IL39063A - Water-insoluble enzyme products - Google Patents

Description

Water-insoluble enzyme products 0*03 0Ό&3 03 'R iWK D'T3K {11313*171 Socie'te' des Produits Nestle* S.A.

This invention is concerned with the preparation o products having enzyme activity which are insoluble in aqueous media · Enzyme reactions such as enzymatic hydrolyses, are generally carried out with one or more enzymes in solution in the medium containing the substrate, the term "substrate" designating the substance subjected to the action of the enzyme. When the enzyme is dissolved in the reaction medium, its separation from the substrate is particularly difficult, and when it is desired to stop the reaction it. is generally necessary to inactivate the enzyme, as by heating, with the consequence that the enzyme is irrecoverable.

To overcome these difficulties, various process&i'-S have been proposed for preparing water-insoluble enzyme products. Using these substances enzymatic reactions may be carried out by passing the substrate over a bed of the insoluble product, or simply by adding the insoluble enzyme to a solution of the substrate and subsequent, separation of the enzyme by mechanical means such as filtration or cent.ri-fugation.

There exist, various methods for preparing insoluble enzymes, including absorption on an insoluble support, encapsulation in a polymeric matrix, cross-linking with bi-functional substances and covalent. bonding to an insoluble carrier .

Products obtained by absorption of the enzyme are however frequently relatively unstable. Enzymes encapsulated in a polymeric matrix, whilst, being fairly stable, units constituting the matrix. Insolubilisati'on by cross- blinking involves formation of a large number of bonds or bridges which generally necessitates the prior protection of the active groups of the enzyme. In addition, insolu-bilisation methods involving cross-linking do not. always afford the possibility of forming the products in shapes, such as beads or granules, allowing for easy mechanical separation.

Covalent bonding of the enzyme to a water-insoluble polymer frequently requires the formation of active sites on the support, by various reactions such as diazotisation, or use of copolymers. As the formation of bonds may however damage some of the active groups of the enzyme, it. is usually necessary first to protect these, and to remove the protecting groups after the bonding which complicates the process .

An object of the present invention is to provide in novel waterVsoluble enzyme products, and a simple process for their preparation.

The invention provides a water-insoluble enzyme product, comprising an enzyme covalently bonded to free aldehyde groups of a water-insoluble oxidised polysaccharide.

By free aldehyde groups are meant, aldehyde groups carried by the oxidised polysaccharide which are capable of forming chemical bonds with other substances.

Oxidation of polysaccharides such as cellulose leads to the formation of free aldehyde groups by the oxidation of alcohol groups carried by the polymer chain. Thus, for example, oxidation of a polysaccharide such as cellulose polymer having 10 - 12% of reducing groups, that- is the number of free aldehyde groups corresponds to 10 - 12% of the number of glucose residues in the polysaccharide.

The detection and estimation of free aldehyde groups may be conveniently effected by the method of Park and Johnson (estimation of reducing sugars) adapted for insoluble substances, as described by J. S. Thompson and G. D. Schockman in Analytical Biochemistry, 22^, 260-268 (1968) .

The process of the present invention may be applied to enzymes containing reactive groups capable of forming co-valent bonds with the free aldehyde groups of the polysaccharide. Free amino groups are one example of such reactive groups, and these may be attached to amino acid residues, a specific example being the £-amino group of lysine.

Bonding of the enzyme to the insoluble oxidised polysaccharide may for example be effected by simply mixing the enzyme with the polysaccharide for a few hours in a buffer solution, at. temperatures below the inactivation temperature of the enzyme. The insoluble product may be separated from the reaction medium mechanically, as by filtration or centrifugation, and washed before being dried (e.g. lyophilised) .

The reaction conditions are by no means critical, provided that the properties of the enzyme or polymer are not. destroyed. Hence the upper temperature limit, is the inactivation temperature of the enzyme, whereas the lower limit, is 0°C The relative proportions of the reactants may be varied within wide limits, and it is preferred to carry out. the reaction in a buffer solution at a pH of 7 not critical provided that the reactants are not' affected. The pH may thus generally lie in the range 1 to 11. Reaction times may range from a few minutes to several hours. Upon completion of the reaction the water-insoluble enzyme product, may be conveniently separated from the medium by filtration, centrifugation or decanting.

The invention is illustrated by the following examples. In these examples the enzyme activities of the products are expressed as percentages of the activity of the amount, of soluble enzyme which is equal to the amount, of insoluble enzyme recovered. If after separation of the product, the reaction medium has no enzymatic activity, the amount, of insolubilised enzyme may be calculated on the basis that the ratios amount, of insoluble enzyme recovered : amount, of enzyme used, and weight of insoluble product, recovered : total weight, of polysaccharide and enzyme used, are equal. Should the residual medium still have some enzyme activity, which means that not. all the enzyme used has been insolubilised, this calculation is not. appropriate and the amount, of enzyme insolubilised is determined experimentally, as by nitrogen analysis of the insoluble product. Example 1 g of cellulose are oxidised at. 4°C in 2 litres of 0.05M aqueous sodium periodate. Progress of the reaction is followed by titration of the periodate.

After 125 hours' reaction, when 0.5 moles of periodate have been used up per glucose unit, of the polysaccharide the oxidation is stopped by addition of ethylene glycol.

The oxidised cellulose is recovered, washed twice with dis- groups. An 0.2M buffer solution of sodium barbital * hydrochloride is prepared and its pH is adjusted to 8.0. 100 mg of oxidised cellulose are then mixed in the buffer solution with 80 mg of crystallised papain. The mixture is maintained at ambient, temperature with stirring for 3 hours and the insoluble product is recovered by centrifu- gation. The supernatant has no enzyme activity.

The insoluble product is washed 3 times with a buffer solution of pH 7.2 containing, per litre, 1.21 g cysteine hydrochloride, 13.5 g potassium phosphate and 20 ml of a 0. IN solution of the disodium salt of ethylene di¬ amine tetraacetic acid, and finally washed twice with dis¬ tilled water.

The enzyme activity of the product is obtained by determination (U.V. absorption spectrum at 405 nm) of the amount of p-nitro-aniline liberated by the enzyme from a -3 solution containing 2.10 mole of o , -benzoyl-DL-argmine 4-nit.roanilide per litre of the buffer solution used for washing, together with 5% by weight of dimethylsulphoxide .

The enzyme activity of the product, determined by this method is 85%.

Example 2 The pH of a 0.2M aqueous solution of trihydroxy- methyl-amino methane is adjusted to 8.0 with, hydrochloric acid. 0.08 moles/litre of calcium chloride are added and the solution is diluted fourfold to give concentrations of 0.05 and 0.02 mole/litre respectively of trihydroxymethyl- amino methane and calcium chloride. 200 mg of crystallised trypsin and 250 mg of oxi then mixed in 50 ml of the solution and the mixture is maintained at. 4°C with stirring for 20 hours. The insolu ble product, is then recovered by centrifugation, washed 3 times with the buffer solution and twice with distilled water and lyophilised. 190 mg of insoluble product are obtained containing 30 mg of trypsin (determined by analysi of total nitrogen, as the supernatant had some enzyme activity) .

The activity of the insoluble product determined a in Example 1 is 33%.

Claims (14)

What we claim is:

1. A water-insoluble enzyme product, comprising an enzyme covalently bonded to free aldehyde groups of a water- insoluble oxidised polysaccharide.

2. A product according to claim 1 in which the oxidised polysaccharide is oxidised cellulose.

3. A product according to claim 1 or claim 2 in which the enzyme contains amino groups which are covalently bonded to the free aldehyde groups.

4. A product, according to any one of the preceding claims in which the enzyme is papain or trypsin.

5. An enzyme product, substantially as herein described with reference to the Examples. as claimed in Claim 1

6. A process for preparing water-insoluble enzyme products/ which comprises reacting an enzyme with a water-insoluble oxidised polysaccharide and recovering a water-insoluble enzyme product.

7. A process according to claim 6 in which the reaction is effected in a buffer solution.

8. A process according to claim 6 or claim 7 in which the reaction is effected at. a pH of 1 to 11.

9. A process according to claim 8 in which the reaction is effected at a pH of 7 to 8.

10. A process according to any one of claims 6 to 9 in which the enzyme is papain or trypsin.

11. A process according to any one of claims 6 to 10 in which the oxidised polysaccharide is oxidised cellulose.

12. A process according to any one of claims 6 to ll in which the recovered enzyme product, is dried.

13. A process for preparing a water-insoluble enzyme product, substantially as herein described wit reference to the Examples.

14. An enzyme product, obtained by a process according to any one of claims 6 to 13. for the Ap licants