CS202373B1

CS202373B1 - A method for preparing immobilized enzymes

Info

Publication number: CS202373B1
Application number: CS736078A
Authority: CS
Inventors: Ludovit Kuniak; Juraj Zemek
Original assignee: Ludovit Kuniak; Juraj Zemek
Priority date: 1978-11-13
Filing date: 1978-11-13
Publication date: 1981-01-30

Abstract

Přihláška vynálezu sa týká přípravy imobilizovaných enzýmov viazaných na perlovej celulóze obsahujúcej karbonylové skupiny. Principy imobilizácie enzýmov na róznych polymérnych nosičoch sú dnes dostatočné známe nielen z literárnych údajov fjíethods in Enzymology, Vol. XLIV, Immobilized Enzames, Ed. Klaus Morbach, Academie Press, New York, San Francisko, London 1976J, ale i mnohých praktických aplikácií tak vo výskume, ako i v priemyslovej praxi. Chemická imobilizácia enzýmov na vhodnom nosiči patří k najrozšírenejším spósobom imobilizácie, pričom ako nosič sa používajú rózne syntetické polyméry, ale i přírodně polysacharidy: škrob, celulóza, dextrany a i. Ak sa použije ako nosič vláknitá alebo práš ková celulóza, je potřebné před imobilizáciou enzýmu vytvořit na celulóze vhodnú funkčnú skupinu, ktorá je schopná v druhom stupni kovalentne intereagovať s příslušnými skupinami enzýmu. Takouto skupinou je napr. karbonylové skupina, ktorá sa velmi jednoducho vytvoří známou oxidáciou celulózy, resp. iného polysacharidů s jodistanom draselným. (T. P. Novell: Methods in Carbohydrate Chemistry New York 1963, str. 165-68). Je však známe, že polymérny nosič, na ktorom je enzým viazaný má zásadný význam na aplikačně možnosti imobilizovaných enzýmov v priemyslovej praxi. Polymérny nosič musí meť dostatočné velký napúčací objem v reakčnom médiu, aby bola zabezpečená dostatočné rýchlosť difúzie substrátu k aktívnym centrám imobilizovaného enzýmu.The application of the invention relates to the preparation of immobilized enzymes bound to pearl cellulose containing carbonyl groups. The principles of immobilization of enzymes on various polymer supports are now sufficiently known not only from the literature data fjíethods in Enzymology, Vol. XLIV, Immobilized Enzymes, Ed. Klaus Morbach, Academie Press, New York, San Francisco, London 1976J, but also many practical applications both in research and in industrial practice. Chemical immobilization of enzymes on a suitable carrier is one of the most widespread methods of immobilization, whereby various synthetic polymers are used as a carrier, as well as natural polysaccharides: starch, cellulose, dextrans, etc. If fibrous or powdered cellulose is used as a carrier, it is necessary to create a suitable functional group on the cellulose before immobilizing the enzyme, which is capable of covalently interacting with the relevant groups of the enzyme in the second step. Such a group is, for example, a carbonyl group, which is very simply formed by the well-known oxidation of cellulose, or of other polysaccharides with potassium periodate. (T.P. Novell: Methods in Carbohydrate Chemistry New York 1963, pp. 165-68). However, it is known that the polymer carrier on which the enzyme is bound is of fundamental importance for the application possibilities of immobilized enzymes in industrial practice. The polymer carrier must have a sufficiently large swelling volume in the reaction medium to ensure a sufficient diffusion rate of the substrate to the active centers of the immobilized enzyme.

Description

CZECHOSLOVAKIASOCIALISTREPUBLIC (19)

DESCRIPTION OF THE INVENTION

TO THE COPYRIGHT CERTIFICATE (61) (23) Show Priority (22) Enrolled 13 11 78 (21) PV 7360-78 202 373 (11) (Bl) (51) IntCl? C 12 N 11/14

OFFICE OFFICE

AND DISCOVERIES (40) Published 30 04 80 (45) Published 01 q4 83 (75)

The inventor of KUNIAK JUDOVÍT ing., CSc., BRATISLAVA and ZEMEK JURAJ ing. CSc., LEHNICA (54) Method of preparation of immobilized enzymes 1 The invention relates to the preparation of immobilized enzymes bound to peroxy cellulose containing carbonyl groups.

The principles of immobilization of enzymes on various polymeric carriers are now well-known not only from fjiethods in Enzymology, Vol. XLIV, Immobilized Enzames, Ed. Klaus Morbach, Academic Press, New York, San Francisco, London 1976J, as well as many practical applications in both research and industry.

The chemical immobilization of enzymes on a suitable carrier is one of the most widely used methods, with the use of various synthetic polymers as well as naturally-polysaccharides: starch, cellulose, dextran and others. If fibrous or powdered cellulose is used as carrier, a suitable functional group on the cellulose which is capable of covalently interacting with the appropriate groups of the enzyme in the second step. Such a group is, for example, a carbonyl group, which is very easily formed by the oxidation of cellulose, respectively. another polysaccharide with potassium periodate. (TP Novell: Methods in Carbohydrate Chemistry New York 1963, pp. 165-68).

It is known, however, that the polymeric carrier on which the enzyme is bound is of crucial importance for industrial immobilized enzymes. The polymeric support must have a sufficiently large swelling volume in the reaction medium to ensure sufficient substrate diffusion velocity to the active immobilized enzyme centers. 202 373 202 373 Of the above-mentioned polymers, the immobilization of polyaaccharides, such as cellulose and starch, is immobilized. Therefore, they may be modified chemically by suitable means so that the swelling volume of the poly-aa-arid gel is perfectly accessible to the larger subatrate molecules. The most suitable cellulose carrier for immobilization of enzymes has been and is to be pearled cellulose (U.S. Pat. No. AO 2.172640 (1976)), which is demanding the requirements of a polymeric carrier for covalent binding of enzymes.

SUMMARY OF THE INVENTION In the first step, the bead cellulose is oxidized in aqueous solution and potassium periodate or sodium at room temperature for 10 to 24 hours using 0.03 to 0.25 mole of potassium periodate to form an anhydroglucose unit of cellulose. in a second step, after washing the product with distilled water to a negative periodate, an aqueous enzyme solution of 10 to 50% by weight of the dry cellulose oxidized carrier is added and left under stirring for 10 to 24 hours at pH 4 to 8 according to pH optima and stability of bound enzyme at 4 to 40 ° C.

In the oxidation of bead cellulose, it is important that the reaction mixture is not mixed with a high-shear mixer as it may result in crumbling of the cellulose beads, resulting in increased resistance to column operation. Therefore, it is expedient to choose very low revolutions needed to not settle the cellulosic suspension during the reaction.

The reaction time required to achieve the desired oxidation step does not exceed 10-24 hours at room temperature. At low levels of oxidation, the reaction time consumes almost 98% of the reagent, ie it is not necessary to chemically eliminate the unreacted periodate after the reaction. Just wash the filter with distilled water and continue to bind the applied enzyme. Bnzyme is added in an aqueous solution at the optimum pH of the applied enzyme. Enzyme binding is completed within 24 hours at 4-40 ° C under the thermal stability of the enzyme. The advantage of the subject immobilization of the enzymes on the bead cellulose is, in particular, that the gelling cellulose due to the precise guT geometry of the particles has very good column performance as well as a sufficiently high swelling volume in the aqueous solution, thus providing good substrate diffusion rates to the active immobilized centers. enzyme. EXAMPLES Example 1 A solution of 40 ml of water with 0.115 potassium iodate was added to 16 g of wet pearl cellulose (10.5% dry matter). The reaction suspension is reacted with stirring at room temperature for 24 hours. Stirring must not be intense, just so that pearl cellulose does not sediment. Upon completion of the oxidation, the pearl cellulose is filtered off and washed with distilled water to give a product with an oxidation degree of 0.05, ie each 20th anhydroglucose unit is oxidized to dialdehyde at position C2, Cy to the washed dialdehyde of cellulose is added 60 ml 0.05 M barate buffer pH 8.6. To this suspension is added a solution of pseudocholinesterase (acylcholine acyl hydrolase EC 3.1.1.8) in an amount of 0.5 µg of 120 U / mg activity in 50 ml of 0.05 M barate buffer pH 8.6 with stirring on an electromagnetic mixer. Reaction and immobilization are continued for 24 hours

Claims

Method for preparing immobilized enzymes, characterized in that, in the first step, the bead cellulose is oxidized in aqueous solution with potassium periodate or sodium at room temperature for 10 to 24 hours using 0.03 to 0.25 moles of potassium periodate per anhydroglucose cellulose unit, then, in a second step, after washing the product with distilled water to a negative periodate reaction, an aqueous solution of the enzyme in an amount of 10 to 50% by weight of the dry oxidized cellulose carrier is added and allowed to react for 10-24 hours at pH = 4-8 under pH of the optimum and stability of the bound enzyme at a temperature of 4-7 ° C

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