Ultrafiltration purification of glucose isomerase

This invention relates to a process for the production of a purified glucose isomerase enzyme which comprises contacting an enzyme extract containing glucose isomerase and impurities with a first polysulfone membrane not normally permeable to glucose isomerase, in the presence of a salt concentration capable of selectively inducing permeation of glucose isomerase through the membrane, and obtaining a glucose isomerase containing permeate.

FIELD OF THE INVENTION 
The present invention relates to a process for enzyme purification. More 
specifically, the invention relates to a method for purification of 
glucose isomerase. 
BACKGROUND OF THE INVENTION 
The use of enzyme extracts from microorganisms in industry is widespread 
and profitable. Among the more common enzymes produced in a larger scale 
are bacterial proteases for use in making detergent powders, gluose 
oxidase for food preservation, and glucanases in the brewing industry. 
Many enzymes isolated for industrial use are extracellular, i.e., excreted 
into the growth medium by the microorganisms; isolation of such enzymes is 
usually a relatively simple matter. However, as is the case with, for 
example, glucose oxidase, many enzymes are produced intracellularly; 
extraction of the enzyme and removal of contaminants such as cellular 
debris and unwanted proteins presents an additional difficulty to the 
larger scale use of such products. 
One particularly valuable intracellularly produced enzyme is glucose 
isomerase. This enzyme is produced by a wide variety of microorganisms, 
and is used to enzymatically catalyze the conversion of glucose, a 
relatively unsweet but inexpensive sugar to the sweeter sugar fructose. 
Examples of known procedures for this conversion may be found in Hamilton, 
et al. ("Glucose Isomerase, a Case Study of Enzyme-Catalyzed Process 
Technology" Immobilized Enzymes in Food and Microbial Processes, Olson, et 
al., Plenum Press, New York, (1974). pp. 94-106, 112, 115-137); and a 
number of other publications (Antrim, et al. "Glucose Isomerase Production 
of High-Fructose Syrups", Applied Biochemistry and Bioengineering, Vol. 2, 
Academic Press (1979); Chen, et al., "Glucose Isomerase (a review)", 
Process Biochem., (1980) 15(6), pp. 36-41; Thompson, et al. "Fructose 
Manufacture from Glucose by Immobilized Glucose Isomerase", Chem. 
Abstracts, Vol. 82, (1975), Abs. No. 110316h; and Takasaki, "Fructose 
Production by Glucose Isomerase", Chem. Abstracts, Vol. 81, (1974), Abs. 
No. 7647a) 
Although the enzyme is water soluble, performing the reaction in an aqueous 
solution presents the difficulty and expense of recovering the enzyme; a 
single use of the enzyme may also be rather costly. There are therefore a 
number of techniques for isomerization which involve immobilizing the 
enzyme so that substantial activity is retained while the enzyme is fixed 
to a water insoluble matrix. This arrangement allows for the repeated use 
of the enzymes for prolonged periods of time and with a number of 
different glucose containing solutions. 
For such a system to function at maximum efficiency the immobilized enzyme 
should preferably be as pure as possible. This allows not only maximum 
loading, but also provides maximum specificity during conversion by 
ensuring a homogeneous enzyme product. A number of types of purification 
methods now exist. U.S. Pat. No. 4,007,842 describes a method in which a 
water insoluble organic solvent is added to an aqueous solution of this 
enzyme, causing precipitation of non-enzyme material, followed by treating 
the remaining solution with a soluble magnesium salt, which then causes 
the precipitation of an enzyme-magnesium complex. While effective, the 
method described therein is timeconsuming and relatively expensive. U.S. 
Pat. No. 4,250,263 describes a system in which a crude glucose-isomerase 
composition is heat-treated to precipitate non-enzyme material, leaving a 
glucose isomerase containing solution. Although this method is somewhat 
simpler than that noted above, the relative purity of the heat-treated 
solution is not very high. U.S. Pat. No. 4,256,838 discloses a method in 
which glucose isomerase is purified by treating an enzyme containing 
solution with a reagent which will precipitate nucleic acids, followed by 
chromatographing the remaining solution on a cellulose column, and eluting 
the enzyme. This method is not only rather complicated, but also provides 
a resulting enzyme solution with a yield of only about 70% of the original 
enzyme activity. 
The present invention teaches a method of glucose isomerase purification 
which provides a final enzyme containing solution of unexpectedly high 
yield and purity, utilizing a technique heretofore unknown for glucose 
isomerase purification. It involves a process of ultrafiltration of an 
enzyme extract in combination with a selective elution of the enzyme by 
use of a salt solution. The salt treatment of the enzyme retained on a 
membrane has the surprising effect of inducing permeation of the enzyme 
through the membrane which would not otherwise allow its passage; the 
mechanism by which this unexpected result is obtained is unknown. The 
resulting enzyme solution contains a yield of at least 75-80% of the 
original crude enzyme extract activity, and which is least 80% pure 
enzyme. Such a product is particularly well suited for immobilization on 
an appropriate support. 
BRIEF DESCRIPTION OF THE INVENTION 
The present invention relates to a process for the production of a purified 
glucose isomerase enzyme which comprises contacting an enzyme extract 
containing glucose isomerase and impurities with a first membrane not 
normally permeable to glucose isomerase, in the presence of a salt 
concentration capable of selectively inducing permeation of glucose 
isomerase through the membrane, and obtaining a glucose 
isomerase-containing permeate. In a preferred embodiment, the 
enzyme-containing permeate is contacted with a second membrane of lower 
molecular weight cut-off to provide a purified enzyme concentrate. In a 
further embodiment, the permeate from the concentration step is recycled 
through a diafiltration reservoir for the salt induced permeation step, 
conserving both salt and water.

DETAILED DESCRIPTION OF THE INVENTION 
The process of the present invention provides an enzyme of sufficient yield 
and purity for further use in immobilization systems. The process 
described herein is equally useful and efficient in both a small-scale 
laboratory procedure as well as in a large-scale industrial application. 
In connection with the present process, it has been unexpectedly 
discovered that permeation of an enzyme which has been retained on an 
ultrafiltration membrane may be induced by the addition of salt to the 
retenate. The addition of the salt then allows the passage of a 
substantially purified enzyme through as a permeate, while most of the 
impurities remain bound to the membrane. The term ultrafiltration as used 
herein is defined as pressure driven filtration on a molecular scale; the 
process of diafiltration is also intended to be encompassed by this term. 
In the present invention, the preferred salt to be used is NaCl. However, a 
number of other salts may also be used to produce the desired permeation. 
Among the alternative salts are K.sub.2 SO.sub.4, Na.sub.2 SO.sub.4, KCl, 
NH.sub.4, Cl, (NH.sub.4).sub.2 SO.sub.4, magnesium, manganese or cobalt 
salts, pyridinium chloride, and various nitrates, acetates, citrates and 
maleates. The latter groups, however, are subject to use under restricted 
conditions of pH. It is also possible, but not particularly practical, to 
utilize cationic or anionic polymers. Specifically not recommended are 
heavy metal or transition metal salts. It is a relatively simple matter, 
however, to determine the suitability of any particular salt by following 
the procedure described herein utilizing the salt of interest. Similarly, 
it is also possible to determine the optimum concentration of any of the 
Osalts by conducting trials similar to those described in Example 2. 
The membrane will be one with a molecular weight cut-off (MWCO) below the 
molecular weight of the enzyme. In a preferred embodiment of this 
invention, the MWCO of the membrane is about 100,000; this is sufficient 
to retain a large proportion of the larger molecular weight impurities, 
especially viable microorganisms, as a retentate on the membrane, while 
the salt allows the enzyme to pass through in the permeate. It is also 
preferred that the membrane be of the polysulfone type, such as, Millipore 
PTAK polysulfide membrane or the Amicon HP 100-20 cartridge used in the 
Amicon CH4 concentrator. In present experience membranes of the cellulose 
or vinyl-acrylic type apparently do not provide the desired results under 
the conditions described herein. 
The appropriate concentration of the necessary salt is dependent upon the 
purity of the starting composition. Significant enzyme permeation will 
occur at concentrations of 0.1-1.0 M NaCl and more; however, at 
concentrations of 1MNaCl, the flux across the membrane will be 
considerably reduced. When a crude enzyme extract is the starting 
material, the optimum NaCl concentration for enzyme permeation is about 
0.5M. Although use of such an extract is feasible (Example 1), the yield 
of enzyme is generally fairly low, as is the purity of the resulting 
permeate. It is thus preferred to use a starting extract which has been 
partially purified and concentrated prior to the ultrafiltration in the 
presence of salt. The most preferred method of initial purification is by 
ultrafiltration in the presence of low salt concentration. Ultrafiltration 
with a 100,000 MWCO membrane will initially remove any lower molecular 
weight impurities, and serves to concentrate the enzyme about twenty-fold; 
it consequently provides a starting material which, in the principle 
ultra-filtration step, will produce a permeate of relatively high purity. 
When such a partially purified, concentrated extract is used, it is 
possible to achieve enzyme permeation with concentrations of NaCl between 
about 0.1-0.3M. The preferred concentration in this circumstance is about 
0.15-0.2M NaCl. When purification is performed prior to the principle 
ultrafiltration, the addition of the salt may be accomplished by direct 
addition of the appropriate amount of solid NaCl to the retentate, or by 
dilution of the retentate with a salt solution to provide the desired 
concentration of NaCl. 
In the case in which a crude extract is used, the NaCl is added directly to 
the extract itself to provide the optimum level prior to ultrafiltration. 
The pH of the salt-containing solution is generally maintained within the 
range of 6-8, but is most preferably maintained around 7. During the salt 
induced permeation, the retentate may be diafiltered at constant volume 
with a salt solution of the required concentration, to replace the 
permeate being removed from the system. 
Following the ultrafiltration-permeation step, the combined enzyme 
containing permeates may be concentrated and desalted by further 
ultrafiltration. This step is preferably achieved using a membrane of 
smaller MWCO than that used in previous ultrafiltrations. The permeate of 
this step may be recycled through the saltsolution diafiltration step. The 
preferred membrane for concentration is one with a MWCO of about 50,000. 
The final concentrated enzyme achieves a purity of at least 80% or more, 
and a recovery of enzyme activity of at least 75%. 
An optional, but often useful, feature of the present invention is that, 
starting with the salt permeation step, all stages of the process may be 
carried out at elevated temperatures. There are a number of advantages of 
this practice. The first is that higher temperatures may reduce the 
chances of microbial contamination during any of the steps of the process. 
It may also serve to reduce the level of salt concentration required to 
induce permeation. Further, particularly at the salt permeation step, high 
temperatures may serve to denature unwanted proteins while not affecting 
the glucose isomerase. Generally speaking, temperatures of 
50.degree.-60.degree. C. are sufficient to accomplish the above 
objectives. If the permeate of the salt-permeation step is heated as 
outlined above, however, it is desirable to filter the heat-treated 
solution to clarify it by removal of any denatured proteins. A general 
summary of the steps of the subject process is presented in FIG. 1. 
Methods to produce the glucose isomerase extracts used as starting 
materials in the process of the present invention are well known in the 
art. For example, an enzyme extract containing glucose isomerase may be 
obtained by fermentation of microorganisms of a species known to produce 
glucose isomerase, extracting the enzyme from the mycelia and removing 
insoluble material by known methods. 
The subject process may be used to purify glucose isomerase produced by any 
of the known glucose isomerase producers. Among the preferred organisms 
are those belonging to the genera Streptomyces, Aerobacter, 
Brevibacterium, Leuconostoc, Paracolobacterium, Nocardia, Micromonospora, 
Microbiospora and Arthrobacter. Also of interest are the glucose 
isomerases which are usually thermostable. Such enzymes will be less 
likely to be affected by the heat-treatment step. Organisms producing such 
heat-stable enzymes are Bacillus stearothermophilus, Ampulariella, and 
Pseudonocardia. The process of the present invention may be better 
understood by reference to the following non-limiting examples. 
EXAMPLE 1 
The following example describes the purification by salt-induced 
ultrafiltration/diafiltration of a crude enzyme extract, having a purity 
expressed in terms of specific activity of about 5 IGIU/mg protein. 
An extract containing about 30 IGIU/ml suitable for this purpose can be 
prepared according to the teachings in Example 1 of U.S. Pat. No. 
3,788,945. The IGIU unit of enzyme activity is defined, and an assay 
method described in "Automated Method for the Determination of D-Glucose 
Isomerase", N.E. Lloyd, et al.; Cereal Chemistry, 49(5) 544-553, 
September-October., 1972. Protein concentration is determined by measuring 
U.V. absorbance at 280 nm and equating 1.0 absorbance unit to a 
concentration of 1.0 mg/ml. 
To determine the optimum conditions for isomerase permeation, portions of 
extract were used to prepare a series of solutions with salt 
concentrations (NaCl) ranging from 0.1 to 1.0 N. The conductivity and 
isomerase activity of each of these solutions was measured and 1000 ml 
portions were used for ultrafiltration under a standard set of conditions 
using an Amicon Model CH 4 concentrator (Amicon Corporation) with a 
100,000 MWCO (HP 100-20) cartridge. Ultrafiltration time was recorded and 
aliquots of the permeate and retentate were taken for analyses. 
The extract used for this study had a conductivity of 12,300.mu.mhos which 
corresponds to about 0.1 M NaCl. In all cases where salt was added at 
least 10% of the activity passed through the membrane and was collected in 
the permeate (Table I). Little activity, 0.4 IGIU/ml or 1.6% of the 
starting activity, permeated where no salt was added. The greatest 
permeation, 7.1 IGIU/ml or 16.4%, was achieved with 0.5 M NaCl addition. 
Increasing salt concentration up to 0.5 M NaCl increased enzyme 
permeation, but decreased flux. At 0.5 M NaCl the flux was 20 ml/min. for 
the cartridge which had a membrane surface area of 550 cm.sup.2, or 400 
ml.times.min.sup.-1 .times.M.sup.2-1. 
Results described above indicated that at the optimum salt concentration, 
0.5 M, for enzyme permeation from crude extract, about 16% of the 
isomerase activity was found in the permeate from a single pass, i.e., 900 
ml of permeate from 1000 ml starting extract. The flux at this salt 
concentration was 20.0 ml/min. To determine if most of the isomerase 
activity in the crude extract could be collected in the permeate from 
repeated diafiltration, a diafiltration series was carried out. 
A portion of the extract was adjusted to pH 7.0, and sodium chloride was 
added (0.5 M). A 1000 ml portion of this solution was ultrafiltered as 
described previously until 900 ml of permeate had been collected. The 
permeate and retentate were sampled for analysis, and 900 ml of fresh 0.5 
M NaCl was added to the retentate before ultrafiltration was resumed until 
900 ml of retentate was collected. This sequence, i.e., ultrafiltration, 
dilution of the retentate, ultrafiltration, was repeated three more times. 
In each step the pressure drop across the system was maintained at 7 psig, 
and the average flux was determined by measuring the time to collect 900 
ml of permeate. 
The results are summarized in Table III. In the original ultrafiltration 
plus four diafiltrations a total of 26,825 IGIU or 65.4% of the starting 
activity was collected in the permeates. Both the flux and the per cent 
activity permeating (based on the starting activity for each step) 
increased with each successive step, probably as a result of decreasing 
concentration of permeable solids in the retentates. The flux during the 
fourth diafiltration was 39 ml/min. or almost double the flux for the 
first step ultrafiltration. 
The permeates from all five steps were combined and ultrafiltered with a 
50,000 MWCO cartridge using the CH4 concentrator. The retentate from this 
step was diafiltered twice with 5 volumes of deionized water. The 
resulting retentate contained a total of 26,230 IGIU with a specific 
activity of 17.2 IGIU/mg. This represents greater than a threefold 
increase in purity. 
EXAMPLE 2 
The following example illustrates the procedure using an enzyme extract 
which has been partially purified. 
A significant portion of the lower molecular weight impurities present in 
crude isomerase extracts can be removed by simple 
ultrafiltration-concentration with a 100,000 MWCO membrane at low salt 
concentration. In this case, the enzyme is almost quantitatively retained 
by the membrane while impurities are removed with the permeate 
To test the effectiveness of salt-induced permeation on such a preparation, 
a portion of extract as in Example 1 was first concentrated .about.20 fold 
by ultrafiltration with an Amicon CH4 concentrator using the 100,000 MWCO 
cartridge. The concentrate was then diluted to 41 IGIU/ml with water and 
the pH was adjusted to 7.0 before the addition of various amounts of NaCl 
to prepare a series of enzyme solutions ranging from 0.1 to 1.0 M salt. A 
1000 ml portion of each solution was then ultrafiltered with the CH.sub.4 
concentrator as described above. The results are shown in Table II. 
In all trials where sodium chloride was added at least 13% of the activity 
passed through the membrane. Increasing salt concentration from 0.2 M to 
0.5 M resulted in decreasing isomerase permeation, while at 1.0 M NaCl, 
31.1% of the activity permeated. In all cases, enzyme permeation was 
significantly greater than from the crude extract (Table I) at similar 
enzyme and salt concentration. This latter observation probably reflects 
the removal by prior ultrafiltration of impurities which would otherwise 
compete with isomerase for permeation through the membrane pores. A trial 
with MgSO.sub.4 addition (0.16 M) was included to demonstrate that the 
salt of a divalent cation and anion would be as effective as sodium 
chloride in promoting enzyme permeation. 
Flux decreased with increasing salt concentration, probably due to a 
competition effect between salt and enzyme for membrane pores. Thus, the 
optimum salt concentration for both enzyme permeation and flux appears to 
be 0.2 M. 
To determine the rate and extent of isomerase permeation from an 
ultrafiltered concentrate, a 40-fold ultrafiltered concentrate (100,000 
MWCO) of extract was prepared. A portion of the concentrate was diluted to 
.about.41 IGIU/ml with 0.2 M NaCl and a 1000 ml aliquot was ultrafiltered 
with the 100,000 MWCO as described in previous experiments. After 
collecting 900 ml of permeate, samples of both the permeate and retentate 
were taken for analyses, and the retentate was diluted with 900 ml of 
fresh 0.2 M NaCl. Ultrafiltration was then resumed until 900 ml of 
permeate had been collected. 
The retentate was then diluted with 100 ml of 0.2 NaCl (total volume-200 
ml) and a constant volume diafiltration was run, as illustrated in FIG. 1, 
by continuous addition of 0.2 M NaCl to replace permeate which was removed 
from the system. A total of 2000 ml of permeate was collected in fractions 
to monitor the progress of the diafiltration. Flux was monitored by 
measuring the time to collect each fraction, and samples of each fraction 
were analyzed for isomerase activity. 
A total of 30,950 IGIU or 75.5% of the starting activity was collected in 
the permeates from ultrafiltration, diafiltration, and constant volume 
diafiltration. The initial flux for the ultrafiltration was 41 ml/min. The 
flux increased constantly over the course of the constant volume 
diafiltration to a final value of 52 ml/min. 
The combined permeates were concentrated and desalted by 
ultrafiltration-diafiltration with a 50,000 MWCO cartridge. The 
concentrated enzyme, 30,980 IGIU total, had a specific activity of 34.4 
IGIU/mg, which represents a 7-fold increase in purity. 
EXAMPLE 3 
The following example illustrates the permeation-diafiltration procedure 
utilizing an undiluted concentrate. 
To reduce the volume of salt solution needed for diafiltration the 
procedure of Example 2 was repeated with several modifications. In this 
case, an enzyme concentrate prepared by 100,000 MWCO ultrafiltration was 
used directly after the addition of solid sodium chloride to a final 
concentration of 0.2 M. A 200 ml portion of this concentrate (432 IGIU/ml) 
was diafiltered at constant volume with 0.2 M NaCl, and the permeate was 
collected in 100 ml fractions. After collecting 1200 ml of permeate the 
retentate volume was reduced to -100 ml by temporarily interrupting the 
influx of 0.2 M NaCl. Diafiltration was then continued at a constant 
retentate volume of .about.100 ml. Permeate fractions were analyzed for 
isomerase activity and protein concentration (U.V.). 
During diafiltration the permeate activity (IGIU/ml) decreased gradually, 
and the flux increased to a high of 38.5 ml/min. When the retentate volume 
was reduced to 100 ml after 1200 ml of permeate had been collected, the 
permeate activity increased temporarily, and the flux dropped to about 33 
ml/min. 
A total of 46,582 IGIU or 54% of the starting activity was collected in 
2000 ml of permeate for an average potency of 23.5 IGIU/ml. The specific 
activity of the permeate ranged from 31.9 to 38.3 IGIU/mg with an average 
of 35.1 IGIU/mg. 
EXAMPLE 4 
The following example illustrates a step-by-step complete procedure, 
including a heat-treatment step, as performed on a relatively larger 
scale: 
A. Concentration and Partial Purification of Crude Extract 
A 25-liter batch of isomerase extract as in Example 1 was clarified by 
filtration through a precoat of filteraid followed by filtration through a 
Gelman 0.45.mu. minicapsule filter. After adjusting the pH to 7.0, the 
filtered extract at a potency of 38.0 IGIU/ml was ultrafiltered with an 
Amicon CH4 concentrator using an HP100-20 cartridge (100,000 MWCO). 
Ultrafiltration was carried out at room temperature, 7 psig pressure drop, 
until the permeate volume was reduced to .about.1200 ml. The retentate was 
then diluted with 6000 ml of deionized water, and ultrafiltration 
(diafiltration) was resumed. Diafiltration increased the final purity by 
about 5% and could be considered as an option in a scaled-up process. The 
retentate from this diafiltration step contained a total of 896,000 IGIU 
at a potency of 854 IGIU/ml. Total volume was 1.8 liters. 
Thus the recovery, based on the total starting activity in the extract 
(950,000 IGIU) was 94.3%. 
B. Salt-Induced Enzyme Permeation Via Constant Volume 
Diafiltration at 60.degree. C. 
A 500 ml portion of the concentrated enzyme was adjusted to a conductivity 
of 15,000 .mu.mhos (.about.0.15 M) by the addition of solid NaCl after 
adding MgSO.sub.4 (1 mM) and MnCl.sub.2 (0.2 mM). This solution was heated 
to .about.62.degree. C. and held for 20 minutes. 
The purpose of the heat step was to precipitate a small amount of protein 
which might otherwise precipitate during furthcr 60.degree. C. operations. 
It may be optional in a scaled-up version. The purpose for operating at 
60.degree. C. was to increase flux by 4 or 5 fold over the low temperature 
alternative of about 15.degree. C., and to prevent microbial 
contamination. 
The slight haze which formed during the heat treatment was removed by 
filtration through a 0.45 .mu. microfilter. The clarified filtrate 
contained a total of 422,000 IGIU at a potency of 824 IGIU/ml for a 
recovery of 98.8% of the activity across the heat treatment step. 
A 250 ml portion of the heat-treated enzyme was used for constant-volume 
diafiltration at 60.degree. C. using an Amicon CH4 concentrator with a 
HP100-20 cartridge (100,000 MWCO). A total of 4000 ml of 0.15 N NaCl 
(15,000 .mu.mhos conductivity) was used for the initial stages of 
diafiltration. The permeate from diafiltration was collected in 1000 ml 
fractions. Flux was estimated by measuring the time to collect each 1000 
ml fraction. After sampling for analysis, the permeate fractions were 
ultrafiltered with a CH4 concentrator using an HlX50-20 cartridge. Flux 
for the 50,000 MWCO ultrafiltration was measured in the usual manner and 
periodic samples of the permeate were taken for analyses. 
The 50,000 MWCO permeate, which contained less than 0.6 IGIU/ml isomerase 
activity, was used to supply the diafiltration reservoir for the 
salt-induced permeation step after the initial 4000 ml of salt solution 
had been used. 
The following table shows the results of the salt-induced permeation for 
the initial 250 ml of enzyme concentrate. 
______________________________________ 
Flux Permeate Activity 
Fraction 
ml/min IGIU/ml IGIU/1000 ml 
IGIU Total 
______________________________________ 
1 23.8 80.3 80,300 80,300 
2 22.2 36.3 36,300 116,600 
3 22.7 18.2 18,200 134,800 
4 23.4 11.5 11,500 146,300 
5* 23.4 10.0 10,000 156,300 
6* 21.4 9.7 9,700 166,000 
______________________________________ 
*Diafiltered with 50,000 MWCO Permeate 
A total of 166,000 IGIU was accumulated in the 6000 ml of permeate for an 
average potency of 27.7 IGIU/ml and a recovery of 80.6% of the starting 
activity. The average flux was about 23 ml/min. 
Near the end of diafiltration the retentate volume was reduced to 
.about.100 ml by interrupting the flow of salt solution. The retentate was 
centrifuged to remove the insoluble haze, and the clear supernate was 
assayed for residual isomerase activity. The clarified retentate contained 
a total of 26,300 IGIU. Thus, the total recovery in the permeate plus 
retentate was 192,300 IGIU or 95.3% of the starting activity. 
The clarified retentate was returned to the constant volume diafiltration, 
and 200 ml of fresh enzyme concentrate (164,800 IGIU) was added. The 
constant-volume diafiltration was then resumed using the 50,000 MWCO 
permeate as diafiltration medium. Fractions were collected and assayed as 
usual. After collecting 3000 ml of permeate an additional 50 ml of fresh 
enzyme solution (41,200 IGIU) was added to the retentate and diafiltration 
was continued. The results are summarized in the following table. 
______________________________________ 
Flux Permeate Activity 
Fraction 
ml/min IGIU/ml IGIU/1000 ml 
IGIU Total 
______________________________________ 
1 25.6 86.1 86,100 86,100 
2 26.7 40.5 40,500 126,600 
3 26.9 20.2 20,200 146,800 
4 25.2 26.0 26,000 172,800 
5 24.8 16.9 16,900 189,700 
______________________________________ 
A total of 189,700 IGIU was collected in the permeate. This represented 
81.7% of the starting activity with an average potency of 34.9 IGIU/ml 
permeate. The final retentate contained a total of 33,200 IGIU so that the 
overall recovery in the permeate plus retentate was 222,900 IGIU or 95.9%. 
Some insoluble material also formed during this second diafiltration. 
However, this did not appear to be a serious problem since the average 
flux was about 26 ml/min. 
In the two diafiltrations described above a total of 500 ml of enzyme 
concentrate of 412,000 IGIU was processed using only 4000 ml of 
.about.0.15 M NaCl solution. A total of about 7000 ml of 50,000 MWCO 
permeate was recycled to the salt permeation step. A total of 11,000 ml of 
100,000 MWCO permeate containing 355,700 IGIU or 86.3% of starting 
activity was collected and concentrated by 50,000 MWCO ultrafiltration. 
C. Permeate Concentration By 50,000 MWCO Ultrafiltration 
The entire permeate from the 100,000 MWCO ultrafiltration was ultrafiltered 
with a 50,000 MWCO cartridge to concentrate the enzyme. Each 1000 ml 
fraction of 100,000 MWCO permeate, after sampling for analyses, was added 
directly to the 50,000 MWCO step. 
The permeate from the 50,000 MWCO ultrafiltration was collected in 1000 ml 
fractions, sampled for analyses, and recycled to the 100,000 MWCO 
permeation step. The flux across the 50,000 MWCO step ranged from a 
starting high of .about.9 ml/min (550 cm.sup.3 membrane area) to a final 
rate of 5.1 ml/min. during the final stages of concentration. No isomerase 
activity (&lt;0.6 IGIU/ml) was found in the 50,000 MWCO permeate. 
The 50,000 ml retentate was reduced to a final volume of 295 ml. The total 
isomerase activity in this concentrate was 336,300 IGIU (1140 IGIU/ml). 
This was 94.7% of the activity in the 100,000 MWCO permeate. The specific 
activity of the concentrated enzyme was 35.7 IGIU/mg, which represents a 7 
fold increase in purity. 
If desired, the final concentrate could be concentrated further by 
ultrafiltration to prepare a potent stable concentrate. The concentrate 
could also be diafiltered to remove residual sodium chloride. 
FIG. 1 summarizes the process in block-flow form. 
TABLE I 
______________________________________ 
THE EFFECT OF SODIUM CHLORIDE 
CONCENTRATION ON ISOMERASE PERMEATION 
FROM CRUDE ENZYME EXTRACT 
Conduc- 
[NaCl] tivity Flux* Permeate Activity 
% Per- 
M mhos ml/min IGIU/ml IGIU Total 
meated** 
______________________________________ 
0 12.3 30.0 0.4 642 1.6 
0.1 19.7 26.5 4.6 4231 10.3 
0.2 30.2 24.3 5.0 4650 11.3 
0.3 37.8 21.4 5.2 4790 11.6 
0.4 45.4 19.6 5.8 5365 13.0 
0.5 52.2 20.0 7.1 6745 16.4 
1.0 84.2 19.6 5.3 5016 12.2 
______________________________________ 
TABLE II 
______________________________________ 
THE EFFECT OF SALT CONCENTRATION 
ON ISOMERASE PERMEATION 
FROM ULTRAFILTERED ENZYME 
Conduc- 
[NaCl] tivity Flux* Permeate Activity 
% 
M mhos ml/min IGIU/ml 
IGIU Total 
Permeated 
______________________________________ 
0 0.7 42.9 0.17 158 0.4 
0.1 9.8 42.9 8.2 7564 18.4 
0.2 19.8 40.9 8.9 8259 20.0 
0.3 29.2 36.0 8.1 7415 19.0 
0.4 37.2 39.1 6.6 6101 14.8 
0.5 45.0 34.6 6.0 5520 13.4 
1.0 79.7 32.1 13.6 12822 31.1 
0.16 13.3 25.7 5.4 4923 11.9 
MgSO.sub.4 
______________________________________ 
*Avg. Flow Rate Determined By Measuring Time To Collect 900 ml Permeate 
**Total Activity In 900 ml Permeate/41,000 IGIU Starting Activity 
TABLE III 
______________________________________ 
ULTRAFILTRATION-DIAFILTRATION 
OF CRUDE ENZYME EXTRACT 
Retentate 
Permeate Activity 
Act. % 
Flux IGIU IGIU Per- 
Step ml/min IGIU/ml Total Total meated* 
______________________________________ 
Ultrafiltration 
20 7.1 6745 33,400 16.5 
Diafiltration 1 
23 5.9 5355 27,400 16.0 
Diafiltration 2 
27 5.7 5130 18,800 18.7 
Diafiltration 3 
32 4.8 4320 13,470 23.0 
Diafiltration 4 
39 5.5 5115 7,066 38.0 
______________________________________ 
*Based On Retentate Activity At The Start Of Each Step