CA1045999A - Insolubilized glucose isomerase - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention discloses an insolubilized glucose isomerase consisting of glucose isomerase which is adsorbed and bound on a porous type anion exchange resin having a porosity of more than 4.5% (measured by aqueous dextran solution method) and an ion exchange capacity of more than 0.035 meq/ml-Resin (measure by polyanion salt decomposition method).
The resin matrix is a styrene-divinyl benzene copolymer, the anion exchange group of the resin being a quarternary ammonium group. The advantages of this invention include (a) more isomerase is adsorbed on the ion exchange resin, (b) higher enzyme activity of the product in the operation, (c) higher resistance to crush and distortion, and (d) less pressure loss as the glucose solution passes through the column containing the insolubilized isomerase.

Description

la4ssss This invention relates to an insolubilized glucose isomerase and more particularly to a glucose isomerase bound by adsorption on an ion exchange resin, the product being ln insolubilized form.
It has already been known that glucose is converted into fructose by glucose isomerase which is an enzyme capable of converting glucose into fructose and vice versa. - :
Such glucose isomerase have been observed in many micro- ::
organisms, including the followings; Streptomyces . ~ -l 10 flavovirens., Stre~ptomyces achrom~enes, StrePt_m~ces ..? echinatus, ~ y~ Albus, Aerobacter cloacae, Bacillus megaterium, Acetobacter suboxydans, ~ aY~
~ructose and Lactobacillus fermenti, and, for practical purpose, an enzyme obtained from ~3~ EY~ Glucose .
isomerase is soluble in water and therefore, where the s conversion of glucose into fructose is enzymatically : ;
conducted, it is convenient that such enzyme is sub~ected to an appropriate treatment to obtain an insolubilized glucose isomerase which can likely be used as a catalyst , :20 in solid ~orm. Various processes for such treatment have !
already been proposed. For example, microbial cells containing glucose isomerase are treated at a specified ,, . , ~
temperature to ef~ect intraccllular fixation o~ the isomerase such as disclosed in U.S.P. 369431~, glucose .:`` ;
; 25 . lsomerase isolated ~rom the cells is adsorbed on an .. ;.: :anione exchanger, such as DEAE-Sephadex [Journal of :
Society of Japan Foodstuff Industry 14-12 p.539 - 540 (1967)~ and DEAE-cellulose (U.S.P. 3708397). However, . such processes involve inherent disadvantages, for example, .
~,~ 30 as ~ollows: The insolubilized glucose isomerase according * Trade Mark "

~45~99 to the former process requires a long time for carrying ou~ the conversion of glucose into fructose and the product solution is hi~hly colored whereby a complicated purifica-tion and decolora-tion treatments are required, thus this process is commercially disadvantageous. On the other hand, in the latter process DEAE-Sephadex and DEAE-cellulose wh,ch are an insoluble carrier but swell in water to become bulkier, this results not only in less isomerase per unit volume of the carrler being adsorbed ~¦ 10 but causes substant~al pressure drop and loss during ~ passage of the glucose solution through a packed column;
¦ thus this process is also disadvantageous~
Another approach is that a synthetic anion exchange resin ia used as a carrier on which glucose isomerase is ~ 15 adsorbed to become in insolubilized form, for example, ;I U.S.P 3788945 discloses a porous anion exchange resin, such as Amberlite IRA938, adsorbed glucose isomerase with which glucose is isomerized. Although an ion exchange resin has less tendency to swell in water, so, if such an ion exchange resin is used as a carrier for the iso-~; merase, it is e~pected that the isomeriza-tion reaction is e~fectively carried out. However, the amount o~ the isomerase to be adsorbed on a conventional ion exchange ~ resin is small and the degree of isomerase retention on ¦ 25 the resin is low. In this connection, the conclusion is that the prior process in which the isomerase is insolu-bilized b~ the adsorption on an ion exchange resin is not satisfactory for commercial purpose.
An object of this inventio~ is, accordingly, to provide an insolubilized glucose isomerase adsorbed and * Tra~e Mark $
~ 3 ~ ~`'' , ' .:

,, . .~ : , 3 (~45~9 bound on an ion exchange resin, such insolubilized iso-merase involves a number of advantages, for example~
more isomerase is adsorbed on the ion exchange resin,

(2) higher enzyme activity o~ the produc-t in the iso-merization operation, (3) higher resistance to crush and distortion, and (4) less pressure loss as the glucose solution passes through the column containing the insolu-bilized isomerase.
A further object is to provide an insolubilized glucose isomerase comprising a glucose isomerase adsorbed and bound on a porous anion exchange resin ha~ing a porosity of more than 4.5%, in particular 7 ~ 50% and preferably 7 ~ 20% measured according to the aqueous dextran solution me-thod and an ion exchange capacity of more than 0.035 ~
~ meq/ml-Resin and preferably more than 0.05 meq/ml-Resin in particular 0.05 ~ 0.1 meq/ml-Resin measured according to the polyanion salt decomposition method.
The procedures for carrying out the aqueous dextran ; ~
solution method and the polyanion salt decomposition method ~- :
referred to in the speci~ication and claims are defined as follows.
A~ueo~ls dex-tran solution metllod ~: ;
Into a jacketed column o~ 11 mm in internal diameter is packed 67 ml of S04 type anion exchange resin having a void ratio of 33~ in wet state to form a resin bed which `:-is maintained at a temperature of 50C to remove any excess water, a 1.5 wt% aqueous solution of dextran having a weight average molecular weight (M~) of2,000,000 determined by a light scattering method is passed through the resin bed at a space velocity ~SV) of 0.4 hr l and each 2 g of , : .

- 4 - ~

.. .. . . ..................... ..
. . . .. . : .

~0 ~ 5 9 ~ 9 the e~fluents is fractionated. The concentration of each effluent fraction is measured by a refractometer. The quotients obtained by dividing the volume of the effluent (Vf) by the volume of the resin bed (Vb) and the quotients obtained by dividing the dextran concentration of each eff}uent fraction (C) by the original dextran concentra-tion of 1.5 wt% (Co) are plotted on the horlzontal axis and the vertical axis, respectively, to obtain a curve.
The same procedures as above are repeated using a 1.5 wt% aqueous solution of dextran having a weight average molecular weight o~ 10,000 determined by a light scatterlng method to obtain a curve. ~ -Then, the porosity is determined by dividing by the factor of 0.67 the difference between the values of Vf/Vb at the points where C/Co is 0.5 for each aqueous dextran solution, the molecular weight being 10,000 and 2,000,000~ usually the porosity is expressed by the term of percentage, so the value determined as above is multi- ~ `
plied by the factor of 100.
The porosity of the resin in the specification and claims is measured by using a Kiriyama column available ~rom Kirlyama Seisakusho,Japan, Dextran T2000 having a weight average molecular weight of 2,000,000 measured by `
light scattering me-thod and ~extran T10 having a wei~ht average molecular weight of 10,500 measured by light scattering method available from Pharmacia Fine Chemicals AB, Uppsala, Sweden and immersion liquid refractometer type T available from Karl Zweiss A.G.
In measuring the porosity of resin, it should be appreciated that the variation in the molecular weight of - 5 - ;~

. . . .

~iL0459~9 dextran by + lO~o from the abovementioned ranges does not essentially affect the result 9 the narrower the distribu-tion of the molecular weight the better the result but no or little substantial e~ect is observed by such ~ ~
variation in distribution ~; ;
Polyanio~ salt decomposltion method A given amount of polystyrene having a number average molecular weight (Mn) of lO,OOO measured by a vapor pressure method and a value of less than 1.06 obtained by di~iding a weight average molecular weight measured by light scattering method by a number avera~e molecular weight is subjected to sulfonation with lO times by weight of 98% sulfuric acid and 0.01 time o~ ~ilver sulfate as catalyst at a temperature of 100C for 5 hours.
The reaction product is brought to neutral by addition of aqueous ammonia9 and water is removed in acuo to obtain a solid product which is then extracted with methanol.
From the extract methanol is removed in vacuo to obtain ammonium poly(stylenesulfonate). One gram of ammonium poly(stylenesulfonate) is dissolved in demineralized water to obtain one liter of an aqueous solution. The solution ~ `
is passed for 5 hours through a jacketed column of 8 mm in internal diameter and packed with lO ml of OH type anion exchange resin maintained at a temperature of 25C at a ra-te of lOO ml/hr. The efM uent from the column in an amount of 500 ml is titrated with l/lON hydrochloric acid using methyl orange as an indicator. The quotient obtained by dividing the amount of the hydrochloric acid required for titration (ml) by the factor of lOO is the ion exchange capacity (meq/ml_Resin).

; - ..... ., . , . . ~ . . , .,. ~ , .
- . . - . . - . -- . . : . . , 1'~)45~99 The resin employed in measuring the ion exchange capacity is Mono-Disperse Polystyrene Standard (Mw = 10,000 and ~w/Mn ~: 1,06) available from Pressure Chemical Co"
Ltd. Variation in the average molecular weigh-t by ~ 10%
from the abovementioned range does not essentially e~fect the results.
Although the mechanism by which glucose isomerase is adsorbed on a macroporous ion exchange resin and the way the enzyme activity is retained have yet not been clarified in detail, it is believed that there are physical adsorption in the macropores of the resin and a certain -chemical bonding force between anion exchange groups and ;
the enzyme employed which results in synerigistio effect.
This seems to be true, since glucose isomerase is hardly adsorbed on a conventional gel type ion exchange resin which does not have any macropores and further a resin which has macropores but no ion exchange group can adsorb only a small amount of enzyme and the activity of enzyme is also low. Thus, it is believed that the amoun-t of ~0 glucose isomerase adsorbed on an anion exchange resin and the degree of the activity retention o~ the isomerase adsorbed are dependent upon the volume of macropores which determine the adsorption of the isomerase and the number .
of ion exchange groups in the maoropores.
The porosity measured by the aqueous dextran solu `
tion corresponds to the total volume of macropores having a specified size into which dextran of Mw being 10,000 can pass but dextran of Mw being 2,000,000 can not pass and such porosity has a close relationship with t~e amount of glucose isomerase which can be adsorbed on the ion ~4S999 exchange resin, therefore, it is believed that the adsorp-tion of glucose isomerase will occur or will occur mainly in such macropores of specified size. It is ~urther believed that the ion exchange capacity measured according to the polyanion salt decomposition method corresponds to the ion exchange capacity at the whole surfaces of such specific macropores.
A conventional anion exchange resin prepared by copolymerizing a vinyl monomer, such as styrene, with a -cross-linkable monomer, such as divinyl benzene, followed by introducing anion exchange group has some micropores and there~ore, it is believed that a polymeric material can contact with only the outer surface but not with the internal sur~ace o~ the micropores. In this connection, the porosity and the ion exchange capacity above specified ., .
o~ such conventional anion exchange resin which is a gel type i,5 far less than that of the resin of this invention.
In contrast, the anion exchange resin employed according to this invention is a macroporous resin and has a large internal surface area.
.. ~ .
Such macroporous anion exchange resin is convenient-ly prepared by any processes which have already been known, for example, a monovinyl monomer and a cross-linkable monomer are copolymerized in the presence o~ any materlal which is removable by a solvent and is not taken place the reaction, such as polystyrene, a~ter the completion o~ the polymerization, the resin obtained is treated with a solvent to extract the material, e.g. polystyrene, and ;~
then anion exchange group is introduced. The anion ~0 exchange resin thus produced has~ in general9 macropores `

: . . .

~ ~ 5 ~ 9 9 the radius of which ranges from about 101 to 104 Angstroms (A). However, it should be noted that among the pores, smaller pores can not adsorb glucose isomerase whereas much larger pores the internal surface of which is similar to the surface of the gel type anion exchange resin also do not take part in adsorption. This is true, since a certain anion exchange resin which has higher porosity measured according to mercury penetration method does not necessarily indicate higher adsorption ability for the isomerase. In general, although the pore size and the total pore volume of the macropores in an anion exchange resin may vary depending upon the conditions under which the resin is prepared, such as the amount of a cross-linkable monomer and the amount of and the molecu-5 lar weight of a polymer which is added initially in the `
polymerization mixture, it is difficult to determine the definite relationship of the pore size and the total pore volume to the preparation conditions.
In this connection, it is desirable to select optimum conditions under which the most appropriate resin is produced by experimental procedures in which various resins are produced by varying preparation conditions and the porosity and the anion exchange capacity are measured according to the abovementioned methods.
In order to lntroduce anion exchange groups into a matrix resin, it is preferable that the matrix resin ;
be subjected to treatment to introduce chloromethyl group followed by treating with various amine compounds, for example, an aliphatic amine such as trimethylamine, dimethylethanolamine~ethylenediamine,diethylenetriamine _ g _ ~:

. ''~ .

~045~9g ::

and triethylenetetramine, and a cyclic amine, such aspyrrolidine, morphorine and piperidine. However, it is appreciated that if a weak basic amine is employed there is observed a tendency to a little release of the adsorbed isomerase from the resin, so it is desirable to use a tertiary amine, such as trimethylamine and dimethyl- ~ ~
ethanol amine, to convert the resin into a quartanary ;
ammonium type.
The glucose isomerase which may be employed accord~
ing to this invention is any of conventional ones produced in the cells of an actinomycete belonging to Streptom ces, ~ -such as St. phaeochromo~enus, St. fradiae, St. roseo-S~ ~3~ St olivaceu_, St. cali~ornicus, St. vinaceus and St. albus. The extraction of glucose isomerase from the microbial cells can be carried out by any suitable known process, such as ultra-sonic treatment, pressurized treatment, mechanical treatment, autolysis and by enzym-atic treatment withl~sozyme7in particular7 the extraction ;~
by lysozymeiS recommended since it results in higher extraction of glucose isomerase from the cell, shorterextraction time and a small amount of impurities in the extracted solution.
The glucose isomerase is adsorbed on the macroporous ion exchange resin by any conventional process which is employed for the treatment of an ion exchange resin. The simplest way is to immerse an anion exchange resin in an aqueous solution of glucose isomerase extracted from the mlcrobial cells ~or a period sufficient to effect adsorp-tion o~ the isomerase (optionally stirring) and then wash with water, however, it is preferable to pass and recycle 1~4599~
the aqueous glucose lsomerase solution upwardly through a column packed with the anion exchange resin to fluidize the resin particles to effect the absorption. ~ ;
Any condition under which glucose isomerase is not ;
inactivated remarkably can be employed according to this invention, in general, at a pH of 5 5 to 9.5, at a temper-ature of 0 to 6~C for 2 to 18 hours, which time, however, ~
may vary depending upon the concentration of the resin ~ ~-slurry, the concentration of glucose isomerase and the ~`
amount of the isomerase solution employed. The anion exchange resin on which the glucose isomerase is to be adsorbed may be of any type, such as a free type or a salt type and, the resin is pretreated with an aqueous solution of, such as, sul~uric acid, hydrochlorio acid, ;~
phosphoric acid and acetic acid to convert it into a ;
salt type capable of effectively adsorbing isomerase.
The anion exchange resin according to this invention ;
posses~es high adsorption ability for glucose isamerase.
The amount of glucose isomerase adsorbed on the resin ~s more than 400U, in particular 700 to 3000U and pre~er-ably 1000 to 2000U of isomerase per 1 ml of the wet resin and in some cases moreO In general, the larger ~he amount of isomerase adsorbed the higher the activity of the insolubilized isomerase, there is observed a tendency to decrease the degree of bonding the isomerase as the amount of the adsorbed isomerase increases. Therefore, the optimum amount of the adsorbed isomerase is determined taking into consideration the degree of bonding, the degree of activity retention and operable life of the product.
The insolubilized glucose isomerase thus produced ~ .

,: . . ~ . . . . . . . .i. .

104S9~9 posse~ses a number o~ advantages which make it possible to carry out the isomerization of glucose into fructose effectively on a commercial basis, for example, higher activity for isomerization of glucose) little or no activity loss for a long time of period, no appreciable ;~
release of the isomerase from the carrier resin and no or little coloration of the resulting product. The iso-merization is conveniently carried out by a fixed bed system but because the insolubilized isomerase according to this invention has high mechanical strength and is easy to separate from a slurry it can be employed in f~ zed bed system, transfer bed system and batch system with agitation.
When, the activity of the insolubilized glucose isomerase according to this invention lowers after being used a long time, it is easy to regenerate by releasing the isomerase from the resin by the treatment of an aqueous salt solution such as aqueous sodium chloride followed by adsorbing fresh isomerase on the resin to obtain insolubilized glucose isomerase wi-thout loss of activity.
The insolubilized glucose isomerP~se according to this lnvention contains a large quantity of isomerase adsorbed and bound on the resin and can be activated to high activity, therefore, the use of such isomerase in the glucose isomerization results in many advantages including (1) less anion exchange resin is required ~or a given amount of fructose, (2) higher space time yield is achieved, (3) due to shorter dwell time of glucose solution in the column, less coloration and lowering of ~ 12 ~

.

~0 4 5 9 9 9 the pH of the product is realized and (4) the pressure drop in the columr.s is decre~sed. Thus the insolubili~ed glucose isomerase according to -this invention is suitable for use in the conversion of glucose into fructose.
This invention will be explained in detail refer-ring to following Examples without intention to limit this invention.
In the Examples, the activity titers of glucose isomerase extracted and insolubilized glucose isomerase, the degree of activity retention and the degree of bound isomerase are determined as follows.
1. Activity titer of ~lucose isomerase extracted 0.2 ml of 1 M aqueous D-glucose solution, 0 2 ml of 0.05 M aqueous MgS04-7H20 solution, 0.2 ml of 0.5 M
aqueous phosphate buffer solution (pH = 7 2) and aqueous solution of extracted glucose isomerase are mixed to prepare an aqueous solution which is diluted with water to make it to 2 ml. The mixture is maintained at a temperature of 70C for 60 minutes to effect the conver-sion which is terminated by the addition of 2 ml of 0.5 M
perchloric acid. The amount of fructose produced is determined by cystein carbaæole method. The value obtained by dividing the amount of fructose produced by the amount of the glucose isomerase solution extracted is actlv~ty titer the unit of which is expressed by the abbreviation "U".
2. Activit titer of insolubilized lucose isomerase F ~
To ~ of an aqueous solution containing, in a ~ ;
concentration, 0.1 M of glucose, 0.005 M of MgS04.7H20 .. :
and 0.05 M of phosphate butter is added. A given amount : ~

~L04599g ~ ~-of an insolubilized glucose isomerase and the mixture thus obtained is slowly stirred at a temperature of 70C
for 60 minutes to e~fect the conversion. Then, the insolubilized isomerase is separated from the reacted solution and the amount of ~ructose so produced ls deter-mined according to the cystein carbazol method. The activity titer is calculated as in above 1 ~;
The unit "U" means the amount of glucose isomerase capable of producing one milligram of fructose under the -~
defined conditions.

3 The degree of bound ~lucose isomerase ., ~
The total titer of glucose isomerase solution to be used ~or adsorption on an anion exchange resin is measured and this value is designated as "A"~ After adsorption of glucose isomerase on an anion exchange resin, the resin is separated by filtratLon and wa~hed with water, then the total titer of the combined ~ilterate and wash water is measured and this value is designated ~ -as "B".
me degree of bound glucose isomerase ~D.B.G.) is given by the following equation:
DBG = ~ x 100 (%)

4. Degree of activity retention of insolubilized isomerase The activity tlter o~ insolubilized glucose iso-merase measured according to 3 above is divided by the activity titer of extracted glucose isomerase solution to be adsorbed on the resin the quotient multiplied by 100 is the degree of activity retention e~pressed as a percentage.

~ .

lV~5999 Examples 1 to 12 One hundred grams of glucose isomerase "NAGASE", which is available from Nagase Sangyo Kabushiki Kaisha, Osaka, Japan and is produced from St. phaeochromogenus~
was suspended in 700 ml o~ demineralized water and, after addition of 80 mg o~ crystallin~of egg white lysozyme, -the suspension was agitated at 40C for 45 hours and centrifuged to obtain an extracted solu~ion of glucose isomerase. It was found that the activity titer of the extracted solution was 200 U/ml. Then, each 50 ml o~ the extracted solution (the total activity titer being 10,000 U) was mixed with each of anion exchange resins listed in Table 1 the bed volume of which was 10 ml in wet condition and the mixture was agitated at 50C ~or 6 hours to effect adsorption of isomerase on the resin to obtain an insolu-bilized glucose isomerase.
The characteristics o~ the insolubilized isomerase are also given in Table 1. ;
Table 1 deals also with Comparative Examples refer-red ~o by letter~.
In Examples 7 and 8 and Comparative Examples ~ toE, the anion exchange resins employed were commercially a~ailable.
The anion exchange resins employed in Examples other than Example l4 were produced by suspension poly-merization technlque using a suspension of styrene, ~ ~ ~
div~nyl benzene, polystyrene and toluene in water and ;
be~zoyl peroxide as catalyst, ~ollowed by subjeçting particles of styrene-divinyl benzene copolymer to chloro~
methylation and introducing each of anion exchange groups * Trade ~lark ~`1~ ' . .

, . - . . . j .. . .

indica-ted in Table 1 The re~in of Example 4 was produced by polymerizing a mixture o.~ styrene, divinyl benzene and n-heptane using benzoyl peroxide as catalyst to obtain particles of styrene-divinyl benzene copolymer followed by chloro~
methylating and introducing anion exchange group. :
Resin types of SO4 , PO4 , OH , Cl and CH3COO
indicated in Table 1 mean that anion exchange resins were treated with 2N-sulfuric acid, lN-phosphurîc acid, 2N
sodium hydroxide, 2N hydrochloric acid and lN acetic acid, respectively.
According to the aqueous dextran solution method, the porosity of the anion exchange resin employed in Example 1 was measured. The curves showing relationsh.~ps between C/Co and Vf/Vb of dextrans oP molecular weights j o~ 10,000 and 2,0007000 are shown in the attached drawing, 1~34S999 Table 1 I Characteristics of ion exchange resin Example and Com-' Degree j ITinal l ExamPle I Cross- IAnion exchange group iexchange ¦Po Y
No. ¦ linkage(~ capacity I (%) I (meq/ml) , (%) , i _ ~
1 10 Trimethyl ammonium ¦O.79 10.4 type 2 10 " 0.95 10.0 3 10 " 0.79 19.4 4 15 ,. 0.57 19.4 " 0.67 7.5 6 25 Dimethylethanol 0.60 ii13.0 ~ :
ammonium type 7 _ Trimethyl ammonium 1.03 16.4 ::

8 _ Dimethylethanol _ 16.4 . .
ammonium type , 9 10 Trimethyl ammonium 0.79 10.4 ;: ~;
~ ~1 ll ll .'; ~
11 ll 1~ ll tl ~ `
12 ~ " ~ "

A _ Dimethylethanol 1.01 8.7 ammonium type B _ ,. 0.91 15.7 . .
C _ Trimethyl ammonium O.53 O.7 :: :
type D 8 ,l ~1.3 0 E !lo . ll ~1.3 ! 3 0 ote (1) The percentage of the weight of divinyl monomer to the total weight of monovinyl monomer and divinyl monomer. `
~, , '., - 17 _ ...

~4S999 Insolubilized .glucose i~omerase _ _ ~
Ion Degree Note exohange Type of Activity capacity of the bound retention : :
matrix ~
tmeq/ml) ~yO) (%) , ~ : ' . . _ , . .
O.074 S042- type ¦ 99.9 91.0 :~

0.088 !1 99.7 92.1 .
0.039 ll 93.7 88.2 `~
0.061 " 99.~ 91.5 ~::
~.070 ll 96.3 91.1 ;
0.096 ll 99.9 88~5 O.038 u 46.7 78.9 Amberlite IRA-900 O.042 n 72,3 61.7 Amberlite IRA-93 0.074 ~ PoL~3 type ~ 99, ~ as . O

OH- type 95.Q 80.2 " Cl- type 100.0 90.3 " CH3C00 type 82.4 84.7 ~.

0~028 S04 type o _ Amberlite IRA-910 O.027 " O _ Amberlite IRA-911 O,034 n 10 45 Amberlite IRA-938 0,028 ll 0 _ Diaion SA-#100 0,030 " . 7 , ~ Diaion PA-320 .

- 17a -, .

.. ~
.. . . . . . -. . .. . .

s~9 Example 1 A glucose isomerase producing strain of ~ æ~Y~
albus YT-No. 5 which had been deposited with The Fermenta-tion Research Institute, Japan as FE~M-P-No. 463 was inoculated on 80 ml of a culture medium which was an aqueous solution containing 1 wt% of polypepton, 0.3 wt%
of K2HP04, 0.1 wt% of MgS04 and 1 wt% of xylose and cultl~ation was effected a-t a pH of 7 and a temperature of 30C for 3 hours. Then 25 ml of the cultivated medium was transferred to 50 ml of a culture medium which was an aqueous solution containing 3% of wheat bran, 2% of corn steep liquor, 0.1% of MgS04.7H20 and 0.024%, by weight, of CoC12-6H20 and cultivation was continued at a temperature of 30C for 30 hours. Subsequently, the -cultivated culture was centrifuged to collect cells from which, after washing with water, glucose isomerase was extracted according to the procedures as in Example 1.
The extracted isomerase was adsorbed on the resin as in Example 1 following the procedures of Example 1.
It was found that in the insolubilized isomerase the degree of bound isomerase was 99 2% and the activity retention was 88.8%.
~ ' ,:
The cells of ~3~ y~ ollvaoeus were sub~ected to a ultrasonic treatment under the waves of 10 kilocycles per second for 10 minutes9 using ultrasonic generator Type N-50-3 available from Toyo Riko Seisakusho, Tokyo Japan, to obtaln extracted glucose isomerase solution~ One ~-hundred milliliters of such extract, the total activity titer being 500 U, was mixed with 4 ml of the resin employed : . . . ; .. .. :. .

~ 04S9g9 in Example 4 and the mixture was agitated at a temperature of 50C for 15 hours. It was found that the insolubilized glucose isomerase thus obtained had a degree of bound iso-merase and an activity retention of 90% and 87%, respec-tively.
Example 15 An extracted glucose isomerase solution obtained following the procedures of Example 1, the activity titer being 200 U/ml and the volume being 120 ml, and 10 ml of the anion exchange resin employed in Example 3 were mixed and agitated at a temperature of 25C for 6 hours. The insolubilized glucose isomerase so obtained had an degree of bound isomerase of 91% and an activity titer of 2026 U/ml, its activity retention being 94%.
Example 16 The procedures of Example 1 were repeated excepting that 190 ml of an extracted isomerase solution (260 U/ml) and 10 ml of the resin werè mixed and agitated at 50C for 18 hours. The degree of bound isomerase and the activity titer of the insolubilized glucose isomerase were 95.4%
and 2400 U/ml with activity retention being 66.2%.

, . : . , . , .: . . , . , . - .
,............ . , . . . ~ .

Claims (13)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An insolubilized glucose isomerase comprising glucose isomerase adsorbed and bound on a porous type anion exchange resin having a porosity of more than 4.5% measured according to the aqueous dextran solution method and an ion exchange capacity of more than 0.035 meq/ml-Resin measured according to the polyanion salt decomposition method, and the resin having a matrix being a styrene-divinyl benzene copolymer and an anion exchange group being a quaternary ammonium group.

2. An insolubilized glucose isomerase according to claim 1, wherein said glucose isomerase of more than 400 U/ml-Resin is adsorbed on said resin.

3. An insolubilized glucose isomerase according to claim 1, wherein said glucose isomerase of from 700 to 3,000 V/ml-Resin is adsorbed on said resin.

4. An insolubilized glucose isomerase according to claim 1, wherein said glucose isomerase of from 1,000 to 2,000 U/ml-Resin is adsorbed on said resin.

5. An insolubilized glucose isomerase according to claim 1, wherein said anion exchange resin has an ion exchange capacity of more than 0.05 meq/ml-Resin.

6. An insolubilized glucose isomerase according to claim 1, wherein said anion exchange resin has a porosity of from 7 to 50% and an ion exchange capacity of from 0.035 to 0.3 meq/ml-Resin.

7. An insolubilized glucose isomerase according to claim 1, wherein said anion exchange resin has a porosity of from 7 to 20% and an ion exchange capacity of from 0.05 to 0.1 meq/ml-Resin.

8. An insolubilized glucose isomerase according to claim 6, wherein said anion exchange group is trimethyl ammonium or dimethylethanol ammonium.

9. An insolubilized glucose isomerase comprising glucose isomerase adsorbed and bound on a porous type anion exchange resin in an amount of from 700 to 3,000 U/ml-Resin, said resin having a porosity of from 7 to 50% measured according to the aqueous dextran solution method and an ion exchange capacity of from 0.035 to 0.3 meq/ml-Resin measured according to the polyanion salt decomposition method, and resin having a matrix being a styrene-divinyl benzene copolymer and an anion exchange group being a quaternary ammonium group.

10. An insolubilized glucose isomerase according to claim 9, wherein said resin has a porosity of from 7 to 20%
and an ion exchange capacity of from 0.05 to 0.1 meq/ml-Resin.

11. An insolubilized glucose isomerase according to claim 9, wherein said quarternary ammonium group is trimethyl ammonium type or dimethylethanol ammonium type.

12. A process for isomerization of glucose into fructose comprising contacting an aqueous solution of glucose with insolubilized glucose isomerase and recovering an aqueous solution containing fructose, said insolubilized glucose isomerase comprising glucose isomerase adsorbed and bound on a porous type anion exchange resin having a porosity of more than 4.5% measured according to the aqueous dextran

Claim 12 continued:

solution method and an ion exchange capacity of more than 0.035 meq/ml-Resin measured according to the polyanion salt decomposition method, and the resin having a matrix being a styrene-divinyl benzene copolymer and an anion exchange group being a quaternary ammonium group.

13. A process for isomerization of glucose into fructose comprising contacting an aqueous solution of glucose with insolubilized glucose isomerase and recovering an aqueous solution containing fructose, said insoluble glucose isomerase comprising glucose isomerase adsorbed and bound on a porous type anion exchange resin in an amount of from 700 to 3,000 U/ml-Resin, said resin having a porosity of from 7 to 20% measured according to the aqueous dextran solution method and an ion exchange capacity of from 0.05 to 0.1 meq/ml-Resin measured according to the polyanion salt decomposition method, and the resin having a matrix being a styrene-divinyl benzene copolymer and an anion exchange group being a quaternary ammonium group.