NO782627L - IMMOBILIZED ENZYME PREPARATION. - Google Patents

Description

11 Immobilized enzyme preparation".

This invention relates to an immobilized enzyme preparation, a method for producing such a preparation and the use of such a preparation in an enzyme-catalyzed reaction.

Enzymes are proteins that can catalyze chemical reactions. However, their usefulness for various applications has been limited since it is difficult to remove enzymes from reaction media, and this limits the purity of the product. Moreover, soluble enzymes can usually only be used once in a batch reaction and, due to the high costs associated with many enzymes, their industrial use has been limited, although they can be very effective catalysts. In recent years, many techniques have been invented to immobilize enzymes by attaching or binding them to water-insoluble support materials in such a way that the enzyme is rendered immobile and yet still catalytically active. Known immobilized enzyme preparations have a number of disadvantages, e.g. some of them, when packed in a column, exhibit unsatisfactory flow properties, some exhibit low enzyme yield and in some the stability of the enzyme is reduced. There is thus a need to develop improved immobilized enzyme preparations.

According to the present invention, we provide an immobilized enzyme preparation which comprises a carrier material which has hydrophobic groups on the surface, these groups containing substituted or unsubstituted aliphatic hydrocarbon chains with at least 6 carbon atoms and/or containing chains of substituted phenyl groups, and having molecules of at least one enzyme attached to the hydrophobic groups.

Furthermore, according to the present invention, we provide a method for the production of an immobilized enzyme preparation which comprises the step of bringing a support material which has hydrophobic groups on the surface which contain substituted or unsubstituted aliphatic hydrocarbon chains with at least 6 carbon atoms and/or contain chains of substituted or unsubstituted phenyl groups, in contact with at least one enzyme, for such a period and under such conditions that the enzyme molecules are attached to the hydrophobic groups.

According to the present invention, we also provide a method for carrying out an enzyme-catalyzed reaction whereby the enzyme used is in the form of an immobilized enzyme preparation comprising a support material that has hydrophobic groups on the surface, these groups containing substituted or unsubstituted aliphatic hydrocarbon chains with at least 6 carbon atoms and/or contain chains of substituted or unsubstituted phenyl groups, and have molecules of at least one enzyme attached to the hydrophobic groups.

The enzyme preparation can be in any suitable physical form, e.g. particle, fiber or matrix form. It is preferably particulate, and particle sizes in the range of 50 mm to 1 mm are particularly suitable. The enzyme-catalyzed reaction is preferably carried out in a column packed with particles of the preparation, or in a fluidized bed. However, other ways of carrying out the reaction are also possible. It can e.g. is carried out in a vessel containing particles of the preparation which are separated from the products when the reaction is complete, e.g. by filtration or sedimentation.

The surface of the support material carries hydrophobic groups that can be described as "arms" that protrude from the surface and form sites for attachment of enzyme molecules. The hydrophobic groups can be an integral part of the carrier material, thus being groups that form part of the compound of which the material is composed, e.g. side chains on a polymer. Alternatively, the hydrophobic groups can be produced by treating the support material with a hydrophobic compound that forms a chemical, e.g. covalently, bonding with the support material or even being attached to the support material in some way.

Suitable support materials are materials which are inert during the enzyme-catalyzed reaction or which are rendered effectively inert when treated with the hydrophobic compound. A very large amount of materials are suitable, e.g. polyvinyl alcohols, dextrans, celluloses, polyacrylamides, agaroses and polyurethanes. To attach the "hydrophobic arm" to these compounds, they can be activated with e.g. cyanogen bromide or cyanuric chloride or a mono-substituted derivative of cyanuric chloride. The resulting derivatives can then be reacted with e.g. hexamethylenediamine to form the "hydrophobic arm". Materials such as glass beads and sand can also be used if they are suitably treated to enable hydrophobic compounds to attach to them.

The hydrophobic "arms" are formed by groups of sufficient length to bind to two of the hydrophobic sites occurring on enzyme molecules. These groups can be groups containing substituted or unsubstituted aliphatic hydrocarbon chains with at least 6 carbon atoms (preferably 6 to 12), groups containing chains of substituted or unsubstituted phenyl groups, e.g. containing 4 phenyl groups, and groups containing aliphatic chains together with phenyl groups. The hydrophobic "arms" have functional groups, e.g. -NH2, -CHO, -CNO or -COOH groups, at their outer ends. Any of these functional groups themselves form bonds with the enzyme or they can be reacted with another compound to form a covalent bond and thus at the end of the "arm" form a group that forms a bond with the enzyme. Suitable modifying compounds for reaction with the "arm" in this manner are compounds containing -CHO and -CNO groups, and preferably contain such a group at each end of the compound, e.g. glutaraldehyde or

CHO (CH 2 ) 6 CNO.

For a very useful immobilized enzyme preparation, the hydrophobic "arm" has the structure -(CH2)gNH2 and reacts with glutaraldehyde to give a glutaraldehyde group at the end of the "arm". When the end of the "arm" is to be modified in this way, the support material with hydrophobic groups can be reacted with the modifying compound in any suitable manner, e.g. by passing a solution of the modifying compound over it.

The strength of the attachment of the enzyme to the hydrophobic group or "arm" can be improved by treatment with a non-ionic surfactant, e.g. "TRITON'X", "TWEENS" or "SPAN". This treatment is most valuable when the end of the "arm" is not modified as described above. The nonionic surfactant should be hydrophobic. The treatment with the nonionic surfactant can be carried out in any suitable manner.

It is most advantageous that the nonionic surfactant is included in the reaction medium during the enzyme-catalyzed reaction.

In order to form the immobilized enzyme preparation, the following steps are preferably carried out: (a) If the support material does not already have hydrophobic groups on its surface, it is treated with a hydrophobic compound. It is of course unnecessary to have such a treatment if the carrier material already has hydrophobic groups on its surface. (b) The ends of the hydrophobic groups are reacted with a covalent

connection to activate them.

(c) The prepared support material is brought into contact with the enzyme in aqueous solution.

If desired, steps (b) and (e) above can be carried out simultaneously or

in reverse order.

Enzymes useful for use in the immobilized enzyme preparation include lactase, glucose isomerase and glucoamylase.

The method of carrying out an enzyme-catalyzed reaction is useful in the conversion of glucose to fructose using glucose isomerase (eg using the methods described in our British Patents No. 1,451,273 and No. 1,492,258),

by conversion of lactose to glucose and galactose in use

of lactase (e.g. using the method described in our co-pending British patent application No. 38002/76, in the conversion of starch to glucose using glucoamylase and in many other enzyme conversions.

The invention is illustrated in the following examples:

Example 1

/3-galactosidase extracted from LT2 bacterium (NCIB no. 11259) described in our co-pending British patent application no..38002/76, was adsorbed on 1 g of aminohexyl-agarose ("SEPHAROSE" - registered trademark - purchased from<p>harmecia) . The agarose suspension was then packed into a column. A 5% lactose solution in 50 mm phosphate buffer pH'6.5 was passed through the column at 50°C and the flow rate was adjusted so that 80% hydrolysis of lactose was achieved. This gave a measure of immobilized β-galactosidase activity. After 36 hours, 100 g of lactose had been hydrolysed.

Example 2

The β-galactosidase was immobilized as in Example 1, except that "TRITON X-100", up to a final concentration of o 10 -5%, was added to the lactose solution. After 150 hours, 400 g of lactose had been hydrolysed.

Example 3

Aminohexyl agarose was incubated with 0.5% glutaraldehyde

for 1 1/2 hours at pH 7.0./3-galactosidase extract, prepared as in Example 1, was reacted for 1 1/2 hours with the resulting aldehyde derivative of the aminohexyl agarose. The covalently linked enzyme was examined as in example 2. After 150 hours, 400g of lactose had been hydrolysed.

Example 4

β-Galactosidase extracted from B. coagulans NRRL B-8100 (obtained from ReynoldsTobaccoCo.) was immobilized and examined as in Example 3. After 1000 hours, 300 g of lactose had been hydrolyzed.

Claims (18)

1. Immobilized enzyme preparation comprising a carrier material that has hydrophobic groups on the surface, characterized in that these groups contain substituted or unsubstituted aliphatic hydrocarbon chains with at least 6 carbon atoms and/or contain chains of substituted or unsubstituted phenyl groups, and have molecules of at least an enzyme attached to the hydrophobic groups. 2. Preparation according to claim 1, characterized in that the carrier material is a material which has been treated with a hydrophobic compound which attaches itself to the material to form hydrophobic groups on its surface. 3. Preparation according to claim 2, characterized in that the carrier material is a polyvinyl alcohol, a cellulose, a polyacrylamine, an agarose or a polyurethane which has been treated to form hydrophobic groups on its surface. 4. preparation according to any of the preceding claims, characterized in that the enzyme is glucose isomerase, lactase or glucoamylase. 5. Process for the production of an immobilized enzyme preparation, characterized by the step of bringing a support material which has hydrophobic groups on the surface containing substituted or unsubstituted aliphatic hydrocarbon chains with at least 6 carbon atoms and/or containing chains of substituted or unsubstituted phenyl groups, into contact with at least one enzyme, for such a period and under such conditions that the enzyme molecules are attached to the hydrophobic groups. 6. Method according to claim 5, characterized in that the carrier material is formed by treating a material with a hydrophobic compound which itself attaches to the material to form hydrophobic groups on its surface. 7. Process according to claim 5 or 6, characterized in that the hydrophobic groups have -NH2/ -CHO, -CNO or -COOH groups as functional groups at their outer ends and where a step is included where these functional groups are reacted with a modifying compound to form an end group to which enzyme is attached. 8. Method according to claim 7, characterized in that the hydrophobic arms have the structure -(CH2 )gNH and are reacted with glutaraldehyde as a modifying compound before they are brought into contact with the enzyme. 9. Method according to any one of claims 5 to 8, characterized in that it comprises the following steps: (a) treating a material with a hydrophobic compound to form hydrophobic groups on its surface; (b) reacting the treated material with a covalent compound which modifies the ends of the hydrophobic groups, and (c) contacting the -treated support material with an enzyme in aqueous solution. 10. Process for carrying out an enzyme-catalyzed reaction where the enzyme used is in the form of an immobilized enzyme preparation comprising a carrier material that has hydrophobic groups on the surface, - characterized in that these groups contain substituted or unsubstituted aliphatic hydrocarbon chains with at least 6 carbon atoms and/or contain chains of substituted or unsubstituted phenyl groups, and have molecules of at least one enzyme attached to the hydrophobic groups. 11. Method according to claim 10, characterized in that during the reaction the enzyme preparation is treated with a hydrophobic, non-ionic surfactant. 12. Method according to claim 11, characterized in that the non-ionic surfactant is "TRITON X". 13. Method according to any one of claims 10 to 12, characterized in that glucose is converted into fructose, starch is converted into glucose or lactose is converted into glucose and galactose. 14. Syrup containing fructose, glucose or glucose and galactose, characterized in that it is produced by a method according to any one of claims 10 to 13. 15. Enzyme preparation, characterized in that it is produced by a method according to any one of claims 5 to 9. 16. Immobilized enzyme preparation substantially as described herein and as shown in any of the examples. 17. Process for preparing an immobilized enzyme preparation substantially as described herein and shown in any of the examples. 18. Process for carrying out an enzyme-catalyzed reaction substantially as described herein and as shown in any of the examples.