10 to about 30 minutes. At 
the completion of the heat denaturation cycle the mixture may be cooled 
rapidly to about 0.degree. C. to about 20.degree. C., the precipitated 
material is then removed by suitable means, centrifugation for example, 
and discarded. 
The subsequent operations in the present process are performed at 
temperatures in the range from about 0.degree. C. to about 20.degree. C., 
and preferably in a range of from about 0.degree. C. to about 5.degree. C. 
The cooled solution is acidified until the pH is in the range from about 
3.5 to about 5.0. In the case of glucose isomerase from A. missouriensis 
the preferred pH range is from about 3.6 to about 4.4. Example of acids 
which may be used include inorganic acids, such as hydrochloric, organic 
carboxylic acids, such as citric, acetic, propionic, butyric and the like, 
and organic sulfonic acids such as benzenesulfonic acid, toluene sulfonic 
acid, methanesulfonic acid, and the like. It is to be understood that 
these examples are not limitations of the invention, and others may be 
used, but not necessarily with equivalent results. In a preferred 
embodiment of the invention the acid employed is acetic acid. The solids 
which form upon acidification are then collected by suitable means, such 
as centrifugation, and subsequently resuspended in a buffer, preferably 
selected from the aforementioned group, at a pH from about 6 to 8. This 
buffer may also contain divalent cobalt ions, or magnesium ions, or any 
combination thereof, in their aforementioned molarity ranges. Such 
resuspension is equivalent to extraction of glucose isomerase because not 
all of the solids obtained upon acidification are soluble in said buffer, 
and these solids are removed by suitable means and discarded. 
The enzyme in solution is now further purified by salt fractionation. A 
salt is added to the cooled solution in an amount corresponding to about 
40% to about 50% of its saturation point (that is, the total amount of 
salt which can be dissolved in the solution). In a preferred embodiment, 
the salt must have a solubility such that about 4 molar solutions at 
0.degree. C. can be prepared, but is otherwise without limitation. 
Examples of such salts include ammonium sulfate, ammonium acid sulfate, 
sodium chloride, potassium acetate, potassium carbonate, and potassium 
chloride. In another preferred embodiment the salt is an alkali metal or 
alkaline earth metal sulfate, such as Li.sub.2 SO.sub.4, Na.sub.2 
SO.sub.4, K.sub.2 SO.sub.4, Rb.sub.2 SO.sub.4, Cs.sub.2 SO.sub.4, and 
MgSO.sub.4. After allowing the solids to precipitate for a period from 
about 5 to about 60 minutes, preferably for a period from about 10 to 20 
minutes, the solids are removed by suitable means and discarded. At this 
point an additional amount of the salt is dissolved such that the total 
amount in solution corresponds from about 41 to about 60% of its 
saturation point, preferably from about 50 to about 60% of its saturation 
point, thereby causing precipitation of glucose isomerase. After a period 
of from about 5 to about 60 minutes, but preferably from about 10 to about 
30 minutes, the precipitate of purified glucose isomerase is collected by 
suitable means, such as centrifugation. 
Salts can be removed from the purified enzyme by any means, such as, for 
example, by dialysis or gel permeation chromatography. The solid which is 
collected as described above is redissolved in a buffer, preferably 
selected from the aforementioned group, at a pH from about 6 to about 8, 
which buffered solution may also contain divalent cobalt and/or magnesium 
ions in their respective aforementioned ranges. This enzyme solution is 
dialyzed overnight against the same solvent used to dissolve the enzyme, 
i.e., a solution of the same buffer containing divalent cobalt and/or 
magnesium ions at the same concentration used for solution of the enzyme. 
The process of the present invention thus affords an enzyme with up to 
about 9-fold purification, by which is meant that the specific activity, 
expressed in units of activity per milligram of protein, is about 9-fold 
greater for the final enzyme preparation that it was for the initial 
enzyme preparation. In addition, total recoveries of glucose isomerase 
range up to about 85%, by which is meant that the total activity of the 
final enzyme preparation is 85% of that of the initial enzyme preparation. 
A surprising discovery is that solutions of certain compositions enhance 
the time periods during which glucose isomerase can be stored without 
losing substantial activity, and that these same solutions impart enhanced 
resistance to thermal deactivation of said glucose isomerase. These 
solutions contain glycerol in amounts ranging from about 5% to about 60% 
on a volume-volume basis, imidzaole as a buffer at a pH of about 6 to 
about 8, divalent cobalt ion in a concentration of about 10.sup.-4 to 
about 10.sup.-2 molar, and magnesium ion in a concentration of about 
10.sup.-3 to about 10.sup.-1 molar. The ability of the aforementioned 
solution to impart these desirable properties to the enzyme will be shown 
in greater detail in the examples which are appended to the specification.

The following examples illustrate the process described in this invention 
and the composition of matter of the storage solution described in the 
invention. These examples are merely illustrative and it is to be 
understood that the present invention is not limited thereto. 
EXAMPLE 1 
Actinoplanes missouriensis (NRRL-3342) was cultured aerobically at 
29.degree. C. in a fermentor using the following medium (per 10 liters): 
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200 g. Casein Hydrolysate 
100 g. Yeast Autolysate 
50 g. NaCl 
5 g. L-Cystine 
5 g. Na.sub.2 SO.sub.3 
30 ml. 1 M MgSO.sub.4 7H.sub.2 O 
500 ml. 1 M Potassium phosphate 
buffer, pH 7.0 
50 g./500 ml. Galactose, autoclaved 
separately 
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Cells were harvested by filtration after an inoculum of 0.05 volume of 
fresh mature cultures. The cell paste was washed once with 0.05 M 
phosphate buffer, pH 7.0, containing 9 g. NaCl per liter to give a yield 
of cells of about 60 g. (dry weight) per 10 liters having an enzyme 
activity of about 1200 units/gram dry cells. 
Enzyme activity was assayed as follows. Glucose isomerase catalyzes 
formation of an equilibrium mixture of glucose and fructose. The method of 
assay utilized in these examples is to measure the initial rate of glucose 
formation from fructose at 60.degree. C. as opposed to the usual assay 
method which measures the initial rate of fructose formation from glucose. 
A 1.0 ml. portion of appropriately diluted enzyme or enzyme-containing 
cells was mixed with an assay solution which contained fructose, 2.5M 
tris(hydroxymethylamino)methane hydrochloride buffer, 2.times.10.sup.-2 M, 
at pH 7.5, magnesium sulfate, 5.times.10.sup.-3 M, and cobaltous chloride, 
5.times.10.sup.-4 M. After incubation at 60.degree. C. for 30-60 minutes 
the reaction was terminated by addition of 1 ml. of 0.1N hydrochloric 
acid, and the glucose formed was measured with a glucose analyzer. One 
unit of glucose isomerase activity corresponds to formation of 1 micromole 
glucose per minute. Specific activity corresponds to micromoles of glucose 
formed per minute per milligram of protein used. 
The freeze-dried cells were suspended in 50 mM imidazole buffer, pH 7.0, 
containing 10.sup.-2 M MgSO.sub.4 and 10.sup.-3 M CoCl.sub.2, to a 
concentration of 5% (w/v) and subjected to sonic disintegration at about 
8.degree. C. for 12 minutes. All subsequent operations were carried out at 
0.degree.-4.degree. C. except heat treatment of the enzyme. The sonically 
disintegrated mixture was centrifuged at 12,000 rpm for 10 minutes and the 
cell debris discarded. The supernatant (780 ml.) was heated at 60.degree. 
C. for 20 minutes, then cooled quickly in an ice bath. Precipitated 
protein was removed by centrifugation (12,000 rpm for 10 min.) and 
discarded. The supernatant (675 ml.) from heat treatment was further 
purified by acid precipitation. To the clear supernatant solution, a 2N 
acetic acid solution was added until the pH of the enzyme solution reached 
pH 4.0. Precipitate was collected after several minutes by centrifugation 
(12,000 rpm). This pellet of protein was resuspended with a tissue 
homogenizer in about 170 ml. of 0.2M of an imidazole buffer at pH 7.0 
containing 10.sup.-2 M MgSO.sub.4 and 10.sup.-3 M CoCl.sub.2. Insoluble 
precipitate was then removed by centrifugation. 
Solid ammonium sulfate was added to the clear supernatant to 45% 
saturation. After 15 minutes, the mixture was centrifuged (20 min. at 
16,000 rpm) and precipitate was discarded. The resulting supernatant 
received additional ammonium sulfate to 55% saturation. After 15 minutes, 
the precipitate was collected by centrifugation and dissolved in about 50 
ml. of 0.05M of an imidazole buffer, pH 7.0, containing 10.sup.-2 M 
MgSO.sub.4 and 10.sup.-3 M CoCl.sub.2. The solution was dialyzed overnight 
against the same buffer. The following table shows that about a 9-fold 
purification with over 80% recovery from crude extract was obtained. 
TABLE I 
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Summary of the Purification of Glucose Isomerase 
Specific 
Volume 
Activity 
Total 
Protein 
Activity 
Yield 
Purifi- 
Fraction 
(ml) Units/ml 
Units 
mg/ml 
(units/mg) 
(%) cation 
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Crude 
Extract 
780 51.6 40,248 
28.3 
1.82 (100) 
Heat 
Treatment 
675 51.6 34,830 
13.9 
3.71 86.5% 
2.0 
Acid 
Precipi- 
tation 
192 135 25,920 
15.8 
8.54 64.4% 
4.7 
45-55% 
(NH.sub.4).sub.2 SO.sub.4 
63 528 33,264 
33 16 82.6% 
8.8 
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The data of Table I clearly shows that initially the enzyme preparation 
showed an activity of 1.82 units/mg protein, whereas after the application 
of the process of this invention the enzyme preparation showed an activity 
of 16 units/mg protein, corresponding to an increase of about 9-fold. 
There was initially present 40,248 units of activity, and after the 
application of the process of this invention there was present 33,264 
units of activity, corresponding to recovery of over 80% glucose isomerase 
activity. 
The glucose isomerase so purified shows unexpected stability in a solution 
of an imidazole buffer, at neutral pH, containing glycerol and divalent 
cobalt ions. When stored in an aqueous solution containing 25% glycerol on 
a volume-volume basis, imidazole buffer at pH 7.0, and 10.sup.-3 molar 
CoCl.sub.2, the glucose isomerase shows no detectable decrease in activity 
after a month at either 4.degree. C. or -40.degree. C. In stark contrast, 
the purified glucose isomerase showed loss of more than 50% of its 
activity when stored for 2 months at -40.degree. C. in 0.1M phosphate 
buffer. Addition of glycerol at a concentration of 25% to 50% on a 
volume-volume basis, to the phosphate buffer did not have appreciable 
beneficial effects. That divalent cobalt ions impart thermal resistance to 
deactivation of purified glucose isomerase is shown dramatically by the 
following observation. Purified enzyme dissolved in an imidazole buffer at 
pH 7.0 containing 10.sup.-3 molar CoCl.sub.2 showed no loss of activity 
when heated to 60.degree. C. for 60 minutes. When the same enzyme was 
dissolved in the same buffer in the absence of divalent cobalt ions, there 
was more than a 30% loss of activity when heated to 60.degree. C. for only 
20 minutes. 
EXAMPLES 2-5 
These examples show the pH range over which acidification is effective to 
purify glucose isomerase from A. missouriensis by precipitation. In all 
examples 30 ml. of a heat-treated enzyme solution were used. To each was 
added a sufficient amount of 1 N acetic acid to adjust the pH in 
increments of 0.5 unit from a pH of 5.0 to 3.5. The resulting precipitate 
was separated from the supernatant solution and dissolved in a phosphate 
buffer. Total enzyme activity was measured for both the supernatant and 
the solution of the formed precipitate with the results shown in Table II. 
TABLE II 
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Total Units of Activity 
Example pH Supernatant Precipitate 
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2 5.0 1922 0 
3 4.5 1972 0 
4 4.0 0 1366 
5 3.5 0 0 
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These data show that this enzyme is not precipitated at pH 4.5 and above, 
and that it is completely deactivated at pH 3.5 and below. Glucose 
isomerase from other microorganisms may have similarly narrow ranges of pH 
within which enzyme precipitation is effectively induced. 
EXAMPLES 6-10 
Experiments to determine the optimum salt concentration for fractionation 
of glucose isomerase from A. missouriensis were performed in the following 
manner. To aliquot portions of equal volume of acid-precipitated enzyme in 
a buffer there was added ammonium sulfate in concentrations ranging from 
35-55% of its saturation point. Solids were separated from the supernatant 
solution and dissolved in about 3 ml. of phosphate buffer. One ml. 
portions each of the supernatant and the solution from precipitated solids 
were assayed for glucose isomerase activity. Results are shown in Table 
III. 
TABLE III 
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Saturation Level Activity per ml 
Example of (NH.sub.4).sub.2 SO.sub.4 
Supernatant Precipitate 
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6 35% 82.9 0 
7 40% 86.8 0 
8 45% 70.4 0 
9 50% 42.7 38.9 
10 55% 10.0 74.8 
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The data of Table III clearly show that when added at levels up to 45% of 
its saturation point ammonium sulfate causes selective precipitation of 
glucose isomerase-inactive proteins. After these are removed, further 
addition of ammonium sulfate causes selective precipitation of glucose 
isomerase-active proteins, thereby effecting a purification of glucose 
isomerase.