GB2034719A - The Recovery of Enzymes Following Their Use in a Reaction - Google Patents

Abstract

The enzyme for use as a catalyst is held in the storage vessel (10) having previously been attached to paramagnetic particles. The enzyme preparation, for example peroxidase on chromic oxide is allowed to flow into the reactor chamber (13) where the reaction process occurs and the paramagnetic particles are then removed from the resulting liquid medium by a high-gradient magnetic separator (15). These particles and the attached enzyme are then returned to the reactor chamber (13) for re-use having been flushed out of the separator (15) by a stream of enzyme preparation flowing through flowline (12) from the storage vessel (10).

Description

SPECIFICATION The Recovery of Enzymes Following Their Use in a Reaction This invention relates to the recovery of enzymes from a liquid medium after they have been used as catalysts in a reaction process. The object of the invention is to produce a method which combines ease of recovery for subsequent re-use with the least possible loss of enzyme activity.

Enzymes are large protein molecules and are the catalysts produced by the cells of living organisms which allow complex chemical transformations to occur in the mild conditions compatible with life. They are, therefore, of great value for use in industrial applications where those transformations might otherwise require the employment of extreme temperature, pressure, acidity or alkalinity, in conjunction with expensive equipment.

Since most enzymes are soluble and sometimes very expensive it follows that in some enzyme-catalysed reactions, large quantities of valuable enzyme may be lost after a reaction process has taken place. It therefore becomes very worthwhile, especially where an industrial process application is involved, to produce an enzyme preparation which makes possible the efficient recovery of the preparation after a reaction process has taken place.

In accordance with the invention there is a method for carrying out enzyme-catalysed reactions in a liquid medium wherein the enzyme used is carried on paramagnetic particles, these particles subsequently being recovered from the liquid medium, enabling the enzyme to be reused.

The particles may be recovered by magnetic means which may take the form of a highgradient magnetic separator.

The paramagnetic particles may comprise a compound of one of chromium, titanium, uranium or platinum. The particles may be particles of chromic oxide.

The enzyme used may be peroxidase or it may be amyloglucosidase.

The enzyme may have been attached to the paramagnetic partidles by adsorption and permanently fixed by cross-linking adjacent enzyme molecules.

Examples of the invention will now be described and reference will be made to the accompanying flow diagram which illustrates a method of recovery of an enzyme from a reaction process.

Experiments have shown that insoluble transition metal oxide powders constitute suitable support materials for enzyme molecules. Any sufficiently paramagnetic oxide may be employed and attachment of trypsin or invertase has been achieved by (a) silanisation of a paramagnetic substrate with an aminofunctional silane coupling agent followed by conversion of the available amino groups to isocyanate with cyanogen bromide and subsequent quiescent coupling of the enzyme; (b) direct adsorption of the enzyme by a paramagnetic suspension followed by crosslinking of the enzyme layer.

The use of a paramagnetic compound as a support for the enzyme allows a high-gradient magnetic separator to be used to recover large quantities of soluble enzyme from the process stream so that the enzyme can be re-used.

The high-gradient magnetic field within the separator allows very small particles i.e. particles less than 5 across, to be captured. To recover such particles by conventional methods, such as filtrtion and centrifuge action, would be economically less attractive.

The use of these small particles increases the surface area per unit weight, i.e. increases the surface area available for enzyme attachment, leading to greater activity of the recoverable enzyme derivative. The use of paramagnetic compounds is preferred over that of ferromagnetic materials since paramagnetic compounds are commonly available in a far more finely-divided form.

In using a preferred enzyme, peroxidase, the method was first applied using a ferromagnetic support of magnetite, an iron oxide. However, after a number of unsuccessful attempts it was appreciated that since peroxidase possesses an iron-containing core moiety essential for activity, it is possible that the proximity of this core to the iron atoms of the magnetite inactivates the enzyme.

Another advantage of paramagnetic compounds over ferromagnetic is that the use of a paramagnetic support is far less likely to damage the enzyme activity.

Chromic oxide (Cr203) was selected as the preferred support material on the basis of its paramagnetic susceptibility of +1 960x 10-6 cgs units; its low cost and insolubility in an aqueous solution.

Peroxidase was selected as the preferred enzyme since it could easily be attached to the chromic oxide particles using the simple technique of adsorption of the enzyme on to the surface of the oxide particles, followed by the cross-linking of adjacent enzyme molecules with 0.25% glutaraldehyde. In this manner 20% of the soluble peroxidase activity was permanently retained by the chromic oxide derivative.

Peroxidase was a preferred enzyme with a view to industrial application since in the presence of hydrogen peroxide it efficiently catalyses the oxidation of phenolic compounds to unstable quinones and is thus applicable to the treatment of industrial effluents produced in the manufacture or processing of wood, plastics, disinfectants, dyes, explosives, resins, etc., and will thus effectively remove phenolic compounds from effluents produced in these processes.

Peroxidase is sufficiently expensive to make its recovery economically attractive. The process operates at 200C and at neutral or slightly acid pH levels.

Amyloglucosidase is a further enzyme with possible industrial application, for example to the conversion of starchy materials to glucose syrups.

this enzyme may be attached with 1 5% retention of activity to a paramagnetic support, will retain good stability and will be recoverable by a high gradient magnetic separator.

In an industrial process, shown schematically in the accompanying flow diagram, the solution or suspension of material for enzyme conversion is contained in a storage vessel 10, the enzyme employed being peroxidase on a support of chromic oxide prepared in accordance with the invention, the storage vessel 10 has two flow paths 11 and 12; 11 leads to an enzyme reactor chamber 13 and then through flow path 14 to a high-gradient magnetic separator 1 5 and through flow path 1 6 to the product or effluent chamber 1 7. Flow path 12 is a flushing stream from the storage vessel and is linked to flow path 14 which leads to the high-gradient magnetic separator the retained enzyme. Valves, not shown, are provided in the flowlines, valve operation is described below.

The high-gradient magnetic separator is itself well known in the art and may consist of an inlet and an outlet of a toroidal chamber containing fine wool entrapment filters surrounded by an electromagnetic coil which creates a highgradient magnetic field within the filters for entrapment and recovery of the enzyme employed, a flow path 18 is provided for directing the enzymes recovered in the high-gradient magnetic separator and may lead back to the enzyme reactor.

The provision of a high-gradient magnetic separator makes possible a continuous process as opposed to a batch process. A timed cycle may be adopted on the basis of the enzyme retention by the high-gradient magnetic separator during the period when the reaction process takes place, the valves in flow paths 12 and 18 are opened, allowing the enzyme retained in separator 1 5 to be returned to the process chamber 13. When this cycle has been accomplished the valves in flow paths 12 and 18 are closed and the valve in flow path 1 6 opened to allow a further process cycle.

The invention is not limited to the specific embodiments described and could be employed for the removal of toxic and/or refractory materials from industrial effluents; for any industrial enzymatic conversion such as starch to glucose; or for biosynthetic steps in pharmaceutical manufacture.

Claims (8)

Claims

1. A method for carrying out enzymecatalysed reactions in a liquid medium wherein the enzyme used is carried on paramagnetic particles, these particles subsequently being recovered from the liquid medium, enabling the enzyme to be re-used.

2. A method as claimed in claim 1 in which magnetic means are used to recover the paramagnetic particles.

3. A method as claimed in claim 1 or 2 in which the paramagnetic particles are recovered by means of a high-gradient magnetic separator.

4. A method as claimed in claim 1 or 2 in which the paramagnetic particles comprise a compound of one of chromium, titanium, uranium or platinum.

5. A method as claimed in claim 1 or 2 in which the paramagnetic particles are particles of chromic oxide.

6. A method as claimed in any of the preceding claims in which the enzyme used is peroxidase.

7. A method as claimed in any of claims 1 to 5 in which the enzyme used is amyloglucosidase.

8. A method as claimed in any of the preceding claims in which the enzyme has been attached to the paramagnetic particles by adsorption and permanently fixed by cross-linking adjacent enzyme molecules.