WO2021186148A1

WO2021186148A1 - Continuous reactor with cross flow filtration loop

Info

Publication number: WO2021186148A1
Application number: PCT/GB2021/050567
Authority: WO
Inventors: Rune Thorbjørn NORDVANG
Original assignee: Dairy Crest Limited
Priority date: 2020-03-20
Filing date: 2021-03-08
Publication date: 2021-09-23
Also published as: AU2021238917A1; GB202004097D0; CA3167997C; AU2021238917B2; EP4121507A1; GB2593223A; CA3167997A1

Abstract

The invention relates to an apparatus for a continuous operation of an enzymatic reaction, to methods of using such an apparatus, and to a process for continuously reacting an enzyme with a substrate. In a specific manifestation, the invention relates to the use of such a reactor in manufacturing galacto-oligosaccharides by transgalactosylation of lactose in the presence of lactase enzymes.

Description

CONTINUOUS REACTOR WITH CROSS FLOW FILTRATION LOOP

Background of the Invention

Continuous flow biocatalysis is a powerful tool in chemical synthesis. In this process, a continuous flow enzyme reactor is packed with a bed of immobilised enzymes over which a solution of a substrate is pumped. This solution passes through the reactor to undergo an enzymatic reaction catalysed by the immobilised enzyme. The exiting solution comprises products that are isolated through any separation or filtration techniques known in the art.

Enzyme immobilisation provides greater productivity due to enzyme stability and efficient usage of the enzymes due to increased activity. However, there are several drawbacks to using immobilisation techniques. The enzymes must be immobilised on appropriate support for the continuous flow reactors to work properly. This set up is costly, and the binding efficiency of the immobilised enzymes decreases over time affecting the product turnover. Moreover, continuous flow reactors comprising immobilised enzymes are extremely difficult to clean, as the cleaning must be gentle enough to not inactivate the enzymes or release them from their support.

Therefore, an object of the present invention is to provide a system for a continuous process of an enzymatic reaction that does not suffer from the above disadvantages.

The present invention provides a crossflow enzyme reactor that allows enzymatic reaction to take place and at the same time allows isolation of the desired products in a continuous process resulting in a faster manner, and in higher yield. This system uses free mobile enzymes instead of immobilised enzymes, avoiding the problems associated with these methods.

In crossflow filtration, a feed liquid is passed across a filter membrane (tangentially) at positive pressure relative to the permeate side. A proportion of the material which is smaller than the membrane pore size passes through the membrane as permeate or filtrate; everything else is retained on the feed side of the membrane as retentate. The principal advantage of crossflow filtration is that the filter cake is substantially washed away during the filtration process, increasing the length of time that a filter unit can be operational. It can be a continuous process, unlike batch-wise dead-end filtration.

Crossflow filtration techniques are known in the field of enzymatic reactions. An example is given in WOOO/24499. In this disclosure, crossflow filtration comprising ultrafiltration membranes have been used with enzyme reactors using mobile enzymes. However, the enzymes in such a system are known to become inactivated due to continuous pumping and recirculation through the ultrafiltration unit. This is because in such a system the enzymatic reaction often takes place in a reactor and then the ultrafiltration takes place to separate the products from the enzymes and other unwanted debris found in the retentate. Prior to filtration, the enzymatic reaction is often terminated, sometimes also through additional measures such as by heat or pH adjustment, further inactivating the enzymes. Thus, although a continuous process, this is a system where two individual processes are combined, namely, the enzymatic reaction taking place in a reactor and filtration of the desired products across the ultrafiltration membrane, as shown in Figure 1. This also slows down the product recovery.

In the present invention, there is provided a system in which the enzymatic reaction and filtration of products occur simultaneously and continuously, thus speeding up the process and limiting enzyme inactivation. As shown in Figure 2, the crossflow enzyme reactor of the present invention does not require a separate reactor tank. Instead, this system provides a feed line to the crossflow filter which is extended in order to provide for an incubation zone in which the enzymatic reaction can take place. By the time the reaction mix arrives at the crossflow filter, most of the reaction has been completed and the separation can take place across the filtration membrane. The flow of the enzymes in the system is controlled such that an adsorbed enzyme layer temporarily forms on the filtration membrane where any remaining enzymatic reaction takes place in order to reduce the risk of any unreacted substrate in the product recovery stream. Enzymes in the present invention are continuously recycled, allowing an extremely high active enzyme dose in the system for very rapid reaction completion, without increasing the cost of the enzyme in the process as enzyme inactivation is limited in the system. Any residual product in the retentate will be returned to a buffer tank for repeated filtration, minimising loss of product.

The crossflow enzyme reactor has been found to be particularly useful in the manufacture of galacto-oligosaccharides (GOS) from lactose digested in the presence of b-D-galactosidase (lactase) enzyme.

GOS resembles oligosaccharides occurring naturally in human milk and is known for its use in relieving the symptoms of constipation in adults and elderly people. GOS has also been found beneficial in infants and thus its use in formula milk is well known.

In the state of the art, the GOS is usually produced in a process in which the starting materials are incubated and then GOS is separated from the mixture using techniques such as ion exchange. This traditional manufacturing process is slow, and the product yield is low. Also, the enzymes used in the GOS manufacture constitute a significant proportion of the manufacturing cost. Thus, it is desirable to have a reactor system in which enzyme inactivation is significantly reduced to save costs by recycling the enzymes. An aim of the present invention is to provide an enzyme reactor that optimises biocatalytic productivity, i.e. amount of product formed per amount of enzyme used.

The crossflow enzyme reactor of the present invention can be used in any enzymatic reaction involving a substrate and an enzyme, particularly for those reactions involving costly enzymes. As an example, the enzymatic reaction taking place between b-fructosidase and sucrose to produce fructose oligosaccharides can be employed in the inventive process.

Summary of the Invention

In a first embodiment of the present invention there is provided a reactor for a continuous process of enzymatic reaction comprising: an extended conduit providing an incubation zone for the enzymatic reaction to take place, a membrane housing in a fluid communication with the extended conduit, the membrane housing comprising a permeable membrane configured to filter desired permeate obtained from the enzymatic reaction, and a retentate loop in fluid communication with the membrane housing and the extended conduit, configured to return retentate from the membrane housing to the extended conduit.

In a second embodiment of the present invention, there is provided a continuous process of enzymatic reaction, process for reacting an enzyme and a substrate in a continuous process comprising steps of: a. bringing the substrate and enzyme into association in a liquid to form a reaction medium; b. flowing the reaction medium at a flow rate along a conduit of sufficient length for reaction to occur between the substrate and the enzyme to form product; c. contacting the reaction medium with a permeable membrane to separate product from the reaction medium; d. returning the retentate to step b; wherein substrate is continuously added into the reaction medium to maintain the concentration of substrate. In yet another embodiment, the invention provides GOS directly obtained by the above process using the crossflow enzyme reactor of the present invention.

Brief description of the Figures

Figure 1 demonstrates a known crossflow filtration setup.

Figure 2 illustrates a reactor of the present invention.

Figure 3 is two flow charts comparing the known and inventive processes.

Figure 4 illustrates an alternative reactor of the present invention.

Figure 5 illustrates an enzyme reactor comprising one membrane housing according to one embodiment of the invention.

Figure 6 illustrates an enzyme reactor according to another embodiment of the invention comprising two membrane housings.

Detailed Description of the Preferred Embodiments

The apparatus and processes of the invention are useful with a range of enzymes. Such enzymes include lipases and esterases, phosphatases, proteases, glycosidases, cellulases, cellobiases, glycosyl hydrolase, and polysaccharide hydrolases. Enzymes may also include enzymes with other specificities such as oxidative enzymes, and the composition of an enzyme cocktail used in the invention may be specifically formulated to meet the needs of specific applications. A nonlimiting list of enzymes and enzyme activities believed to be useful in the invention includes the following: alkaline phosphatase, esterase (C-4), esterase-lipase (C-8), lipase (C-14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, alpha galactosidase, beta galactosidase, beta glucuronidase, alpha glucosidase, N-acetyl-beta- glucosaminidase, alpha mannosidase, and alpha fucosidase. Suitable substrates for use in connection with the abovementioned enzymes will be known to the person skilled in the art.

In a preferred embodiment, the enzyme is a galactosidase, especially a b- galactosidase such as b-galactosidase of Aspergilus oryzae, b-galactosidase of Aspergilus niger, b-galactosidase of Bacillus circulans, b-galactosidase of bifidobactaerium bifidum, b-galactosidase of Kluvermyces lactis, b- galactosidase of Lactobacillus helveticus, b-hexosyltransferase of Sporobolomyces singularis, and b-galactosidase of PapHiotrema terrestris. As is known, these enzymes effect the transfer of the glycosyl group of one or more D-galactosyl onto the D-galactose moiety of lactose, in a process known as transgalactosylation. In this embodiment, a preferred substrate is lactose.

Galactooligosaccharides (GOS) are traditionally formed by the enzyme b- galactosidase in a so-called double displacement reaction initiated by a nucleophile backside attack on the C1 carbon of a galactose moiety at the non reducing end of a (donor-)saccharide (e.g. lactose), resulting in a galactosyl moiety covalently bound to the catalytic amino acid of the enzyme and the release of the remaining (donor-)saccharide (e.g. glucose). In this so-called transgalactosylation reaction the galactosyl moiety is transferred to another sugar by a subsequent nucleophile attack on the C1 carbon of the galactosyl moiety by a lactose molecule or a (previously formed) GOS molecule (instead of a nucleophile attack by water) resulting in oligosaccharides of different chain lengths and glycosidic linkages. Because their structures are similar to oligosaccharides present in human breast milk, galactooligosaccharides act as prebiotics, and are of significant commercial value.

In industrial enzymatic processes, the enzyme is generally either immobilized or free. The uses of immobilized enzyme eliminates the enzyme separation step from the main process thus simplifying and increasing the overall process yield. The use of immobilized enzyme has some advantages as compared with an application of free enzymes. Immobilized enzymes can be recovered from reaction mixture and can be made available for reuse. However, immobilized enzymes are considerably more expensive than native enzymes, and processes using free enzymes would be advantageous. The apparatus and process of the present invention concern free enzymes.

The apparatus and methods are described as of the continuous flow type, as opposed to batch processes. Continuous in this context means that the process can be run without interruption for extended periods of time, merely by the addition of substrate and extraction of product.

As will be understood by the skilled person, the enzyme/substrate reaction necessarily occurs in a liquid medium, which will usually be an aqueous medium. The concentration of enzyme and substrate, temperature, and presence of other adjuvants (such as surfactants and co-factors) will be within the judgement of the skilled person.

In prior art systems, as shown in Figure 1 , contact between enzyme and substrate occurs in a reaction vessel or reactor. This is agitated to effect reaction. In the present system, such a reactor is not present, and reaction occurs instead in a conduit, which forms part of a loop including the filtration membrane, as shown in Figure 2.

The term “conduit” as used herein means a channel or passage through which a liquid can flow. The exact dimensions of the conduit will depend on a number of factors, such as the overall scale, the type of enzyme and substrate being used, the reaction temperature, and in particular the flow rate used. The critical parameter is that the residence time of the reaction medium in the conduit is sufficient that a significant proportion of the substrate is converted into product by the time the reaction medium reaches the permeable membrane. As used herein, the term “significant proportion” means at least 10%, more preferably at least 25%, still more preferably at least 50%, still more preferably at least 60%, still more preferably at least 70%, still more preferably at least 80%, still more preferably at least 90%, still more preferably at least 95%, most preferably at least 99%. The skilled person, using common general knowledge and routine trial and error, will be able, starting from the disclosure herein, to determine the suitable length, diameter and other dimensions of a conduit for any particular parameters of enzyme, substrate, reaction conditions and concentration.

The conduit may be made from any suitable material, such as metal, glass or an engineering plastics material. The configuration of the conduit may be linear, or helical, or any other shape to minimize the overall size of the unit.

Product is separated from the reaction medium by bringing it into contact with a permeable membrane. The permeable membrane is configured to allow reaction products (for example, galactooligosaccharides) to pass through, whilst preventing passage of enzyme and unreacted substrate. The permeable membrane is configured such that there is no rejection of the product passing through the membrane and there is 100% rejection of the enzyme from passing through. The permeable membrane is selective to the enzyme to an extent that the rejection coefficient for products is (in the order of decreasing favourability) most favourably substantially 0%, <1% ,<5%, <10%, <20%, <30%, <40%, <50%, <60%, <70%, <80%, <90%, and configured such that at least 50%, more preferably 60% more preferably 70%, more preferable 80%, more preferably 90%, more preferably 95%, most preferably substantially all of the products are captured in the permeate.

In the apparatus and processes of the invention, cross flow filtration is preferred. Cross flow filtration, also known as tangential flow filtration, is a filtration technique in which the starting solution passes tangentially along the surface of the filter. A pressure difference across the filter drives components that are smaller than the pores through the filter. Components larger than the filter pores are retained and pass along the membrane surface, flowing back to the feed reservoir. Cross-flow filtration devices consist of a central sample channel with a filtration mechanism separating adjacent filtrate channels. In a preferred embodiment of the invention, the permeable membrane is configured as a spiral-wound module. Spiral-wound modules are composed of a combination of flat membrane sheets separated by a thin meshed spacer material which serves as a porous screen support. These sheets are rolled around a central perforated tube and fitted into a pressure vessel casing. The feed solution passes over the membrane surface and the permeate spirals into the central collection tube.

In an additional preferred embodiment of the invention, where high temperatures are required for the reaction or for efficient cleaning, the spiral wound membrane elements are substituted for ceramic membranes.

The permeable membrane is selected principally on the basis of pore size which is determined by the molecular weight cut-off (MWCO). In one embodiment, the mwco of the permeable membrane is between 1 kDa to 500 kDa, preferably between 5 kDa to 500 kDa. Preferably, the mwco of the membrane is 10 kDa to 200 kDa. More preferably, the mwco of the membrane is between 20 kDa to 100 kDa. Still more preferably, the mwco is 50 kDa.

In another embodiment of the invention, there may be multiple membrane housings in the enzymatic reactor. The additional membrane housings may be placed in a series or separately.

In yet another embodiment, the membrane housing may comprise multiple permeable membranes.

Further details of the invention will become apparent from the following description referring to a specific embodiment of the invention.

The reactor of the present invention is provided for a continuous process of enzymatic reaction which obtains a high yield of product turnover in a cost- effective manner. An example of the crossflow enzyme reactor is shown in Figures 2, 5 and 6. As shown in Figure 2, the crossflow enzyme reactor (10) is provided with an inlet (11 ) for introducing the enzymes and the substrates to the system. The inlet is in a fluid communication with an extended conduit (12) which provides an incubation zone for the enzymatic reaction to take place between the enzymes and the substrate. The conduit (12) may be provided with a valve and/or a pump for controlling the flow of a stream of substrates and enzymes flowing through it, thereby controlling the rate of product recovery.

The conduit (12) is in a fluid communication with a membrane housing (13). The membrane housing comprises a porous filtration membrane that allows selected components (permeate) to pass through the membrane to be collected in a collection tank (not shown). The permeate includes components such as the product(s) of the enzymatic reaction and water.

The membrane housing may also be provided with a control for controlling the filtration rate across the membrane (cross-flow) and/or for controlling the flow of the stream of retentate to flow through it.

The filtration membrane may have properties such as a particular pore size, electrochemical charge, hydrophobicity, etc. such that only the reaction products are able to cross the membrane through the pores. Membranes may have properties such that more than one type of reaction product can cross the membrane. Suitable pore sizes are mentioned above. The permeates can then be separated using any known separation techniques. Thus, the filtration membrane can be adapted according to the desired permeate. The permeate may be the product of the enzymatic reaction.

During the process the pores may become blocked by the enzymes or other components in the reaction mixture. The valve and/or the pump mentioned above may be used to increase the flow rate in the membrane housing. Such high flow rates can be used periodically to unblock the pores and thus restore the membrane permeability. The apparatus is suitably provided with a circulation pump to enable the flow of liquid around the circuit, at a controlled flow rate.

The filtration membrane may be any known flat sheet membrane or a spiral wound membrane. The filtration membrane may be a ceramic membrane.

The membrane housing (13) is in a fluid communication with a retentate loop (14) that retains retentate (filtrate) of the enzymatic reaction leaving the membrane housing (13). The retentate loop returns the retentate of the enzymatic reaction from the membrane housing back to the extended conduit to be recycled. The retentate loop may be in a fluid communication with the extended conduit via a buffer tank (15). The buffer tank (15) may be in a fluid communication with the inlet (11 ) thus the reactor forms a cyclic loop of inlet- conduit-membrane housing-retentate loop-buffer tank-inlet.

In an alternative embodiment, the conduit may be provided with one or more of a static mixer.

In one embodiment, the conduit is longer than the retentate loop. Preferably, the conduit is up to 1000 times longer than the retentate loop. More preferably, the conduit is up to 500 times longer than the retentate loop. Still, more preferably, the conduit is up to 100 times longer than the retentate loop. In some embodiments, the conduit is between 100 and 1000 times longer than the retentate loop.

In some embodiments, as shown in Figure 4, the apparatus of the invention does not include a buffer tank. In some embodiments, the extended conduit may also comprise the permeable membrane configured to filter out the products immediately as they are formed in an enzymatic reaction taking place in the conduit. In some embodiments, the extended conduit is a filtration membrane configured to immediately remove the products of the enzymatic reaction taking place therein. The buffer tank (15) may also be useful in maintaining the flow rate of the reactor and remove any blockages from time-to-time if any. The buffer tank may thus be provided with a valve for controlling the flow of the stream flowing into the inlet (11 ).

The buffer tank (15) may be provided with an outlet for removing any unwanted debris. The unwanted debris may include inactivated enzymes, by-products, or a mixture thereof.

The retentate loop may also be provided with an outlet for removing any inactive enzymes when necessary. This outlet may be manifested as a microfiltration membrane to remove coagulated enzymes.

The buffer tank (15) may be provided with an inlet for introducing fresh enzyme to the crossflow reactor. The enzymes may be stored in a separate feed tank wherein the outlet of this feed tank may be in a fluid communication with the inlet for introducing enzymes in the buffer tank. The enzymes may be prepared for the enzymatic reaction in this feed tank, including pH regulation, temperature adjustment, etc.

The reactor may also be provided with a feed tank for storing substrate (16). The substrate solution may be prepared in this tank where pH adjustments, temperature control, the addition of other stability compositions, etc. can take place. This feed tank may comprise an outlet (17) connected to the buffer tank (15) for transporting the substrate to the buffer tank (15).

In use, prior to the addition of substrate, the reactor is loaded with enzymes. The enzymes are retained by the filtration membrane and recirculated until a steady state is achieved, with the enzymes evenly distributed throughout the buffer-tank, extended conduit, membrane housing, and retentate loop.

In an embodiment where no additional enzyme is added during filtration, the preloading of enzyme furthermore serves to prevent any of the enzyme formulation ingredients to end up in the product and (potentially) to interfere with the reaction, as small components are washed out of the system (in this case permeate is discarded) prior to addition of substrate. This is particularly useful if the enzyme contains formulation ingredients which are unwanted in the final product (e.g. glycerol is often used as an enzyme stabiliser but is not allowed in infant formula).

A proportion of the enzymes form a temporary layer on the filtration membrane by adsorption, thereby mimicking the immobilisation technique where enzymes are adsorbed on a membrane as a fluid (containing the enzymes) flows directly through (as oppose to partly tangentially to) the membrane. This technique helps in reducing the risk of any unreacted substrates entering the product recovery stream, as the remaining substrate can undergo the enzymatic reaction with the enzyme layer adsorbed on the membrane immediately before filtration.

Once the reactor has been loaded with the enzymes, the substrates are fed continuously to the reactor where the enzymatic reaction occurs, primarily in the incubation zone in the extended conduit. The reaction time can be controlled by adjusting the flow rate in the incubation zone. The trans membrane flow and the flow in the membrane housing can also be controlled separately as mentioned above, preferably by diafiltration. Diafiltration can in addition be used for a complete wash out of the products through the membrane.

The filtered products are collected in the collection tank which comprises an outlet for the products to exit the reactor or can be fed directly to any additional processes such as carbon filtration, evaporation etc.

In an embodiment as illustrated in Figure 6, the membrane housing (13) may be connected to another membrane housing connected to the retentate loop (14) wherein one of the two membrane housing comprises a filtration membrane that allows by-products to be removed from the reactor. The additional membrane housing can be placed upstream or downstream of the primary membrane housing used for the product recovery, depending on the requirement of the enzymatic reaction taking place in the reactor.

In another embodiment, one crossflow enzyme reactor of the present invention is connected to another crossflow enzyme reactor through the collection tank such that the permeate filtered out of the first reactor are fed into another reactor as substrates for another enzymatic reaction.

The crossflow enzyme reactor of the present invention aims to overcome many limitations associated with traditional continuous flow enzyme reactors including that of WOOO/24499. As the enzymatic reaction and filtration of products occur simultaneously in the crossflow reactor of the present invention, the costs associated with this process are significantly reduced. This is demonstrated in Figure 3.

Additionally, the simple setup of this system enables easy cleaning using standard clean-in-place procedures after the enzymes have been removed from the reactor.

Traditional membrane reactors suffer from membrane fouling as the enzymes get adsorbed on the filtration membranes leading to reduced filtration rate. This can be avoided in the crossflow reactor of the present invention as the flow rate in the membrane housing can be increased through the control mentioned above which unblocks the membrane. Additionally, as the enzyme concentration flowing inside the reactor can be controlled, the membrane fouling can also be controlled accordingly.

The crossflow enzyme reactor allows easier maintenance of enzyme activity as the enzymes can be removed and added into the system separately to the substrates. Thus, inactive enzymes flowing into the system can be limited. This also increases the recyclability of the enzymes further bringing down the costs.

The incubation zone may also be provided with a temperature control so as to regulate the temperature inside the extended conduit. As most enzymatic reaction takes place in the conduit, the temperature fluctuation must be regulated in order to avoid enzyme denaturation due to heat. The temperature control may be a heat exchanger.

The retentate loop may also be provided with a temperature control to avoid denaturation of the enzymes retained in the retentate loop. A heat exchanger may be used to control the temperature inside the retentate loop in order to preserve the enzyme and/or limit reaction taking place during recirculation. In most cases the temperature is often lowered.

The invention is further described in the following examples.

Example 1

The crossflow enzyme reactor of the present invention can be used in the manufacture of galacto-oligosaccharides (GOS) from lactose in the presence of lactase. Lactase is a galactosidase, especially a b-galactosidase such as b- galactosidase of Aspergilus oryzae, b-galactosidase of Aspergilus niger, b- galactosidase of Bacillus circulans, b-galactosidase of bifidobactaerium bifidum, b-galactosidase of Kluvermyces lactis, b-galactosidase of Lactobacillus helveticus, b-hexosyltransferase of Sporobolomyces singularis, and b-galactosidase of PapiHotrema terrestris.

In one embodiment, the reactor is charged with lactase such that a temporary layer of lactase is formed on the filtration membrane and the lactase is evenly distributed in the buffer tank, the extended conduit, the membrane housing, and the retentate loop. Thereafter, the lactose is continuously added to the reactor such that in the incubation zone inside the extended conduit, transgalactosylation of lactose catalysed by lactase results into GOS. The filtration membrane in the membrane housing is adapted to continuously filter GOS from the reactor into the collection tank. The retentate is returned through the retentate loop into the buffer tank for reprocessing. In another embodiment, the filtration membrane may also be adapted to further remove by-products such as glucose, carbon dioxide, etc. In such a case another membrane housing may be required to separate GOS from the other permeates.

GOS produced using the crossflow reactor of the present invention faster and cheaper compared to the traditional enzyme reactors.

Example 2

The crossflow enzyme reactor of the present invention can also be used in the manufacture of fructose-oligosaccharides (FOS) from sucrose in the presence of b-fructosidaseby means of enzymatic transfructosylation. The method of manufacturing FOS using the crossflow enzyme reactor of the present invention is the same as that of GOS.

Example 3

The crossflow enzyme reactor of the present invention can also be used in the manufacture of fructose-oligosaccharides (FOS) from Inulin in the presence of an endo-inulinase by the means of enzymatic hydrolysis. This method of manufacturing FOS using the crossflow enzyme reactor enables un-hydrolysed inulin to react further (by recirculation to the extended conduit with the enzyme) by selection of a suitable membrane

Claims

1 . A reactor for a continuous process of enzymatic reaction comprising: an extended conduit providing an incubation zone in which the enzymatic reaction to take place; a membrane housing in fluid communication with the extended conduit, the membrane housing comprising a permeable membrane configured to filter desired permeate obtained from the enzymatic reaction, and a retentate loop in fluid communication with the membrane housing and the extended conduit, configured to return retentate from the membrane housing to the extended conduit.

2. A reactor according to claim 1 , wherein the extended conduit is longer than the retentate loop.

3. A reactor according to any preceding claims wherein the molecular weight cut-off (mwco) of the permeable membrane is between of 1 kDa to 500 kDa.

4. A reactor of any preceding claims further comprising a buffer tank located between the retentate loop and the extended conduit wherein the buffer tank is configured to receive retentate for reprocessing and is in a fluid communication with the extended conduit through the inlet.

5. A reactor of claim 4 wherein the buffer tank further comprises an inlet for receiving further enzymes into the reactor.

6. A reactor of any of claims 4 or 5 further comprising a feed tank for holding substrate, the feed tank comprising an outlet connected to the buffer tank for feeding substrate into the reactor.

7. A reactor of any preceding claims wherein the retentate loop further comprises an outlet for removing enzymes.

8. A reactor of any preceding claims wherein the membrane housing is connected to a second membrane housing via a second retentate loop such that the second membrane housing comprises a second permeable membrane that is configured to allow other permeate components found in the retentate from the first membrane housing of the enzymatic reaction to pass through.

9. A reactor of claim 8, wherein the second membrane housing is placed upstream or downstream of the first membrane housing.

10. A reactor of any preceding claims wherein the reactor is further provided with a collection tank for collecting permeate filtered across the permeable membrane.

11. A reactor of any preceding claims wherein the extended conduit further comprises a pump for controlling the flow rate in the incubation zone.

12. A reactor of any preceding claims wherein the membrane housing further comprises a pump for controlling the flow rate in the membrane housing.

13. A reactor of any preceding claims wherein the permeable membrane is a spiral wound membrane, or a ceramic membrane.

14. A reactor of any preceding claims wherein the extended conduit is further provided with a temperature control for controlling the temperature inside the incubation zone.

15. A reactor of any of claims 10 to 14, connected to a further crossflow enzyme reactor to form a chain of crossflow enzyme reactors through the collection tank such that the permeate of the first reactor is fed into an inlet of the second reactor as a substrate.

16. A process for reacting an enzyme and a substrate in a continuous process using the reactor of any of claims 1 to 15, comprising steps of: a. loading the enzymes into the reactor such that the enzymes are evenly distributed across the reactor; b. flowing the substrate medium at a flow rate along the extended conduit of sufficient length for reaction to occur to form product; c. filtering the product across the permeable membrane in the membrane housing; d. returning retentate from the membrane housing to the extended conduit of step b; wherein substrate is continuously added into the reaction medium to maintain the concentration of substrate.

17. A process of claim 16 wherein the flow rate of the membrane housing is increased periodically to restore the permeability of the membrane.

18. A process of any of claims 16 or 17 wherein the substrate is lactose.

19. A process of any of claims 16 to 18 wherein the enzyme is a lactase selected from b-galactosidase of Aspergilus oryzae, b-galactosidase of Aspergilus niger, b-galactosidase of Bacillus circulans, b-galactosidase of bifidobactaerium bifidum, b-galactosidase of Kluvermyces lactis, b- galactosidase of Lactobacillus helveticus, b-hexosyltransferase of Sporobolomyces singularis, and b-galactosidase of PapiHotrema terrestris.

20. A process of claim 19 wherein the lactase is selected from a fungal acid lactase, a yeast neutral lactase or a mixture thereof.