CA1107669A - Enzyme-immobilization carrier and preparation thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A novel enzyme-immobilization resin and process for its manufac-ture is provided herein. The resin comprises a macroporous, synthetic anion-exchange resin having a specific surface area of at least 1 m2/g-dry resin and containing macropores with a pore size of 100 .ANG. to 2,000 .ANG., of which total volume is at least 0.1 cc/g-dry resin; the resin comprising (a) a phenol-formaldehyde condensate matrix and diethylaminoethyl groups linked to the matrix through an ether linkage, wherein the ion-exchange capacity of the resin is not less than 1 meq/g-dry resin, (b) a phenol-formaldehyde condensate matrix having primary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and diethylaminoethyl groups linked to the matrix through an ether linkage or to the primary or secondary amino groups to form a secondary or tertiary amino group respectively, wherein the ion-exchange capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g-dry resin, or (c) a crosslinked polystyrene matrix having pri-mary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and diethylaminoethyl groups linked to the primary or secondary amino group to form a secondary or tertiary amino group respectively, wherein the ion-exchange capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g-dry resin. This provides an immobilized enzyme high in activity with good activity retentivity and also high in the amount of the immobilized enzyme per unit weight of the carrier. The resin is provided by reacting the diethylaminoethyl derivative or its salt with granules of the above defined synthetic resin of a macroporous type. The reaction takes place in the presence of an alkaline compound.

Description

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~'his invention reJates to arl enz~r~e-imn,obil~
zation carrier anc'~ a procec-.s ~or ~roducin~r the same.
ore particularly, this invention perta-ins to an enzyme-il~mobilizatlon carrier compr;sin~ a s~nthetic resin containinr~; dieth~/laminoethvl r~roups obtajne-i bv reactin~ '~r.. ' a ctiethvlaminoeth!/l derivative or :its salt l~i.th a macro~orou~s svnthetic resin (hereinafter sometimes referred to merely as "resin' all Or the resins herein mr~entioned refer to pure synthetic resins, excluclinrr polyr;accharide derivatives) having functional groups reactive with said diethvlaminoethyl derivative or its salt in the r~resence of an allrc?.line com~ound, and also to a process for producin~r~ the sa~e.
'['here have been cleveloped in recent years enz~/me immobilization technioues because of the useful-ness of immobilized enzvmes in comrnercia]. application (refer to, for examp]e, C.R. Zal)orsk!~ Immob lized En%~/mes, C.R.C. Press, 1973) and thererore various --irnrno~)ilization carriers are Icno~/n in the art. Arnong these carriers, ~olvsaccharides and their derivatives, for example, crosslinlced dextran~ have been freauentlv -.Ised and they proved to be successful as carriers for .several enz~/mes (lnzymolo~-r,ia, Vol. 31~ p. 214, 19~
These polysaccharides~ however, are insurficient in rlechanical strenrrth and therefore it is difficult to -~-obtain a suffic:ient flolil ve]ocity in column operation.
rther, thev are liable to cause clorrr~rinrr durin~, the operatiorl and wea!c .n resistance to microbial attack. ~-~nothe:r ~rreat pro~)lern is that ionicall~ boncted enzv~les ,G on tnese carriers are easil~ released ~lhen a ]ar~e amount ..~

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of electrolytes are present in a reaction mixture.
On the other hand, in the late nineteen fifties, it had been known to use synthetic resins, especially ion-exc'nange resins as en-zyme-immobilization carriers. But these resins have been evaluated to be of little practical value due to the small quantity of enzymes carried per unit weight of carrier and the low activity of the immobil-ized enzymes obtained. Ion-exchange resins having a synthetic resin matrix, however, have several outstanding features superior to poly-saccharides and their derivatives. They generally maintain sufficient mechanical strength and can be durable for continuous running for a long term in a large scale column without suffering from a great ex-tent of damages. Further, they have a suitable particle size to ensure sufficient flow velocity in column operation and are strongly resistant to microbial attack.
It has been found that specifically modified ion-exchange resins in which diethylaminoethyl (hereinafter referred to as "DEAE") groups having specific affinity with a large number of enzyme proteins introduced into synthetic resins having specific physical and chemical properties are excellent enzyme-immobilization carriers.
Thus, an ob;ect of a broad aspect of the presen~ invention is to provide an enzyme-immobilization carrier which can give an immobilized enzyme having a high immobilized enzyme activity with ex-cellent activity reten~ivity and/or containing a large quantity of im-mobilized enzymes per unit weight of the carrier and a method for pro-ducing the same.
An object of anotller aspect of the present invention is to provide an enzyme-immobilix~tion carrier suitable for commercial appli-cation which can stablli~e enzymes by immobilization and enable re-peated and continuous uses of enzymes which are in themselves catalysts for homogenous aqueous reActions, and a process for producing the same.

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By one broad aspect of this invention, an enzyme-immobilization carrier is provided comprises a microporous, synthetic anion-exchange resin having a specific surface area of at least 1 m /g-dry resin and containing macropores with a pore size of 100 A to 2,000 A, of which total volume is at least 0.1 cc/g-dry resin; the resin comprising (a) a phenol-for~aldehyde condensate matrix and diethylaminoetllyl groups linked to the matrix throigh an ether linkage, ~lherein the ion-exchange capaciLy of the resin is not less than 1 meq/g-dry resin, (b) a phenol-formaldehyde condensate matrix having primary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and diethylaminoethyl groups linked to the matric through an ether linkage or to the primary or secondary amino groups to form a secondary or tertiary amino group respectively, wherein the ion-exchange capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g-dry re-sin, or (c) a crosslinked polystyrene matrix having primary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and di-ethylaminoethyl groups linked to the primary or secondary amino group to form a secondary or tertiary amino group respectively, wherein the ion-exchange capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g--dry resin.
By one variant thereof~ the specific surface area of the resin is Sm2/g or more.
By another variant, the average pore size of the pores in the synthetic resin is from 150 A to 1,000 A.
By still another variant, the volume of macropores Witil pore O O
sizes from 100 A to 2,000 A is 0.2 cc/g or more.
By another aspect of this invention, the carrier as above de-scribed further contains other anion exchange groups.

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r By another variant, the synthetic anion-e~change resin comprises a phenol-formaldehyde condensate matrix having primary ainino-groups, secon-dary amino groups and a mixture thereof and diethylaminoethyl groups linked to the matrix through an ether linkage or to the primary or secondary amino group to form a secondary or tertiary amino group respectively, the ion-exchange capacity provided by the d-iethylaminoethyl groups is not ]ess thln 1 meq/g-dry resin, and the total ion-exchange capacity of the resin is not less than 2 meq/g-dry resln.
By yet another variant, the synthetic anion-exchange resin com-prises a phenol-formaldehyde condellsate matrix and diethylaminoethyl groups linked to the matrix through an e~her linkage and the ion-exchange capacity of the resin is not less than 2 meq/g-dry resin.
By still a further variant, the synthetic an;on-exchange resin comprises a crosslinked polystyrene matric having a primary amino groups, secondary amino groups or a mixture thereof and diethylaminoethyl groups linked to the primary or secondary amino group to form a secondary or ter-tiary group respectively, the ion-exchange capacity provided by the di-ethylaminoethyl groups is not less than 1 meq/g-dry resin and the total ion-exchange capacity of the resin is not less than 2 meq/g-dry resin.
By a variation thereof, the ion-exchange capacity provided by the diethylaminoethyl groups is not less than 1.5 meq/g-dry resin.
By another variation thereof, the ion-exchange capacity provided by tha diethylaminoethyl groups is not less than 1.5 meq/g-dry resin.
By still another aspect a process is provided for producing an enzyme-immobilization carrier comprising a synthetic anion exchange resin having diethylaminoethyl groups, which comprises reacting a diethylamino-ethyl derivative of the iormula C2 5 \

2 5 A s . .
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..

wherein X is halogen or hydrogensulfate or its salts with granules of a solid phenol-formaldehyde resin.of a macroporous type having no other functional g-roups besides OH groups, primary amino groups, and/or secondary amino groups and tertiary amino groups in addltion to OH group (not including the diethylaminoethyl groups), or ~ith bea~s of a cros~l;nked polystyrene resin of a macroporous type i~aving prilllary ~m:ino gro~lps .~nd/or ~econdary amino groups and tertiary amino groups having a specific surface area of at least 1 m /g-dry resin and containi.ng macropores of l~hich the total volume of those with pore sizes from 100 A to 2,000 A is at least 0.1 cc/g-dry resin in the presence of an alkaline compound.
sy a variant thereof, the diethylaminoethyl derivative or its salt is ~ -diethylamirceth~lchloride hydrochloride, ~ -diethylaminoethyl bro~ade hydrobromide or ~ -diethylamin~ethyl hydlcgensulfate, especially wherein the diethylamin~ethyl derivative salt is ~ ~diet.hylamunoethyl chloride hydrochloride.
By an~ther variant thereof, the amount of the diethylaminoethyl derivative or its salt is used in an am~unt of 1/3 to 10 p~rts per one part of the dry resinJespecially wllere the diethylalninoethyl derivaki-~e or its salt is used in an amount o~ 1/2 to 3 parts per one part of the dry resln.
By still another variant, the alkali~e ccm~ound is an aL~ali ~tal hydrQxiae, an alkaline earth metal hydroxide or an organic amine particularly ~ere ik is sodium hydroxide.
By yet another variant, the amounk of the alkaline com~ound added is frc~ 1/10 to 5 ti~es moles as ~uch as that of khe diethyla~uL~o-ethyl der.ivative or its salt.
By a further variant, the synthetic resin has primary amuno group, second~ry amino group, hydroxyl yrvup, imino group or sulfh~ryl gr~up as functional groups reactive with the diethylaminoethyl deri~tive or its salt.
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By a further variant, the synthetic resin is so selected that i~
has a specific surface ar.ea of 5 m /g-dry resin or more.
By still a further variant, the macroporous resin is so selected O
that the total volume of macropores with pore sizes from 100 A to 2,000 A
in the synthetic resin is 0.2 cc/g or more.
By yet another vari.ant, the macroporous resin is so selected that the average pore size of the pores in the synthetic resin is from 150 A to 1,000 A.
By another variant, the anion exchange capacity on the basis of primary, secondary and/or tertiary amino group excluding diethylaminoethyl groups is at least 1 meq g-dry resin.
By a further aspect of this invention, a process is provided for immobiliæing an enzyme which comprises carrying the enzyme on a synthetic resin which comprises a macroporous, synthetic anion-exchange resin having a specific surface area of at least 1 m /g-dry resi.n and containing macro~

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pores with a pore size of 100 A to 2,000 A, of which total volume is at least 0.1 cc/g-dry resin; the resin comprising (a) a phenol-formaldehyde con-densate matrix and diethylaminoethyl groups linked to the matrix through an ether linkage, wherein the ion-exchange capacity of the resin is not less than 1 meq/g-dry resin, (b) a phenol-formaldehyde condensate matrix having primary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and diethylaminoethyl groups linked to the matric through an ether linkage or to the primary or secondary amino groups to form a secondary or tertiary amino group respectively, wherein the ion-exchange capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion--exchange capacity is not less than 1 meq/g-dry resin, or (c) a crosslinked polystyrene matrix having primary amino groups, - secondary amino groups or a mixture thereof as an anion-exchanger. and di-ethylaminoethyl groups linked to the primary or secondary amino group to form a secondary or tertiary amino group respectively, wherein the ion-ex-- ,, 1~ 6a : ~
.

change capacity due to the diethylaminoethyl groups is nat less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g-dry resin.

According to ~an aspect of the present invention, then, an en-zyme-ir~obilizacion carrier is provided ccmprising a DE~E-m~dified resin obtained by reacting a diethyla~i~loeth~l derivative of the formLla:

C2H5 ~
~ N-C2H4X

(wherein X is halogen or hydrvgensulfate) or its acid addition salt, for lo e~a~ple, ~ -diethylarninoethyl hydrogensulfate, ~ -diethylaminoethyl chlor-ide, under alkaline conditions with a synthetic resin having functional groups reactive with the DE~E derivatives; e.g. hydl~ yl group, primary a3nLno group, secondary amino grou~, irnLno group or sulfydryl group, especi-ally a macro~orous resin having specific surface area of 1 m2/g or rnDre and containing]~cropores with pore sizes of 100 A to 2000 A, the total volume of said rnacropores being at least 0.1 cc/g-dxy resin. The DEAE-mDdified resin of an aspect of the present invention is found to have not 03~y the excellent characteristics of ion-exchange resins as rnentioned above, e.g. m~chanical strength, column operability and resistance co mi-- 6 b -,~

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crobial a-t-tack, but also it ls an extrernely excellent enzyrne-ilr,rr.ohiliza-tion carrier which can give an imm~bilized enzyrne having a large quan-tity of imrrobilized enzymes, a high specific activity of immobilized enzyrnes as well as good activity retentivity.
The DE~E.-rnodified resin carrier of an aspect of the present in-vention is a very good carrier in prac-tical application especially because it can give an immobilized enzyme having good activity retentivity which is the raost important factor in carrying out con-tinuous operation for a long time by using imrr,obilized enzymes. Other groups similar to DE~E groups, e.g. dimethylaminoethyl groups or dii.sopropylaminoethyl groups can al.so be introduced into the resins to prepare similar resins. DEAE groups are found to be the best of all frorn standpoint of their easier modifica-tion reaction and affini-ty with enzy.mes.
Accordingly, it seerns that the excellen-t characteristics of the DE~E-rnodified resin as an enzyme-imrr.obilization carrier are not owing to the anion exchange capacity of DE~E groups b~t to a specific affinity with enzy.me molecules which is an inherent characteristic of DE~E group. This is further evidenced hy the following fact: a macroporous phenol-formalde-hyde polycondensate resin having an anion exchange capacity of 7.10 rneq/g-dry resin (ion-exchange capacity is estimated by batchwise neutralization titration after conditioning the resin to OH-form; the dry resin weight is obtained by weighing the resin after conditioning the resin to OH-form, followed by vacuurn drying at the temperature of 60C for 10 hours and standing at room temperature from 18 to 25C for more than 2 hours; the ion-exchange capacities mentioned hereinafter in the specification are all measured according to this method) modified with polyeth.ylene polyamine ~the number of recurring ethyleneamine units heing not more than 4) has hydroxyl groups, primary amino gr.oups and secondary ~ ~no groups on the resin and suitahle for DEAE-rrodification. When this resin is suhjected to .

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DEAE-mDdifica-tion reaction under a]k~line conditions, tne increase in weight of the resin is som~e-times greater than the increase in anion-eYchange capacity hy DE~E groups for some reason which lla~ no-t yet been made very clear and it occurs thk~t the -total anlon exchange capacity per unit weight of dry resin is decreased to less than 7 10 meq/g. For e-xample, while there is increase of weight as much as 57.2~ by the DE~E-modification reac-tion, the to-tal anion exchange capacity is 6.34 mec~/g-dry resin with the anion exchange capacity of DE~E groups being 1.82 meq/g-dry resin. Never- ~
theless when this DE~E-rnodified resin is used as a carrier, the quantity of imrrobilized enzymes per unit weigl~t is greater as compared with the case when the original carrier without DE~E-modification having 7.10 meq/g-dry resin is used as carrier. Further, the DEAE-modified resin can give im-mobilized enzymes having high ac-tivity with good activi-ty retentivity, ex-hibiting superior charac-teristics of DE~E-modified resins as carrier. The effect of modification with DEAE groups is not therefore due to an increase in anion exchange capacity but apparently is due to a specific affinity with enzymes inherent in DE~E groups, the nature of which is not yet clarified i~ de~tail.
There is also an increase in the volume of the resin together with increase in weight by DE~E-modification. m erefore, there is little change in volurr,e Fer unit weight. For example, phenol-formaldehyde poly-condensate resins with sizes of 16 m2sh to 60 rr2sh screened through a sieve are packed in a column and a liquid is passed therethrough, the volume of the resin is between 3.0 ml and 4.0 r~ per one gram of dry resin before and after rrodification with DEAE groups.
I'he DE~E-modified resin of an aspect of the present inven-tion can be prepared ~y reacting a ccmpo~md of the formula: C2H5~ N-C2H4X (wherein X is halogen or hydrogensulfate)or its acid addition salt wi-th a macro-porous synthetic resin having functional groups reactive with such compound in the presence of an alkaline compound. It is important to carry out the " ' . , ~ ~

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reaction so that the resin may be wetted with the reaction mixtures even at the internal portion of macropores in order to obtain DEAE-modi-fied resins with a high degree of substitution.
DEAE-modification reagents represented by the formula C2H5 ~ N-C2H4X or its acid addition salt are preferably ~ -diethylamino-ethyl chloride hydrochloride,, ~ -diethylaminoethyl bromide hydrobromide and ~-diethylaminoethyl hydrogensulfate, etc. From the standpoint of reactivity and economy, ~ -diethylaminoethyl chloride hydrochloride is the most advantageous.
The amount of the DEAE-modification reagents to be used de-pends on the desired amount of DEAE groups introduced (degree of sub-stitution), but a resin containing too small an amount of those groups is of little significance according to aspects of the present invention.
It is generally advantageous to use an amount in excess of stoichiome-try to carry out the reaction smoothly. But it is difficult to know precisely the number of functional groups reactive with the DEAE-modi-fication reagents on the surface of the resin. Accordingly, in many cases, the empirically preferred amount of the DEAE-modifications rea-gent, i.e., the preferred amount of the DEAE-modification reagent, is 1/3 part of 10 parts per one part of the dry resin, more preferably from 1/2 to 3 parts.
The alkaline compound to be used in the process of an aspect of the present invention includes al~ali metal hydroxides, e.g., sodium hydroxide or potassium hydroxide; alkaline earth metal hydroxides, e.g.
calcium hydroxide or magnesium hydroxide; and ths like. In some cases, there may also be used organic amines, e.g. triethylamine, etc. Pre-ferable alkaline compounds are alkali metal hydroxides, among which sod-ium hydroxide is the most preferred.

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The alkaline compound is added in an amount of 1/10 to 5 times as much as the moles of the DEAE-modification reagent. ~hen it is necessary to remove the acid addition salt during the reaction, the alkaline compound is used in an amount which is sufficient to neutra-lize the acid.
The synthetic resins to be used in the process of an aspect of the present invention may be any resin capable modification with DEAE groups having such groups as, e.g., primary amino group, secondary amino group, hydroxyl group, imino group or sulfhydryl group. In compliance with an object of one aspect of the - 9a -., , .

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" ' ' present invention to provide a practica]ly excellent enzyme-immobiliza-tion carrier Eor catalysts in industrial uses, the resin is desired to have in addition to the above functional groups a large number of macropores with pore sizes of 100 A to some 1000 A as well as micro-pores which are formed dependlng on the extent of crosslinking, thus having a large pore volume and a large specific surface area. Such macropores are imparted physically by a specific polymerization method.
The resins containing macropores are better in mechanical strength than those containing only micropores and suitable for continuous running.
Macroporous resins are also referred to as MR type9 macro-reticular structure or high-porous structure, etc. For the purpose of exhibiting a remarkable effect in immobilization of enzymes, the macroporous resins is desired to have a specific surface area of at least 1 m /g-dry resin, very preferably 5 m /g-dry resin or more (surface area of dry resin is measured by nitrogen adsorption method by means of a surface area measurlng instrument, produced by Carlo-Erba Co., followed by data analysis by BET method to evaluate the values), a total volume of macropores with sizes from 100 A to 2000 A being at least 0.1 cc/g-dry resin, preferably 0.2 cc/g-dry resin (pore size and pore volume are measured by a mercury penetration type porosimeter produced by Carlo-Erba Cl., data analysis being made to determine the values by supposing the shape of macropores as cylindrical with spherical cross-sectional area). Larger pores with sizes - 9b -: ' .

of 2000 A or more will contribute little to dimensional stability of immobilized enzyme. The average pore size, which is different depend-ing on the enzymes to be immobilized, is preferably from 150 A to 1000 A.
The macroporous resins satisfying the above requirements can be prepared by well-known methods but there are already many commercial-ly available macroporous resins. ~lost of the commercially available resins, however, have hydroxyl groups, aliphatic primary amino groups or aliphatic secondary amino groups and there can scarcely been found those having sulfhydryl groups, imino groups or aromatic amino groups.
Some examples of these macroporous resins are set forth in the following Table 1 together with physical and chemical properties thereof. The surface area and the pore volume of a resin do not undergo substantial changes after DEAE-modification reaction except for those within ex-perimental errors.

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While there is no particular upper limit to the specific surface area and the total pore volume, the mechanical strength is in-sufficient when they are too great. Thus, the specific surface area ls desired to be not more than 120 m2/g and the total pore volume not more than 80% of the total resin volume.
The reaction solvent is not particularly limited but an aqueous solvent, a non-aqueous solvent and a mixture of water with a non-aqueous solvent may employed. The reaction is carried out generally at a temperature not higher than 200C. The resins are liable to be broken and undesirable side reactions are liable to occur at too high a temperature; the preferable reaction temperature is less than 100C.
The reaction rate is retarded at too low temperature. Thus, the most preferable temperature range is from 10C to 80C. Stirring may be conducted so that the reactants may well be mixed but the resins may not be broken.
The anion exchange capacity based on DEAE groups (indicated by CD) is represented by the following formula when the synthetic resin before introduction of DEAE groups is an anion exchange resin (of which ion-exchange capacity is indicated by CA) and the total anion ex-change capacity after introduction of DEAE groups is indicated by CT:

CD = CT ~ CA (X/Y)wherein X represents the total weight of dry anion-exchange resin be-fore DEAE-modification reaction used and Y the total weight of dry resin after DEAE-modificatlon reaction. The thus obtained CD is 0 to

5 meq/g-dry : - .~, . . .

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~'r'~ 3 resin and may sometimes show a negative value due to too great an in-crease in weight by the DEAE-modification reaction. As mentioned above and apparently seen from the Examples, excellent enzyme-immobilization carriers can be obtained even wehn the value of CD is approximately zero.
Thus, it cannot simple be determined from CD value only whether the DEAE-modiEied resin carrier is good or not. Another criterion for judgment of effectiveness, the DEAE-modified resin carrier is the propor- j tion Z of increase in weight by the DEAE-modification reaction: i Z e (Y/X ~ 1) X 100 (%) In consequence, when the resin to be subjected to DEAE-modification has already an anion exchange capacity, it is preferred to satisfy at least one of the requirements CD ~ 0.5 meq/g and z ~ 10, more preferably CD > 1.0 meq/g and Z ~ 20. ~lost preferably, at least one of the re-quirements CD 1.5 meq/g and Z ~ 30 should be satisfied.
On the other hand, when DEAE groups are introduced into a synthetic resin having only hydroxyl groups or sulfhydryl groups as functional groups reactive with DEAE-modification reagents and no anion exchange capacity in itself (irrespective of whether it has cation ex-change capacity), it naturally follows that CD is equal to CT, and CD
is desired to be not less than 1.0 meq/g, more preferably not less than 2.0 meq/g to give an excellent enzyme-immobilization carrier. When a large quantity of DEAE groups cannot be introduced by one reaction, the modificatioh reaction is preferably repeated twice or three times.
When the resin to . .. :

, be subjected to DEAE-modification in an anion exchange resin of strongly basic type II containing no functional group other than hydroxyl groups in quaternary ammonium salts, it is difficult to in-troduce a large quantity of DEAE groups thereinto. A resin having DEAE groups as well as other anion exhcange groups is generally better as an en~yme-immobilization carrier than a resin having only DEAE
groups as anion exhcange groups.
One of the particular features of the DEAE-modified macro-porous resin in aspects of the present invention as an enzyme-immobili-zation carrier is that immobilization by the covalent attachment method as well as by the adsorption method is possible, so long as some of the hydroxyl groups primary amino groups or secondary amino groups remain unaltered in the resin.
Preparation of the immobilized enzymes by the adsorption method can be carried out according to conventional methods. For ex-ample, DEAE-modified resin is treated with an aqueous acid or an alkali solution with concentraiton of 0.02 M to 3 M to activate the DEAE
groups or alternatively it is buffered at near the pH range for enzyme action with a buffer solution (with a concentration of 0.02 M to 3M), followed by thorough washing, and then the DEAE-modified resin is immersed in a solution of enzymes to be immobilized for a sufficient time (it is thereby important that the resin should be wetted to the internal portion of macropores wlth the solution), followed by stirring if desired and further by filtration and washing to immobilization.
The temperature for adsorption ~i ; ~ , ., ., :
, , .: . ., :.:. ,.
~, ~: : ;: :

::

~ ~ ~ 7 ~f~ ~

immobilization should be no-t higher than 40C unless the enzyrne is heat-resistant. It is more preferably 10C or lower. m e thus pre~ared im~o-bilized enzyme can generally contain 100 rng or rnore of enzyme proteins per one gram of dry carrier and is stable if it is not washed with a saline so-lution having s-trong ionic strength. me enzymes to be immobilized on the carrier according -to an aspect of the present invention rnay include not only those consisting only of simple proteins bu-t also enzymes which re- ~-quire co-enzymes. Further, not only one kind of enzymes but also t~ or more different kind of enzymes can simultaneously be imrnobilized.

On the other hand, in the case of the covalent attachment rnethod, there can be applied various attachrnent methods in which the reactivity of hydroxyl groups, priunary amino groups or secondary amino groups present in DE~E-modified resin carrier is utilized. In particular, there rnay be rnen-tioned such r~thods as relatively simple methods among covalent attachment methods to give stable immobilized enzymes as e.g. (l) an attachment method by s-triazynyl derivative using cyanuric chloride or its derivative, (2) an attachrnent method using glutaraldehyde and ~3) an attachment method using a car~odiimide reagent. In the case of imrnobilization by the co-valent attachment rnethod, the amount of enzymes to be imnobilized per unit wieght of the carrier is srnaller than in case of immobilization by the ad-sorption method, but there can be obtained a carrier excellent in specific activity of inmobilized enzymes and activity retentivity.
Typical examples of enzymes which can be immobilized on the pre-sent carrier include, e.g. alcohol dehydro-.

.: :
' ~

, . : ' .

~, :
' ~ ., ' ~fJ~7~f~

genase, aspartate aminotransferase, asparaginase, aspartate decarboxy-lase, amino acid racemase, asparagine synthetase, D-amino acid oxidase, amino acylase, ~- and ~-amylase, adenosine deaminase, amylo-glucosi-dase, aspartase, bromelain, catalase, cellulase, cholinesterase, chymo-trypsin, colagenase, deoxyribonuclease, dextranase, ficin, fumarase, galactose oxidase, ~ -galactosidase, glucose isomerase, glucose oxidase, glutaminase, glutamate dehydrogenase, hesperidinase, hexokinase, inver-tase, inulase, lactase, esterase, lactate racemase, lipase, lysozyme, papain, pronase, pepsin, penicillin amidase, pectinase, phosphatase, phosphatase, phosphorilase, maltase, protease, ribonuclease, me]lbiase, phenol oxidase, tannase, tryosinase, trypsin, urease and uricase.
When the adsorption method is used, enzymes with isoelectric points more acidic than the optimum pH range are preferred. For example, there may be mentioned pronase, amino acylase, glucose isomerase, /~-galactosidase (lactase), ribonuclease, f3-amylase, iso-amylase, pullula-nase, urease, deaminase, lipase, esterase, etc. In the covalent attach-ment method, any enzyme can be used except for those which lose enzyma-tic activity by immobilization.
The present invention in its various aspects is illustrated in further detail with reference to the following Examples, ,'' ~, - . : . :

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

~7~9 Example 1 In a pressure beaker, 13.8 g of sodium hydroxide is dissolved in 60 ml of water (the word "water" hereinafter refers to distilled water).
In this solution is immersed 20.0 g of dry DUOLITE A-7 and degasing is con-ducted by an aspirator for 50 minutes while cooling the mixture with ice-water. Then, while rinsing the resin and the soclillln hydroxide solution with 200 ml of water, they are transferred into a flask of 500 ml capacity.
Stirring slowly the mixture, 180 ml of aqueous solution having 50.0 g of ~-diethylaminoethyl chloride hydrochloride (DF~EC-HCl) dissolved therein is added dropwise by a dropping funnel over 2 hours. The reaction tempera-ture is raised gradually from initiation of the dropwise addition from 20C
to 50C after one hour and thereafter mainta;ned at such temperature.
After completion of the dropwise addition, stirring is further continued for three hours before the reaction mixture is filtered and the resin is washed with water, 0.5 M aqueous nitric acid solution, water, 0.5 M aqueous sodium hydroxide solution, water, 0.5 M aqueous nitric acid solution, water and 0.5 M aqueous sodium hydroxide solution, successively in the order just mentioned. The resin is further washed thoroughly with water until the pH
of the waste washing is 6.9. The resin is then dried by the method as described above and the total resin is weighed to be 28.9 g, namely Z=44.5~.
From the measured total anion exchange capacity of 6.74 meq/g, the anion exchange capacity based on DEAE groups is calculated as 1.80 meq/g.

- ~ , , f,~

Example 2 The same Duorite A-7 and amount of DEAEC HC~ as in Example 1 are used. In the following, there are set forth only the amounts and conditions different from Example 1. 14.0 g of sodium hydroxide is dissolved. While rinsing the resin together wlth the sodium hydroxide solution with 50 ml of water, they are yransferred into a flask of 500 ml capacity. In a reaction mixture already maintained at 62C, a solution of 50 g of DEAEC-HC~ dissolved in 70 ml of water is added dropwisely over 2 hours~ The temperature of the reaction mixture is raised up to 68C one hour after initiation of the dropwise addition.
After completion of the dropwise addition, the reaction mixture is maintained at from 55 to 60C to carry out the reaction for total of 7 hours. Then, as in Example 1, the resin is filtered, washed and dried.
The total amount of the resin is measured to be 39.5 g, Z=97.5%. The total anion exchange capacity is found to be 3.50 meq/g and CD calcu-lated to be -0.09 meq/g. Considering experime.ntal errors, CD is approxi-mately 0 meq/g. Measurement of the water content at the time of drying shows that the starting Duorite A-7 contains 0.084 g water/g-dry resin and said DEAE-modified Duorite A-7 0.0837 g water/g-dry resin, indicat-ing substantially the same value within experimental errors. Hence, the great increase in weight in this Example seems to be due to un-known side reactions as well as the normal DEAE-modification reaction.
Example 3 ~hile cooling a solution of 7.2 g of sodium .. ...
':, . , , : `

hydroxide dissolved in 35 ml of water with ice-water, 10.0 g of DUOLITE
A-7 is immersed therein and degasing is conducted by an aspirator for one hour. Then the resin and the sodium hydroxide solution are transferred into a flask of 300 ml caoacity~ while rinsing with 35 ml of water, and 60 ml of an aqueous solution having ]8.1 g of DEARC-HC dissolved therein is added dropwi~sely over one half hour into the resin-sodium hydroxide solution maintained at 55 to 60C under continuous stirring. Seven hours after initiation of the dropwise addition, the reaction is discontinued, followed by filtration, washing and drying similarly as in Example 1 to fiDd the lQ total weight=15.72 g and Z=57.2%. CT is found to be 6.34 meq/g, from CD
is calculated to be 1.82 meq/g.
Example 4 In 25 ml of ~ N aqueous sodium hydroxide solution is immersed 4.0 g of thoroughly washed and dried D~ILITE S-30. After degassing for 30 minutes by an aspirator under cooling at 2C to 4C, the mixture is transferred into a flask by rinsing with 20 ml of water. I~ile stirring is continued slowly, and maintaining the reaction temperature at 50C to 60C, 28 ml of an aqueous solution having dissolved 7.6 g of ~-diethyl-- aminoethyl bromide hydrobromide (DEAEB.HBr) is added dropwisely to the mix-ture over one hour. The reaction is discontinued 6 hours after initiation of the dropwise addition, followed by filtration, washing and drying as in Example 1. The proportion of increase in weight is found to be 10.5 %, with CT = CD ~ 1.57 meq/g-,~ ,............................. .

, . . . .

Using the thus DEAE-modified DUOLITE S-30 as starting material(4.42 g), a second reaction is repeated similarly as in the above first reaction, whereby the total amount of resin obtained is found to be 4.73 g with CD=2.48 meq/g. Further, a third DEAE-modifi-cation reaction is repeated using the DEAE-modified DUOLITE S-30 ob-tained in the second reaction as starting material, whereby the total amount of the resin is found to be 5.04 g, with CD=2.74 meq/g.
_xamples 5-20 In the following Table 2 are shown in the conditions for pre-paration of various carriers and the results are obtained. The amount of water used shows the total amount of water used in the reaction. Un-less otherwise specifically noted, the starting resin is immersed in an aqueous alkaline compound solution, followed by degasing under cooling with ice-water for 30 minutes to one hour. The reaction time shows that from initiation of the dropwise addition to filtration of the resin.
Filtration, washing and drying are conducted in the same manner as in Example l.

, , '` ~ `

In the following, imrnob:ilization of enzymes on the cc~riers of aspects of the present invention and some reaction experiments are shc~
In the Experiments set forth below, the specific activity of glucose isGmer-ase, protein content and fructose content are determined by the following methods:
(1) ~RaSUrement of activity:
Tb a glucose isomerase solu-tion are added 0.1 M D-glucose, 0.05 M
phosphate buffer solution and 0.005 M MgS04.7H20 cmd, while maintaining the pH at 7.0, the reaction is carried out at 70C for one hour. m e amount of fructose formed is measured. One unit of activity is expressed as the amount of enzymes necessary for forming 1 mg of fructose under the above conditions.
The activity of the immobilized glucose isomerase is determined by measuring the fruc-tose contained in the filtrate when the reaction is carried out with slow stirring under the sarne reaction conditions as de-scribed above except that imrnobilized glucose isomerase is added in place of glucose isomerase solution, followed by filtration of the reaction mix- _ ture. The amount of immobilized glucose isomerase necessary for forming 1 mg of fructose is determined as one unit.
(2) Measuremen-t of fructose content:
Fructose content is determined quantitatively fr~m adsorption strength at 560 nm after 30 minutes by the cysteine carkazol sulfate method at 30C. Under such conditions, the percentage of color formation by glucose is 1/200 of that by fructose and negligible.

.. , , : .
, (3) Measurement of protein content:
With reference to "Seikagaku Jikken Koza" (Biochemical Ex-periment Course) Vol. 5, page 27, quantitative analysis is carried out by Lowry method. The calibration curve is made by using crystalline bovine serum albumin (250 llg/ml-solution).
Experiment 1 There is prepared 30 ml of a 0.05 M phosphate buffer solution \ (pH 7.65, containing 0.005 M MgS04-7H20) containing 45,330 units of glucose isomerase, (protein content: 399 mg), which is purified by acetone fractionation from a u]trasonic crushed suspension of living microorganism cells belonging to genus ~ tomyces (produced by Nagase Sangyo Co.). In this solution is immersed 3.0 g of the DEAE-modified Duorite A-7 prepared in Example 1 and immobilization is carried out at 20C for 16 hours under shaking at 80 r.p.m.
After immobilization, immobilized glucose isomerase is separated by filtration and washed with 0.2 M phosphate buffer solu-tion (pH 7.65) and water. From the activity of the resultant filtrate, the amount of immobilized enzyme is calculated to find that 41,750 units of glucose isomerase are immobilized. The percentage of immobilization of activity is 92.1%.
Similarly, from the protein content in the filtrate, the amount of protein immobilized is found to be 333.4 mg, with an immobili-zation percentage of 83.3%. This immobilized enzyme is packed in a column (diameter: 15 mm) equipped with a jacket and 3 M purified glu-cose solution is flowed at SV=2.5 hr 1 while maintaining .. .

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

~L~.f~3r~

the column temperature at 60C. Conversion to fructose 20 hours after commencement of flowing is found to be 50.4%.
Reference experiment 1 Experiment 1 is repeated except that unmodified Duorite A-7 is used in place of the DRAE-modified Duorite A-7 prepared in Example 1, whereby the immobilized activity is found to be 19,800 units, with immobilized protein content being only 167 mg. When this immobilized enzyme is packed in a column and a 3 M purified glucose solution is passed through such column at 65C at SV=5.5 hr ], the conversion is found to be 19.0%.
Experiments 2 - 11 Purified glucose isomerase extracted from living microorganism cells belonging to genus Streptomyces is immobilized on various carriers under the same conditions as in Experiment 1 to give the results as shown in Table 3. In Table 3, the carrier numbers correspond to those of Examples.

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G) 4~ U~
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o o U h O U) u~ .
011 ~U ~ ~ O O O t~l O O O ~ O O

~ ~1 O ~1 0 r~ O
~:) l O ~U~ o O U') o o o O O O
S~l ~3 h O ~ D ~ ~ D `D .C
orC:~`~ . 3 O O
~,_1 ! U~
~ o ~ Iu~ o n o o u~ u~ o ~r~
.~
U) U~
P~ ~ ~D ~ ~D ~ ~ ~ ~`I
~ r~ r~ 0r~ 0 r~ 0 ~ 0 1--1~

~ rl ~
rl ~I) rl ~ C O O O O O O O O O
,D ~ ,~ ~ rl 0 1~ ~ a~ o o o o o o ~ o ;~ ~ 1~ ~ o1~ ~ 0 ~ ~ ~
E-l ~ O `_ ~1 o ô ~ D O (~ 0 1~
~ G) ~d ~ ~ ~ ~ ~1 ~

~ ~ A C~ O ~) ~ ~1 N ~J sd 1:
U-) ~ ~1 ~ 0 t~ ~ ~ ~ ~~

O O O O C O O O O O
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~ o ~ 0 .~
a) ~c~ OD ~ O r{
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Z

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Experiment 12 In 20 ml N aqueous sodium hydroxide solution is immersed 2.0 g of the DEAE-modified Duolite A-7 prepared in Example 1 and, after degasing at 4C for 15 minutes, excessive alkaline solution is removed by filtration. ~llis resin is immersed in 25 ml of dioxane at room tem-perature for 5 minutes and, after stirring the mlxture, 20 ml of dioxane solution containing 4 g of cyanuric chloride prepared beforehand is added thereto, fol]owed by vigorous stirring at room temperature. After three minutes, 25 ml of cold water is-added to the reaction mixture and, after 5 seconds 25 ml of acetic acid is further added thereto to terminate the reaction. The mixture is quickly washed with cold water and cold acetone.
The resultant resin is added to a phosphate buffer solution of pH 7.8 containing 20,400 units (protein content 148 mg) of glucose isomerase.
Immobilization reaction is conducted with stirring for 5 hours while maintaining the temperature at 2C to 4C and maintaining the pH at 7.8 by the addition of 0.2 N sodium hydroxide solution. Immobilized enzyme is separated by filtration and washed with ice-cooled 5 M sodium chlor-ide solution, 0.1 M phosphate buffer solution and ice-cooled water in this order until there is detected no protein in the wash. From the activities and protein content in the wash, the immobilized activity is found to be 17,500 units (86%) with an immobilized protein content of 121 mg (81%).
Experiment 13 In a solution having 200 mg of commercially available enzyme pronase E originated from ~tr~p_omy~ griseus dissolved in 25 ml of a 0.05 M phosphate buffer solution of pH 7.0 maintained at 4C, there is immersed 2.0 g of the DEAE-modified DUOLITE A-7 prepared in Example 1.
The mixture is slowly stirred at 4C for 8 hours under suction by an aspirator to .

immobilize the enzyme by adsorption. The immobioized pronase E is thoroug'n-ly washed wi th 0.05 M phosphate buffer solution of pH 6 0 and then with water. The inunobilized enzyme protein is found to be 182 mg. The specific activity of the inunobilized enzyme is mèasured at 40C and p}l 6.0 by a pH-stat (liiranuma Seisakusho, Model pS-ll) using 20% DL-Iysine methyl ester as a substrate to be 3.42,~ moles/mg.min, wllich corresponds to 57% of the specific acitivity of the solution enzyme.
Experiment 14 Pronase E is immobilized by the covalent attachment method under the same conditions and procedure as in Experiment 12 except that 2.0 g of the DEAE-modified DUOLITE A-4 prepared in Example 12 is used in place of 2.0 g of the DEAE-modified DUOLITE A-7 in Experiment 12 and 120 mg of pro-nase E in place of 20,400 units of glucose isomerase. The amount of immo-bilized enzyme is found to be 95.8 mg/g-carrier and the specific activity to be 2.90 ~t moles/mg.min, as measured by pH--stat at pH 6.0 and 40C using 20% DL-lysine methyl ester as substrate.
Experiments 15 - 19 Several examples of pronase E immilbilized on the carriers of aspects of the present invention are shown below in Table 4. The adsorption method in the column of ': ;

~76~
immobilization method in such Table refers to a similar procedure as described in Experiment 13 and the covalent attachment method to a simi-lar procedure as in Experiment 14 using cyanuric chloride. The numbers of carriers correspond to those of Examples. All of the specific activi-ties are measured by pH-stat under the conditions of pH 6.0 at 40C, using 20æ DL-lysine methyl ester solution as a substrate.
Table 4 Amount Amount of Immo- Amount of enzymes Specific bili- of enzymes immobilized activity Experi- Carrier zation carrier charged (mg/g- (umoles/
ment No. No. method _ ) (mF~) carrier) _min~m 13 Adsorp- 2.0 200 65 2.72 tion method 16 14 " 2.0 200 61 2.75 17 7 covalent 1.0 120 105 1.91 attachment method 18 1 " 1.0 115 73 4.86 19 5 " 1.0 100 78 3,48 Experiment 20 Immobilization of lactase is effected according to the same covalent attachment method with cyanuric chloride as in Experiment 12 using 100 mg of lactase produced by Asperigillus oryzae and 1.0 g of the DEAE-modified DUOLITE A-7 prepared in Example 2. After thorough washing of the immobilized enzyme, the amount of immobilized enzyme is measured to be 66.7 mg protein/g-carrier and the specific activity is measured at pB 4.5 and 30C using 5 ~ lactose as substrate to be .

~:
.~ ., :
, J~ ,r,"~3 5.2fumoles/mg min, which corresponds to 30 % of the activity o~ the solution enzyme (solution activity: 17.3 ~moles/mg-min).
Experiment 21 Lactase produced by ~p__&~ oryzae (different in degree of purification from that of E~periment 20, with a solution activity of 10.2 ~moles/mg-min at pH 4.5 and 30C), 165 mg, is dissolved in 10 ml of a phosphate-citrate buffer solution of pH 4.5 with a concentration of 0.02 M which is maintained at 4C. In this solution is immersed 1.0 g of the DEAE-modified DUOLITE-37 prepared in Example 5 and immobiliza-tion is effected under shaking at 80 r.p.m. for 16 hours while maintain-ing the temperature at 4C. After immobilization, the immobilized en-zyme is washed with phosphate-citrate buffer solution with a concentra-tion of 0.05 M and the same pH, and then with water. The amount of the immobilized enzyme is found to be 123 mg and the specific activity9 at pH 4.5 and 30C using 5 % lactose as substrate, is measured to be 2.1 ,umoles/mg min~
Experiment 22 Lactase produced by ~ gillus oryzae (enzyme activity in solution of 17.3~umoles/mg-min, at pH 4.5 and 30C), 200 mg, is dissolved in 10 ml of a phosphate-citrate buffer solution maintained at 4C with a concentration of 0.02 ~l and pH S.5. In this solution, there is immersed 1.0 g of the DEAE-modified D~OLITE A-7 prepared in Example 3 and immobiiization is effected under shaking at 80 r.p.m. for 18 hours while maintaining . .

' ~ ' , ~ ` ~ ' ' the temperature at 4C. The amount of the immobilized en7yme, after washing thoroughly with a phosphate-citrate buffer solution with a con-centration of 0.05 M and pH 5.5 and then with water, is found to be 149 mg. The specific activity is measured to be 2.1 ~Imoles/mg-min. at pH 5.5 and 30C using 5 % lactose as a substrate.
Rxperiment 23 _ _ _ _ A commercially available ~ -amylase produced from soybean (produced by Nagase Sangyo Co.; 1.5 x 104 unit*/g), 300 mg, is dissolved in 30 ml of a 0.02 M acetate buffer solution of pH 5.0 and insolubles are separated by centrifugation. In the resultant supernatant lS
thrown 2.0 g of the DEAE-modified DIAION WA-21 prepared in Example 13.
After degasing by aspirator, immobiliæation is effected under shaking at 80 r.p.m. for 16 hours while maintaining the temperature at 4C.
After thorough washing with the above acetate buffer solution and water, the amount of the immobilized enzyme is found to be 121 mg/g-carrier.
This inmobilized enzyme is packed in a column equipped with a jacket and, while maintaining the column at 50C~ a substrate of 2~ soluble starch solution is flown at SV=0.4 hour 1. The increase in amount of reduc-ing sugar is measured by the Somogyi-Nelson method to determine the amount of maltose formed. The amount of maltose in the effluent is found to be 11.5 mglml-solution. Vnder these conditions, continuous reaction is performed for 10 days. The amount of maltose in the effluent on the 10th day is found to be 11.3 mg/ml. showing that there is little change.
*) 1 unit represents activity which can form ~ 32 -;:~
` , ' ' ' a reducing power correspondlng to 100 r of glucose at pH 4.5, 40~C
for one minute.
Experiment 24 Experiment 23 is repeated except that the DEAE-modified DUOLITE A-7 prepared in Example 1 is used in place of the carrier pre-pared in Example 13. The amount of immobilized enzyme is found to be 129 mg/g-carrier. ~hen continuous reaction is performed in the same manner as in Experiment 23, the amount of maltose in the first day is found to be 11.9 mg/ml-solution, while that on the 10th day to be 12.0 mg/ml.
Experiment 25 Pullulanase produced by _r_ acter aerogenes (produced by Nagase Sangyo Co.), 200 mg, is immobili~ed on ~he DEAE-modified AMBERLITE IR-45 prepared in Example 14 by the same method as in Experi-ment 23. The amount of enzyme immobilized is found to be 58.5 mg/g-carrier. This immobilized enzyme, 2 g, is immersed in 25 ml of a 0.5 %
pullulane solution. After shaking at 80 r.p.m. at pH 5.0 and 40C for 30 minutes, the amount of maltotriose in the filtrate is measured by the Somogyi-Nelson method to find that the conversion to maltotriose is 97 %.
Experiment 26 The DEAE-modified Duorite A-4 prepared in Example 8, 2.0 g, is equilibrated with 0.05 M Veronal buffer solution of pH 7.0 and tl-en immersed in 30 ml of 0.05 M Veronal buffer solution containing 300 mg of aminoacylase produced by ~ gillus _ryzae to effect to effect immobili-zation at 4~C under shaking at 80 r.p.m.

~ ~ .
' ~ ; , -for 15 hours. The amount of the immobilized enzyme is calculated frGm the filtrate as 97 mgi/g-carrier. This aminoacylase is packed in a column of 10 mm in diameter equipped with a jacket and, while maintaining the column f temperature a-t 40C, 0.2 M N-ace-tyl-DL-methionine solution (pII 7.0, con-taining 1 x 10 4 M CoC 2) is flowed t~ough the column continuously. m e percentage of hydrolysis of L-isc~r after 24 hours is 100% and there is observed no loss in activit~v even af-ter 10 days.
Experiment 27 A cammercially available aden~lic deamlnase (produced by Amano Seiyaku Co., 50,000/g) for producing inosinic acid, 400 mg, is dissolved in 50 ml of a phosphate buffer solution of 0.02 M concentration (pH 5.6) m~ltained at 4C. In ~his solution is immPrsed 4.0 g of the DEAE-modified Duorite A-7 prepared in E~ample 3. After degasing by an aspirator, inmo~
bilization is effected under shaking at 80 r.p.m. at 4C for 16 hours. The amount of the en~yme immobilized is found to be 72 mg/g-carrier. This immobilized enzyme is packed in a column ~diameter: 15 mm) equipped with a jacket and, while maintaining the temperature at 50C, 1 % adenylic acid solution is flowed through the column at SV=l.l hour for 4 days, whereby there is observed no substantial loss in activity even after 100 hours.
Application examples:
In the following, there are shcwn various app].ication examples.
Application examples 1 to 5 are the experiments of the continuous isomeri-zation reaction using a column of glucose isamerase Lmmobilized on ~ 34 --: .' , . - :

. ~:
': ~ ` ' '' : -.:

- , . :
, the carrier of the present invention. Application examples 6 to 8 are experiments in which batch reactions using immobilized pronase E
are repeated. Application examples 9 and 10 are experiments in which batch reactions using immobilized lactase are repeated. The results are shown in Tables 5 to 7. The numbers of Experiments show that the imrnobilized enzymes prepared and measured for their activities in the Experiments as set forth above are used.
Glucose isomerase is packed ln a column and 3 M (54 I~/V %) purified glucose is passed therethrough under the same conditions as described in the above Experiments. Accordingly, SV is maintained con-stant until the end of the column operation and the time at which con-version to fructose becomes half of that at the initial stage (shown in Table 3 as that after 20 - 24 hours) is determined as the half-life time.
The amount of purified glucose solid components treated per 1 g of the immobilized enzyme by the time at which conversion to fructose becomes 1/4 of that at the initial stage is determined as the productivity.
When the carrier according to an aspect of the present invention is em-ployed, activity retentivity is very good and productivity is very high as seen in Table 5.
For immobilized pronase, 10 ml of 10 ~ L-lysine methyl ester is used as a substrate and each 100 mg of the immobilized enzyme pre-pared in corresponding Experiments is used. The experiments are re-peated by batch methods. Tha experiment time is 40 minutes per one experiment and the number of experiments by which ~, - 35 -~7~

the reaction rate measllred b~ p~l~rn.eter 'r~ecom~ half of -,-that of the fi.rst experimerlt is determined as the half-lire number.
For irnrnobi].izecl lactase, ].00 mgr o~` :i.n1rnobi.liz~(1 enz~/rne is ~)sed al~d 5 usin,rr 10 ml 0~` 5 ~
lactase solution as substrate~ the reaction is carried -~ -out b~y batcll met;hod under shaking at c~O r,p.ln. for 30 ~;
rninutes for each experiment. The activit,y is meclsllred from the arnollnt o~ glucose formecl. This experiment is repeated uncler otherwise the sarne condition~ as described in the above Fx~eriments.
~able 5 Cc~ntinuouc; isomerization reaction through column Productivity A~plicatior! ~,x~eri- ~lalf-life time (g~ sugar solid/g example ment f'or activity immobilized No. _ _ !lo. _ (da~;s) enz,yme) r~
2n ~ 500 L
4 3 l 3 9 1 5 0 3 3 53 l~,~
ll 5 ~lc' 12,c~OC
c~ l~6 ~ c300 q'ab].e 6 .r'epeated reaction~, usin~, i.rnmobilized pronase ~'he nwnlier of' experiments at ~pr)li.cal,:Lon E~perimer]t which the acti.vity is r~duced exarnnle ~lo. J!o. to half-~alue (half-life nurmber)

6 l~l 90

7 1, 50 19 25 ~:~

Tclble 7 ~.
I~e,r)eated reaction~ usinr immobili~ed lactase r~i The nu~nber of exl-eriment at icat-ion I~ erlrnent ~ hl~.h the ~ct:iv;ty is reduced a~ nl.e l~lo. ~lo. __ t,( hal.r`-vallle (ha -1-lf`e nurr.ber) :L0 22 38 ~.
~, ..

6, .' . .

~".

\

Claims (23)

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, synthetic anion-exchange resin having a specific surface area of at least 1 m2/g-dry resin and containing macropores which a pore size of 100 .ANG. to 2,000 .ANG., of which total volume is at least 0.1 cc/g-dry resin;
said resin comprising (a) a phenol-formaldehyde condensation matrix and diethylaminoethyl groups linked to the matrix through an ether linkage, wherein the ion-exchange capacity of the resin is not less than 1 meq/g-dry resin, (b) a phenol-formaldehyde condensation matrix having primary amino groups, secondary amino groups or a mixture hereof as an anion-exchanger and diethylaminoethyl groups linked to the matrix through an ether linkage or to said primary or secondary amino groups to form a secondary or tertiary amino groups respectively, wherein the ion-ex-change capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g-dry resin, or (c) a cross-linked polystyrene matrix having pri-mary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and diethylaminoethyl groups linked to said primary or secondary amino group to form a secondary or tertiary amino group respectively, wherein the ion-exchange capacity due to the diethylamino-ethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exhange capacity is not less than 1 meq/g-dry resin.

2. An enzyme-immobilization carrier as claimed in claim 1, wherein the specific surface area of said resin is 5 m2/g or more.

3. An enzyme-immobilization carrier as claimed in claim 1, wherein the average pore size of the pores in said synthetic resin is from 150 .ANG. to 1,000 .ANG..

4. An enzyme-immobilization carrier as claimed in claim 1, wherein the volume of macropores with pore sizes from 100 .ANG. to 2,000 .ANG.
is 0.2 cc/g or more.

5. An enzyme-immobilization carrier as claimed in claim 1, wherein the resin further contains other anion exchange groups.

6. The enzyme-immobilization carrier according to claim 1, wherein said synthetic anion-exchange resin comprises a phenol-formalde-hyde condensation matrix having primary amino groups, secondary amino groups and a mixture thereof and diethylaminoethyl groups linked to the matrix through an ether linkage or to said primary or secondary amino group to form a secondary or tertiary amino group respectively, the ion-exchange capacity provided by the diethylaminoethyl groups is not less than 1 meq/g-dry resin, and the total ion-exchange capacity of the resin is not less than 2 meq/g-dry resin.

7. The enzyme-immobilization carrier according to claim 1, wherein said synthetic anion-exchange resin comprises a phenol-formalde-hyde condensate matrix and diethylaminoethyl groups linked to the matrix through an ether linkage and the ion-exchange capacity of the resin is not less than 2 meq/g-dry resin.

8. The enzyme-immobilization carrier according to claim 1, wherein said synthetic anion-exchange resin comprises a crosslinked polystyrene matrix having primary amino groups, secondary amino groups or a mixture thereof and diethylaminoethyl groups linked to said pri-mary or secondary amino group to form a secondary or tertiary amino group respectively, the ion-exchange capacity provided by the diethyl-aminoethyl groups is not less than 1 meq/g-dry resin and the total ion-exchange capacity of the resin is not less than 2 meq/g-dry resin.

9. The enzyme-immobilization carrier according to claim 5, wherein the ion-exchange capacity provided by said diethylaminoethyl groups is not less than 1.5 meq/g-dry resin.

10. The enzyme-immobilization carrier according to claim 8, wherein the ion-exchange capacity provided by said diethylaminoethyl groups is not less than 1.5 meq/g-dry resin.

11. A process for producing an enzyme-immobilization carrier comprising a synthetic anion exchange resin having diethylaminoethyl groups, which comprises reacting a diethylaminoethyl derivative of the formula:

wherein X is halogen or hydrogensulfate or its salts with granules of a solid phenol-formaldehyde resin of a macroporous type having no other functional groups besides OH groups, primary amino groups, and/or secon-dary amino groups and tertiary amino groups in addition to OH group (not including said diethylaminoethyl groups), or with beads of a crosslinked polystyrene resin of a macroporous type having primary amino groups and/
or secondary amino groups and tertiary amino groups, having a specific surface area of at least 1 m2/g-dry resin and containing macropores of which the total volume of those with pore sizes from 100 A to 2,000 A
is at least 0.1 cc/g-dry resin in the presence of an alkaline compound.

12. A process as claimed in claim 11, wherein said diethyl-aminoethyl derivative or its salt is .beta.-diethylaminoethyl chloride hy-drochloride, .beta.-diethylaminoethyl bromide hydrobromide or .beta.diethylamino-ethyl hydroensulfate.

13. A process as claimed in claim 12, wherein said diethylamino-ethyl derivative salt is .beta.-diethylaminoethyl chloride hydrochloride.

14. A process as claimed in claim 11, wherein said diethylamino-ethyl derivative or its salt is used in an amount of 1/3 to 10 parts per one part of the dry resin.

15. A process as claimed in claim 14, wherein said diethylamino-ethyl derivative or its salt is used in an amount of 1/2 to 3 parts per one part of the dry resin.

16. A process as claimed in claim 11, wherein said alkaline compound is an alkali metal hydroxide, an alkaline earth metal hydroxide or an organic amine.

17. A process as claimed in claim 16, wherein said alkaline com-pound is sodium hydroxide.

18. A process as claimed in claim 11, wherein the amount of said alkaline compound added is from 1/10 to 5 times moles as much as that of said diethylaminoethyl derivative or its salt.

19. A process as claimed in claim 11, wherein said synthetic resin is selected so that it has a specific surface area of 5 m2/g-dry resin or more.

20. A process as claimed in claim 11, wherein said macroporous resin is selected so that the total volume of macropores with pore sizes from 100 .ANG. to 2,000 .ANG. in the synthetic resin is 0.2 cc/g or more.

21. A process as in claim 11, wherein said macroporous resin is selected so that the average pore size of the pores in the synthetic resin is from 150 .ANG. to 1,000 .ANG..

22. A process as claimed in claim 11, wherein an on exchange capacity on the basis of primary, secondary and/or tertiary amino group excluding diethylaminoethyl groups is at least 1 meq/g-dry resin.

23. A process for immobilizing an enzyme which comprises carrying said enzyme on a synthetic resin which comprises a macroporous, synthetic anion-exchange resin having a specific surface area of at least 1 m2/g-dry resin and containing macropores with a pore size of 100 .ANG. to 2,000 .ANG., of which total volume is at least 0.1 cc/g-dry resin; said resin comprising (a) a phenol-formaldehyde condensate matrix and diethylaminoethyl groups linked to the matrix through an ether linkage, wherein the ion-exchange capacity of the resin is not less than 1 meq/g-dry resin, (b) a phenol-formaldehyde condensate matrix having primary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and diethylaminoethyl groups linked to the matrix through an ether linkage or to said primary or secondary amino groups to form a secondary or tertiary amino group respec-tively, wherein the ion-exchange capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g-dry resin, or (c) a crosslinked polystyrene matrix having primary amino groups, secondary amino groups or a mixture thereof as an anion-exchanger and diethylaminoethyl groups linked to said primary or secondary amino group to form a secondary or tertiary amino group respec-tively, wherein the ion-exchange capacity due to the diethylaminoethyl groups is not less than 0.5 meq/g-dry resin and the total ion-exchange capacity is not less than 1 meq/g-dry resin.