GB2200637A

GB2200637A - Process and apparatus for immobilizing enzymes

Info

Publication number: GB2200637A
Application number: GB08702512A
Authority: GB
Inventors: Mikhkel Oskarovich Mandel; Evgeny Ivanovich Khristoforov; Natalia Mikhailovna Samoshina; Levon Arutjunovich Nakhapetian
Original assignee: VNII BIOTEKHNOLOGII; Tallinn University of Technology
Current assignee: VNII BIOTEKHNOLOGII; Tallinn University of Technology
Priority date: 1985-04-02
Filing date: 1987-02-04
Publication date: 1988-08-10
Anticipated expiration: 2007-02-04
Also published as: GB2200637B; DE3704041A1; FR2610640A1; FR2610640B1; BG47580A1; DK8700430A; SU1317024A1; GB8702512D0

Abstract

The process comprises successive recycling through a carrier of a chemical reagent for activation of the carrier surface and for combining the enzyme, and then the enzyme or alternatively, recycling an enzyme and then the chemical reagent, said recycling being effected until full saturation of the carrier pores, whereafter the enzyme immobilized on the carrier is subject to purification. Apparatus for carrying out the above process has a conveyor having holders 2 secured to a drive chain 6, said holders supporting perforated vessels 3 containing the carrier. Chambers 8 provide treatment of the carrier with a chemical reagent, an enzyme, or with a solution for purification of the immobilized enzyme. Means 22, 23 charges the carrier into the vessels 3. <IMAGE>

Description

Process and apparatus for producing immobilized enzymes This invention relates to the microbiological industry and, more specifically, to a process and apparatus for producing immobilized enzymes. The invention is useful in the preparation of immobilized enzymes, in parti.cular, immobilized P -galactosidase employed in whey processing.

yeast invertase employed in hydrolysis of solutions of saccharose bacterial i - and gluco-amylase employed in starch processing, and the like.

According to the present invention there is provided a process for producing immobilized enzymes comprising the steps of successively recycli.ng, in either order, a chemical reagent and an enzyme, through a stationary carrier, said chemical reagent activating the surface of the stationary carrier to enable combination with the enzyme, said recycling being effected until the pores of the carrier are fully saturated with said reagent immobilizing said enzyme on the carrier, and purifying said immobilized enzyme.

The process of the present invention enables an increase of productivity of 2-5 times over that of prior art processes, improves the activity of immobilized enzymes, simplifying the production process so that it may be implemented on a commercial scale.

The invention also provides apparatus for producing immobilized enzymes comprising vessels for receiving a carrier, means for introducing carrier into a vessel, means for successively recycling a chemical reagent and an enzyme through the carrier until the pores of the carrier are fully saturated with said reagent and said enzyme is immobilized' on said carrier, purifying means for supplying a purifying solution to the immobilized enzyme, and conveyor means for conveying said vessels in turn from the carrier introducing means to said recycling means and said purifying means.

In accordance with a preferred embodiment of the present invention, an apparatus for carrying out,the process of the invention comprises a perforated vessel for the carrier, means for charging the carrier into the vessel. means for supplying the chemical reagent, enzyme and the solvent for purification of the immobilized enzyme and for discharging the enzyme immobilized on the carrier which apparatus in accordance with the present invention comprises at least one additional perforated vessel for a carrier, a conveyor with holders secured to its drawing organ and intended for positioning, thereon, of perforated vessels for the carrier and chambers for the treatment of the carrier contained in the perforated vessels with a chemical reagent, an enzyme or a solution for purification of immobilized enzymes mounted along the movement of the perforated vessels; each of the chambers has a tube with opening for admission of one of the above-mentioned reagents i.nto the perforated vessel, said tube being positioned in the upper section of the chamber; and a tank provided on the bottom of the chamber for collection of the above-mentioned reagents fed from the perforated vessels, which communicates with the tube by means of a pipe and a pump to ensure recycling of the reagents through the perforated vessel till a complete saturation of the carrier pores with these reagents; the upper and end-face walls of the chambers have slots and ports for moving the holders with the perforated vessels from a preceding chamber into the next one.

Preferably the above-mentioned holders are secured to the drawing organ of the conveyor at a distance from one another corresponding to the distance between centers of the two adjacent chambers thus enabling carrying out all process opera tions in different chambers, whereby the process duration is reduced and the procedure is simplified at the same time.

The apparatus according to the present invention is suitable for a commercial implementation, it operates under continuous production condition and features a high productivity of, for example, in the production of immobilized '3-galactosidase, up to 70 ton a year. The apparatus makes it possible to fully automate the entire process of imaobi- lization of enzymes.

The present invention will now become more fully apparent from the following detailed description of the process and apparatus for producing immobilized enzymes with reference to the accompanying draw ings, wherein: Fig. 1 shows a general isometric view with a partial elevation of the apparatus for producing immobilized enzymes according to the present invention; Fig. 2 - section of one branch of the conveyor along II -II in Fig. 1 of the apparatus according to the present invention.

The process according to the present invention can be effected both continuously and discontinuously.

As carriers for immobilization of enzymes use is made of inorganic and organic carriers. As inorganic carriers use is made of large-pore silica gels (silica) with a particle size of from 0.1 to 1.0 mm and with a pore size of 30 to 200 nm, porous ceramics, various types of clays, activated coal and the like.

As organic carriers use is made, for example, of na turally-occurring polysaccharides, cellulose and derivatives thereof, agarose and its derivatives with a pore size of from 30 to 100 nm and the like.

The process of immobilization according to the present invention can be used for any enzymes being present in solutions.

Immobilization of enzymes is effected by first recycling a chemica; reagent through a stationary carrier for activation of the carrier surface. Then the enzyme is recycled through the carrier. In doing so, a chemical bonding of the enzyme with the activated carrier surf ace takes place.

As the chemical reagent for activation of the surface of the carrier use can be made of silane derivatives, for example g -aminopropyltriethoxysilane or their combinations with bifunctional reagents.

Recycling is carried out till complete saturation of the carrier pores with these reagents.

Immobilization of enzymes can be effected by first recycling an enzyme through a stationary carrier till a full saturation of the carrier pores with the enzyme, whereafter recycling of a chemical reagent through the carrier is effected to combine the carrier on the enzyme. As the above -mentioned chemical reagent use can be made of glutaric aldehyde. Purification of the enzyme immobilized on the carrier is effected bl recycling water and various solutions, for example buffer solutions, solution of sodium chloride and the like, depending on the immobilized enzyme, through the above-mentioned enzyme.

The process according to the present invention makes it possible to simplify the production procedure owing to elitnination of mechanical stirring, repeated discharge of the carrier from one process cycle into the other, elimination of abrasion and disintegration of the carrier, reduction of losses thereof, as well as to a reduced duration of the process of immobilization of enzymes.

The process according to the present invention makes it possible to improve the process productivity and activity of imsobilized enzymes.

The apparatus for producing immobilized enzymes (Fig. 1) comprises a conveyor provided with electric drive means 1 with a chain, rope, rotary, screw or a similar drawing organ. On the drawing organ of the conveyor at an equal distance from each other holders 2 are secured to fis and move perforated vessels 3 for the carrier along the convenor path. Perforations should be preferably made over the entire surface of a vessel 3.

In the case of use of a chain drawing organ it is made in the form of a monorail 4 with carriages 5 movably mounted thereon with holders 2 connected by means of a chain 6 with a gear wheel 7 fixed on an output shaft of the drive means 1.

The apparatus comprises a number of chambers 8 for the treatment of a carrier placed in perforated vessels 3, with chemical reagents, an enzyme or a solution for parification of the imLobilized enzyme.

Chambers 8 are placed along the movement of perforated vessel 3. A slot 9 is provided along the entire upper wall of each chamber 8; this slot ensures a free movement of holders 2 from one chamber 8 into another. On both end-face walls of each chamber 8 ports 1 are provided for a free passage of vessel 3 from one chamber 8 into another.

The outside walls of chambers 8 are provided with looking windows 11 to control the progress of the process of in chambers 8. On the bottom of each chamber 8 a tank 12 is provided (Fig. 2) for accumulation of the reagents. The tank 12 is connected with a tube 13 by means of a pipe 14 and a pump 15 (Fig. 2). The pipe 14 is provided with a solenoid-type valve 16 (Fig. 2).

Tubes 13 function as means for the supply, into per orated vessel 3 (Fig. 1), of reagents; these tubes are po positioned in the upper portion of chambers 8.

Tanks 12 (Fig. 2) are connected by means of pipes 17 (Fig. 1) with external accumulation containers (not shown) for the employed components and by means of a pipe 18 (Fig.2) provided with a solenoid valve 19 (Fig. 2) to sewerage.

The apparatus according to the present invention also comprises a band conveyor 2O (Fig. 1) with a pusher 21 for the supply of empty vessels 3 to the means for charging the carrier into vessels 3. The means for charging the carrier into vessels 3 is made as a bin 22 with a screw-type feeder 23.

The apparatus is also provided with a pusher 24 for discharging the enzyme immobilized on the carrier onto the band conveyor 25.

The enzyme comprises chambers 8 in a number corresponding to the number of process steps. The distance between centers of two adjacent chambers 8 is chosen equal to the distance between two neighbouring holders 2 which defines the conveyor a pitch.

It goes without saying tut t the apparatus according to the present invention, likewise all those on-stream plants in the art, is provided with all necessary devices and instruments of a known design enabling visual observations, control of the process parameters within predetermined limits and an automatic performance of the process.

The apparatus according to the present invention operates in the following manner.

Into a bin 22 of a charging means a granulated carrier, for example silica gel, is introduced, a band conveyor 20 for the supply oS empty perforated vessels 3 by means of pushers 21 on holders 2 to the bin 22 is switched on. At the same time, an electric drive means 1 starting the conveyor is switched on. When the conveyor is made as a chain transporter, its chain 6 engaged with a gear 7 moves carridges 5 with holders 2 -transportlng vessel 3 from the bin 22 of the char ging means to the first of successively positioned chambers 8 along the monorail 4. Ov;ing to ports 10 and the slot 9 the vessel 3 on the holder 2 passes freely into the chamber 8.During the movement of the vessel 3 to the chamber 8 a system of supply of the enzyme or the chemical reagent for activation of the carrier surface along the line 17 into the tank 12 is switched on. After filling of the tank 12 with the required one of the above-mentioned solutions, their supply is stopped. At the moment when the vessel 3 is in the chamber 8 the electric drive means 1 is switchedeff and the conveyor movement is stopped. The solenoid-type valve 16 is opened and the above-mentioned required solution is fed by means of the pump 15 via the line 14 from the tank 12 into the tube 13 with perforations.

Through the perforations 13 tbe solution employed in a given chamber 8 is contacted with the carrier contained in the vessel 3. Raving passed the layer of the carrier, the employed solution flows back into the tank 12, wherefrom it is recycled as described hereinabove onto the carrier till a complete saturation of the carrier pores viith the employed solution. Thereafter the valve 16 is closed and circulation of the employed solution from the tank 12 to the tube 13 is stopped. The valve 19 is opened and the exhausted solution is withdrawn by means of the pump 15 via the line 18 from the tank 12 into sewerage.

During the treatment of the carrier in the first of the successively arranged chambers 8 the carrier is charged into the subsequent vessels 3 moved on the band transporter 20 towards the bin 22.

After closing of the valve 16 the electric drive means 1 is switched on to start the conveyor and the vessel 3 with the spent carrier is moved to the center of the second of the successively arranged chambers 8.

In doing so, the next vessel 3 with the carrier charged from the bin 22 is transferred into the first of the successively arranged chambers 8 and into its tank 12 again fed is the enzyme or the chemical reagent for activation of the carrier surface. The process of the carrier treatment in the first chamber 8 is effected in the manner similar to that described hereinabove.

Owing to the fact that the holders 2 are fixed on the oonveyor at a distance from one another corresponding to the distance between centers of two adjacent chambers 8, it is possible, during the carrier treatment in a preceding chamber 8, to carry out treatment of the carrier fed from the preceding chamber 8 in a following chamber 8. In said following chamber 8 a solution of a chemical reagent is used for combining the enzyme, or a solution of the enzyme.

The supply of the required reagent, its accumulation, circulation through the carrier already treated in the preceding chamber 8 and discharging of the reagent are carried out in a manner similar to that described hereinbefore for the treatment procedure in the first chamber 8.

After a full saturation of the carrier pores with the employed solutions in the first and second chambers 8, the valves 16 provided in each chamber 8 are closed and circulation of the employed solutions from the tanks 12 to the tubes 13 is stopped. The valves 19 are opened and the exhausted solutions from the tanks 12 are withdrawn by means pumps 15 via lines 18 into the sewerage.

Then a carrier-filled vessel 3 is delivered into the first of series-positioned chambers 8 along the conveyor operated. by means of the drive 1, while into the second chamber 8 a vessel 3 with the carrier already treated in the first chamber 8 is moved and into the third chamber 8 a vessel 3 is passed with the carrier subjected to the treatment in the second chamber 8.

After positioning vessels 3 in the center of each chamber 8 the electric drive means 1 is switched-off, the conveyor is stopped and in each chamber the carrier is treated with a solution necessary according to the process for the preparation of immobilized enzymes.

The vessels 3 with the carrier having passed all steps of the process of treatment in chambers 8 with the reagents enabling the production of the enzyme immobili-zed on the carrier are delivered, one-by-one, by the conveyor to the pusher 24 for the unloading of the enzyme immobilized on the carrier onto the band transporter 25.

For a better understanding of the present invention some specific examples illustrating the process for producing immobilized enzymes are riven hereinbelow.

Example 1 Into each of perforated vessels there is charged 0.45 kg of dry silica gel with a particle size of 0.3-0.5 mm, pore diameter of 40-60 nm and the specific surface area of 80 m2 /g from a bin. The carrier bed height in the perforated vessels is 75 mm. The carrier-filled perforated vessels are delivered by means of a conveyor into the first of successively positioned chambers. In the first chamber a 0.1M acetate buffer with the pH of 4.6 is passed through the carrier bed at the recycling rate of 4.0 1/mien for 15 minutes to impregnate the carrier granules.Then the perforated vessel with the carrier impregaated by the acetate buffer is delivered into the second chamber, wherein it is treated with 0.9 1 of an enzymatic solution of p-galactosidase at the recycling rate of 4.0 1/mien for 45 min. The initial activity of the enzymatic solution of p -galactosidase is 175 U/ml.A unit of activity of p -galactosidase represents such an amount of the enzyme which forms in a 5% solution of lactose 1 vumol of products of lactose hydrolysis for 1 minute at the pH = 4.6 and at the temperature of 300C. The recycling is effected till a full saturation of the pores of the carrier granules, whereafter from the second chamber the perforated vessel with the above me ntioned carrier is delivered into the next chamber, wherein the carrier is treated by way of recycling, there through, a 1 aqueous solution of glutaric aldehyde in a 0.1M phosphate buffer with the pR of 6.85 at the rate of 4 1/min. At the recycling rate of 4.0 1/min the process lasts for 45 minutes.During this period there occurs a comp lete bonding of p -galactose on the surface of the carrier granules. Then the perforated vessels with p -galactosidase immobilized on the carrier are delivered into the. following chamber, wherein immobilized / -galactosidase is purified to remove traces of the chemical reagent and enzyme by way of recycling water through the carrier with A -galactosidase immobilized thereon at the rate of 3 1/min for 15 minutes, then a 2M solution of NaCl at the rate of 3 1/min in the next chamber for 15 minutes.Thereafter, the washing with water is repeated for 15 minutes, whereafter this vessel is moved into the following chamber, wherein / -galaoto- sidase immobilized on the carrier is treated with a 0.1M acetate buffer at the recycling rate of 3 1/min for 15 minutes to extend the storage life of the immobilized enzyme.

There are thus obtained 2.2 kg of immobilized ss -galasto- sidase with the activity of 253 U/g (65.5%) as calculated for dry solids.

Example 2 The process is carried out in a manner similar to that described in Example 1 hereinabove. Into each of perforated vessels there is charged 1 kg of silica gel with a particle size of 0.3-0.5 mm, pore diameter of 4; nm and the specific surface area of 80 m2/g. The height of the granule bed is 125 mm. The recycling process is carried out as described in the foregoing Example 1. 2.2 1 of an euzymatic solution of ss-galactosidase with the initial activity of 255 U/ml are used. There are thus obtained 3.6 kg of immobilized fi -galactosidase with the activity of 255 U/g (66.0X) as calculated for dry solids.

Example 3 The process is conducted in a manner similar tb that described in the foregoing Example 1. Into each of perforated vessels there are charged 2.4 kg of silica gel similar to that of Example 1. The recycling in the chambers is effected following the procedure described in Example 1 hereinbefore.

Use is made of 5.5 1 of an enzymatic solution of ss-galacto- sidase with the initial activity of 175 U/ml. There are thus obtained 8 kg of immobilized ss -galactosidase with the acti- vity of 245 U/g (65%) as calculated for dry solids.

Example 4 Into each of perforated vessels from a bin there is charged 0.56 kg of silica gel with a particle size of 0.6-0.8 mm with a pore diameter of 40-6o nm and the specific surface area of 80 m2/g. The height of the carrier bed in the per forated vessels is 80 mm. The carrier-packed perforated vessels are delivered by means of a conveyor into the first of series-positioned chambers. In the first chamber a 5 aqueous solution of -aminopropyltriethoxysilane is passed for 30 minutes through the carrier bed at the recycling rate of 4.0 1/min. In doing so, g -aminopropyltriethosysi- lane is chemically combined with the carrier.Then the per forated vessel with this carrier combined with g -aminopro- pyltriethoxysilane is fed into the second chamber, wherein it is treated with a 1% glutaric aldehyde at the recycling rate of 2.0 1/min for 30 minutes, where after in the subsequent chamber this carrier is treated with 2.0 1 of an enzy matic solution of ss -galactosidase at the recycling rate of 4.0 1/min for the period of 60 minutes. The initial activity of the enzymatic solution of ss -galactosidase is 56 U/ml. The recycling is effected till a complete saturation of pores of the carrier granules; simultaneously therewith there occurs a chemical bonding of the enzyme with the carrier surface activated by the abovementioned chemical reagents.Then the vessel with p -=alactosidsse immobilized on the carrier is fed into the following chamber, wherein it is washed with water at the recycling rate of 2.0 1/min for 10 minutes; afterwards, in the subsequent chamber A- galactosidase immobilized on the carrier is treated with a 0.1M acetate buffer with the pR of 4.5 for 10 minutes.

Then the washing with water is repeated in the next chamber for 10 minutes, then in still another chamber - with a 1M solution of NaCl under the same conditions. As a result, 1.8 kg of immobilized ss -glactosidase are produced with the activity of 40 U/g (52.35) as calculated for dry solids.

Example 5 The process is conducted in a manner similar to that described in the foregoing Example 4. Into each vessel there are charged 2.6 kg of silica gel with a particle size of 0.3-0.5 mm, pore diameter 40-60 nm and the specific surface area of 80 m2/g; the carrier bed height is 180 mm. The carrier is treated with a 1% solution of #-aminopropylt- riethoxysilane -for 30 minutes at the recycling rate of 4 1/min.

Use is made of 7.8 1 of an enzymatic solution of y-alacto- sidase with the initial activity of 140 U/ml. The process is carried out in a manner similar to that of Example 1.

There are thus obtained 8.2 kg of immobilized y5-galactosi- dase with the activity of 40 U/g (52.3%) as calculated for dry solids.

Example 6 The process is carried out following the procedure described in Example 4 hereinbefore. Into each perforated vessel 1.0 kg of dry silica gel is charged with a particle size of 0.3-0.5 mm, pore diameter of 40-60 nm and the specific surface area of 80 m2/g. The carrier bed height in the vessel is 125 mm. The carrier treatment is effected in a manner similar to that described in Example 4. Use is made of 4 1 of a 1% aqueous solution of t-aminopropyltriethoxysilane.

As the enzymatic preparation use is made of a preparation of invertase obtained from brewerts yeast. The activity of the employed solution of invertase is 1,920.0 U/ml in a 0.1S acetate buffer with the pH of 4.6.- The activity in U/ml means mol of the products of saccharose hydrolysis formed during 1 minute at the pH of 4.6 and at the temperature of 3000 in a ? solution of saccharose. 3.0 1 of an enzymatic solution of invertase are used.The process of iumobilization of invertase is conaucted in a manner similar to that described in Example 4 hereinbefore to give 3.5 kg of immobilized invertase with the activity of 2,050 U/g (55.6%) as calculated =or dry solids.

Example 7 For the preparation of immobilized glucoamylase the process is carried out as described in Example 4. Into each perforated vessel 1.0 kg of dry silica gel is charged with a particle size of 0.3-0.5 mm, pore diameter of 40-60 nm and the specific surface area of 80 m2/g. The carrier treatment is performed as described in the foregoing Example 4.

Use is made of 4.0 1 of an aqueous solution of #-aminopropyl- triethoxysilane. As the enzymatic preparation a solution of glucoamylase with the activity of 2,380 U/ml is used.

A unit of activity (U) of glucoamylase means such an amount of the enzyme which in a 1% solution of sluble starch catalazes its hydrolysis at the pH of 4.6 and at the temperature of 50 C with the formation of 1 mol of glucose during 1 minute. 1.5 1 of the enzymatic solution of glucoamylase is used. The immobilization process is carried out in a manner similar to that described in Example 4 hereinbefore. There are thus obtained 3.5 kg of immobilized glucoamylase with the activity of 990 U/g (31.2ss) as calculated for dry solids.

Example 8 The process for the production of O6-amylase is conducted in a manner similar to that described in Example 4 hereinbefore, except that as the enzymatic preparation use is made of 1.5 1 of a solution of α-amylase in a 0.1M phosphate buffer with the pE of 6.2 and the activity of 2,520 U/ml.

For a unit of activity (U) of α-amylase such an amount of products of hyarolysis (as calculated for maltose) is assume in mol which is formed in hydrolysis of a 1%-aqueous solution of starch under the effect of 7 ml of the enzyme at the temperature of 5000, pfl = 6.2, for 1 minute. The process of immobilization of α -amylase is conducted as described in Example 4. There are thus obtained 3.5 kg of imtnobilized α-amylase with the activity of 720 U/g (19.05) as calculated for dry solids.

Claims

1. A process for producing immobilized enzymes com- prising the steps of successively recycling,in either order, a chemical reagent and an enzyme, through a stationary carrier, said chemical reagent activating the surface of the stationary carrier to enable combination with the enzyme, said recycling being effected until the pores of the carrier are fully saturated with said reagent, immobilizing said enzyme on the carrier, and purifying said immobilized enzyme.

2. Apparatus for producing immobilized enzymes comprising vessels for receiving a carrier, means for introducing carrier into a vessel, means for successively recycling a chemical reagent and an enzyme through the carrier until the pores of the carrier are fully saturated with said reagent, and said enzyme is immobilized on said carrier, purifying means for supplying a purifying solution to the immobilized enzyme, and conveyor means for conveying said vessels in turn from the carrier introducing means to said recycling means and said purifying means.

3. A process for producing immobilized enzymes comprising successively recycling, through a stationary carrier, a chemical reagent for activation of the carrier surface and combining of the enzyme, then an enzyme, or an enzyme and then a chemical reagent for combining an enzyme on the carrier said recycling being effected till a full saturation of the carrier pores with said reagents, whereafter the enzyme immobilized on the carrier is subjected to purification.

4. An apparatus for carrying out the process according to claim 3 which comprises perforated vessels for the carrier, means for charging the carrier into a vessel, means for the supply of a chemical reagent, enzyme and a solution for purification of the enzyme immobilized on the carrier into the vessel with the carrier and for discharging the enzyme immobilized on the carrier, a conveyor with holders secured to it and having drawi.ng organ and intended for placing on them, of perforated vessels for the carrier; chambers mounted along the movement of said perforated vessels and intended for the treatment of the carrier located in said perforated vessels by a chemical reagent an enzyme or a solution for purification of the immobilized enzyme, each chamber having a perforated tube for the supply into a perforated vessel of one of said reagents positioned in the upper zone of the chamber and a tank mounted on the bottom of the chamber and intended for collections of said reagents supplied from the perforated vessels which tank is connected with the tube by means of a pipe and a pump for recycling the reagents through the perforated vessel till a. complete saturation of the carrier pores with said reagents, the upper and end-face walls of said chamberseing provided with slots and ports for moving the holders with the perforated vessels from a preceding chamber i.nto a subsequent one.

5. An apparatus according to claim 4, wherein said holders are secured to the drawing organ of the conveyor at a distance from one another corresponding to the distance between the centers of two adjacent chambers.

6. A process for producing immobilized enzymes according to claim 3, substantially as described in the specification and examples hereinbefore.

7. An apparatus as herein described with reference to the accompanying drawings.

7. An apparatus for performing the process according to any of the foregoing claims 4 and 5 substantially as described in the specification and illustrated in the drawings attached.

Amendments to the claims have been filed as follows CLAIMS: 1. A process for producing immobilised enzymes comprising the steps of successfully recycling, in either order, a chemical reagent and an enzyme through a substantially stationary mass of carrier material until the pores of the carrier material are fully saturated therewith, the chemical reagent activating the surface of the carrier material to enable combination with the enzyme whereby the enzyme becomes immobilised on the carrier material, and purifying said immobilised enzyme, each of said reagent and said enzyme during the recycling thereof being supplied to the mass of carrier material so as to flow therethrough and being collected downstream of the mass of carrier material for re-supply.

2. A process according to claim 1 in which the step of recycling said chemical reagent takes place first.

3. An apparatus for carrying out the process of claim 1, comprising vessels for receiving carrier material, means for introducing a mass of carrier material into a vessel, means for successively recycling a chemical reagent and an enzyme through the carrier material until the pores of the carrier material are saturated therewith, purifying means for supplying a purifying solution to the carrier material for purifying immobilised enzyme which has become immobilised on the carrier, conveyor means for conveying said vessels in turn from the carrier introducing means to said recycling means and said purifying means, said recyling means including means for introducing said reagent and enzyme into said vessel, to flow through the substantially stationary carrier material therein and means for collecting reagent and enzyme downstream of the carrier material, and returning said reagent and enzyme to the introducing means.

4. An apparatus according to claim 3 comprising chambers mounted along the movement of said vessels, each chamber including slots and ports in the end face walls to allow entrance and exit of said vessels when carried by the conveyor, recycling means within each said chamber comprising a perforated tube positioned in an upper zone of the chamber to introduce said reagent, enzyme or purifying solution into said vessels, said vessels being perforated to allow said reagent enzymes or purifying solution to drain and collect into a tank mounted on the bottom of the chamber, said tank being connected by a pipe and a pump for recycling the reagent, enzyme or purifying solution to the perforated tube and through the carrier.

5. An apparatus according to claim 4 in which said vessels are suspending form a drawing organ of the conveyor and each vessel is separated by a distance equal to the distance between the centres of the chamber.

6. A process for producing immobilised enzyme as herein described and substantially as claimed in claim 1.