CA1104110A - Enzyme-immobilization carriers and preparation thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Enzyme-immobilization carriers are provided herein.
They comprise a macroporous amphoteric ion-exchange resin having a cation-exchange capacity due to carboxymethyl groups of 0.5 meq/g-dry resin or more, an anion-exchange capacity of 1 meq/g-dry resin or more, and a specific area of 1 m2/g-dry resin or more, the total volume of macropores having a diameter of 100 .ANG. to 2000 .ANG. being 0.1 cc/g-dry resin or more.
A novel process for producing such carriers is also provided.
Such resins are superior as carriers for enzyme immobilization, and provide immobilized enzymes having great stability.

Description

The present inyention relates to carriers ~or immobilizing enzyme thereto (referred ~~o as "enzyme-immobiliza~

tion carriersl' hereina~er~ and to processes for the prepara-t1on thereof. More par-ticularly~ it relates to enzyme-immobiliæation carriers which comp~ise a macroporous syntheti~
polymer having both anion~ex~hange groups of 1 meq/g-dry resin or more and carboxymethyl groups of 0.5 me~!g-dry resin or - more (in the present invention, all the terms "resin" mean synthetic resin other than polysaccharides and derivatives thereof) and to processes for the preparation thereof.
Because of the usefulness of immobilized enzymes in industrial uses, a number of -techniques for enzyme-immobiliza~ion have recently been developed /C~R. Zaborsky: Immobilized Enzymes (published from C.R.C. Press, 1973)/ . Therefore a large number of enzyme-immobilization carriers are well kn~wn.
Of these carriers, polysaccharides and their derivatives, for example, celluloses, crosslinked dextrans and ionic derivatives thereof, are widely used and some of them gain a fair success.
Such polysaccharides have many drawbacks e.g., as follows:

mechanical strengths are poor; in column operation~ a sufficient flow rate is difficultly obtained and blocking is easy to occur; and polysaccharides are easily attacked by microorganisms.
~Further, when enzymes are immobilized to ionic polysaccharide derivatives through an ionic lihkage~ -they are easily released therefrom by reaction solutions or product solutions having a little high concentration of electrolyte. This is a serious problem in practical use.
On the other hand, using ion-exchange resins as \

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-2-an en~me-immobilization carrier are already known in the latter half o~ 1950 /J. Amer, Chem. Soc, ? Vol. 81, 5133~5136 (1959~ . The'amoun-t of enzyme immobilized per u~it weight of carrier is very small and the activity of the resulting immobilized enzymes ]s verY low so tha-t prac-tical value was regarded as very low. Ion-exchange resins made of synthetic resins as matrices are however more-superior to polysaccharides and derivatives thereof in many points described below: mechanical strengths are high; the resins withstand long-term operation in large columns with a relatively low degree of damage;
sufficien-t flow rate can be ensured in column operation on account of a suitable particle size; resistance to attack of microorganisms is high; and cost is low.
An object of one aspect of the present invention is to provide enzyme-immobilization carriers which provide immobilized enzyme-immobilization carriers which provide immobilized enzymes having both high activity and long life time (stability), and besides which enable enzymes to be immobilized ,in large amounts per unit weight of carrier, and to provide processes for the preparation thereof.
An objec-t of another aspect of the present inven-tion is to provide enzyme-immobilization carriers suitable for ,industrial use which immobilize enzymes which are in -themselves a catalyst for ~action in homogeneous aqueous media, thereby -- enhancing the stability of the enzymes and making -them suita-ble for repeated or continuous use, and to provide processes for the preparation thereo.~.
By one broad aspect of this invention, an enzyme~
immobilization carrier is p~ovided which comprises a macroporous ' 30 amphoteric ion-exchange resin having a cation-exchange capacity

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due to carboxymethyl groups of 0.5 me~/g-dry resin or more, an anion-exchange capacity of l meq/g~dry resin or more, an a specific area of l m2~g~ry resin or mo~e, the -total volume of macropores having a diametbr of lO0 ~ to 2000 A being 0.1 cc!g~dry resin or more, By one variant thereof, the specific ~urface area of the resin ~s 5 m ~g~dry resin or more.
By another variant, the mean pore diameter of the pores in the resin is from 150 A to lO00 A.

By yet another variant, the total volume of macropores having a diameter of lO0 A -to 2000 A is 0.2 cc/g-dry resin or more.
By another aspect of this invention, a process is provided for producing an enzyme-immobiliza-tion carrier, the process comprising reacting a compound of the formula, wherein X is ahalogen atom and '~ is a hydrogen a-tom or an alkali metal, with a macroporous anion-exchange resin having a functional group capable of reacti~g with such compound, an anion-exchange capacity of 1 meq/g-dry resin or more, and a specific surface area of l m2/g-dry resin or more, the total volume of macropores having a diameter of lO0 A to 2000 A
being 0.1 cc/g-dry resin or more, in the presence of an alkaline compound, whereby 0.5 meq/g-dry resin or more of carboxymethy~
groups is introduced into the macroporous anion-exchange resin to obtain an amphoteric ion~exchange resin.
By one variant thereof, the compound is chloroacetic acid or sodium chloroacet~te.
By another varian-t, the compound is used in an amount of l/2 to 10 parts preferably in an amount of 2/3 -to 3 parts based on l part of -the dry resin.

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By another varian-t, the alkaline compound is an alkali met~l hydroxide,preferably sodium hydroxide~ an alkaline earth metal hydroxide or an organic amine, By yet another ~ariant, the alkaline compound is used in an amount of 1~3 to 2 times by mole ~ased on said compound.
By s-till another-variant, the anion-exchange resin has a hydroxyl, a primary amino, a secondary amino, an imino or an sulfhydryl group.
By a s-till further variant, the specific surface area of the resin is 5 m2!g~dry resin or more.
By yet another var1an-t a the mean pore diameter of the pores in said resin is from 150 A to 1000 A .
By a further ~riant, the total vo]ume of macropores having a diameter of 100 A to 2000 A is 0.2 cc/g-dry resin or more.
By another aspect of this inven-tion, a process is : provided for immobilizing an enzyme on an enzyme-immobilization carrler which comprises immobilizing an enzyme on the enzyme-immobillzation carrier as described hereinabove.

By variants of this process, the enzyme may be carried on the resin by an adsorption method or by a covalent attachment method.
Extensive s-tudies have been carried to by the inventors to develop carriers, by making use of the advantages of ion-exchange resins, which provide immoblized enzymes havlng high activity and long life time as catalysts 7 and moreover which enable enzyme$ to be immobilized thereto in large amoun-ts.
As a result, i-t was found that the so-called macroporous resins or macroreticular resins which are made of amphoteric -- 30 ion~exchange resins having bo-th carboxymethyl (referred to as ~'C~" herein~ter~ groups haYing unique a~finity to many enzyme proteins and anion-exchange g~oups, e,g., a primary ?
secondary or tertiary amino group or quaternary ammonium group, and besides which have a large number of macropores o~ 100 A to several thousand A in diameter and therefore have large specific surface area and pore volume, are a superior carrier satisfying the aforesaid requirements. Par-ticularly, such macroporous or macroreticular resins proved to be a superior carrier in practical use since they provide immobilized enzymes which are very long in life time (s-tability in operation) regarded as par-ticularly an important factor in a long-term continuous operation with emmobilized enzymes.
It is theorized tha-t amphoteric ion-exchange resins having both CM groups and anion-exchange groups (e.g., amino group ) are superior as carriers for enzyme-immobilization and provide immo~ilized enzymes having long life time (stability), for the Following reasons. Because of the similarity of the ion-exhange groups in the carrier to the ionic groups (i.e., carboxyl groups and amino groups) in the enzyme molecules, I
a~inity between carrier and enzyme probably becomes large. I
The ampho-teric ion-exchange resins used as a carrier in aspects of the present invention are produced by various processes, but a simple and desirable process is -the introduction of CM groups into macroporous anion-exchange resins.
The introduction of CM groups is achieved by reacting a compound of the formula, XCH2 CooY
wherein X is a halogen atom and Y is a hydrogen atom or an alkali metal, with a macroporous synthetic resin having an hydroxyl, a primary amino, a secondary amino, an imino or a sulfhydryl group, or a deriva~ive thereof in the presence o:F an - alkaline co~pound, In orde~ -to obtain highly CM-subs-tituted resins~ it is essenti~al to wet the resin with the reaction solution far into the ~nner parts-of the ~acropores. When the CM_substitution is ins-ufficient a-t one reaction, it is well achieved by~repeating the reac-tion ewice, three times and so on. 0~ the CM-reagents monochl~roacetic acid and i-ts ~odium salt are most favorable in terms o~ reactivity and economy.
The amount of CM-reagent used depends upon the degree of CM-substitution of the resins, but less CM-substi-tuted resins are not important in accordance with aspects of the present invention. For allowing the reaction to proceed rapidly, it is generally favorable to use CM-reagents in excess of stoichimetric amounts. It is very difficult to know the exact mole numbers of the resin to be CM-substituted.
A preferred amoun-t of CM-reagent empirically obtained is 1~2 to 10 parts, preferably 2/3 -to 3 parts, based on 1 part - of dry resin. In the disclosure of aspects of the present invention, the dry weight of resin is measured as follows:
Anion-exchange resins and amphoteric ion-exchange resins are converted to the OH-form and H-form, respectively, and neutral resins having no ion-exchange groups are washed progressively with an acid, alkali and then a large amount of water; after conditioning, the resins are vacuum-dried at 60C for more than 6 hours and allowed to achieve constant~
weight at room temperature of 18 to 25C for more -than 2 hours; and then the resins are weighed. In -the description given hereina~ter? the weights of resin and carrier always mean-a dry weigh-t obtained by the above described procedure, îf not particularly referred to, As the alkaline compound, alkali metal hydroxides (e.g.
sodlum hydroxide, potassium hydroxide), alkaline earth metal -6a :: `
hydroxides (e.g, ~a~nesium hydroxide ? calcium hydx~oxide ?
and ln some cases organic amines Ce.g. triethylamine) are used. Among those, sodium hydroxide is most preferred.
The amount a~ alkaline compound used is preferably 1~3 to 2 times by mole based on t~e CM-reagent. In the CM
substitution, a side reaction producing glycolic acid proceeds.
When the amount of alkaline compound is too large, the side reaction increases and the efficiency of CM~reagent becomes poor. Accordingly, the most preferred amount of alkaline compound is 1/3 to 2 times by mole based on -the CM-reagent.
As the resins to be CM-substituted, the macroporous resins as described below meet the concept of aspects of the presen-t.invention ~hat the resins are a carrier for immobïlized enzymes having excellent~practical performances as industrial catalysts. That is the macroporous resins are such that they have one or more functional .
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groups selected from the group consisting of hydroxyl, ~-primary amino, secondary amino~ imino and ~lfhydryl groups, have a large number of macropores of 100 o to several thousand A in diameter in addition to micropores 5produced depending upon the degree o~ crosslinking and therefore have large pore volumes and large specific surface areas.
The macropores are physically produced by special polymerization method and are suitable for 10continuous operation since -they have physical strengths superior to gel type resins having micropores only~ 1 rl`he macroporous reslns are also called a MR type resin~ r MP type resin, macroreticular type resin or highly porous type resin. In order that the macroporous resins may 15clearly display an enzyme-immobilizing effect~ it is necessary that the specific surface area of the resins is at least 1 m2/g-resin, more-pre~erably 5 m2/g-resin or more, and besides that the total pore volume of the macro- i-pores having a diameter of 100 ~ to 2000 ~ is at least 200.1 cc/g-resin, more preferably 0.2 cc/g-resin. 'Lhe specific surface area is o~tainedby mea~suring the sur~ac~
area of dry resin according to the nitrogen adsorption method using the surface area measuring in.strumen~ of Carlo-Erbo Co. and calculating according to the B~T me~hod~
25The pore diameter and pore volume are obtained by measurement on the Hg-penetration porosi~eter of Carlo-~'rbo Co. and calculation on the assumption that the macropores are of` a cylindrical form having a circular crosssection.
Pores having a dlameter of more than 2000 ~ ~o not con- ~
tribute to the stabilization of immobilized enzymes since r . .

they have much la~ger dimensions than the enzymes. A _ preferred mean pore diameter depends upon the kind of enzyme to be immobilized, but generally it is often within the r range of 150 ~ to 1000 A.
r~acroporous resins meeting the above-described requirements can be produced by well-known processes, but ready-made articles (mainly made of anion-exchange r resins) are on the market and easily available. Many of such articles mainly have a hydroxyl, aliphatic primary amino or aliphatic secondary amino group, and those having .
a sulfhydryl, imino or aromatic amino group are very few.
Some examples of the articles and their physical and ohemical pro~ertie are snown in the iol1owing table.

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_~Et c/~ ~ a) ~ ¢ ~ a z ": -The upper limits of the spec-ific surface area and total pore volume can not strictly be determined, but ~oo large values lower the mechanical strengths of resin. Accordingly, the specific surface area is preferably 120 m Ig-resin or less and the total pore volume is preferably 80% or less of the resin volume.
As to the reaction solvent, using a large amount of water is not desirable, and it is desirable to confine the amount of water to 1 to 10 times by mole based on the alkaline compound and to add a water-miscible nonaqueous solvent in order to promote mixing of reagents. Such the solvent includes, for example, methanol, ethanol, acetone, dioxane, ; tetrahydrofuran and the like. The reaction temperature is preferably 50C or less in order to minimize side-reactions, and it is more prefera-bly 5 to 30C in order to elevate the efficiency of the C~-reagent as much as possible. Stirring is desirably carried out to such an extend that the reagents are well mixed but the resin is not crushed.
In view of various aspects of the present invention, the ion-exchange capacity due to CM groups of the present resins needs to be at least 0.5 meq/g~resin, more preferably 1.0 meq/g-resin or more. ~esides, the resins need to have anion-exchange groups of at least 1 meg/g-resin.
In the description of aspects of the present invention, the ion-exchange capacity was measured batchwise as follows: For measuring anion-exchange capacity, the resin was converted to the OH-form by conditioning, and the capacity was measured by neutralization titration and expressed in a value per unit weight of the resin which was dried and weighed in the same manner as described hereinbefore.

~easurement of ca-tion~exchange capacity was ~ame as aboye except tha-t the resin was converted to the ~I~form b~ condi-tion-ing, When the CM groups are introduced in-to anion-exchange resins, it is natural t~at the apparent anion-exchange capacity per unit weight of d~y resin becomes smaller than the original resins on accoun-t of weight increase due to introduced CM groups.
One of the features of -the macroporous amphoteric ion-exchange resins used in aspects of the present invention as an enzyme-immobilization carrier is that, so long as unreacted hydroxyl, primary amino or secondary amino groups are present in the resins, enzyme-immobiliza-tion by covalent attachment methods is also possible in addition to enzyme-immobilization by adsorption methods. The most remarkable feature of the resins as an immobilization carrier according to aspects of the present invention is that the stabili-ty ~long life time) of enzymes immobilized by adsorption method is very superior as is shown in the experimental examples described hereinafter.
This is very advantageous in -terms of indus-trial use of immobilized enzymes.
Preparation of immobilized enzymes by adsorption methods is achieved by the usual ways. For example, the resins of aspects of the present invention are first ac-tivia-ted with 0.0?M to 3M acid or alkali solution, or buffered with 0.02M
to 3M buffer solution showing a buffer action in -the vicinity of pH wherein an enzyme to be immobilized works well; the resins are well washed with water and well immersed in the enzyme solution so as -to wet the resins far into the inner parts of macropores, and then~ after s-tirring it necessary, the resins having immobilized `

enzyme thereon are filtered and washed with water. Temperature for adsorption-immobilization should be 40C or less, particularly preferably ; 10C or less, so long as enzymes to be immobilized are not e~tremely heat-resistant. The immobilized enzymes thus obtained generally contain enzyme protein of lO0 mg/g-resin or more, and they are stable so long as they are(not washed wtth salt solutions having a very high ionic strength.
Enzymes immobllizable to the carriers of aspects of the preseDt invention include not only enzymes comprising simple proteins alone but also enzymes requiring coenzymes and mixtures of one or more enzymes. Further, since the carriers of aspects of the present invention are amphoteric ion-ex-- change resins, both enzymes of acidic proteins and those of basic ones may be immobilized thereto.
In the enzyme-immobilization by covalent attachment methods, on the other hand, various attachment methods making use of the reactivity of hydroxyl, primary amino, secondary amino, sulfhydryl or imido groups in the present resins may be applied. Of these methods, the following ones are particularly suitable in the respects that they are relatively simple in operation and provide stable immobilized enzymes:
(1) Attachment method with s-triazinyl derivatives e.g. cyanuric chloride and its derivatives;
(2) Attachment method with glutaraldehyde;
(3) Attachment method by an azide linkage; and (4) Attachment method with monohaloacetyl derivatives.
In enzyme immobilization by covalent attachment methods, the amount of immobilized enzyme per unit weight of carrier is generally smaller than in the adsorption-- 13 _ ~ Y

immobilization. The immobili~ed enzymes by this method are superior in specific activity and stability in the presence of electrolyte solution of high concentration.
The enzymes immobilizable to the carriers of aspects of the present invention are not particularly limited, and any enzyme except those which lose enzy~ne activity by the immobilization may be used. For example, Pronase~ aminoacylase, glucose isomerase, lactase, nuclease, -amylase, isoamylase, pullulanase, urease, deaminase, lipase, esterase, trypsin and the like may be exemplified.
The present invention will be illustrated in more detail with reference to the following examples.
~xample 1 _ __ __ _ lO.0 g of DUO~IT~ A-7 was immersed in 70 ml of methanol, and a concentrated solution of 6.23 g of sodium hydroxide in 7 ml of water was added thereto. After well mixîng by stirring, the mixed solution was degassed, while being cooled with ice water, for 35 minutes by an aspirator to wet the resin far in the inner part of macropores.
Thereafter~ a solution of 8.68 g of monochloroacetic acid in 10 ml of methanol was added, and reaction was carried out at room temperature (24 + 1C.~ for 7 hours with slow stirring.
~ fter the reaction was finished, the resin was filtered, washed with two cycles of water, 0.5M aqueous sodium hydroxide solution, water and 0.5M aqueous nitric acid solution, and finally washed with water sufficiently.

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The resin was thlls converted to the H-form. Ion-exchange capacity of the introduced carboxymethyl groups was then measured.
Ion-exchanger capacity 4.82 meq/g-resin Weight increase by ~-substitut:ion 3.87 g (38.7%) It was confirmed by this weight increase that anion-exchange capacity was apparently decreased Erom 7.10 meq/g-resin to 5.17 meq/g-resin.
Example 2 C~-substitution was started under the same condition as in Example 1. After 3 hours, a solution of 1.42 g of sodium hydroxide and 3.35 g of monochloroacetic acid in a mixture of 5 ml of water and 10 ml of methanol was added, and reaction was continued for a further 4 hours. Cation-exchange capacity was 5.47 meq/g-resin and weight increase was 34.2~. (The reason why the ion-exchange capacity is not proportional to the weight increase in this example is not clear at present).
Example 3 Ten grams of ~UOLITE A-6 was immersed in a solution of 11 g of sodium monochloroacetate in a water (LO ml)-methan~l (60 ml) mixture, and the solution was degassed for 20 minutes by an aspirator while being cooled with ice water. Thereafter, a concentrated solution of 6.0 g of sodium hydroxide in 8 ml of water was added thereto, and reaction was carried out at 22 + 2C. for 6 hours with slow stirring. ~fter the reaction was finished, the resin was filtered, washed uith water and converted to the H-form by conditioning.
Ion-exchange capacity.of the introduced carboxymethyl groups was '`' :j ~

--3.10 meq/g-resin. Weight increase was 22.3%. Anion-exchange capacity after CM-substitution was 4.38 meq/g-resin.
Example 4 10.0 g of Dl~OLITE S-30 which was previously well wasl~ed with an acid, alkali and water and then dried was immersed in 35 ml of an aqueous solutlon containing 7.2 g of sodium hydroxide. The solution was degassed for 40 minu~es by an aspirator while being cooled with ice water and then 35 ml of water was added additionally. Thereafter, 50 ml of an aqueous solution containing 18.1 g of ~ -diethylaminoethyl chloride hydrochloride was added dropwise, over 2 hours with stirring, to the resin-sodium hydroxide mixed solution kept at room temperature of 22 + 1C. Reaction was continued for a further 7 hours (9 hours in total). After 9 hours, the resin was filtered, washed with 0.5M
aqueous nitric acid solution, 0.5M aqueous sodium hydroxide solution and then water, and immediately (without drying~ subjected to CM-substitution as follows All the resin obtained was imlnersed in a solution of 6.6 g of sodium hydroxide in a water (5 ml) - ethanol (40 ml) mixture, and the solution was slowly stirred for 30 minutes while being cooled with ice water; and 70 ml of a methanol solution containing 13 g of sodium monochloroacetate was added and reaction ~as carried Ollt at 21 + 2C. for 7 hours with slow stirring. After the reaction was finished, the resin was washed with two cycles of water, 0.5M aqueous sodium hydroxide solution, water and 0.5~ aqueous nitric acid solution, and divided into two. The one was well washed repeatedly with water to convert to the H-form. The other was washed once more .

` l with 0.5M aqueous ~odium hydroxide solution, and then L~
washed repeatedly with water to convert to the OII-form.
Cation-exchange capacity due to the introduced carboxymethyl F
group~ was 1.85 meq~g-resin/ and anion-exchange one due to the introduced diethylaminoethyl groups was 1.49 meq/g resin.
In using this amphoteric ion-exchange resln as a carrier for immobili~ation, all the resins were converted to the H-form.
Exam ~ L
Reaction coditions in which amphoteric ion-exchange resins were produced by introduction of CM groups inta resins having anion-exchange groups, and properties of the r resulting resins are shown in the following table. Prior to reaction, the matexial resins were immersed in an alkali solution or a Cl~l-reagent solution, and the solution was 1egassed for 30 to 60 minutes while being cooled with ice ~Y
water. lor measuring cation-exchange capacity due to Ciq groups, the resulting resins were con~erted to the H-form by washing the resins in the same manner as in Example 1.
For measuring anio~-exchange capacity, the resulting resins were converted, after CIJI-substitution was finished, ; to the OH-form by was~ing.

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- 19 - j - Some e~rperimental examples of in~obilization of enzymes to the carriers of aspects of the prese~t invention will be shown hereinafter.
Experiment 1 800 nlg of lactase originated from Asper Q lus _yzae [produced by Shinnihon ~aguku K-ogyo Co.; activity of the enzyme in solution, 24.1~1moles/mg min. (ph, 4.5; 40C.; subs~rate, 13.3 w/v% purified lactose)] was dissolved in 40 ml of 0.02M acetate buffer solution (pH 5.5) kept at 4C. 4.0 g of the CM-substituted DUOLITE A-7 produced in E~ample l was immersed in this solution, and the enzyme w~s immobilized at 4C. for 16 hours while shaking the solution at 80 rpm. After immobilization, tlle product was thoroughly washed with 0.05~1 acetate buffer solution (pH 4.5) until the enzyme protein was no longer detected in the washing solution. lhe amount of immobilized enzyme was 149 mg/g-carrier as calculated from the amount of protein in the washing solution measured by the Lowry method. The specific activity of immobilized enzyme was 4.8~umole/mg min. as calculated from the amount of glucose which was produced by shaking the immobilized enzyme at 80 rpm, at 40C. and pH of 4.5, for 15 minutes with 13.3 ~/v% purified lactose as a substrate.
The amount correspond;ng to 3.0 g of this immobilized enzyme was packed in a column equipped with a jacket of 12 mm in inside diameter, and a solution of 7 w/v% purified lactose in 0.02M acetate buffer (pH 4.5) ~as passed down through the column at SV (space velocity) of 2.3 hr while maintaining the column temperature at 40C. The decomposition rate of lactose was found to be 100% by analysis of glucose in the effluent.

When a lactose solution of the same concentration was passed down for 100 days continuously under the same conditions, the decomposition rate of lac-tose at the 100th day was also 100%. Thus, it was found that the activity of the immobilized enzyme did not drop at all. When the same specific activity measurement as above was repeated 25 times using lO0 mg of this immobilized enzyme, the activity at 25th time was 4.7 ~moles/mg.min, whlch means that the activity did not drop even by the batch method.
Experiment 2 1200 mg of substantially the same lactase as in Experiment 1 was dissolved in 60 ml of 0.05M acetate buffer solution (pH 5.5). 6 g of the C~l-substituted Duolite A-7 produced in Example 2 was immersed in this solu-tion, and the enzyme was immobilized at 20 + 2C for 6 hours while stirring the solution at 180 rpm. After immobilization, the immobilized enzyme was washed in the same manner as in Experiment l. The amount of immobilized en-zyme was 170 mg/g-resin as calculated from the amount of protein in the washing solution. The specific activity of immobilized enzyme was 5.2 ~moles/mg.min as measured under the same condition as in Experiment l. The immobilized enzyme thus obtained was divided into two of the same amount.
The one was packed in a column equipped with a jacket of 14 mm in inside diameter, and the same purified lactose solution as in Experiment l was passed do~n through the column at SV of 8.0 hr 1. The decomposition rate of lsctose by the immobilized enzyme was 95%. Decomposition of lactose was continued for 30 days through using the column in which the immobilized enzyme was packed.

3~
.

The decomposition rate at the 30th day was 94%. Thus, it may be said that, considering a sllght experimental error, the activity of immobilized enzyme did not drop at all even though used for 30 days continuously.
Experiment 3 _ __. _ ~e other half of`the immobilized lactase prepared in Experiment 2 (corresponding to 3 g of the carrier) was packed in a column equipped with a jacket of 14 mnl in inside di~neter. A solution of 12 w/v% purified lactose in the same buffer solution as in 10 Experiment 1 was passed do~n through the column at SV of 5.0 hr 1 for 30 days continuously. The decomposition rate of lactose calculated from the amoullt of glucose in the e~fluent was 91 + 2.5% over 30 days, which means that the activity of immobilized enzyme did not drop at all.
Experiment 4 600 mg of lactase originated from ~ el~illus oryzae [produced by Shinnihon Kagaku Ko-gyo Co.; activity of the enzyme in solutionS
50.9 ~moles/mg-min. (pH, 4.5; 40C.; su~strate, 13.3 w/v% purified lactose)~ was dissolved in 30 ml of 0.05M acetate buffer solution 20 (pH 5.5). 3.0 g of the CM-substituted DUOLITE A-4 produced in Example 5 was innnersed in this solution, and the enzyme was immobilized at 20C. for 7 hours while stirring the solution at 180 rpm. After immobilization~ the immobilized enzyme was washed in the same manner as in Experiment 1. The amount of immobilized enzyme was 118 mg/g-carrier, ~le specific activity of immobilized enzyme was 9.2 umoles/
mg-min~ as measured under the same condition as in Experiment 1. This immobilized enzyme was packed in a column equipped with a jaclcet o 13 mm in inside diameter, and the same , purified lactose solution as in Experiment 1 was passed do~n through the column at 40C. and ae SV of 5.0 hr~l or 30 days continuously.
The decomposition rate of lactose was kept 100% over 30 days, and loss in enzyme activity was not observed. Continuous decomposition of lactose was continued for a urther 20 days in the same manner as above except that SV was increased to 8.0 hr 1, Thlls, it was folmd that the activity did not drop at all even though the immobilized enzyme was used for 50 days continuously.
Experiment 5 100 mg of a commercially available papain was dissolved in 30 ml of 0.02M phosphate buffer solution (pH 6.2) kept at 4C. 1.0 g of the C~l-substituted DUOLITE A-4 produced in Example 6 was immersed in this solution, and the enzyme was imrnobilized at 4 to 10C. for lO
hours while stirring the solution at 150 rpm. Ihe immobilized enzyme was washed thoroughly with O.OS'~ phosphate bufer solution (pH 6.2), O.lM aqueous sodium chloride solution and ion exchange water in this order. The washing solution was recovered and measured for the amount of protein by the Lowry method. By calculation from that amount, the amo~mt of immobilized enzyme was found to be 76 mg/g-carrier. The specific activity of immobilized enzyme was measured at 40C. and pH 6.2 by means of a pH-stat (Hiranullla pH--stat sp-ll) using 0.2SM N-benzoyl-L-arginine ethyl ester (BAEE) as a substrate, and it ~as found to be 2.7,umoles/mg min. This value corresponds to 35%
of the specific activity of the original enzyme in solution. ~easure-ment of the specific activity of this immobilized papain was repeated 15 times in the same manner as above, and the activity at the l5th time was 98~o of that a~ the first time, which means that there was little loss in activity. 'i'he above measurements of the activities of the immobilized enzyme and the enzyme in solution was carried out in the presence of 2 x 10-3M
ethylenediamine tetraacetic acid, 5 x 10-3~,1 cysteins and 0.llvi sodium chloride~
Experlment 6 60 mg of a commercially available purified trypsin was dissol~ed in 15 ml of 0.05M Tris-l~Cl buffer solution (pH 7.5) kept at 4C. l.0 g of the Cl~-substituted Duolite S-37 produced in ~xample 8 was added to this r ~ol~tion, and the enzyme was immobilized at 4C while slowly stirring the solution at 60 rpm. After lO t hours, the immobilized trypsin was iltered ancl well washed with 0,05M Iris~HCl buffer solution~ O.liii sodium chloride solution and distilled water in this or:ler. mhe washing solution was recovered and measured for the amount of ~:
protein by ultraviolet absorption intensity. By calculation from that amount, the arnount of immobilized enzyme was found to be 56 mg/g-carrier. The specific activi-ty of immobiliæed trypsin was measured by means of a p~ tat at 30C and pH 7.5 in the presence of 0,02~ calcium chloride using BAEE as a substrate, and it was found to be 5.8 ~moles/mg-min. This value corresponds to 227, of the r specific activity of original enzyme in solution, Using , lO0 mg of this immobilized trypsin, measurement of ~pecific activity was repeated 5 -times under the same con~ition as above with BA~ as a substrate. A~ a result, the specific actiyity at the 5th time was 4.6 ymoles/mg-min. r .

~ . ...

Experiment 7 20 ml of 0~L~ phosphate buffer solution (pH 6.7) containing 1250 Sumner unit of a commercially available urease (purchased from Tokyo Kasei Co.) was prepared. 1.0 g of the C~-substituted DIAION
I~A-21 produced in Exa1nple 9 was added to the solution, and stirred at 60 rpm at ~1C. for 16 hours. AEter immobilizatlon, the immobilized enzyme was fil'Lered and tl-orollghly washed with 0.05M phosphate buffer solution and then distilled water until protein was no longer detected in the filtrate. The amount of immobilized enzyme was ca]culated as 382 Sumller ~mit/g-carrier. The activity of immo~ilized enzyme was measured from a time required for Q.L~ phosphate buffer solution containing 3.0% by weight of urea to change its pH from 6.7 to 7.7 at 20C., and it was found to be 412 Sumner ~mit/g-carrier.
Note (1): One Sumner unit refers to the amount of enzyme which decomposes urea corresponding to 1 mg of ammonia nitrogen at 20C.
and pH 7.0 during 5 minutes in a phosphate buffer solution.
Experiment 8 Glucose isomerase extracted from Streptomyces sp. (produced hy ~agase Sangyo Co.~ and purified which has activity of 36,000 unit and 255 mg of protein (measured by the Lowry method), was dissolved in 30 ml of 0.05M phosphate buffer solution (pH 7.65). 3.0 g of the CM-substitllted D~OLITE A-7 produced in Example 1 was added to the solution, and immobilization was carried out at room temperature (18C.~ for 9 hours while stirring the solution at 120 rpm. After immobilization, the immobilized enzyme was filtered and well washed with 0.~I

, ~ - 25 _ :
phosphate buffer solution (pH 7~ 65)o By measurements of ---the activity of the filtrate and the amount of protein in the filtrate, it was found that the activity of ir~mobiliæed protein was 9500 unit/g-carrier and the amount of immobilized protein was 61 mg/g-carrier. '~he immobilized gluco~e isomerase thus obtained was filled in a column e~uipped with a jacket of 12 mm in inside diameter, and 54 w/v~ib aqueous purified glucose solution (pH 7a 65, containing 5 x 10 ~ ~/1gS04 7H20) was passed down through the column at SV of 2.0 hr~l while maintai~ing the column temperature at 60C, whereby isomerization of glucose was carried out.
Conversion was kept at 50 to 51'i~ for 500 hours after r beginning of the isomeriYation, and then it gradually decreased.
Note (2): One unit of glucose isomerase refers to the amount of enzyrne which produces 1 mg of fructose when reactiOn is carried out at 70C and pl~ 7.0 for 1 hour, with O.lM D-glucose solution as a subetrate, in 0.05~d pho~phate bu~`fer solution containing 0.0051~ MgSO~ 7H20.
Note (3): I~easurement of fractose wa~ carried out by the cysteine-carbazole~sulfuric acid method according to JAS.
Experiment 9 200 mg of pullulanase originated from Aerobacter ~ e~ (produced by liagase Cangyo Co ) was dissolved in 30 ml of 0,02~. acetate buf~er solution (p~ 5.0). 3 g of the Cl~-substituted Diaion WA-21 produced in ~xample 9 was added to the solution, and the enzyrr.e was immobiliYed at 10C for 16 hours with slow ~tirring. 'rhe amount of the immobiliYed enzyrne was calculated from the recovered washing solution and it was 43 rng/g-carrier. All of the immobilized enzyme was packed in a column equipped with a jacket of 12 mm in inside diameter, and a 1.0~ purified pullulane solution (pH 5.0~ was passed down through the column at SV of 1 hr for 10 days continuously while maintaining the column temperature at 40C.
The amount of maltotriose in the effluent was measured by the Somogyi-Nelson method, and it was Eound that conversion to maltotriose was kept unchanged at 95 + 4% for lO days.
Ex~eriment 10 Using 600 mg of the same lactase as used in Experiment l, the lactase was immobilized to 3.0 g of a carrier, the CM-substituted DIAION I~A-20 produced in Example 11, under ~he same condition as in Experiment 1. The amount of immobilized enzyme was 98 mg/g-carrier as calculated from the amount of protein in the washing solution.
The specific activity of immobilized enæyme was 4.5 ~moles/mg-min.
as measured under the same conditions as in Experiment 1. Using 100 mg of this immobilized lactase, the specific activity measurement was repeated 15 times using a new lactose solution at every measure-ment, and the activity at the 15th time was 3.5 ~Imoles/mg min. It is known from the results that the carrier produced in Example 11 was a little inferior to that produced in Example 1 in operational 11 stability of immobilized enzyme. One of the reasons may be con- ¦
sidered due to that the amount of introduced CM groups in Example ll is smaller. It is, however, clear that the immobilized enzyme pro-duced in Example ll is more stable (i.e., longer in life time) than that produced in the follol~ing reference example.

Reference Experiment The experiment was carried out in the same manner as in Experiment lO, except that a weakly basic macroporous ion-exchange resin, DIAION WA-20, was used as a carrier taking into account that lactase origlnated from A~ illus oryzae is an acidic protein. The .
amount of immobilized enz~7me was 79 mg/g-carrier and the specific activity of i;~obilized enzyme was 3.6~umoles/mg min. Th-~s specific activity measurement was repeated lO times, but the activity dropped to 1.4 ~moles/mg-min. at the 5th measurement and to 0.3~umoles/mg-min.
at the 10th measurement. As is clear from the above results, stable ln~nobilized enzymes withstanding industrial use could not be obtained.
Experiment 11 2.0 g of the CM-substituted DUOLITE A-4 produced in Example

6 was immersed in 20 ml of lN sodium hydroxide solution. The solution was degassed at 4C. for 15 minutes and the excessive al~ali solution was removed by filtration. This resin was immersed in 25 ml of dio~ane at room temperature (2~C.) for 5 m;nutes with stirring, and then 20 ml of a previously prepared dioxane solution containing ~ g of cyanuric chloride was added thereto, followed by vigorous stirring at room temperature. After 3 minutes, 25 ml of cold water was added to the reaction solution and, after 5 seconds, 25 ml of acetic acid was added to stop the reaction. The mixed solution was filtered, and the resin was immediately washed with cold water and cold acetone. The resin was then added to 30 ml of 0.05~I phosphate buffer solution (pH 7.8) containing 220 mg of a co~nercially available enzyme, Pronase E, origin-ted :

: .

: ```

.

from Streptomyces gr _eus. The solution was stirred at 4C. and kept at pH 7.8 with addition of 0.2N sodi-um hydroxide solution. Immobili-zation was then carried out for 5 hours, The immobilized enzyme was filtered and thoroughly washed with ice-cooled lM sodium chlor;de solution, 0.1M phosphate buffer solution and ice-cooled water in this order until protein was no longer detected in the washing solution.
The amount of immobllized enzyme was 86 mg/g-carrier. The specific activity of immobilized enzyme was measured by means of a pH-stat at 40C. and pW 6.0 with a 20%-DL-lysine methyl ester as a substrate, and it was found to be 2.7 ~moles/mg-min.
Next, using 100 mg of this immobilized enæyme, experiment was repeated batchwise at 40C. and pH 6.0 with lO ml of 10%-L-lysine methyl ester as a substrate. Required time per experiment was fixed to 40 minutes and reaction rate was measured by a pH-stat. In this way, the number of experiments which were repeated until reaction rate dropped to half of the first reaction rate was obtained. The number thus obtained was called "half life number". The half life number of this immobilized enzyme was ~3.
E~eriment 12 -Two grams of the C~-substituted DUOLITE A-7 produced in ; Example 2 was immersed in methanol, converted to methyl ester with hydrogen chloride gas, thereafter to hydrazide with hydrazine hydrate and finally to azide w;th 3~ sodium nitrite solution. Immediately, the product was immersed in 20 ml of a phosphate buffer solution containing 1000 Sumner units of a com~ercially available urease ` ' ~

' (purchased from Tokyo Kasei Co.). Thereafter, the enzyme was immobilized at 4C. for 16 hours with mild shaking. The immobilized enzy~ne was waslled with 5M sodium chloride solution, O.lM phosphate buffer solution (pH 6.7) and ion-exchange water in this order. The activity of immobiliæed enzyme w~s measured by colorimetry at 20C.
using a 3.0~ urea solution as a substrate, and it was found to be 290 Sumner unit/g-carrier. ~sing this immobilized urease, ~he e~per;ment was repeated 10 times but loss in activity was hardly observed.
Experlment 13 2.0 g of the Amphoteric-ionized DUOLITE S-30 produced in Example 4 was immersed in 20 ml of a dioxane solution containing 25 g of bromoacetic acid, followed by slow stirring at room temperature for 8 hours. Thereafter, 17 ml of bromoacetyl bromide was gradually added drop~ise, followed by stïrring for 6 hours. After the reaction was finished, bromoacetylated resln was obtained by washing with ice-cooled O.lM sodium carbonate solution and then ice-cooled water. This resin was immersed in 0.2M phosphate buffer solution (pll 8.5) containing 100 mg of aminoacylase (produced by Amano Seiyalcu Co., 15,000 unit/~
and the enzyme was immobilized at 4C. for 18 hours with slow stirriDg.
The resulting immobilized enzyme was washed repeatedly. The activity of this immobilized enzyme was measured at 37C. with 0.2M N-acetyl-DL-methionine solution (pH 7.0, containing 1 x 10 4 mole of CoC12) as a substrate, and it was Eound that the activity was 270 unit/g-carrier from the amount of produced L-methionine. All the immobilized enzyme thus obtàined was packed in a , . .

.
` ` ' ' ` . ' ` ~ ' .

column equipped with a jacket of 10 mm in inside diameter, and the same N-acetyl-DL-methionine solution as used in the activity measurement was continuously passerl down through the column at SV of lo 1 hr~l while maintaining the column temperature at 40C. The hydrolysis rate of the L-isomer after one day was 99'jo and loss in enzyme activity was hardly observed even after 10 days.

' :'

Claims (18)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An enzyme-immobilization carrier which comprises a macro-porous amphoteric ion-exchange resin having a cation-exchange capacity due to carboxymethyl groups of 0.5 meq/g-dry resin or more, an anion-ex-change capacity of 1 meq/g-dry resin or more, and a specific surface area of 1 m2/g-dry resin or more, the total volume of macropores having a dia-meter of 100 .ANG. to 2000 .ANG. being 0.1 cc/g-dry resin or more.

2. An enzyme-immobilization carrier according to Claim 1, wherein said specific surface area of the resin is 5 m2/g-dry resin or more.

3. An enzyme-immobilization carrier according to Claim 1, wherein the mean pore diameter of the pores in said resin is from 150 .ANG.
to 1000 .ANG..

4. An enzyme-immobilization carrier accord;ng to Claim 1, wherein said total volume of macropores having a diameter of 100 .ANG. to 2000 .ANG. is 0.2 cc/g-dry resin or more.

5. A process for producing an enzyme-immobilization carrier which comprises reacting a compound of the formula, wherein X is a halogen atom and Y is a hydrogen atom or an alkali metal, with a macroporous anion-exchange resin having a functional group capable of reacting with said compound, an anion-exchange capacity of 1 meq/g-dry resin or more, and a specific surface area of 1 m2/g-dry resin or more, the total volume of macropores having a diameter of 100 .ANG. to 2000 .ANG. being 0.1 cc/g-dry resin or more, in the presence of an alkaline compound, whereby 0.5 meq/g-dry resin or more of carboxymethyl groups is introduced into said macroporous anion-exchange resin to obtain an am-photeric ion-exchange resin.

6. A process according to Claim 5, wherein said compound is chloroacetic acid or sodium chloroacetate.

7. A process according to Claim 5, wherein said compound is used in an amount of 1/2 to 10 parts based on 1 part of the dry resin.

8. A process according to Claim 7, wherein said compound is used in an amount of 2/3 to 3 parts based on 1 part of the dry resin.

9. A process according to Claim 5, wherein said alkaline com-pound is an alkali metal hydroxide, alkaline earth metal hydroxide or organic amine.

10. A process according to Claim 9, wherein said alkaline com-pound is sodium hydroxide.

11. A process according to Claim 5, wherein said alkaline com-pound is used in an amount of 1/3 to 2 times by mole based on said com-pound.

12. A process according to Claim 5, wherein said anion-exchange resin has a hydroxyl, primary amino, secondary amino, imino or sulfhydryl group.

13. A process according to Claim 5, wherein said specific sur-face area of the resin is 5 m2/g-dry resin or more.

14. A process according to Claim 5, wherein the mean pore dia-meter of the pores in said resin is from 150 .ANG. to 1000 .ANG..

15. A process according to Claim 5, wherein said total volume of macropores having a diameter of 100 .ANG. to 2000 .ANG. is 0.2 cc/g-dry resin or more.

16. A process for immobilizing an enzyme on the enzyme-immobili-zation carrier which comprises immobilizing an enzyme on the enzyme-immobilization carrier of Claim 1.

17. A process as in Claim 16, wherein the enzyme is carried on the resin by adsorption method.

18. A process as in Claim 16, wherein the enzyme is carried on the resin by covalent attachment method.