NL2017064B1 - Cross-linked enzyme aggregate comprising magnetizable particles - Google Patents

Abstract

The invention relates to a CLEA comprising magnetizable particles, characterized in that the magnetizable particles comprise particles of a zerovalent metal selected from the group of iron, nickel, cobalt and any mixture thereof, which particles are fully covered with a coating. The CLEAs of the invention were applied in catalytic processes, wherein it was demonstrated that the CLEAs can be recycled without any substantial loss of activity and with a virtually complete catalyst recovery. In addition, no leaching of metal and enzyme from the CLEAs was observed during their application.

Description

OctrooicentrumPatent center

NederlandThe Netherlands

(21) Aanvraagnummer: 2017064 © Aanvraag ingediend: 29/06/2016 © 2017064(21) Application number: 2017064 © Application submitted: 29/06/2016 © 2017064

BI OCTROOI (51) Int. CL:BI PATENT (51) Int. CL:

C12N 11/14 (2016.01) C12N 9/96 (2016.01)C12N 11/14 (2016.01) C12N 9/96 (2016.01)

Aanvraag ingeschreven: Application registered: (73) Octrooihouder(s): (73) Patent holder (s): 05/01/2018 05/01/2018 CLEA Technologies B.V. CLEA Technologies B.V. te Delft. in Delft. (43) Aanvraag gepubliceerd: (43) Application published: θ Uitvinder(s): θ Inventor (s): Octrooi verleend: Patent granted: Sander van Pelt te Delfgauw. Sander van Pelt in Delfgauw. 05/01/2018 05/01/2018 Michiel Hubertus Arnold Janssen Michiel Hubertus Arnold Janssen te Den Haag. in The Hague. (45) Octrooischrift uitgegeven: (45) Patent issued: Jo-Anne Michelle Rasmussen te IJmuiden. Jo-Anne Michelle Rasmussen in IJmuiden. 25/01/2018 25/01/2018 Menno Jort Sorgedrager te Schipluiden. Menno Jort Sorgedrager in Schipluiden. Roger Arthur Sheldon te Hoog-Keppel. Roger Arthur Sheldon in Hoog-Keppel. Pieter Koning te Dordrecht. Pieter Koning in Dordrecht. (74) Gemachtigde: (74) Agent: dr. T. Hubregtse te Beek-Ubbergen. Dr. T. Hubregtse in Beek-Ubbergen.

© Cross-linked enzyme aggregate comprising magnetizable particles (57) The invention relates to a CLEA comprising magnetizable particles, characterized in that the magnetizable particles comprise particles of a zerovalent metal selected from the group of iron, nickel, cobalt and any mixture thereof, which particles are fully covered with a coating. The CLEAs of the invention were applied in catalytic processes, wherein it was demonstrated that the CLEAs can be recycled without any substantial loss of activity and with a virtually complete catalyst recovery. In addition, no leaching of metal and enzyme from the CLEAs was observed during their application.© Cross-linked enzyme aggregate comprising magnetizable particles (57) The invention relates to a CLEA comprising magnetizable particles, characterized in that the magnetizable particles contain particles of a zerovalent metal selected from the group of iron, nickel, cobalt and any mixture thereof, which particles are fully covered with a coating. The CLEAs of the invention were applied in catalytic processes, it was demonstrated that the CLEAs can be recycled without any substantial loss or activity and with a virtually complete catalyst recovery. In addition, no leaching or metal and enzyme from the CLEAs was observed during their application.

NL BI 2017064NL BI 2017064

Dit octrooi is verleend ongeacht het bijgevoegde resultaat van het onderzoek naar de stand van de techniek en schriftelijke opinie. Het octrooischrift komt overeen met de oorspronkelijk ingediende stukken.This patent has been granted regardless of the attached result of the research into the state of the art and written opinion. The patent corresponds to the documents originally submitted.

Cross-linked enzyme aggregate comprising magnetizable particlesCross-linked enzyme aggregate comprising magnetizable particles

The invention relates to a cross-linked enzyme aggregate (CLEA), to a method for making such CLEA and to a process that makes use of the CLEA.The invention relates to a cross-linked enzyme aggregate (CLEA), to a method for making such a CLEA and to a process that makes use of the CLEA.

In enzymatic processes that are performed at an industrial scale, it is important that the enzyme has a long-term operational stability and that recovery and re-use of the enzyme are efficient and convenient. These requirements can be met by using enzymes in an immobilized form. Efficient recovery of an immobilized enzyme from a reaction medium can be achieved by means of standard separation techniques, such as filtration, centrifugation or settling.In enzymatic processes that are performed on an industrial scale, it is important that the enzyme has a long-term operational stability and that recovery and re-use of the enzyme are efficient and convenient. These requirements can be met by using enzymes in an immobilized form. Efficient recovery of an immobilized enzyme from a reaction medium can be achieved by means of standard separation techniques, such as filtration, centrifugation or settling.

There are processes, however, wherein other solids are present in the reaction medium besides the immobilized enzyme. In such cases, separation only by filtration, centrifugation or settling is not sufficient, since this does not separate the immobilized enzyme from the other solid(s). It is known that this problem can be overcome by providing the immobilized enzyme with magnetizable components. In this way, the separation from other solid components in the mixture is possible by making use of a magnet.There are processes, however, in which other solids are present in the reaction medium besides the immobilized enzyme. In such cases, separation only by filtration, centrifugation or settling is not sufficient, since this does not separate the immobilized enzyme from the other solid (s). It is known that this problem can be overcome by providing the immobilized enzyme with magnetizable components. In this way, the separation from other solid components in the mixture is possible by making use of a magnet.

Generally, methods of enzyme immobilisation can be divided into three categories (R.A. Sheldon, S. van Pelt, Chem. Soc. Rev., 2013, 42, 6223 - 6235)·, 1) binding the enzyme to a support (carrier); 2) entrapment of the enzyme (encapsulation); and 3) cross-linking of enzyme aggregates (CLEAs) or enzyme crystals (CLECs). The advantage of the third method - as compared to the first and the second method - is that it produces carrier-free macroparticles wherein non-catalytic material can be virtually absent. Therefore, CLEAs have a particularly high activity per weight unit as compared to carrier-based and encapsulated enzymes. When a CLEA is to be provided with a magnetizable component, however, the absence of any carrier or other non-catalytic auxiliary material makes it more difficult to introduce and immobilize magnetizable components in the immobilized enzyme matrix.Generally, methods of enzyme immobilization can be divided into three categories (R. A. Sheldon, S. van Pelt, Chem. Soc. Rev., 2013, 42, 6223 - 6235) ·, 1) binding the enzyme to a support (carrier); 2) entrapment of the enzyme (encapsulation); and 3) cross-linking or enzyme aggregates (CLEAs) or enzyme crystals (CLECs). The advantage of the third method - as compared to the first and the second method - is that it produces carrier-free macroparticles of non-catalytic material that can be virtually absent. Therefore, CLEAs have a particularly high activity per weight unit as compared to carrier-based and encapsulated enzymes. When a CLEA is provided with a magnetizable component, however, the absence of any carrier or other non-catalytic auxiliary material makes it more difficult to introduce and immobilize magnetizable components in the immobilized enzyme matrix.

A known method to prepare magnetizable CLEAs is to incorporate (nano)particles of magnetite in the CLEA. The magnetite is usually functionalized (e.g. with (3-aminopropyl)triethoxysilane or (3aminopropyl)trimethoxysilane) to be able to co-crosslink the magnetic particles with the enzyme aggregates, so that the magnetite particles are fixated in the final CLEA. Although reports on the catalytic action of CLEAs thus obtained do not seem to mention any undesired leaching of iron, caused by the dissolution of magnetite, it has been found by the present inventors that iron leaching indeed takes place under certain conditions, such as a lower pH of reaction (pH < 7) and can be accelerated in presence of free carboxylic acids. The dissolution of magnetite has undesirable effects. Such effects concern the presence of iron ions in the reaction medium with undesirable properties, a decrease in the catalytic activity of the CLEA and an incomplete recovery of the CLEA using magnetic recovery techniques. It appears that the undesirable effects of iron leaching become apparent only after long residence times of the CLEA in the reaction medium and after a plurality of CLEA recovery cycles. This is indeed different from the conditions wherein the magnetizable CLEAs known in the art are applied (i.e. neutral or basic pH, organic solvent, or short residence times and only a few or no recovery cycles). Since industrial processes often require a long catalyst lifetime and comprise multiple catalyst recoveries, the use of conventional magnetizable CLEAs poses serious problems for their application on an industrial scale.A known method to prepare magnetizable CLEAs is to incorporate (nano) particles or magnetite into the CLEA. The magnetite is usually functionalized (e.g. with (3-aminopropyl) triethoxy silane or (3 aminopropyl) trimethoxy silane) able to co-crosslink the magnetic particles with the enzyme aggregates, so that the magnetite particles are fixed in the final CLEA. Although reports on the catalytic action of CLEAs thus obtained do not seem to mention any undesired leaching or iron, caused by the dissolution or magnetite, it has been found by the present inventors that iron leaching indeed takes place under certain conditions, such as a lower pH of reaction (pH <7) and can be accelerated in the presence of free carboxylic acids. The dissolution of magnetite has undesirable effects. Such effects concern the presence of iron ions in the reaction medium with undesirable properties, a decrease in the catalytic activity of the CLEA and an incomplete recovery of the CLEA using magnetic recovery techniques. It appears that the undesirable effects of iron leaching become apparent only after long residence times of the CLEA in the reaction medium and after a multiple of CLEA recovery cycles. This is indeed different from the conditions of the magnetizable CLEAs known in the art are applied (i.e. neutral or basic pH, organic solvent, or short residence times and only a few or no recovery cycles). Since industrial processes often require a long catalyst lifetime and include multiple catalyst recoveries, the use of conventional magnetizable CLEAs poses serious problems for their application on an industrial scale.

Another clear disadvantage of the conventional magnetizable CLEA is the relatively low saturation magnetizability of magnetite, which makes it challenging to separate the conventional magnetizable CLEA from large scale applications with high flow rates and/or challenging media using cheap permanent magnet recovery set-ups.Another clear disadvantage of the conventional magnetizable CLEA is the relatively low saturation magnetizability or magnetite, which makes it challenging to separate the conventional magnetizable CLEA from large scale applications with high flow rates and / or challenging media using cheap permanent magnet recovery set-ups.

It is therefore an object of the present invention to provide improved magnetizable CLEAs, in particular with respect to their tendency to leach iron (or any other metal present in the magnetizable particles), their magnetizability, the decay of their activity in catalytic processes, and/or their recovery in catalytic processes.It is therefore an object of the present invention to provide improved magnetizable CLEAs, in particular with respect to their tendency to leach iron (or any other metal present in the magnetizable particles), their magnetizability, the decay of their activity in catalytic processes, and / or their recovery in catalytic processes.

Therefore, the invention relates to a CLEA comprising magnetizable particles, characterized in that the magnetizable particles comprise particles of a zerovalent metal selected from the group of iron, nickel, cobalt and any mixture thereof.Therefore, the invention relates to a CLEA comprising magnetizable particles, characterized in that the magnetizable particles consist of particles or a zerovalent metal selected from the group of iron, nickel, cobalt and any mixture thereof.

A CLEA of the invention is in principle present as a collection of CLEA particles. The dimensions of CLEA particles of the invention are usually within the range of those of CLEA particles described in the art. Typically, the average diameter of the CLEA particles in a CLEA of the invention is 0.5 pm or more. Preferably, it is 1 pm or more. It may also be 2 pm or more, 3 pm or more, 5 pm or more, 7 pm or more, 10 pm or more, 15 pm or more, 20 pm or more, 30 pm or more, 40 pm or more or 50 pm or more. The average diameter of the CLEA particles is usually 100 pm or less, 80 pm or less, 60 pm or less, 40 pm or less, 30 pm or less, 20 pm or less, 15 pm or less or 10 pm or less. By “average diameter” is meant that the diameter is the average of the diameters of all CLEA-particles in a particular CLEA. Usually, the average diameter of the CLEA-particles is in the range of 1-100 pm, preferably it is in the range of 1-50 pm. The diameter of the CLEA-particles are measured with laser diffraction, using e.g. a Malvern MasterSizer 3000 Hydro MV.A CLEA of the invention is in principle present as a collection of CLEA particles. The dimensions of CLEA particles of the invention are usually within the range of those of CLEA particles described in the art. Typically, the average diameter of the CLEA particles in a CLEA or the invention is 0.5 pm or more. Preferably, it is 1 pm or more. It may also be 2 pm or more, 3 pm or more, 5 pm or more, 7 pm or more, 10 pm or more, 15 pm or more, 20 pm or more, 30 pm or more, 40 pm or more or 50 pm or more. The average diameter of the CLEA particles is usually 100 pm or less, 80 pm or less, 60 pm or less, 40 pm or less, 30 pm or less, 20 pm or less, 15 pm or less or 10 pm or less. By "average diameter" is meant that the diameter is the average of the diameters of all CLEA particles in a particular CLEA. Usually, the average diameter of the CLEA particles is in the range or 1-100 pm, preferably it is in the range or 1-50 pm. The diameter of the CLEA particles are measured with laser diffraction, e.g. using a Malvern MasterSizer 3000 Hydro MV.

The size distribution of CLEA particles of the invention usually has a D10 value in the range of 1-9 pm, a D50 value in the range of 10-30 pm, and a D90 value in the range of 35-75 pm (measured with laser diffraction using a Malvern MasterSizer 3000 Hydro MV). The values of D10, D50 and D90 indicate that 10 percent of the particle population has a particle diameter below the D10 value; half of the population has a particle diameter below the D50 value; and 90 percent of the population has a particle diameter below the D90 value.The size distribution of CLEA particles of the invention usually has a D10 value in the range or 1-9 pm, a D50 value in the range or 10-30 pm, and a D90 value in the range or 35-75 pm (measured with laser diffraction using a Malvern MasterSizer 3000 Hydro MV). The values of D10, D50 and D90 indicate that 10 percent of the particle population has a particle diameter below the D10 value; half of the population has a particle diameter below the D50 value; and 90 percent of the population has a particle diameter below the D90 value.

In some cases, CLEA particles of the invention tend to form clusters, likely due to protein-protein interactions. Such clusters may have a size of up to 500 pm. The breaking up of such clusters in smaller clusters or in separate CLEA particles is possible, yet sometimes difficult. Such clustering appears to have no negative influence on the catalytic properties of the CLEAs of the invention, such as their leaching of metal ions, their leaching of magnetizable particles, their catalytic activity or their recovery efficiency in catalytic processes.In some cases, CLEA particles of the invention tend to form clusters, likely due to protein-protein interactions. Such clusters may have a size or up to 500 pm. The breaking up of such clusters into smaller clusters or into separate CLEA particles is possible, yet sometimes difficult. Such clustering appears to have no negative influence on the catalytic properties of the CLEAs of the invention, such as their leaching of metal ions, their leaching or magnetizable particles, their catalytic activity or their recovery efficiency in catalytic processes.

CLEA particles of the invention may in principle have any shape. Usually, they are compact, i.e. not of a flat or elongated form. They may for example be essentially spherical. The largest dimension (largest diameter) of the CLEA particles may also be up to 1.1 times, up to 1.3 times, up to 1.5 times, up to 2 times or up to 3 times larger than its smallest dimension. A CLEA particle of the invention may also deviate more from a compact shape in that its largest dimension is up to 5, up to 7, or up to 10 times larger than its smallest dimension.CLEA particles of the invention may have any shape in principle. Usually, they are compact, i.e. not or a flat or elongated form. They may for example be essentially spherical. The largest dimension or the CLEA particles may also be up to 1.1 times, up to 1.3 times, up to 1.5 times, up to 2 times or up to 3 times larger than its smallest dimension. A CLEA particle of the invention may also deviate more from a compact shape in that its largest dimension is up to 5, up to 7, or up to 10 times larger than its smallest dimension.

Figure 1 displays a Scanning Electron Microscope (SEM) picture of CIP-silica particles measured at 5 kV and 15000 x magnification on a TableTop SEM Hitachi TM3030Plus by Sysmex.Figure 1 displays a Scanning Electron Microscope (SEM) picture of CIP silica particles measured at 5 kV and 15000 x magnification on a TableTop SEM Hitachi TM3030Plus by Sysmex.

Figure 2 displays a Microscope image of a CLEA of the invention, comprising particles of silica-coated CIP and cross-linked glucose amylase. The cross-linked protein content of the CIP-silica mCLEA is 34 wt%.Figure 2 displays a Microscope image of a CLEA of the invention, including particles or silica-coated CIP and cross-linked glucose amylase. The cross-linked protein content of the CIP-silica mCLEA is 34 wt%.

Figure 3 displays a Microscope image of a CLEA of the invention, comprising particles of silica-coated CIP and cross-linked glutaminase. The cross-linked protein content of the CIP-silica mCLEA is 12 wt%.Figure 3 displays a Microscope image of a CLEA of the invention, including particles or silica-coated CIP and cross-linked glutaminase. The cross-linked protein content of the CIP-silica mCLEA is 12 wt%.

The entities that are visible on the photographs of Figures 2 and 3 are the CLEAs. The black areas of the CLEAs are the magnetizable particles or clusters thereof, while the lighter areas of the CLEAs between the magnetizable particles are the cross-linked proteins that hold together the CIP particles in a CLEA of the invention. The CLEA particles in Figure 3 have a lower cross-linked protein content than those in Figure 2.The entities that are visible on the photographs of Figures 2 and 3 are the CLEAs. The black areas of the CLEAs are the magnetizable particles or clusters of, while the lighter areas of the CLEAs are between the magnetizable particles that are the cross-linked proteins that hold together the CIP particles in a CLEA of the invention. The CLEA particles in Figure 3 have a lower cross-linked protein content than those in Figure 2.

The cross-linked protein content of a CLEA of the invention is defined as the weight percentage of cross-linked protein in the CLEA. The lower loading in Figure 3 is confirmed by the presence of less cross-linked protein (i.e. light areas) between the magnetizable particles, than in Figure 2.The cross-linked protein content of a CLEA or the invention is defined as the weight percentage or cross-linked protein in the CLEA. The lower loading in Figure 3 is confirmed by the presence of less cross-linked protein (i.e. light areas) between the magnetizable particles, than in Figure 2.

Some magnetizable CLEA particles only comprise a thin layer of cross-linked protein. Figures 2 and 3 demonstrate that magnetizable particles are indeed present in a CLEA of the invention and that such CLEAs can have various shapes.Some magnetizable CLEA particles only contain a thin layer or cross-linked protein. Figures 2 and 3 demonstrate that magnetizable particles are indeed present in a CLEA or the invention and that such CLEAs can have various shapes.

Further evidence for the incorporation of magnetizable particles was obtained by staining the protein of the CLEA with Coomassie Blue. In the recorded photographs (not shown), the black magnetizable particles were embedded in a blue environment of stained protein.Further evidence for the incorporation of magnetizable particles was obtained by staining the protein of the CLEA with Coomassie Blue. In the recorded photographs (not shown), the black magnetizable particles were embedded in a blue environment or stained protein.

The enzyme that is immobilized in a CLEA of the invention is usually selected from the group of hydrolases, such as esterases, proteases, amidases, cellulases, nitrilases, xylanases and glycosylases; lyases, such as hydroxynitrile lyases and aldolases; oxidoreductases, such as alcohol oxidases, peroxidases, keto reductases and imine reductases; and transferases, such as transaminases.The enzyme that is immobilized in a CLEA or the invention is usually selected from the group of hydrolases, such as esterases, proteases, amidases, cellulases, nitrilases, xylanases and glycosylases; lyases, such as hydroxynitrile lyases and aldolases; oxidoreductases, such as alcohol oxidases, peroxidases, keto reductases and imine reductases; and transferases, such as transaminases.

A CLEA of the invention may also contain two or more different enzymes in the aggregate (i.e. in the same CLEA particle). Such a CLEA is also known as a “combi-CLEA”. Using combi-CLEAs allows multiple process steps in a single operation (a one-pot process), which minimizes solvent usage, waste generation and energy.A CLEA or the invention may also contain two or more different enzymes in the aggregate (i.e. in the same CLEA particle). Such a CLEA is also known as a "combi-CLEA". Using combi-CLEAs allows multiple process steps in a single operation (a one-pot process), which minimizes solvent usage, waste generation and energy.

A CLEA of the invention comprises magnetizable particles of a zerovalent metal selected from the group of iron, nickel, cobalt and mixtures thereof. Preferably, the amount of the zerovalent metal(s) in the magnetizable particles is as high as possible, for example 90 wt% or more, 95 wt% or more, 96 wt% or more, 97 wt% or more, 98 wt% or more, 98.5 wt% or more, 99 wt% or more, 99.5 wt% or more or 99.8 wt% or more. More preferably, the magnetizable particles essentially consist of the zerovalent metal or a mixture of the zerovalent metals. In the event that the magnetizable particles comprise other components, these components are typically non-zerovalent metals, in particular selected from the group of iron, nickel and cobalt; and more in particular oxides and/or mixed oxides thereof. The magnetizable particles may also contain one or more of the elements nitrogen, carbon and oxygen.A CLEA of the invention comprises magnetizable particles of a zerovalent metal selected from the group of iron, nickel, cobalt and mixtures thereof. Preferably, the amount of zerovalent metal (s) in the magnetizable particles is as high as possible, for example 90 wt% or more, 95 wt% or more, 96 wt% or more, 97 wt% or more, 98 wt% or more, 98.5 wt% or more, 99 wt% or more, 99.5 wt% or more or 99.8 wt% or more. More preferably, the magnetizable particles essentially consist of the zero-metal or a mixture of the zero-metal. In the event that the magnetizable particles include other components, these components are typically non-zerovalent metals, particularly selected from the group of iron, nickel and cobalt; and more in particular oxides and / or mixed oxides thereof. The magnetizable particles may also contain one or more of the elements nitrogen, carbon and oxygen.

In a CLEA of the invention, the magnetizable particles in particular comprise zerovalent iron. More in particular, the particles comprise so-called “carbonyl iron particles” (CIP) or are derived thereof. CIP is an iron powder that is prepared by thermal decomposition of iron pentacarbonyl. This wellknown method produces spherical iron particles of high purity, typically with particle sizes of a few micrometers.In a CLEA of the invention, the magnetizable particles in particular include zerovalent iron. More in particular, the particles include so-called “carbonyl iron particles” (CIP) or are derived. CIP is an iron powder that is prepared by thermal decomposition or iron pentacarbonyl. This well-known method produces spherical iron particles or high purity, typically with particle sizes or a few micrometers.

Alternatively, when the magnetizable particles are made of nickel or cobalt, these metals may also be used in a form analogous to that of CIP, i.e. nickel particles prepared from “carbonyl nickel” (e.g. nickel tetracarbonyl) and cobalt particles prepared from “carbonyl cobalt” (e.g. dicobalt octacarbonyl).Alternatively, when the magnetizable particles are made from nickel or cobalt, these metals may also be used in a form analogous to that or CIP, ie nickel particles prepared from carbonyl nickel and cobalt particles prepared from carbonyl cobalt (Eg dicobalt octacarbonyl).

In a CLEA of the invention, it is preferred that the particles of the zerovalent metal are covered with a coating. More preferably, the metal particles are completely covered by such a coating. The main function of such a coating is to maintain the physical properties of the particles and to protect the particles against corrosion by the aqueous and/or oxidative environments wherein a CLEA of the invention usually resides during its synthesis and/or during the catalytic processes wherein the CLEA is used.In a CLEA or the invention, it is preferred that the particles of the zero-metal are covered with a coating. More preferably, the metal particles are completely covered by such a coating. The main function of such a coating is to maintain the physical properties of the particles and to protect the particles against corrosion by the aqueous and / or oxidative environments of a CLEA or the invention usually resides during its synthesis and / or during the catalytic processes the CLEA is used.

Thus, the coating is preferably of a compound that is capable of protecting the zerovalent metal from degradation by water, oxygen and/or acid. The coating in particular comprises silica (i.e. SiO₂), more in particular non-mesoporous silica, or a material comprising a silicate anion, such as orthosilicate (i.e. SiO₄ ⁴). The coating may also comprise a compound selected from the group of carbon, metal oxides such as AI₂O₃, and polymers such as polystyrene. Preferably, the coating consists of any of the materials mentioned above.Thus, the coating is preferably or a compound that is capable of protecting the zero-metal from degradation by water, oxygen and / or acid. The coating in particular comprises silica (ie SiO ₂ ), more in particular non-mesoporous silica, or a material including a silicate anion, such as orthosilicate (ie SiO ₄ ⁴ ). The coating may also comprise a compound selected from the group of carbon, metal oxides such as AI ₂ O ₃ , and polymers such as polystyrene. Preferably, the coating consists of any of the materials mentioned above.

The coating usually has a thickness of at least 1 nm. It may in particular be 5 nm or more, 10 nm or more, 20 nm or more, 30 nm or more, nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 125 nm or more, 150 nm or more, 200 nm or more or 300 nm or more.The coating usually has a thickness of at least 1 nm. It may in particular be 5 nm or more, 10 nm or more, 20 nm or more, 30 nm or more, nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 125 nm or more, 150 nm or more, 200 nm or more or 300 nm or more.

The coating usually has a thickness of 400 nm or less. It may in particular be 300 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 75 nm or less or 50 nm or less.The coating usually has a thickness of 400 nm or less. It may in particular be 300 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 75 nm or less or 50 nm or less.

The thickness may be in the range of 10-500 nm, in the range of 25150 nm, in the range of 50-100 nm, or in the range of 100-150 nm.The thickness may be in the range of 10-500 nm, in the range of 25150 nm, in the range of 50-100 nm, or in the range of 100-150 nm.

Optionally, the coating is functionalized. By such functionalization of the coating is meant that particular functional goups are provided (e.g.Optionally, the coating is functionalized. Such functionalization or coating means that particular functional groups are provided (e.g.

grafted) at the surface of the magnetizable particles via a spacer of a particular length, which can realize an interaction (in particular a covalent interaction) between the magnetizable particles and the enzyme.grafted) at the surface of the magnetizable particles via a spacer or a particular length, which can realize an interaction (in particular a covalent interaction) between the magnetizable particles and the enzyme.

A covalent interaction can be realized by allowing a functional group to participate in the cross-linking reaction of the CLEA synthesis. For example, the surface may be functionalized with alkoxysilanes having a pendant amine functionality that can co-cross-link with the enzyme aggregates. The surface is for example functionalized with (3-aminopropyl)triethoxysilane or (3-aminopropyl)ethyldiethoxysilane. In this way, the binding strength of the magnetizable particles with the cross-linked protein can be increased, which diminishes the release (i.e. leaching) of the magnetizable particles from the CLEA according to the invention.A covalent interaction can be realized by allowing a functional group to participate in the cross-linking reaction or the CLEA synthesis. For example, the surface may be functionalized with alkoxy silanes having a pendant amine functionality that can co-cross-link with the enzyme aggregates. The surface is for example functionalized with (3-aminopropyl) triethoxysilane or (3-aminopropyl) ethyldiethoxysilane. In this way, the binding strength of the magnetizable particles with the cross-linked protein can be increased, which diminishes the release (i.e., leaching) or the magnetizable particles from the CLEA according to the invention.

A covalent interaction between the magnetizable particles and the cross-linked protein can also be realized by epoxide or carboxylic acid groups.A covalent interaction between the magnetizable particles and the cross-linked protein can also be realized by epoxide or carboxylic acid groups.

Other means of functionalization result in a hydrophobic interaction between the cross-linked protein and the magnetizable particles (by using e.g. hydrophic groups such as phenyl, octyl or octadecyl groups) or in an ionic interaction between the cross-linked protein and the magnetizable particles (by using e.g. charged groups such as tertiary and quaternary amines).Other means of functionalization result in a hydrophobic interaction between the cross-linked protein and the magnetizable particles (by using eg hydrophic groups such as phenyl, octyl or octadecyl groups) or in an ionic interaction between the cross-linked protein and the magnetizable particles ( by using eg charged groups such as tertiary and quaternary amines).

In the invention, however, functionalities on the coating are in principle not necessary. It appeared for example that smooth, non-porous, silica-coated magnetizable particles can effectively be included in a CLEA to yield a CLEA of the invention and that leaching of the magnetizable particles from a CLEA of the invention in essence does not occur during its synthesis and neither when the CLEA of the invention is applied in a catalytic process. Thus, in a CLEA of the invention, a functionalization of the magnetizable particles with bifunctional molecular entities that provide a covalent attachment of the magnetizable particles to the cross-linked protein, in particular those entities having an alkyl chain, such as aminoalkylalkoxysilanes, may be absent. This absence does however not exclude any other covalent interaction between the cross-linked protein and the particles, since it may indeed be possible that the surface of the magnetizable particles as such is capable of forming a covalent interaction with a particular group of the cross-linked protein.In the invention, however, functionalities on the coating are in principle not necessary. It appears for example that smooth, non-porous, silica-coated magnetizable particles can effectively be included in a CLEA to yield a CLEA or the invention and that leaching of the magnetizable particles from a CLEA or the invention in essence does not occur during its synthesis and neither when the CLEA or the invention has been applied in a catalytic process. Thus, in a CLEA of the invention, a functionalization of the magnetizable particles with bifunctional molecular entities that provide a covalent attachment of the magnetizable particles to the cross-linked protein, in particular those entities having an alkyl chain, such as aminoalkylalkoxysilanes, may be absent. However, this absence does not exclude any other covalent interaction between the cross-linked protein and the particles, since it may indeed be possible that the surface of the magnetizable particles as such is capable of forming a covalent interaction with a particular group of the cross- linked protein.

As described above, the magnetizable particles in a CLEA of the invention comprise a zerovalent metal particle that is preferably surrounded by a coating. In case of the coating, the magnetizable particles can be regarded as particles comprising a core of the zerovalent metal and a surface layer of another material.As described above, the magnetizable particles in a CLEA or the invention consist of a zerovalent metal particle that is preferably surrounded by a coating. In case of the coating, the magnetizable particles can be viewed as particles including a core of the zero-metal and a surface layer of another material.

For the purpose of the present invention, the description of the size of the magnetizable particles is based on the outer dimensions of the magnetizable particles. When the magnetizable particles comprise a coating, their outer dimensions are defined by the size of the metal particle and the thickness of the coating. When the magnetizable particles do not comprise a coating, their outer dimensions are defined by the size of only the metal particle.For the purpose of the present invention, the description of the size of the magnetizable particles is based on the outer dimensions of the magnetizable particles. When the magnetizable particles comprise a coating, their outer dimensions are defined by the size of the metal particle and the thickness of the coating. When the magnetizable particles do not include a coating, their outer dimensions are defined by the size of only the metal particle.

Typically, the average diameter of the magnetizable particles in a CLEA of the invention is in the range of 10 nm - 20 pm, in particular it is in the range of 1-15 pm. For example, it is 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 75 nm or more, 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 750 nm or more, 1 pm or more, 2 pm or more, 3 pm or more, 4 pm or more, 5 pm or more, 6 pm or more, 7 pm or more, 8 pm or more, 9 pm or more, 10 pm or more, 12 pm or more, 14 pm or more, or 20 pm or more. The average diameter of the magnetizable particles is 20 pm or less, 16 pm or less, 13 pm or less, 10 pm or less, 8 pm or less, 7 pm or less, 6 pm or less, 5 pm or less, 4 pm or less, 3 pm or less, 2 pm or less, 1 pm or less, 800 nm or less, 600 nm or less, 400 nm or less, 200 nm or less, 100 nm or less, 80 nm or less, 60 nm or less, 40 nm or less, 30 nm or less, or 20 nm or less. By “average diameter” is meant that the diameter is the average of the diameters of all magnetizable particles in a particular CLEA of the invention.Typically, the average diameter of the magnetizable particles in a CLEA or the invention is in the range of 10 nm - 20 pm, in particular it is in the range or 1-15 pm. For example, it is 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 75 nm or more, 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 750 nm or more, 1 pm or more, 2 pm or more, 3 pm or more, 4 pm or more, 5 pm or more, 6 pm or more, 7 pm or more, 8 pm or more, 9 pm or more, 10 pm or more, 12 pm or more, 14 pm or more, or 20 pm or more. The average diameter of the magnetizable particles is 20 pm or less, 16 pm or less, 13 pm or less, 10 pm or less, 8 pm or less, 7 pm or less, 6 pm or less, 5 pm or less, 4 pm or less, 3 pm or less, 2 pm or less, 1 pm or less, 800 nm or less, 600 nm or less, 400 nm or less, 200 nm or less, 100 nm or less, 80 nm or less, 60 nm or less, 40 nm or less, 30 nm or less, or 20 nm or less. By "average diameter" is meant that the diameter is the average of the diameters of all magnetizable particles in a particular CLEA of the invention.

The magnetizable particles may in principle have any shape. They may have a flat shape (e.g. flake-like), an elongated shape (e.g. rod-like) or a more compact shape (e.g. spherical, ovoid or cubic-like).The magnetizable particles may have any shape in principle. They may have a flat shape (e.g., flake-like), an elongated shape (e.g., rod-like) or a more compact shape (e.g., spherical, ovoid, or cubic-like).

When the magnetizable particles have a compact shape, the largest dimension (largest diameter) of the particles is typically up to 2 times, up to 3 times, or up to 5 times larger than their smallest dimension. When a magnetizable particle is nearly spherical, the largest dimension (largest diameter) of the magnetizable particle is typically up to 1.05 times, 1.1 times,When the magnetizable particles have a compact shape, the largest dimension or the particle is typically up to 2 times, up to 3 times, or up to 5 times larger than their smallest dimension. When a magnetizable particle is nearly spherical, the largest dimension or the magnetizable particle is typically up to 1.05 times, 1.1 times,

1.2 times, 1.3 times or 1.5 times larger than its smallest dimension.1.2 times, 1.3 times or 1.5 times larger than its smallest dimension.

In an embodiment, the magnetizable particles have an essentially spherical shape. In such case, the ranges of the average diameter as provided hereinabove are the ranges of the actual diameter of the sphere. When the magnetizable particles are spherical or nearly spherical, they preferably comprise “carbonyl iron particles” (CIP), as described hereinabove.In an embodiment, the magnetizable particles have an essentially spherical shape. In such a case, the ranges of the average diameter as provided are the ranges of the actual diameter of the sphere. When the magnetizable particles are spherical or nearly spherical, they preferably include carbonyl iron particles (CIP), as describedabove.

The magnetizable particles, and in particular magnetizable particles that have a spherical shape, may be present in a particle size distribution having a median value D50 in the range of 25 nm - 15 pm, the D50 value being the particle size that splits the distribution with half above and half below this size. The relative span of the distribution of magnetizable particles is usually in the range of 1-10, wherein the relative span is defined by dividing the absolute span D10-D90 by the D50 value, D10 and D90 being the particle size that splits the distribution with 10% of the particles below the size of D10 and 90% of the particles below the size of D90, respectively (i.e. the relative span is (D90-D10)/D50). The relative span may also be 10 or less, 7 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less.The magnetizable particles, and in particular magnetizable particles that have a spherical shape, may be present in a particle size distribution having a median value D50 in the range of 25 nm - 15 pm, the D50 value being the particle size that splits the distribution with half above and half below this size. The relative span of the distribution of magnetizable particles is usually in the range of 1-10, the relative span is defined by dividing the absolute span D10-D90 by the D50 value, D10 and D90 being the particle size that splits the distribution with 10% of the particles below the size of D10 and 90% of the particles below the size of D90, respectively (ie the relative span is (D90-D10) / D50). The relative span may also be 10 or less, 7 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less .

In a particular embodiment, the particle size distribution has a D10 value in the range of 1-3 pm, in particular 1.7-2.8 pm, a D50 value in the range of 3.5-6 pm, in particular in the range of 3.9-5.3 pm and a D90 value in the range of 6.5-10 pm, in particular 7.2-9.3 pm. The thickness of the coating of such magnetizable particles is usually in the range of 10-500 nm, in the range of 25-150 nm, in the range of 50-100 nm, or in the range of 100-150 nm.In a particular embodiment, the particle size distribution has a D10 value in the range or 1-3 pm, in particular 1.7-2.8 pm, a D50 value in the range or 3.5-6 pm, in particular in the range or 3.9-5.3 pm and a D90 value in the range of 6.5-10 pm, in particular 7.2-9.3 pm. The thickness of the coating or such magnetizable particles is usually in the range of 10-500 nm, in the range of 25-150 nm, in the range of 50-100 nm, or in the range of 100-150 nm.

The magnetizable particles may also be present in a CLEA of the invention in an aggregated form. For example, when compact particles, in particular particles comprising CIP, stick together, they may be incorporated in the CLEA of the invention as a small cluster of particles, e.g. of 2 or more, or more, 10 or more, 20 or more, 50 or more or 100 or more particles. It is also possible that during a coating process of the magnetizable particles, the metal particles are not completely separated, so that the resulting coating embraces a small cluster wherein a plurality of metal particles are present, e.g. 2 or more, 5 or more, 10 or more, 20 or more, 50 or more or 100 or more. An aggregate of magnetizable particles as described above (i.e. wherein each magnetizable particle is separately coated or wherein an aggregate of metal particles is coated as a whole) may as such be incorporated into a CLEA of the invention. The skilled person is capable of finding the appropriate conditions to reach any of these conditions wherein the magnetizable particles reside (e.g. as completely separated particles or as one of the aggregates as described hereinabove), without undue burden and without exerting an inventive effort.The magnetizable particles may also be present in a CLEA or the invention in an aggregated form. For example, when compact particles, in particular particles including CIP, stick together, they may be incorporated in the CLEA or the invention as a small cluster of particles, eg or 2 or more, or more, 10 or more, 20 or more, 50 or more or 100 or more particles. It is also possible that during a coating process of the magnetizable particles, the metal particles are not completely separated, so that the resulting coating embraces a small cluster of a variety of metal particles are present, eg 2 or more, 5 or more, 10 or more, 20 or more, 50 or more or 100 or more. An aggregate of magnetizable particles as described above (i.e. each magnetizable particle is separately coated or if an aggregate of metal particles is coated as a whole) may be such as incorporated into a CLEA of the invention. The skilled person is capable of finding the appropriate conditions to reach any of these conditions of the magnetizable particle reside (e.g., completely separated particles or one of the aggregates as describedabove), without undue burden and without exerting an inventive effort.

It is also possible that the magnetizable particles are obtained by the sintering of smaller metal particles, followed by an eventual coating the sintered product under appropriate conditions. In this way, particles with an irregular shape can be incorporated into a CLEA of the invention. For example, the magnetizable particles in a CLEA of the invention may comprise sintered particles that comprise CIP. The number of sintered particles may be 2 or more, 5 or more, 10 or more, 20 or more, 50 or more or 100 or more.It is also possible that the magnetizable particles are obtained by sintering or narrower metal particles, followed by an eventual coating of the sintered product under appropriate conditions. In this way, particles with an irregular shape can be incorporated into a CLEA of the invention. For example, the magnetizable particles in a CLEA or the invention may comprise sintered particles that comprise CIP. The number of sintered particles may be 2 or more, 5 or more, 10 or more, 20 or more, 50 or more or 100 or more.

It was surprisingly found that the zerovalent magnetizable particles could effectively and permanently be incorporated into a CLEA of the invention by cross-linking a precipitated enzyme in the presence of the magnetizable particles, even when the particles were not functionalized.It was surprisingly found that the zerovalent magnetizable particles could be effectively and permanently incorporated into a CLEA or the invention by cross-linking a precipitated enzyme in the presence of the magnetizable particles, even when the particles were not functionalized.

Accordingly, the invention further relates to a process for preparing a CLEA, comprising providing a solution of an enzyme; then precipitating the enzyme by forming an enzyme aggregate; then cross-linking the enzyme aggregate with a cross-linking agent in the presence of magnetizable particles comprising a particle of a zerovalent metal selected from the group of iron, nickel, cobalt and any mixture thereof.For example, the invention further relates to a process for preparing a CLEA, including providing a solution of an enzyme; then the enzyme precipitates by forming an enzyme aggregate; then cross-linking the enzyme aggregate with a cross-linking agent in the presence of magnetizable particles including a particle or a zerovalent metal selected from the group of iron, nickel, cobalt and any mixture thereof.

The magnetizable particles are present during the cross-linking step, and thus are added to the enzyme before this step is carried out. The magnetizable particles may be added before, during or after the step of precipitating the enzyme.The magnetizable particles are present during the cross-linking step, and thus are added to the enzyme before this step is carried out. The magnetizable particles may be added before, during or after the step or precipitating the enzyme.

In a method of the invention, the magnetizable particles are preferably completely covered by a coating so as to ensure that the zerovalent metal in the particles does not degrade under the conditions that are present during the CLEA synthesis or during a catalytic process wherein the CLEA is used. Usually, such conditions are aqueous and/or aerobic conditions, under which the zerovalent metal in the particles is prone to oxidation followed by dissolution.In a method of the invention, the magnetizable particles are preferably completely covered by a coating so as to ensure that the zero-metal metal in the particles does not degrade under the conditions that are present during the CLEA synthesis or during a catalytic process according to CLEA used. Usually, such conditions are aqueous and / or aerobic conditions, under which the zero metal in the particles is prone to oxidation followed by dissolution.

In a method of the invention, it is preferred to use CIP-based magnetizable particles, i.e. iron particles derived from CIP. It is also preferred that these particles comprise a coating comprising silica (i.e.In a method of the invention, it is preferred to use CIP-based magnetizable particles, i.e. iron particles derived from CIP. It is also preferred that these particles comprise a coating comprising silica (i.e.

non-(meso)porous silica). The coating in particular consists of silica (i.e. non-(meso)porous silica). It appears not necessary to use a functionalization on the coating, thus the coating may be unfunctionalized.non- (meso) porous silica). The coating in particular consists of silica (i.e. non- (meso) porous silica). It does not appear necessary to use a functionalization on the coating, thus the coating may be unfunctionalized.

The CIP-based magnetizable particles in particular have a particle size distribution wherein D10 is in the range of 1-3 pm, in particular 1.7-2.8 pm, D50 is in the range of 3.5-6 pm, in particular in the range of 3.9-5.3 pm and D90 is in the range of 6.5-10 pm, in particular 7.2-9.3 pm. The thickness of the coating on the CIP is usually in the range of 10-500 nm, in the range of 25-150 nm, in the range of 50-100 nm, or in the range of 100-150 nm.The CIP-based magnetizable particles in particular have a particle size distribution of D10 is in the range of 1-3 pm, in particular 1.7-2.8 pm, D50 is in the range of 3.5-6 pm, in particular in the range of 3.9 -5.3 pm and D90 is in the range of 6.5-10 pm, in particular 7.2-9.3 pm. The thickness of the coating on the CIP is usually in the range of 10-500 nm, in the range of 25-150 nm, in the range of 50-100 nm, or in the range of 100-150 nm.

It is surprising that a CLEA of the invention comprising non-functionalized magnetizable particles, did not measurably leach magnetizable particles into the medium of this CLEA, while this CLEA contained a high loading of protein on the magnetizable particles. This is surprising because the silica coating on the particles has a low surface area as compared to mesoporous silica materials that are generally used in the art as a support for catalyst immobilization, so the area for interaction of the cross-linked protein with the magnetizable particles is more limited than in the art. In addition, no functionalization was present, which - if present - would compensate for that. Further, given that the size of the magnetizable particles is in general above 1 pm, the surface area per gram of particle material is even more remote from that of commonly used silica supports. For these reasons, it was not expected that the use of silica-coated CIP would yield a CLEA with magnetizable particles that has improved properties, in particular a virtually absent leaching of CIP in combination with a high protein loading. A further advantage is that functionalization of the magnetizable particles is not necessary in a CLEA of the invention. In contrast, the magnetite used in the art often requires functionalization for a successful and long-lasting incorporation into a CLEA.It is surprising that a CLEA of the invention comprising non-functionalized magnetizable particles, did not measurably leach magnetizable particles into the medium of this CLEA, while this CLEA contained a high loading of protein on the magnetizable particles. This is surprising because the silica coating on the particles has a low surface area as compared to mesoporous silica materials that are generally used in the art as a support for catalyst immobilization, the area for interaction of the cross-linked protein with the magnetizable particles is more limited than in the art. In addition, no functionalization was present, which - if present - would compensate for that. Further, given that the size of the magnetizable particles is in general above 1 pm, the surface area per gram or particle material is equally more remote from that or commonly used silica supports. For these reasons, it was not expected that the use of silica-coated CIP would yield a CLEA with magnetizable particles that has improved properties, in particular a virtually absent leaching or CIP in combination with a high protein loading. A further advantage is that functionalization of the magnetizable particles is not necessary in a CLEA of the invention. In contrast, the magnetite used in the art often requires functionalization for a successful and long-lasting incorporation into a CLEA.

Iron leakage experiments were carried out on CLEAs of the invention, by incubating them in an acid environment and performing an iron detection assay on the incubation supernatant. Although the conditions chosen for incubation were extreme with respect to common enzyme applications, iron leakage from the CIP-silica mCLEA was found to be negligible.Iron leakage experiments were carried out on CLEAs of the invention, by incubating them in an acid environment and performing an iron detection assay on the incubation supernatant. Although the conditions chosen for incubation were extreme with respect to common enzyme applications, iron leakage from the CIP-silica mCLEA was found to be negligible.

It is further surprising that CLEAs of the invention were not substantially affected by a magnetic field used for their recovery. In particular, no leaching of the magnetizable particles could be detected. Due to the relatively low surface area of the magnetizable particles and the lack of functionalization thereon, it was considered a likely possibility that the magnetizable particles would be pulled out of a CLEA of the invention by an external magnetic field, leaving the CLEA of the invention in a state in which it cannot be magnetized anymore. This not at all being the case, it can be concluded that the magnetizable particles in a CLEA of the invention do not only withstand the forces resulting from vibration and other thermally induced movements, but that they also withstand the magnetic force of an external magnetic field.It is further surprising that CLEAs of the invention were not significantly affected by a magnetic field used for their recovery. In particular, no leaching or the magnetizable particles could be detected. Due to the relatively low surface area of the magnetizable particles and the lack of functionalization thereon, it was considered a likely possibility that the magnetizable particles would have been pulled out of a CLEA or the invention by an external magnetic field, leaving the CLEA of the invention in a state in which it cannot be magnetized anymore. The magnetizable particles in a CLEA or the invention do not only withstand the forces resulting from vibration and other thermally induced movements, but that they also withstand the magnetic force of an external magnetic field .

It further appears that the CIP-based magnetizable particles are not only prevented from leaching into the medium, but they also maintain a fixed position within a CLEA of the invention. By a fixed position is meant that the particles do not undergo substantial translational movement within a CLEA of the invention.It further appears that the CIP-based magnetizable particles are not only prevented from leaching into the medium, but they also maintain a fixed position within a CLEA of the invention. By a fixed position is meant that the particles do not undergo substantial translational movement within a CLEA of the invention.

Further investigations revealed that there is essentially no interaction between the free dissolved enzyme and the CIP-based particles. In view of these findings, it is even more surprising that the particles are so well incorporated in a CLEA of the invention.Further investigations revealed that there is essentially no interaction between the free dissolved enzyme and the CIP-based particles. In view of these findings, it is just as surprising that the particles are so well incorporated in a CLEA or the invention.

The leakage of enzyme from CLEAs of the invention into the reaction medium was also investigated. Surprisingly, no leaching of enzyme could be demonstrated, even after stirring the CLEA for proloned times, such as 10 days, in an aqueous medium at 32 °C.The leakage of enzyme from CLEAs or the invention into the reaction medium was also investigated. Surprisingly, no leaching or enzyme could be demonstrated, briefly after stirring the CLEA for prone times, such as 10 days, in an aqueous medium at 32 ° C.

Recycling experiments were also carried out with CLEAs of the invention, to mimic their application on an industrial scale. It was found that at least 10 cycles could be made without any substantial loss of activity and with a virtually complete catalyst recovery.Recycling experiments were also carried out with CLEAs of the invention, to mimic their application on an industrial scale. It was found that at least 10 cycles could be made without any substantial loss or activity and with a virtually complete catalyst recovery.

The measured saturation magnetizations of CLEAs of the invention were surprisingly high. Values of up to 200 emu-g'¹ were found, while known CLEAs containing APTES functionalized magnetite generally have a saturation magnetization in the range of 10-40 emu-g'¹.The measured saturation magnetizations or CLEAs or the invention were surprisingly high. Values of up to 200 emu-g ' ¹ were found, while known CLEAs containing APTES functionalized magnetite generally have a saturation magnetization in the range or 10-40 emu-g' ¹ .

Generally, the saturation magnetization of a CLEA of the invention is lower when it contains more protein, since protein is a non-magnetizable material. Thus, a large amount of protein would result in a poorer separability and recovery of the CLEA in a catalytic process. On the other hand, it is an advantage that the amount of protein in a CLEA of the invention is high, because this increases the catalytic activity per gram of CLEA. It has been found that the use of the zerovalent metal (i.e. iron, nickel, cobalt) in the magnetizable particles provides a good balance between the saturation magnetization and the catalytic activity per gram of CLEA.Generally, the saturation magnetization of a CLEA or the invention is lower when it contains more protein, since protein is a non-magnetizable material. Thus, a large amount of protein would result in a poorer separability and recovery of the CLEA in a catalytic process. On the other hand, it is an advantage that the amount of protein in a CLEA or the invention is high, because this increases the catalytic activity per gram of CLEA. It has been found that the use of zerovalent metal (i.e. iron, nickel, cobalt) in the magnetizable particles provides a good balance between the saturation magnetization and the catalytic activity per gram of CLEA.

The number of magnetizable particles that is incorporated in a CLEAparticle of the invention is 1 or more. The number of magnetizable particles that is on average incorporated in a CLEA-particle of the invention (the “average number”) may be in the range of 1-10,000, in particular it is in the range of 1-100. The average number may be 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more or 80 or more.The number of magnetizable particles that is incorporated in a CLE Particle of the invention is 1 or more. The number of magnetizable particles that is on average incorporated in a CLEA particle of the invention (the "average number") may be in the range of 1-10,000, in particular it is in the range of 1-100. The average number may be 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more or 80 or more.

The content of magnetizable particles in a CLEA of the invention may also be defined as a weight percentage of the entire CLEA. The content is usually in the range of 1-99 wt%. It may also be in the range of 25-90 wt%, in the range of 50-95 wt% or in the range of 70-90 wt%. It may be 3 wt% or more, 5 wt% or more, 10 wt% or more, 15 wt% or more, 20 wt% or more, 30 wt% or more, 40 wt% or more, 50 wt% or more, 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, or 85 wt% or more.The content of magnetizable particles in a CLEA or the invention may also be defined as a weight percentage of the entire CLEA. The content is usually in the range of 1-99 wt%. It may also be in the range or 25-90 wt%, in the range or 50-95 wt% or in the range or 70-90 wt%. It may be 3 wt% or more, 5 wt% or more, 10 wt% or more, 15 wt% or more, 20 wt% or more, 30 wt% or more, 40 wt% or more, 50 wt% or more , 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, or 85 wt% or more.

The cross-linking agent may be selected from the group of formaldehyde, glyoxal, glutaraldehyde and aldehyde cross-linkers derived from polysaccharides.The cross-linking agent may be selected from the group of formaldehyde, glyoxal, glutaraldehyde and aldehyde cross-linkers derived from polysaccharides.

The invention further relates to a CLEA obtainable by the preparation method described hereinabove.The invention further relates to a CLEA available by the preparation method described belowabove.

The invention further relates to a process comprising the use of a CLEA as described hereinabove for a catalytic conversion, wherein the CLEA is separated from the reaction medium by collecting the CLEA with the aid of a magnetic field, in particular with the aid of a permanent magnet.The invention further relates to a process including the use of a CLEA as described readabove for a catalytic conversion, the CLEA is separated from the reaction medium by collecting the CLEA with the aid of a magnetic field, in particular with the aid of a permanent magnet.

EXAMPLESEXAMPLES

GeneralGeneral

Magnetic strengthMagnetic strength

Magnetic strength measurements were carried cut cn a Mcdel 2000 Alternating Gradient Magnetcmeter cf the ccmpany Princetcn Measurement Cccperaticn. The repcrted values are an average cf three measurements.Magnetic strength measurements were carried out cn a Mcdel 2000 Alternating Gradient Magnetcm cf the ccmpany Princetcn Measurement Cccperaticn. The replied values are an average or three measurements.

SEMSEM

Scanning Electron Microscope (SEM) measurements were performed on a TableTop SEM Hitachi TM3030Plus by Sysmex.Scanning Electron Microscope (SEM) measurements were performed on a TableTop SEM Hitachi TM3030Plus by Sysmex.

Iron detection assayIron detection assay

The organic ligand 2,2’-bipyridine forms stable, strongly colored complexes with Fe(ll). Very small quantities of iron in solution can be detected by using the intense light absorption properties of these complexes. Hydroxylammonium chloride was used as a reducing agent to convert Fe(lll) into Fe(ll) as Fe(lll) and 2,2’-bipyridine form a complex that absorbs less strongly and at a different wavelength. Using this method, Fe(ll) concentrations of 1 to 5 ppm (1-5 mg-L'¹) could easily be detected (Vogel, A Textbook of Quantitative Inorganic Analysis, 3rd Ed., p. 294, 310 and 787).The organic ligand 2,2'-bipyridine forms stable, strongly colored complexes with Fe (11). Very small quantities of iron in solution can be detected by using the intense light absorption properties of these complexes. Hydroxylammonium chloride was used as a reducing agent to convert Fe (III) into Fe (III) as Fe (III) and 2,2'-bipyridine form a complex that absorbs less strongly and at a different wavelength. Using this method, Fe (II) concentrations of 1 to 5 ppm (1-5 mg-L ' ¹ ) could easily be detected (Vogel, A Textbook of Quantitative Inorganic Analysis, 3rd Ed., Pp. 294, 310 and 787) .

Example 1. Synthesis of silica coated CIP (CIP-silica)Example 1. Synthesis of silica coated CIP (CIP silica)

Preparation of CIPPreparation or CIP

CIP particles were prepared according to known methods of iron carbonyl decomposition, such as described in e.g. GB684054A. Besides, the CIP particles were also directly obtained from BASF.CIP particles were prepared according to known methods of iron carbonyl decomposition, such as described in e.g. GB684054A. Besides, the CIP particles were also obtained directly from BASF.

Providing the CIP with a silica coatingProviding the CIP with a silica coating

The silica coating layer is introduced in order to maintain the physical and physicochemical properties of the CIP, such as to protect the CIP against oxidation and subsequent dissolution, or as to serve as a matrix that allows for chemical modification for the introduction of functional groups. The majority of these CIP-silica particles have been produced relying on a method known as the Stöber method, which was originally reported by W. Stöber, A. Fink, and E. Bohn, Journal of Colloid Interface Science, 1968, 26, 62-68, or a sol-gel method.The silica coating layer has been introduced in order to maintain the physical and physicochemical properties of the CIP, such as to protect the CIP against oxidation and subsequent dissolution, or as a serve as a matrix that allows for chemical modification for the introduction of functional groups. The majority of these CIP-silica particles have been produced relying on a method known as the Stöber method, which was originally reported by W. Stöber, A. Fink, and E. Bohn, Journal of Colloid Interface Science, 1968, 26, 62 -68, or a sol-gel method.

The reaction was performed under a nitrogen atmosphere. A 500 ml_ reaction vessel was charged with 30 g of CIP (OM grade, BASF), 210 ml_ of ethanol, 2.9 g of TEOS (tetraethyl orthosilicate), 33 ml_ of water and 6 ml_ of 25% ammonia. The mixture was vigorously stirred with an overhead stirrer for 30 minutes after which it was heated to 60 °C. Next, 15 g of TEOS was added over a period of 2 hours after which stirring was continued for another 4 hours. The mixture was then cooled to room temperature and the particles were repeatedly collected with a handheld magnet and resuspended in ethanol for washing. Finally, the CIP-silica particles were dried under reduced pressure at 80 °C for 3 days. Figure 1 shows a Scanning Electron Microscope (SEM) picture of the CIP-silica particles measured at 5 kV and 15000 x magnification on aTableTop SEM Hitachi TM3030Plus by Sysmex.The reaction was performed under a nitrogen atmosphere. A 500 ml reaction vessel was charged with 30 g of CIP (OM grade, BASF), 210 ml of ethanol, 2.9 g of TEOS (tetraethyl orthosilicate), 33 ml of water and 6 ml of or 25% ammonia. The mixture was vigorously stirred with an overhead stirrer for 30 minutes after which it was heated to 60 ° C. Next, 15 g or TEOS was added over a period of 2 hours after which stirring was continued for another 4 hours. The mixture was then cooled to room temperature and the particles were repeatedly collected with a handheld magnet and resuspended in ethanol for washing. Finally, the CIP silica particles were dried under reduced pressure at 80 ° C for 3 days. Figure 1 shows a Scanning Electron Microscope (SEM) picture of the CIP silica particles measured at 5 kV and 15000 x magnification on aTableTop SEM Hitachi TM3030Plus by Sysmex.

Iron leakageIron leakage

Iron leakage from CIP-silica particles was determined by using the iron detection assay as described above.Iron leakage from CIP silica particles was determined by using the iron detection assay as described above.

The absorption of the supernatant in the iron detection assay at 100 wt% iron leakage was determined by completely dissolving 6 mg of CIP-silica particles in concentrated HCI and subsequent dilution.The absorption of the supernatant in the iron detection assay at 100 wt% iron leakage was determined by completely dissolving 6 mg or CIP-silica particles in concentrated HCI and subsequent dilution.

The iron leakage from CIP-silica particles was then studied by contacting 6 mg of the CIP-silica particles with 18 ml of a 1wt% aqueous lactic acid solution of pH 3. The iron leakage (wt%) after incubation was determined by directly comparing the absorption of the incubation supernatant of the CIP-silica particles to that of the absorption after total dissolution (100 wt% iron leakage). After gently shaking at 32 °C for 72 h, leakage of Fe(l 1,111) was <1 wt%.The iron leakage from CIP-silica particles was then studied by contacting 6 mg of the CIP-silica particles with 18 ml or a 1wt% aqueous lactic acid solution or pH 3. The iron leakage (wt%) after incubation was determined by directly comparing the absorption of the incubation supernatant of the CIP-silica particles to that of the absorption after total dissolution (100 wt% iron leakage). After gently shaking at 32 ° C for 72 hours, leakage of Fe (1.111) was <1 wt%.

Commercial CIP without any protective coating, on the other hand, appeared to dissolve completely in these incubation mixtures within 24 hours (i.e. 100 wt% leakage).Commercial CIP without any protective coating, on the other hand, appeared to dissolve completely in these incubation mixtures within 24 hours (i.e. 100 wt% leakage).

Magnetic strengthMagnetic strength

The saturation magnetization of dry CIP-silica particles was measured at 235 emu-g'¹ (Am²/kg).The saturation magnetization of dry CIP silica particles was measured at 235 emu-g ' ¹ (Am ² / kg).

Example 2. CIP-silica magnetizable CLEA (mCLEA) of glucose amylase - high protein loadingExample 2. CIP-silica magnetizable CLEA (mCLEA) or glucose amylase - high protein loading

SynthesisSynthesis

In a 2 L plastic beaker, a mixture of 0.727 L of saturated ammonium sulfate and 0.225 kg of CIP-silica particles (prepared according to Example 1) was stirred with an overhead stirrer (Velp Scientifica ES overhead stirrer) at room temperature for 1 h, to form a suspension. Thereafter, 0.3 L of glucose amylase (Zibo Guoao, Shandong, China) was added slowly to the suspension, followed by stirring the suspension at room temperature for 1 hour. After the addition of 0.153 L of 25 wt% glutaraldehyde, the reaction mixture was stirred at room temperature for 18 h. The resulting CIP-silica mCLEA was removed with a handheld magnet (ERIEZ Mega Rare Earth Tube Magnet 150 mm, 10700 Gauss) and washed five times with 4.5 L of water. The final CIP-silica mCLEA was suspended in 1 L of water. The total dry weight of the CIP-silica mCLEA was 341 grams. Figure 2 shows a Microscope image of the CIP-silica mCLEA of glucose amylase, obtained with a Bresser microscope using a 40x magnification lens and Mikro CamLab software (Version 6.1.4.0).In a 2 L plastic beaker, a mixture of 0.727 L or saturated ammonium sulfate and 0.225 kg or CIP-silica particles (prepared according to Example 1) was stirred with an overhead stirrer (Velp Scientifica ES overhead stirrer) at room temperature for 1 h , to form a suspension. Thereafter, 0.3 L of glucose amylase (Zibo Guoao, Shandong, China) was added slowly to the suspension, followed by stirring the suspension at room temperature for 1 hour. After the addition of 0.153 L or 25 wt% glutaraldehyde, the reaction mixture was stirred at room temperature for 18 h. The resulting CIP-silica mCLEA was removed with a handheld magnet (ERIEZ Mega Rare Earth Tube Magnet 150 mm, 10700 Gauss) and washed five times with 4.5 L of water. The final CIP-silica mCLEA was suspended in 1 L of water. The total dry weight of the CIP-silica mCLEA was 341 grams. Figure 2 shows a Microscope image of the CIP-silica mCLEA or glucose amylase, obtained with a Bresser microscope using a 40x magnification lens and Mikro CamLab software (Version 6.1.4.0).

Activity assayActivity assay

In a 100 mL conical flask were added 30 mL of maltodextrin (33% w/v in water) and 0.2 mL of glucose amylase CIP-silica mCLEA suspension. The suspension was shaken at 150 rpm at 32 °C for 3 hours in aIn a 100 mL conical flask were added 30 mL of maltodextrin (33% w / v in water) and 0.2 mL of glucose amylase CIP-silica mCLEA suspension. The suspension was shaken at 150 rpm at 32 ° C for 3 hours in a

Stuart Orbital Incubator SI500. After 1,2 and 3 h, a 0.1 mL sample was taken from the suspension and added to 0.9 mL of water. The mixture was then centrifuged, filtered (0.2 pm Phenex™ syringe filter), transferred to an HPLC vial and subjected to HPLC analysis under the following conditions to determine the glucose content:Stuart Orbital Incubator SI500. After 1.2 and 3 h, a 0.1 mL sample was tasks from the suspension and added to 0.9 mL of water. The mixture was then centrifuged, filtered (0.2 pm Phenex ™ syringe filter), transferred to an HPLC vial and subjected to HPLC analysis under the following conditions to determine the glucose content:

HPLC: Shimadzu LC-20AT Prominence LiquidHPLC: Shimadzu LC-20AT Prominence Liquid

Column:Column:

Mobile phase: Flow rate: Temperature: Detector: Sample volume Runtime:Mobile phase: Flow rate: Temperature: Detector: Sample volume Runtime:

Chromatograph using a SIL-20AC Prominence auto samplerChromatograph using a SIL-20AC Prominence auto sampler

Bio-Rad Aminex® HPX-87H 300x7.8 mm 5 mM H₂SO₄ 0.6 mL-min'¹ 60 °CBio-Rad Aminex® HPX-87H 300x7.8 mm 5 mM H ₂ SO ₄ 0.6 mL-min ' ¹ 60 ° C

Shimadzu RID 10 A pL minutesShimadzu RID 10 A pL minutes

Retention time: 9.2 min. (glucose)Retention time: 9.2 minutes (glucose)

The HPLC analysis demonstrated 18% activity recovery versus the free enzyme in this assay. Cross-linked protein content of the CIP-silica mCLEA was 34 wt%. Glucose amylase activity was neither detected in the supernatant of the CLEA preparation nor in the washing water.The HPLC analysis demonstrated 18% activity recovery versus the free enzyme in this assay. Cross-linked protein content of the CIP-silica mCLEA was 34 wt%. Glucose amylase activity was neither detected in the supernatant of the CLEA preparation nor in the washing water.

Enzyme leakage studyEnzyme leakage study

In a 30 mL glass bottle were added 20 mL of maltodextrin (33% w/v in water) and 2 mL of CIP-silica mCLEA. The resulting suspension was shaken at 150 rpm at 32 °C for 10 days in a Stuart Orbital Incubator SI500. After this time the CIP-silica mCLEA was removed with a magnet (40 x 20 x 10 mm, neodymium magnet) and the supernatant was kept. The supernatant (0.2 mL) was added to 30 mL fresh maltodextrin (33% w/v in water) and shaken at 150 rpm at 32 °C for 3 hours in a Stuart Orbital Incubator SI500. After 1,2 and 3 h, a 0.1 mL sample was added to 0.9 mL water, centrifuged, filtered (0.2 pm Phenex™ syringe filter) and transferred to an HPLC vial and subjected to HPLC analysis for glucose determination. No additional glucose formation could be detected and therefore it was concluded that no enzyme was leaking from the CIP-silica mCLEA.In a 30 mL glass bottle were added 20 mL of maltodextrin (33% w / v in water) and 2 mL of CIP-silica mCLEA. The resulting suspension was shaking at 150 rpm at 32 ° C for 10 days in a Stuart Orbital Incubator SI500. After this time the CIP-silica mCLEA was removed with a magnet (40 x 20 x 10 mm, neodymium magnet) and the supernatant was kept. The supernatant (0.2 mL) was added to 30 mL of fresh maltodextrin (33% w / v in water) and shaken at 150 rpm at 32 ° C for 3 hours in a Stuart Orbital Incubator SI500. After 1.2 and 3 hours, a 0.1 mL sample was added to 0.9 mL of water, centrifuged, filtered (0.2 pm Phenex ™ syringe filter) and transferred to an HPLC vial and subjected to HPLC analysis for glucose determination. No additional glucose formation could be detected and therefore it was concluded that no enzyme was leaking from the CIP-silica mCLEA.

RecyclesRecycles

In a 100 ml_ conical flask were added 30 ml_ of maltodextrin (33% w/v in water) and 0.2 ml_ of CIP-silica mCLEA. The resulting suspension was shaken at 150 rpm at 32 °C for 48 hours in a Stuart Orbital Incubator SI500. Samples of 0.1 ml_ were taken after 24 and 48 h. The 0.1 ml_ sample was added to 0.9 ml_ water, centrifuged, filtered (0.2 pm Phenex™ syringe filter) and transferred to an HPLC vial and subjected to HPLC analysis. After 48 h, the CIP-silica mCLEA was removed with a magnet (40 x 20 x 10 mm, neodymium magnet), washed five times with 10 mL water and was then reused by adding another 30 mL maltodextrin mixture. This reuse constitutes the first recycle. In total, ten of such recycles were performed without loss of glucose amylase activity of the CIP-silica mCLEA, indicating excellent immobilised enzyme stability and highly effective magnetic separation.In a 100 ml conical flask were added 30 ml of or maltodextrin (33% w / v in water) and 0.2 ml of or CIP-silica mCLEA. The resulting suspension was shaking at 150 rpm at 32 ° C for 48 hours in a Stuart Orbital Incubator SI500. Samples of 0.1 ml were tasks after 24 and 48 h. The 0.1 ml sample was added to 0.9 ml of water, centrifuged, filtered (0.2 pm Phenex ™ syringe filter) and transferred to an HPLC vial and subjected to HPLC analysis. After 48 h, the CIP-silica mCLEA was removed with a magnet (40 x 20 x 10 mm, neodymium magnet), washed five times with 10 mL of water and then reused by adding another 30 mL of maltodextrin mixture. This is the first recycle. In total, such recycles were performed without loss or glucose amylase activity of the CIP-silica mCLEA, indicating excellent immobilized enzyme stability and highly effective magnetic separation.

Iron LeakageIron Leakage

Iron leakage experiments were carried out with an amount of CIP-silica mCLEA that contains 6 mg of CIP-silica particles. This amount was added to 18 mL of incubation mixture (1 wt% lactic acid of pH 3). Incubation of the CIP-silica mCLEA was performed by shaking the mixture for 72 hours at 32 °C at 150 rpm in a Stuart Orbital Incubator S1500. Absorption in the iron detection assay after dissolution of the total amount of CIP-silica particles was determined by dissolving the sample in concentrated HCI and subsequent dilution. The iron leakage (%) after incubation was determined by directly comparing the absorption of the incubation supernatant of the CIP-silica mCLEAto that of the absorption after total dissolution. Leakage of Fe(l 1,111) was <1%. Although the conditions chosen for incubation were extreme with respect to common enzyme applications, iron leakage from the CIP-silica mCLEA was negligible.Iron leakage experiments were carried out with an amount of CIP-silica mCLEA that contains 6 mg or CIP-silica particles. This amount was added to 18 mL or incubation mixture (1 wt% lactic acid or pH 3). Incubation of the CIP-silica mCLEA was performed by shaking the mixture for 72 hours at 32 ° C at 150 rpm in a Stuart Orbital Incubator S1500. Absorption in the iron detection assay after dissolution of the total amount of CIP-silica particles was determined by dissolving the sample in concentrated HCI and subsequent dilution. The iron leakage (%) after incubation was determined by directly comparing the absorption of the incubation supernatant of the CIP-silica mCLEAto that of the absorption after total dissolution. Leakage or Fe (1.111) was <1%. Although the conditions chosen for incubation were extreme with respect to common enzyme applications, iron leakage from the CIP-silica mCLEA was negligible.

Magnetic strengthMagnetic strength

The saturation magnetization of the dry glucose amylase CIP-silica mCLEA was measured at 161 emu«g'¹ (Am²/kg).The saturation magnetization of the dry glucose amylase CIP-silica mCLEA was measured at 161 emu ¹ (Am ² / kg).

Example 3. CIP-silica magnetizable CLEA (mCLEA) of glutaminase - low protein loadingExample 3. CIP-silica magnetizable CLEA (mCLEA) or glutaminase - low protein loading

SynthesisSynthesis

In a 500 mL beaker were added 100 mL of a glutaminase enzyme solution, which was prepared by dissolving 10 grams of glutaminase (Amano glutaminase SD-C100S) in 50 mM potassium phosphate buffer of pH 6 to a total volume of 100 mL. An amount of 20.05 g of CIP-silica particles (prepared according to Example 1) was added to the enzyme solution. The resulting suspension was stirred for 15 minutes with an overhead stirrer (Velp Scientifica ES overhead stirrer). Subsequently 400 mL of saturated ammonium sulfate solution was added to the suspension to precipitate the enzyme in the presence of the CIP-silica particles. To allow full precipitation of the enzyme, the suspension was stirred for 1 hour at room temperature. After precipitation, 20 mL of a 25 wt% glutaraldehyde solution were added and the mixture was cross-linked overnight. The CIP-silica mCLEA was removed with a hand held magnet (ERIEZ Mega Rare Earth Tube Magnet 150 mm, 10700 Gauss) and was washed 4 times with H₂O (500 mL end volume). Each wash was stirred for 15 minutes with the overhead stirrer. After this, the CIP-silica mCLEA was stored in 100 mL 50 mM potassium phosphate buffer pH 6. Total dry weight of the CIP mCLEA was 22.7 grams. Figure 3 shows a Microscope image of the CIP-silica mCLEA of glutaminase obtained from a Bresser microscope using a 40x magnification lens and Mikro CamLab software (Version 6.1.4.0).In a 500 mL beaker were added 100 mL or a glutaminase enzyme solution, which was prepared by dissolving 10 grams or glutaminase (Amano glutaminase SD-C100S) in 50 mM potassium phosphate buffer or pH 6 to a total volume or 100 mL. An amount of 20.05 g or CIP-silica particles (prepared according to Example 1) was added to the enzyme solution. The resulting suspension was stirred for 15 minutes with an overhead stirrer (Velp Scientifica ES overhead stirrer). Subsequently, 400 mL of saturated ammonium sulfate solution was added to the suspension to precipitate the enzyme in the presence of the CIP silica particles. To allow full precipitation of the enzyme, the suspension was stirred for 1 hour at room temperature. After precipitation, 20 mL or a 25 wt% glutaraldehyde solution were added and the mixture was cross-linked overnight. The CIP-silica mCLEA was removed with a hand-held magnet (ERIEZ Mega Rare Earth Tube Magnet 150 mm, 10700 Gauss) and was washed 4 times with H ₂ O (500 mL end volume). Each wash was stirred for 15 minutes with the overhead stirrer. After this, the CIP-silica mCLEA was stored in 100 mL of 50 mM potassium phosphate buffer pH 6. Total dry weight of the CIP mCLEA was 22.7 grams. Figure 3 shows a Microscope image of the CIP-silica mCLEA or glutaminase obtained from a Bresser microscope using a 40x magnification lens and Mikro CamLab software (Version 6.1.4.0).

Activity assayActivity assay

An L-glutamine substrate solution of 250 mM was prepared by dissolving 3.65 g of L-glutamine in 100 mL of 50 mM potassium phosphate buffer of pH 6. In a glass reaction vial, 10 mL of substrate solution were added. The vial was placed in a Stuart Orbital Incubator SI500 at a temperature of 50 °C. The vial was left to shake for 5 minutes to allow reaching the desired temperature. Hereafter, 10 pL of the enzyme solution or CIP-silica mCLEA suspension were added to start the catalytic conversion. After 5, 10 and 20 minutes, a sample of 500 μΙ_ was taken and placed in an Eppendorf tube. The sample was heated in a water bath at 95 °C for 10 minutes. Glutaminase-catalysed L-glutamic acid release from L-glutamine was determined using an L-glutamic acid assay kit (K-GLUT) from Megazyme International, Ireland.An L-glutamine substrate solution or 250 mM was prepared by dissolving 3.65 g or L-glutamine in 100 mL or 50 mM potassium phosphate buffer or pH 6. In a glass reaction vial, 10 mL or substrate solution were added. The vial was placed in a Stuart Orbital Incubator SI500 at a temperature of 50 ° C. The vial was left to shake for 5 minutes to allow reaching the desired temperature. Hereafter, 10 pL of the enzyme solution or CIP-silica mCLEA suspension were added to start the catalytic conversion. After 5, 10 and 20 minutes, a sample of 500 μΙ_ tasks and placed in an Eppendorf tube. The sample was heated in a water bath at 95 ° C for 10 minutes. Glutaminase-catalyzed L-glutamic acid release from L-glutamine was determined using an L-glutamic acid assay kit (K-GLUT) from Megazyme International, Ireland.

The glutaminase CIP-silica mCLEA showed 81% activity recovery versus the free enzyme in this assay. Cross-linked protein content of the CIPsilica mCLEA was 12 wt%. Glutaminase activity was neither detected in the supernatant of the CLEA preparation nor in the washing water.The glutaminase CIP-silica mCLEA showed 81% activity recovery versus the free enzyme in this assay. Cross-linked protein content of the CIPsilica mCLEA was 12 wt%. Glutaminase activity was neither detected in the supernatant of the CLEA preparation nor in the washing water.

Enzyme leakage study and CIP-silica mCLEA stabilityEnzyme leakage study and CIP-silica mCLEA stability

A part of the CIP-silica mCLEA suspension prepared as described above was incubated for 7 days at 50 °C, while shaking in a Stuart Orbital Incubator SI500. After incubation the supernatant was separated from the CIP mCLEA and the CIP mCLEA was washed three times with 50 mM potassium phosphate buffer of pH 6. Activity assays where then carried out on the supernatant and the CIP-silica mCLEA. No activity was detected in the supernatant and the glutaminase CIP-silica mCLEA had a residual activity of 98%, indicating that the CIP-silica mCLEA is stable at 50 °C and that no or negligible leakage takes place during 1 week of incubation at 50 °C.A part of the CIP-silica mCLEA suspension prepared as described above was incubated for 7 days at 50 ° C, while shaking in a Stuart Orbital Incubator SI500. After incubation the supernatant was separated from the CIP mCLEA and the CIP mCLEA was washed three times with 50 mM potassium phosphate buffer or pH 6. Activity assays where then carried out on the supernatant and the CIP-silica mCLEA. No activity was detected in the supernatant and the glutaminase CIP silica mCLEA had a residual activity of 98%, indicating that the CIP silica mCLEA is stable at 50 ° C and that no or negligible leakage takes place during 1 week of incubation at 50 ° C.

RecyclesRecycles

A 250 mM glutamine solution was prepared by dissolving 3.5 g of glutamine in 100 mL of 250 mM potassium phosphate buffer of pH 6. In a 250 mL round bottomed flask, 100 mL of the substrate solution were added. The solution was heated to 50 °C in an oil bath, while stirring with an overhead stirrer (Velp Scientifica ES overhead stirrer). Once the desired temperature of 50 °C was reached, 2.26 mL of glutaminase CIP-silica mCLEA suspension (513 mg of CIP-silica mCLEA) as prepared above was added to the reaction. After stirring for 15 hours a sample of 1 ml_ was taken from the reaction mixture. Glutaminase-catalysed L-glutamicacid release from L-glutamine was determined using an L-glutamic acid assay kit (K-GLUT) from Megazyme International Ireland. The glutaminase CIP-silica mCLEA was removed from the reaction mixture using a magnet (40 x 20 x 10 mm, neodymium magnet) and washed once with 50 mM potassium phosphate buffer pH 6, after which it was used in the next cycle. Five consecutive cycles were performed under the same conditions, all leading to a conversion of 68 ±2%, indicating a stable immobilized enzyme and an effective magnetic separation.A 250 mM glutamine solution was prepared by dissolving 3.5 g or glutamine in 100 mL or 250 mM potassium phosphate buffer or pH 6. In a 250 mL round bottomed flask, 100 mL of the substrate solution were added. The solution was heated to 50 ° C in an oil bath, while stirring with an overhead stirrer (Velp Scientifica ES overhead stirrer). Once the desired temperature of 50 ° C was reached, 2.26 mL of glutaminase CIP-silica mCLEA suspension (513 mg or CIP-silica mCLEA) as prepared above was added to the reaction. After stirring for 15 hours a sample or 1 ml was tasks from the reaction mixture. Glutaminase-catalyzed L-glutamicacid release from L-glutamine was determined using an L-glutamic acid assay kit (K-GLUT) from Megazyme International Ireland. The glutaminase CIP-silica mCLEA was removed from the reaction mixture using a magnet (40 x 20 x 10 mm, neodymium magnet) and washed once with 50 mM potassium phosphate buffer pH 6, after which it was used in the next cycle. Five consecutive cycles were performed under the same conditions, all leading to a conversion of 68 ± 2%, indicating a stable immobilized enzyme and an effective magnetic separation.

Magnetic strengthMagnetic strength

The saturation magnetization of the dry glutaminase CIP-silica mCLEA was measured at 208 emu-g'¹ (Am²/kg).The saturation magnetization of the dry glutaminase CIP-silica mCLEA was measured at 208 emu-g ' ¹ (Am ² / kg).

Example 4. APTES functionalized magnetite magnetic CLEA preparation glucose amylaseExample 4. APTES functionalized magnetite magnetic CLEA preparation glucose amylase

APTES functionalized magnetite was produced as described in WO2012023847A2. The magnetic particles were washed with (NH₄)₂SO₄ before use. In a 2 L plastic beaker, a mixture of 0.7 L of saturated ammonium sulphate and 0.124 kg of APTES functionalized magnetite was stirred with an overhead stirrer (Velp Scientifica ES overhead stirrer) at room temperature for 1 h. Thereafter, 0.3 L of glucose amylase (Zibo Guoao, Shandong, China) was added slowly and the mixture stirred at room temperature for 1 hour.APTES functionalized magnetite was produced as described in WO2012023847A2. The magnetic particles were washed with (NH ₄ ) ₂ SO ₄ before use. In a 2 L plastic beaker, a mixture of 0.7 L or saturated ammonium sulphate and 0.124 kg or APTES functionalized magnetite wax stirred with an overhead stirrer (Velp Scientifica ES overhead stirrer) at room temperature for 1 h. Thereafter, 0.3 L of glucose amylase (Zibo Guoao, Shandong, China) was added slowly and the mixture stirred at room temperature for 1 hour.

After the addition of 0.153 L of 25 wt% glutaraldehyde, the reaction mixture was stirred at room temperature for 18 h. The resulting mCLEA was removed with a hand held magnet (ERIEZ Mega Rare Earth Tube Magnet 150 mm,After the addition of 0.153 L or 25 wt% glutaraldehyde, the reaction mixture was stirred at room temperature for 18 h. The resulting mCLEA was removed with a hand held magnet (ERIEZ Mega Rare Earth Tube Magnet 150 mm,

10700 Gauss) and washed five times with 4.5 L of water. The final mCLEA was suspended in 1 L of water. The total dry weight of the APTES functionalized magnetite mCLEA was 245 grams.10700 Gauss) and washed five times with 4.5 L of water. The final mCLEA was suspended in 1 L of water. The total dry weight of the APTES functionalized magnetite mCLEA was 245 grams.

Activity assayActivity assay

Following the activity assay and HPLC analysis as described in Example 2, it was found that the glucose amylase APTES functionalized magnetite mCLEA showed 20% activity recovery versus the free enzyme in this assay. Cross-linked protein content of the APTES functionalized magnetite mCLEA was approximately 50 wt%. Glucose amylase activity was neither detected in the supernatant of the CLEA preparation nor in the washing water.Following the activity assay and HPLC analysis as described in Example 2, it was found that the glucose amylase APTES functionalized magnetite mCLEA showed 20% activity recovery versus the free enzyme in this assay. Cross-linked protein content or the APTES functionalized magnetite mCLEA was approximately 50 wt%. Glucose amylase activity was neither detected in the supernatant of the CLEA preparation nor in the washing water.

Iron LeakageIron Leakage

Iron leakage experiments were carried out with an amount of APTES functionalized magnetite mCLEA that contains 6 mg of APTES functionalized magnetite in 18 mL of incubation mixture (1 wt% lactic acid of pH 3). Incubation of the CIP-silica mCLEA was performed by shaking for 72 hours at 32 °C and 150 rpm in a Stuart Orbital Incubator S1500. Absorption in the iron detection assay after dissolution of the total amount of APTES coated magnetite was determined by dissolving the sample in concentrated HCI and subsequent dilution. The iron leakage (wt%) after incubation was determined by directly comparing the absorption of the incubation supernatant of the APTES functionalized magnetite mCLEA to that of the absorption after total dissolution. After 72 h of incubation in 1% lactic acid of pH 3, 35 wt% of the magnetite in the CLEA had dissolved.Iron leakage experiments were carried out with an amount of APTES functionalized magnetite mCLEA that contains 6 mg or APTES functionalized magnetite in 18 mL or incubation mixture (1 wt% lactic acid or pH 3). Incubation of the CIP-silica mCLEA was performed by shaking for 72 hours at 32 ° C and 150 rpm in a Stuart Orbital Incubator S1500. Absorption in the iron detection assay after dissolution of the total amount of APTES coated magnetite was determined by dissolving the sample in concentrated HCI and subsequent dilution. The iron leakage (wt%) after incubation was determined by comparing directly the absorption of the incubation supernatant of the APTES functionalized magnetite mCLEA to that of the absorption after total dissolution. After 72 h or incubation in 1% lactic acid or pH 3, 35 wt% of the magnetite in the CLEA had dissolved.

Magnetic strengthMagnetic strength

The saturatien magnetizatian gf the APTES functianalized magnetite was measured at 37.3 emu«g'¹ (Am²/kg) and dry glucese amylase APTES functianalized magnetite mCLEA was measured at 16.1 emu«g'¹ (Am²/kg)The saturation magnetizatian gf the APTES functianalized magnetite was measured at 37.3 emu ¹ (Am ² / kg) and dry glucese amylase APTES functianalized magnetite mCLEA was measured at 16.1 emu g ¹ (Am ² / kg)

Claims (15)

ConclusiesConclusions 1. Een gecrosslinkt enymaggregaat (CLEA) omvattende magnetiseerbare deeltjes, met het kenmerk dat de magnetiseerbare deeltjes deeltjes omvatten van een zerovalent metaal gekozen uit de groep van ijzer, nikkel, kobalt en alliages daarvan, welke deeltjes volledig zijn bedekt met een coating.A crosslinked enymaggregate (CLEA) comprising magnetizable particles, characterized in that the magnetizable particles comprise particles of a zerovalent metal selected from the group of iron, nickel, cobalt and alloys thereof, which particles are completely covered with a coating. 2. Een CLEA volgens conclusie 1, omvattende een meervoud van CLEA-deeltjes met een gemiddelde diameter van 1 pm of groter, in het bijzonder in het bereik van 2-100 pm.A CLEA according to claim 1, comprising a plurality of CLEA particles with an average diameter of 1 µm or larger, in particular in the range of 2-100 µm. 3. Een CLEA volgens conclusie 1 of 2, waarin de gemiddelde diameter van de magnetiseerbare deeltjes in het bereik ligt van 0.010-20 pm, in het bijzonder in het bereik van 1 -15 pm.A CLEA according to claim 1 or 2, wherein the average diameter of the magnetizable particles is in the range of 0.010-20 µm, in particular in the range of 1 -15 µm. 4. Een CLEA volgens een der conclusies 1 - 3, waarin de magnetiseerbare deeltjes een sferische vorm hebben, of afwijken van een sferische vorm in dat hun grootste dimensie tot twee keer groter is dan hun kleinste dimensie.A CLEA according to any one of claims 1 to 3, wherein the magnetizable particles have a spherical shape, or deviate from a spherical shape in that their largest dimension is up to twice as large as their smallest dimension. 5. Een CLEA volgens een der conclusies 1 - 4, waarin de magnetiseerbare deeltjes afgeleid zijn van carbonylijzerpoeder (CIP).A CLEA according to any one of claims 1 to 4, wherein the magnetizable particles are derived from carbonyl iron powder (CIP). 6. Een CLEA volgens een der conclusies 1 - 5, waarin het gemiddelde aantal magnetiseerbare deeltjes in een CLEA-deeltje in het bereik ligt van 1-100 en/of waarin het gehalte van magnetiseerbare deeltjes in het bereik ligt van 50-95 gew%.A CLEA according to any one of claims 1 to 5, wherein the average number of magnetizable particles in a CLEA particle is in the range of 1-100 and / or wherein the content of magnetizable particles is in the range of 50-95 wt% . 7. Een CLEA volgens een der conclusies 1 - 6, waarin de coating gefunctionaliseerd is.A CLEA according to any one of claims 1 to 6, wherein the coating is functionalized. 8. Een CLEA volgens een der conclusies 1 - 7, waarin de coating silica omvat.A CLEA according to any of claims 1 to 7, wherein the coating comprises silica. 9. Een CLEA volgens een der conclusies 1 - 8, waarin de coating een dikte heeft in het bereik van 10-500 nm.A CLEA according to any one of claims 1 to 8, wherein the coating has a thickness in the range of 10-500 nm. 10. Een CLEA volgens een der conclusies 1 - 9, waarin de verzadigingsmagnetisatie van de CLEA in het bereik ligt van 80-200 emu-g'¹.A CLEA according to any one of claims 1 to 9, wherein the saturation magnetization of the CLEA is in the range of 80-200 emu- ¹ . 11. Een CLEA volgens een der conclusies 1-10, waarin het enzym gekozen is uit de groep van hydrolases, zoals esterases, proteases, amidases, cellulases, nitrilases, xylanases en glycosylases; lyases, zoals hydroxynitrile lyases en aldolases; oxidoreductases zoals alcohol oxidases, peroxidases, ketoreductases en imine reductases; en transferases, zoals transaminases.A CLEA according to any one of claims 1-10, wherein the enzyme is selected from the group of hydrolases such as esterases, proteases, amidases, cellulases, nitrilases, xylanases and glycosylases; lyases, such as hydroxynitrile lyases and aldolases; oxidoreductases such as alcohol oxidases, peroxidases, ketoreductases and imine reductases; and transferases, such as transaminases. 12. Werkwijze voor het vervaardigen van een CLEA volgens een der conclusies 1-11, omvattendeA method of manufacturing a CLEA according to any of claims 1-11, comprising - het voorzien in een oplossing van een enzym; dan- providing an enzyme solution; then - het neerslaan van het enzym om een enzymaggregaat te vormen; dan- precipitating the enzyme to form an enzyme aggregate; then - het crosslinken van het enzymaggregaat met een crosslinkend agens in de aanwezigheid van magnetiseerbare deeltjes omvattende een deeltje van van een zerovalent metaal gekozen uit de groep van ijzer, nikkel, kobalt en alliages daarvan, welk deeltje volledig is bedekt met een coating.crosslinking the enzyme aggregate with a crosslinking agent in the presence of magnetizable particles comprising a particle of a zerovalent metal selected from the group of iron, nickel, cobalt and alloys thereof, which particle is completely covered with a coating. 13. Werkwijze volgens conclusie 12, waarin het crosslinkend agens gekozen is uit de groep van glutaaraldehyde en aldehyde crosslinkers afgeleid van polysaccharides.The method of claim 12, wherein the crosslinking agent is selected from the group of glutaraldehyde and aldehyde crosslinkers derived from polysaccharides. 14. CLEA verkrijgbaar door de werkwijze volgens conclusie 12 of 13.CLEA obtainable by the method according to claim 12 or 13. 15. Werkwijze omvattende het gebruik van een CLEA volgens conclusies 1-11 en 14 voor een katalytische omzetting, waarin de CLEA gescheiden wordt van het reactiemedium door de CLEA te verzamelen met behulp van een extern magnetisch veld, in het bijzonder met behulp van een permanente magneet.Method comprising the use of a CLEA according to claims 1-11 and 14 for a catalytic conversion, wherein the CLEA is separated from the reaction medium by collecting the CLEA with the aid of an external magnetic field, in particular with the aid of a permanent magnet. Figure 1Figure 1 Figure 2Figure 2 20 pm20 pm Figure 3Figure 3