IMMOBILIZED CHOLINESTERASE ENZYME PREPARATIONS AND A PROCESS FOR THE PREPARATION THEREOF

Abstract: Immobilized cholinesterase enzyme preparations are prepared by treating a polymeric resin, built up from acrylic acid and/or methacrylic acid and acrylamide and/or methacrylamide monomers with an acryl or allyl type cross-linking agent and contains at least 0.1 meq/g of -COOH funtional groups, with a carbodiimide derivative which is soluble in water or is soluble in an organic solvent at temperatures below 0<o>C, applying a solution of cholinesterase enzyme with a pH of 4.5 to 8.5 onto the resulting activated support, washing the resulting product, and drying it then, if desired.

Description: IMMOBIIIZBD CH0I_I-H3ST.SR S3 -SNZYMB ER3PARATI0US AHD A PROCESS _? OR 1533 PREPARATION THEREOF The invention relates to new immobilized cholin¬ esterase enzyme preparations as well as to a process for the preparation thereof. It is known that cholinesterase enzyme, isolated most frequently from horse serum, can be applied to detect neo otoxins of phosphate ester, carba ate and sulfate ester type, and to determine the quantity of ne rotoxins occurring in air or water. The basis of de¬ tection and measurement is the inhibiting effect exβrt- ed on the enzyme by neuro oxins. (Thus, immobilized cholin¬ esterase enzyme preparations may play the central role of continuously operating measuring systems. The prin¬ ciples of such measuring systems are described, among others, in Anal. Che . 2£, 1378 (1965) and Anal. Biochem. £1, ? 6 (1973). Several methods have been described in the litera¬ ture for the immobilization of cholinesterase isolated from horse serum. According to Anal. Chem. 2Z » 1378 (1965), Anal. Biochem. lg, 8 (1967), Anal. Biochem. ' 21, 3 1 (1970) and the U.S. patent speci ica ion Ho. 3,2231593 the enzyme is entrapped into various gels, • primarily starch gels. The U.S. patent speci ication HO. 3,223 > 593 also discloses the entrapment of the enzyme into agar and carrageen gels. The use of polyacrylamide as entrapping medium is suggested in Biochi . Biophys. Act a 212, 352 (1970) and Anal. Biochem. 22, 3-+1 (1970). The enzyme can also be coupled through covalent bonds to polymeric macro olecules with appropriate functional groups. Thus e.g. according to Biochim. Biophys. Acta OMPI - 2 - 191. 478 (1969) the enzyme is immobilized on Sepharose 2B activated previously with cyanogen bromide, whereas according to Biochim. Biophys. Acta 2ZZ, 297 (1975) polymaleic anhydride is applied as support. According to Glin. Chim. Acta 121, 125 (1980) the enzyme is immobiliz -. . on non-porous glass by carbodiimide or glutaric aldehyde -co pling, whereas according to the USSR patent specifica tion Ho. 707,923 the enzyme is coupled to porous glass or silica gel activated with cyanuric acid chloride. As described in Can. J. Biochem. 48, 1314 (1970) , cholin¬ esterase isolated from horse serum can also be attached to a Procion brilliant orange DEAB-cellulose complex through covalent bonds. A disadvantage of the known cholinesterase enzym preparations in which the enzyme is entrapped into a gel is that the enzyme dissolves continuously from it. -There ■= fore, such preparations are not suitable for continuous, prolonged application. The known cholinesterase enzyme preparations immobilized on the substrate by covalent ^ bonds have adverse throughflow properties from the aspects of application in automatic systems, moreover some of the supports, -such as Sepharose 23, are sensitiv to icrobial effects. The invention aims at the eliminarjion of the above disadvantages. According to the invention im¬ mobilized cholinesterase enzyme preparations are provid¬ ed which see the requirements of prolonged use, and contain the enzyme bound to a chemically and xicrobially inert support with appropriate mechanical properties ensuring a high throughflow rate. As a further advantage the enzyme can be coupled to the suppers under mild reaction conditions. It has been observed that polymeric resins built up from acrylic acid and/or methacrylic acid and acryl amide and/or ethacryl amide monomers with acryl or allyl type cross-linking agents /such as H^lT'-methyl- 5 ene-bis(acrylamide) , ethylene diacrylate or ,IP-di- allyl-tartaric amide/, containing at least 0.1 meq/g, preferably 2 to 8 meq/g, of -COOH functional groups, completely meet the requirements set forth in connection with the supports. These supports can be prepared by 10 methods known per se. One can proceed e.g. so that acryl amide and/or methacryl amide is copolymerized with acrylic acid and/or methacrylic acid in the presence of a cross-linking monomer, whereas according to another possible method a cross-linked polymer is prepared first 15. from acryl amide and/or methacryl amide using the above cross-linking agents, and the acid amide groups of the resulting polymer are subjected then to partial hydro¬ lysis to provide the required amount of -COOH unctional grou ≤. Akriles C type enzyme supports, marketed by 0 Reanal Finomvegyszergyar (Budapest, Hungary), prepared by subjecting Akrile-x ? type acryl amide - E,23 " '-methy1- ene-bis(acrylamide) copolymer beads (e.g. Akriles P-30, P-100 or P-200) to partial hydrolysis with an acid (e.g. hydrochloric acid or another strong acid) or a base 5 (e.g. sodium hydro-xide, sodium carbonate or another strong base) , belong to this latter type of support polymers. In these substances about 0 % of the -COM- functional groups present are converted into carboxy groups upon hydrolysis, * .vhereas the remaining -C02TH 2 0 groups do not hy rolyze even under severe reaction con¬ ditions. Consequently, unchanged -QQI ^ groups are situated between she carbo-zy groups cf the hydrolyzed copolymer, which fix the carboiϊy groups in ' favourable steric positions. If an enzyme is coupled to she carbo-sy - -+ - groups by a carbodiimide activation method known per se, the functions of the individual immobilized enzyme mole¬ cules do not interfere with each other due to the favourable steric positions of the carboxy groups; thus the immobilized enzyme preparation may have a very high specific activity. Polymers containing acrylic acid and/or methacrylic acid and acryl amide and/or methacryl amide monomers connected to each other through a bi- functional cross-linking monomer and carrying at least 0.1 meq/g of -COOH functional groups, prepared by other methods, possess similar advantageous properties. These polymers have bead shape, thus they enable a great throughflow rate, and at the same time they are chemical inert and completely resistant to the effects of micro— organisms. The carbo-xy functional groups of these supports can be activated by the well-known carbodiimide method, and the resulting activated supports can bind cholin¬ esterase enzyme. This reaction can be performed even under mild conditions (at temperatures of 0- °C in a medium of pΞ r 7.0) • Based on the above, the invention relates to im¬ mobilized cholinesterase enzyme preparations which contain the cholinesterase enzyme bound to a polymeric resin, built up from acrylic acid and/or methacrylic acid and acryl amide and/or methacryl amide monomers with acryl or allyl type cross-linking agents, contain¬ ing at least 0.1 meq/g of -CCOH functional groups, previously activated with a carbodiimide. The invention also relates to a process for the preparation cf these iπmobilized enzyme preparations. According to the invention a polys-eric resin, built up from acrylic acid and/or methacrylic acid e d acryl amide and/or methacryl amide monomers with an acryl or allyl type cross-linking agent and containing at least 0.1 meq/g of -COOH functional groups, is treated with a carbodiimide derivative which is soluble in water or is soluble in an organic solvent at a temperature below 0°C, a solution of cholinesterase enzyme with a pH of . to 8.5 is applied onto the resulting activated support, the resulting product is washed, and then dried, if desired. Akrilez C type polymers discussed above can be applied to particular advantage as supports for the cholinesterase enzyme. Cholinesterase of any origin,, isolated by any method, can be applied as enzyme in the process s ccord- ing to the invention. To activate the polymeric support, e.g. N-cyclo- hθxyl-ST * - β-(N-meth lmorpholino)-ethyl7-carbodiimide p-toluenesαlfonate or H-eth l-H , -(5-dime hylaminopropyl.)- carbodii ide hydrochloride can be applied as carbodi- imide derivative. It is preferred to utilize water- soluble carbodiimides for this purpose. Of the carbodi¬ imide derivatives soluble only in organic solvents those substances can be applied in the process of the inven¬ tion which have appropriate solubility in the given ■ organic solvent even at low temperatures, i.e. below 0°C. Cholinesterase is applied onto the activated support from a solution of pH . to 8.5, preferably from an almost neutral solution (pH about 7.0) • Cholin- estarase is applied preferably as a solution forced with a 0.1 molsr potassium phosphate buffer (pH = 7.C) . The immobilized enzyme preparation formed in this coupling reaction is washed in a manner known per se, and then dried, if desired. The enzyme preparation is, however, also storable in aqueous suspensions at 0 to 4°C. The specific activity of the immobilized enzyme preparations according to the invention is 100-120 units/1 g of -zerogel, which, when compared to the activit data disclosed in Anal. Chem.2Z, 1378 (1965) > Anal. Bio- chem. 1°/, 58 (1967), Biochim. Biophys. Acta 212, 362 (1970) and Anal. Biochem. 1, 362 (1973) » s favourable for practical use. The immobilized enzyme preparations according to the invention are more stable in slightly alkaline media, i.e. at the optimum pE of their application (pH 8.0), than the soluble enzyme itself, which is a significant advantage in their prolonged use. The invention is elucidated in detail by the aid " ' of the following non-limiting S∑amples. Example 1 100.6 mg of Akrilex C-100 xerogel are suspended in 5»0 ml of a 0.1 molar potassium phosphate buffer (pH 7.0) , and a solution of 102.5 mg of Jϊ-ethyl-F , -(5-di- e thylaminopropyl) -carbodiimide hydrochloride in 2.5 ml of a cold (+4°C) buffer solution is added to the suspen¬ sion at 0°C under steady stirring. After 10 minutes of stirring 47.4 g of cholin sterase , dissolved in 2.5 nil of a cold (+4°C) buffer solution, are added to the reac¬ tion mixture . The mixture is maintained at 0 to 4°C for 43 hours; during this period the suspension is stirred twice f or 6 hours , each. The gel is filtered off , washed thrice with 10 ml oortions of a 0.1 molar no assi phosphate buffer (pH 7»0) , thrice with 10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7»0) also containing 0.5 moles of sodium chloride, and then thrice with 10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7.0) . Thereafter the gel is washed four times with 25 ml portions of distilled water, and the result¬ ing salt-free gel is subjected to freeze-drying. 106.2 mg of an immobilized cholinesterase preparation are obtained. Activity: hydrolysis of 120 u oles of butyryl'-thiocholine iodide/min/g of dry substance. Example 2 102 mg of A rilβx C-100 xerogel are suspended in 5.0 ml of a . 0.1 molar potassium phosphate buffer (pH 7.0) , and a solution of 202 mg of ϊϊ-cyclohexyl-lT-'- I- (IT-methylmorpholino)-ethyl7-carbodiimide p-toluenesπl- fonatβ in 2.5 ml of a cold (+4°C) buffer is added to the suspension at 0°C under steady stirring. After 10 minutes of stirring 49 mg of cholinestersse, dissolved in 2.5 ml of a cold (+4°C) buffer solution, are added to the reaction mixture . The mixture is maintained at 0 to 4°C for 48 hours j during this period the suspension is stirred twice for 6 hours, each. The gel is filtered off, washed thrice with 10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7.0) , thrice with 10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7«0) also containing 0.5 moles of sodium chloride, and then thrice with 10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7.0) . Thereafter the gel is washed four times with 25 ml portions of distilled water, and the resulting salt-free gel is subjected to freeze-drying. 110 mg of an i-imobilized cholinesterase are obtained. Activity: hydrolysis of 110 ,umoles of butyryl-thiocholine iodide/min/g of dry substance . Comparative tests and results The activity and specific activity of the im¬ mobilized enzyme preparations according to the invention were compared to the respective data of cholinesterase enzyme preparations immobilized on other supports. The following reference substances were applied: a) An Snzacryl AA type support (acryl amide - UjJS " '- -oa th lene-bis-acrylamide copolymer containing aromatic amine functional groups, produced by Koch-Light Laboratories Ltd. , φlπbrook, Bucks, Great Britain) was diazotatβd according to the prescriptions of the producer firm 2och-Light Laboratories Ltd. , EL 3 » page 343 { 1970 7- A mixture of 100 mg of the resulting di- azotated Snzacryl AA support and 2.5 in of cholinester- ase was stirred in 0.5 ml of a 0.1 molar potassium phosphate buffer (pH 7-»5) at 0-4°C for 8 hours, the suspension was allowed to stand in a refrigerator at 4°C for 16 hours , then it was stirred again at 0-4°C for 8 hours, and kept again in a refrigerator at 4°C for 16 hours. b) An -Snzacr l AH type support ^acryl amide - acrylic hydrazide - 23 " , N' -me thylene-bis ( acryl amide) co¬ polymer, produced by Koch-Light Laboratories Ltd. , Coin- brook, Bucks , Great Britain/ was activated with sodium nitrite in acidic medium according to the prescriptions of the producer firm ^och-Light Laboratories Ltd. , KL 3, page 344 ( 1970)7 • iOO mg of the activated support, which contains the functional groups in the f orm of acid azide groups, were admixed with 2.5 s of cholin- esterase enzyme in a 0.1 molar borate buffer (pH 8.C) , and the resulting suspension was stirred under the conditions described in point a ) above . OMH c) 93 mg of carboxymethyl cellulose with a bind¬ ing capacity (-COOH content) of 2.3 meq/g, produced by Eeanal Fino vβgyszergyar, Budapest, Hungary, were suspen ed in ml of a 0.1 molar potassium phosphate buffer (pH 7.0), and 0 mg of tf-ethyl-N'-(3-dimβthylaminopropyl carbodii idβ hydrochloride, dissolved in 2.5 nil of a . cold (+4°C) buffer solution, were added to the suspen¬ sion under steady stirring. After 10 minutes of stirring 25 mg of cholinesterase, dissolved in 2.5 ml of a cold (+4°C) buffer solution, were added to the suspension. The enzyme was reacted with the activated support at 0-4°C for 43 hoursj during this time the mixture was stirred twice for 6 hours, each. d) A polymaleic anhydride support produced by the firm Merck (Darmstadt, German Federal Hepublic) was coupled with cholinesterase according to the prescrip¬ tions of the producer firm Biochemics Merck: Polyfmalei anhydride) crosslinked enzyme support/. 1 g of the support was suspended in 25 ml of a 0.05 molar potassium phosphate buffer (pH 7.0) at 0°C, and after one minute of stirring 0.5 g of cholinesterase, dissolved in 5 ml of a cold (+4°C) buffer solution, were added to the suspension. The pH of the reaction mixture was maintaine between 8 and 9 by adding continuously a 1 n aqueous sodium hydroxide solution to the mixture. The introduc¬ tion of the base was continued until the pH of the mix¬ ture remained constant for about 0.5 hours. The immobilized enzyme preparations, obtained in the reactions described in points a) to d) above, were filtered off or centrif ged, and washed then with the buffer applied at immo ilizetion, a 0.5-1.0 molar sodium chloride solution prepared with the. same buffer, and then again with the buffer applied at i mobiliza- tion, in order to remove the proteins not bound by co- valent bonds. Thereafter the ions of the buffer solution were removed by washing the solids with water, and the immobilized enzyme preparations were subjected to freeze-drying. The freeze-dried products were stored in a refrigerator at 4°C. The amounts of proteins bound in the immobilized enzyme preparations was determined indirectly, as the difference of the amount of protein introduced into the reaction mixture and remained in the supernatant and wash after immobilization. The protein content of the cholinesterase solutions was determined by the biuret method ^A .G. Gornell, C.S. Bardawill, M.M. David: J. Biol. Chem. 1 2, 751 (194917 * The activity of dissolved and immobilized cholin- esterases was determined by measuring the sulfhydryl groups liberated upon the decomposition of butyryl-thio- ■ choline iodide substrate . The sulfhydryl groups were measured with bis (5-carboxy-4-nitrophenyl)-disulfidβ ^G.L. Silman, K.D. Courtney, V. Andres Jr . , E.M. Peathsr- stone : Biochem. Pharmacol. £, 88 (1 6117. The amount of enzyme capable of catalyzing the conversion of 1 ,umole of butyryl-thiocholinβ iodide per minute at 25°C and at the optimum pH of the catalytic activity (pH 7.6 for the dissolved enzyme and 8.0 for the immobilized enzyme) was regarded as one activity unit . The results of the tests are summarized in Table 1. Immobilized Immobilized Activity re¬ Loss in Activity of Specific Support protein, activity, covered in activity, the product activity, % % dissolved % * state , % finzacr l AA 100 0 0 100 0 0 Enzacryl AH 61.8 0.2 60,7 39.1 0.2 0.3 Akrilex C-100 (Example 1) 24.5 ' 7.5 60 32. 120 30.6 Carbox me thyl cellulose 5.5 0.2 3.5 96.3 2 3.6 Polymaleic anhydride 23.3 7.5 31.3 61.2 106 32.2 unit/mg of dry substance *xthe specific activity of the dissolved enzyme was regarded as 100 . H It appears from the data of Table 1 that the pre¬ paration according to the invention hβs the greatest activity related to both dry weight (protein + support) and protein content * The activity of the enzyme prepara- tion immobilized on poly aleic anhydride support approaches that of the new product; polymalβic anhydride is, however, a support prepared by bulk polymerization, thus the throughflow characteristics of a column made of cholinesterase enzyme immobilized on polymaleic an- hydride are much poorer than those of a column made of the product according to the invention, in which the enzyme is immobilized on polymer beads.

Claims: What we claim is: 1. An immobilized cholinesterase enzyme prepara¬ tion characterized in that it contains the cholinester¬ ase enzyme bound to a polymeric resin, built up from acrylic acid and/or methacrylic acid and acryl amide and/or . methacryl amide monomers with acryl or allyl type cross-linking agents and contains at least 0.1 meq/g of -COOH functional groups, previously activated wi h a carbodiimide * 2 * A process for the preparation of an immobilize cholinesterase enzyme preparation, characterized in that a polymeric resin, built up from acrylic acid and/or methacrylic acid and acryl amide and/or methacryl amide monomers with an acryl or allyl type cross-linking agent and contains at least 0.1 meq/g of -COOH functional groups, is treated with a carbodiimide derivative which is soluble in water or is soluble in an organic solvent at temperatures below 0°C, a solution of cholinesterase . . enzyme with a pH of 4.5 to 8.5 is applied onto the resulting activated support, the resulting product is washed, and then dried, if desired. 3. A process as claimed in claim 2, characterized in that a polymeric resin containing 2 to 8 meq/g of -COOH functional groups is applied as starting substance. 4. A process as claimed in claim 2 or 3 > char¬ acterized in that an Akrilex C type polymer, prepared by the partial acidic or alkaline hydrolysis of Akrilex P type acryl amide - IT,ir , -methylene bis(acryl amide) copolymer beads, is applied as starting substance. 5» A process as claiaed in any of claims 2 to 4, characterized in that cholinesterase is applied onto the activated support as a solution in a 0.1 molar potassium phosphate buffer (pH 7.0). 6. A process as claimed in any of claims 2 to 5> characterized in that cholinesterase is reacted with the activated support at 0-4°C. OMH

Inventors: BOROSS LASZLO, KOVACS KAMILLA NEE SZENTES, SZAJANI BELA, BOROSS, LASZLO, KOVACS, KAMILLA, NEE SZENTES, SZAJANI, BELA
Applicants: REANAL FINOMVEGYSZERGYAR
Application year: 1981
Main classification: C12N9
IPC codes: C12N9, C12N11, C12Q1