This application claims the benefit of the European Patent Application EP20382797.7 filed on September 8, 2020.

Technical Field

The present invention relates to the field of chemistry, more particularly to immobilization of enzymes. In particular, the invention relates to composite beads containing immobilized enzymes, and to a process for their preparation. It also relates to the use of the immobilized enzyme-containing composite beads for their use in industrial processes, particularly in the production of pulp for the manufacture of paper, more particularly in the process of paper deinking.

Background Art

In the pulping and paper processes enzymes are used to improve the production of the right desired paper. Current methods of producing virgin pulp utilize large manufacturing facilities (pulp mills) that produce pulp for multiple end users. Pulp mills process pulp from softwood, hardwood logs, chips or non-wood sources into fibers that are used in the manufacture of paper. The use of enzymes in the process manufacturing is currently applied because of the strength enhancement and drainage improvement of the obtained paper. However, the main drawback of this use is the loss of the enzymes due to their solubility in the aqueous media during this process.

One of the most challenging areas in improvement of the process efficiency is related to recovery of enzymes applied in the process of paper deinking. REFINASE® commercial products are currently used for this purpose. REFINASE® products contains enzyme mixtures with at least one of the enzymes being a cellulase, endo-glucanase, cellobiohydrolase, hemicellulase, mannanase, xylanase, pectinase, pectin esterase, pectin and pectate lyase, gamanase, esterase, laccase or lipase. The main drawback of these products is their water-solubility, which makes impossible their recovery from the process tanks and generates a huge amount of waste.

Thus, there is still a need of providing further solutions for providing enzyme products that can be used in the recycling paper industry which overcome the problems of the prior art.

Summary of Invention The present inventors have developed a new composite bead comprising an immobilized enzyme that allows reducing enzyme consumption by up to a 90% through the attachment of the enzymes to a composite polymer bead making them water-insoluble. In particular, the inventors have found that by immobilizing an enzyme in composite beads comprising a polymer matrix and an adsorption material, stable water-insoluble beads having a size in the range from 200 pm to 1 cm can be prepared, namely no leaching of the enzyme is produced when the beads are added to an aqueous solution, such as in pulping and paper processes. Furthermore, the water-insoluble immobilized enzyme-containing composite beads are easily recoverable and can be reused since they maintain their activity.

Therefore, a first aspect of the present disclosure relates to an enzymatic bead that is a porous water-insoluble immobilized enzyme-containing composite bead comprising:

- a polymer matrix;

- an adsorption material; and

- an enzyme selected from the group consisting of cellulase, endo-glucanase, cellobiohydrolase, hemicellulase, mannanase, xylanase, pectinase, pectin esterase, pectin and pectate lyase, gamanase, esterase, laccase, lipase, and mixtures thereof, the composite bead having a surface and pores containing the adsorption material, wherein the enzyme is immobilized by the absorption material on the surface of the bead or by the absorption material on the surface of the bead and within the pores, and wherein the bead has an average particle diameter from 200 pm to 1 cm.

A second aspect of the present disclosure relates to a process for the preparation of the enzymatic bead that is a porous water-insoluble immobilized enzyme-containing composite bead as defined herein above, the process comprising the following steps: a) providing a polymeric solution of a polymer in a suitable solvent; b) adding to the polymer solution an adsorption material in order to obtain a polymeric mixture; c) dropwise dosing the polymeric mixture into a non-solvent or into a solvent containing a chelating agent in order to obtain composite beads having an average diameter in the range from 200 pm to 1 cm; d) adding the composite beads to an enzyme aqueous solution in order to obtain composite beads comprising immobilized enzyme on the surface of the beads; e) isolating the immobilized enzyme-containing composite beads; or, alternatively, comprising the following steps: a) adding an adsorption material to an enzyme aqueous solution in order to obtain an enzyme immobilized on an adsorption material; b) providing a polymeric solution of a polymer in a suitable solvent and adding thereto the enzyme immobilized on an adsorption material in order to obtain a polymeric mixture; c) dropwise dosing the polymeric mixture into a non-solvent or into a solvent containing a chelating agent in order to obtain immobilized enzyme-containing composite beads having a diameter in the range from 200 pm to 1 cm; d) isolating the immobilized enzyme-containing composite beads.

The immobilized enzyme-containing composite beads of the present disclosure can be used in a number of applications, particularly in the paper industry, more particularly in the production of pulp for the manufacture of paper, and even more particularly in the process of paper deinking. They can be used as such or forming part of a composition comprising further components. Thus, it also forms part of the invention a composition comprising the immobilized enzyme-containing composite beads as defined herein, and one or more excipients or carriers.

Examples of excipients or carriers include, without being limited to, acetate buffer, phosphate buffer, citrate buffer, or any other buffer solution in which the immobilized enzyme is stable. The pH of the buffer is related to the optimum pH in which the enzyme is recommended to be stored.

A further aspect of the invention relates to the use of the enzymatic beads that are porous water-insoluble immobilized enzyme-containing composite beads as defined herein or a composition comprising it for the production of pulp for the manufacture of paper products.

Brief Description of Drawings

FIG. 1 shows optical micrographs of polysulfone beads with a 20 wt% activated carbon and of polysulfone beads without activated carbon.

FIG. 2 shows ESEM representative cross section micrographs of polysulfone/active carbon beads with a 20% of activated carbon and without activated carbon.

FIG. 3 shows the ESEM representative cross section micrographs of polysulfone/active carbon beads with a different carbon content (0 wt%, 2.4 wt%, 4.8 wt%, 9.1 wt%, and 20 wt% active carbon relative to the weight amount of polymer (polysulfone), respectively).

FIG. 4 shows the ESEM representative surface micrographs of polysulfone/active carbon beads with different carbon content (0 wt%, 2.4 wt%, 4.8 wt%, 9.1 wt%, and 20 wt% active carbon relative to the weight amount of polymer (polysulfone), respectively).

FIG. 5 shows optical micrographs of alginate/active carbon beads with a 20% of activated carbon.

FIG. 6 shows the enzymatic bead activity of: A) polysulfone beads of Example 4 (see Examples 36 to 40); and B) alginate beads of Example 35 (see Examples 41-45).

FIG. 7 shows the magnetic properties of polysulfone beads containing activated carbon and iron magnetic microparticles (iron powder).

Detailed description of the invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply throughout the description and claims.

The term "adsorption" as used herein refers to the adhesion of molecules (the adsorbate; i.e. , the enzymes, in the context of the present disclosure) to the surface of an adsorbent material. It is a surface phenomenon consequence of surface energy wherein a film of the adsorbate is formed on the surface of the adsorbent material.

The term “bead(s)” as used herein is intended to include spherical or spheroid (of approximately spherical shape) particles. In the present disclosure, the size range may cover from 200 pm to 1 cm in diameter. As can be seen from the images (FIGs. 2-4) the internal structure of the beads is essentially uniform.

The terms "enzymatic bead", " immobilized enzyme-containing composite bead" and "porous water-insoluble immobilized enzyme-containing composite bead" are intended to indicate the same and are used interchangeably.

As used herein, the term "essentially uniform" is taken to mean a dispersion of the adsorbent material (either containing an immobilized adsorbed enzyme or not) in the polymer matrix in a manner that exhibits minimal perceptible clumping or agglomeration.

The term “matrix” is a well-known term in the art and generally refers to a solid, semi-solid, or undissolved or still undissolved material that imparts structure and volume to the composition. In the context of the present disclosure, the polymer matrix is a polymer capable of providing support to the absorption material and to the enzyme immobilized by the absorption material.

The term “polymeric solution” as used herein refers to a solution comprising one or more polymers dissolved in a liquid solvent.

The term 'non-solvent' as used herein, when referred to the dropwise dosing of a polymeric mixture in a non-solvent, refers to a solvent that does not dissolve the polymer, that is a solvent in which the polymer precipitation occurs.

The term "average", as used herein, refers to the arithmetic mean, that is the sum of the values divided by the number of values being averaged.

The term "about" as used herein refers to a range of values ± 10% of a specified value. For example, the expression "about 10" includes ± 10% of 10, i.e. from 9 to 11.

The expression “one or more” as used herein referred to excipients or carriers, means that there can be one or more of such entities, in particular 1 , 2, 3 or 4.

As mentioned above, a first aspect of the present disclosure relates to an enzymatic bead having an average particle diameter from 200 pm to 1 cm, the enzymatic bead comprising a polymer matrix, an adsorption material, and an enzyme as defined above immobilized by the absorption material, wherein the enzyme is immobilized by the absorption material on the surface of the bead or on the surface of the bead and within the pores.

The enzyme is selected from the group consisting of cellulase, endo-glucanase, cellobiohydrolase, hemicellulase, mannanase, xylanase, pectinase, pectin esterase, pectin and pectate lyase, gamanase, esterase, laccase, and lipase, and mixtures thereof. In an example, the enzyme is a mixture comprising arabanase, cellulase, p-glucanase, hemicellulase, and xylanase.

The average particle diameter can be determined in several different ways, for example by using a digital calliper or by means of a scanning electron microscope (SEM)

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the enzyme is adsorbed by the absorption material within the pores of the composite bead and on the surface of the bead. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the enzyme is adsorbed by the absorption material on the surface of the bead.

In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the absorption material is selected from the group consisting of activated carbon, activated silica, celite, zeolite, cordierite ceramic, crosslinked phenol-formaldehyde polycondensate resin (duolite A568), and mixtures thereof. Particularly, the absorption material is activated carbon.

In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the weight amount of adsorption material is from 1 to 30 wt%, or from 2 to 25 wt%, or from 2 to 20 wt%, such as 1 wt%, 2 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, or 30 wt%, with respect the weight amount of polymer.

In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the amount of enzyme is from 0.5 to 50 mg/gbeads, or from 0.5 to 20 mg/g _b eads, or from 0.5 to 10 mg/g _b eads.

Examples of polymer matrices include, without being limited to, polysulfone, polyethersulfone, polyvinylidene difluoride, polystyrene, polyacrylonitrile, and mixtures thereof; alginate, carrageenan, gellan gum, carboxyl methyl cellulose, hyaluronic acid, and mixtures thereof.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polymer is selected from the group consisting of polysulfone, polyethersulfone, polyvinylidene difluoride, polystyrene, polyacrylonitrile, and mixtures thereof, particularly, polysulfone. More particularly, polysulfone has an average molecular weight from 10 to 90 kg/mol, or from 15 to 50 kg/mol, or from 16 to 37 kg/mol. In a more particular embodiment, the amount of enzyme is from 0.5 to 10 mg/g _be ads.

In an example of the porous water-insoluble immobilized enzyme-containing composite bead of the present disclosure, the enzyme is adsorbed by the absorption material within the pores of the composite bead and on the surface of the bead; the polymer is polysulfone; the absorption material is activated carbon and it is in a weight amount from 9 to 25 wt%, particularly from 9 to 20 wt%, more particularly of about 20 wt%, with respect the weight amount of polymer; and the amount of enzyme is from about 0.5 to about 10 mg/g _b ead, such as about 7.5 mg/g _be ad. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polymer is selected from the group consisting of alginate, carrageenan, gellan gum, carboxyl methyl cellulose, hyaluronic acid, and mixtures thereof, particularly, alginate. More particularly, alginate has an average molecular weight from 10 to 500 kg/mol, or from 60 to 380 kg/mol, or from 100 to 300 kg/mol. In a more particular embodiment, the amount of enzyme is from 20 to 50 mg/g _b eads, or from 30 to 45 mg/g _b eads.

In an example of the porous water-insoluble immobilized enzyme-containing composite bead of the present disclosure, the enzyme is adsorbed by the absorption material within the pores of the composite bead and on the surface of the bead; the polymer is alginate; the absorption material is activated carbon and it is in an amount from 10 to 20 wt%, particularly 20 wt%, with respect the weight amount of polymer, and the amount of enzyme is from about 40 to about 50 mg/g _bea d, such as about 45 wt%.

In another embodiment, the porous water-insoluble immobilized enzyme-containing composite bead of the present disclosure is capable of liberating an amount of reducing sugars from about 400 to about 70 mg/L, or from 375 to about 70 mg/L, or from about 200 to about 70 mg/L, or from about 150 to about 70 mg/L or from about 100 to about 70 mg/L, during cellulose hydrolysis.

In a particular embodiment, optionally in combination with one or more features of the various embodiments described above, the polymer is selected from the group consisting of polysulfone, polyethersulfone, polyvinylidene difluoride, polystyrene, polyacrylonitrile, and mixtures thereof, and the porous water-insoluble immobilized enzyme-containing composite bead is capable of maintaining an enzymatic activity without significantly decreasing from the second cycle of use onwards. In an example, the porous waterinsoluble immobilized enzyme-containing composite bead is capable of liberating an amount of reducing sugars from about 200 to about 70 mg/L, or from about 150 to about 70 mg/L, or from about 100 to about 70 mg/L, during cellulose hydrolysis.

In a particular embodiment, optionally in combination with one or more features of the various embodiments described above, the polymer is alginate and the porous waterinsoluble immobilized enzyme-containing composite bead is capable of maintaining an enzymatic activity without significantly decreasing from the second cycle of use onwards. In another particular embodiment, the polymer is alginate and the porous water-insoluble immobilized enzyme-containing composite bead is capable of liberating an amount of reducing sugars from about 400 to about 70 mg/L, or from about 375 to about 70 mg/L, or from about 200 to about 70 mg/L, or from about 150 to about 70 mg/L or from about 100 to about 70 mg/L, during cellulose hydrolysis.

In another embodiment, optionally in combination with one or more features of the various embodiments described above, the enzymatic bead defined herein above further comprised magnetic microparticles such as having a particle size from 1 pm to 900 pm, particularly from < 400 pm (< 35 mesh), or magnetic nanoparticles such as having a particle size from 1 nm to 999 nm.

Examples of magnetic microparticles or nanoparticles include, without being limited to, iron magnetic microparticles or nanoparticles such as of metal iron, iron oxides such as magnetite (Fe ₃ O4, also referred to as FeO x Fe2O ₃ ) and maghemite (y-Fe2C>3), barium ferrite (BaFe2C>4), cobalt ferrite (CoFe2C>4), nickel ferrite (NiFe2C>4), manganese ferrite (MnFe2C>4), strontium ferrite SrFe^O , and zinc ferrite (ZnFe2C>4) with magnetic properties. Particularly the magnetic particles are iron magnetic microparticles such as of metal iron, or iron magnetic nanoparticles such as of iron oxide, magnetite or maghemite (y-Fe ₂ O ₃ ).

As an instance, iron magnetic nanoparticles of Fe2O ₃ can be prepared and characterized following the procedures of Massart R. “Preparation of aqueous magnetic liquids in alkaline and acidic media”, IEEE Trans Magn. 1981 ; 17(2): 1247-8; and Bee A, Massart R, Neveu S. “Synthesis of very fine maghemite particles”, J Magn Magn Mater. 1995; 149; 6- 9.

Magnetic microparticles and nanoparticles are commercially available. Some examples of iron magnetic microparticle are IOTHINKS-POWDER, particle size < 400 pm (< 35 mesh); Hawa magnetic strontium ferrite microparticles (https://www.magnetische- compounds.com/en/ferritpulver.php), particle size between 0.92 and 2 pm; Advanced metal (https://www.amtmetaltech.eom/StandardProducts/mim.html7http s://www.amtmetaltech.co m/StandardProducts/mim.html&gclid=CjwKCAjwOqOIBhBhEiwAyv Vcf6DIFUFkltlZFX_BR2 KzZY-MuZVNocxWpZqlRN7h3AEmeL-YCVMJSBoCUG8QAvD_BwE); and MAGRON magnetic powders with different particles size provided by MAGRON CO., LTD (https://bf37347f-33b0-4de9-8f44-

51 adc9d780ca.filesusr.com/ugd/8d6a0e_6acfe848f82b473eb6106185a c529a5d.pdf).

In an embodiment, optionally in combination with one or more features of the various embodiments described above, magnetic microparticles or nanoparticles are in an amount from 0.1 to 30 wt% with respect the weight amount of polymer. Advantageously, when the immobilized enzyme-containing composite beads comprise iron magnetic microparticles or nanoparticles, they can be easily and selectively recovered from the reaction medium using a magnet and, subsequently, reused in more catalytic cycles.

As mentioned above, the porous water-insoluble composite bead of the present disclosure can be prepared by two alternative processes. In a first option, the process comprises the following steps: a) providing a polymeric solution of a polymer in a suitable solvent; b) adding to the polymer solution an adsorption material and, optionally, iron magnetic microparticles or nanoparticles in order to obtain a polymeric mixture; c) dropwise dosing the polymeric mixture into a non-solvent or into a solvent containing a chelating agent in order to obtain composite beads having an average diameter in the range from 200 pm to 1 cm; d) adding the composite beads to an enzyme aqueous solution in order to obtain composite beads comprising immobilized enzyme on the surface of the beads; e) isolating the immobilized enzyme-containing composite beads.

The composite beads obtained in step c) or the immobilized enzyme-containing composite beads can be isolated according to methods known in the art, including, without being limited to, filtration, particularly filtration under vacuum. Then, the filtered beads can be washed with water and dried. When the immobilized enzyme-containing composite beads comprise iron magnetic microparticles or nanoparticles, they can also be recovered using a magnet.

Alternatively, the porous water-insoluble composite bead of the present disclosure can be prepared by a process comprising the following steps: a) adding an adsorption material to an enzyme aqueous solution in order to obtain an enzyme immobilized on an adsorption material; b) providing a polymeric solution of a polymer in a suitable solvent and adding thereto the enzyme immobilized on an adsorption material in order to obtain a polymeric mixture; c) dropwise dosing the polymeric mixture into a non-solvent or into a solvent containing a chelating agent and, optionally, iron magnetic microparticles or nanoparticles in order to obtain immobilized enzyme-containing composite beads having a diameter in the range from 200 pm to 1 cm; d) isolating the immobilized enzyme-containing composite beads.

The enzyme immobilized on an adsorption material obtained in step a) and the immobilized enzyme-containing composite beads can be isolated as mentioned above.

Depending on the solvent wherein the polymer is dissolved, the non-solvent could be water, an organic solvent or a mixture of both. In case polymer precipitation does not occur, a chelating agent is required to cross-link the polymer. Chelating agents include multivalent metallic cations, particularly divalent cations, in the form of a salt. Non-limiting examples of chelating agents include salts of multivalent metallic cations, particularly salts of divalent cations such as salts of calcium (Ca ²⁺ ), magnesium (Mg ²⁺ ), copper (Cu ²⁺ ), barium (Ba ²⁺ ), beryllium (Be ²⁺ ), chromium (Cr ²⁺ ), cobalt (Co ²⁺ ), iron (Fe ²⁺ ), nickel (Ni ²⁺ ), and zinc (Zn ²⁺ ).

In an example, when the polymer is a water-soluble polymer such as alginate, carrageenan, gellan gum, carboxyl methyl cellulose, hyaluronic acid, or a mixture thereof, precipitation of the beads is carried out by coacervation. In a particular example, in order to obtain composite alginate beads, dropwise dosing of the alginate mixture can be carried out into a water bath containing CaCh.

In another example, when the polymer is soluble in an organic solvent and non-soluble in water, such as polysulfone, beads are obtained by phase inversion precipitation. Thus, in a particular example, in order to obtain the composite polysulfone beads, dropwise dosing of the polymeric mixture is carried out into water. Dropwise dosing can be carried out with a dropping device.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the process of the present disclosure dropwise dosing is carried out with a dropping device having an exit tube or a nozzle having an internal diameter from 0.2 to 1 .5 mm and at a rate from 0.05 to 1 mL/min, such as 0.4 mL/min. In an example, the distance between the dropping device and the surface of water in the coagulation bath is from 2 to 10 cm, such as 3.5 cm.

The dropping device comprises a liquid reservoir and an exit tube or a nozzle downward the liquid reservoir so that liquid from the reservoir flows through the exit tube, such as a needle, or the nozzle. An example of dropping device is a syringe pump equipped with a syringe and a needle.

In an example, the dropping device is capable of discharging uniform droplets of liquid, particularly at equal drop intervals.

In the processes above, once the immobilized enzyme-containing composite beads have been isolated, they are stored in an aqueous buffer solution at a pH adjusted as required for the stability of the specific enzyme or mixture of enzymes.

The porous water-insoluble immobilized enzyme-containing composite bead of the present disclosure may be defined by its preparation process. Thus, it also forms part of the invention a porous water-insoluble immobilized enzyme-containing composite bead comprising:

- a polymer matrix;

- an adsorption material;

- optionally, iron magnetic microparticles or nanoparticles; and

- an enzyme is selected from the group consisting of cellulase, endo-glucanase, cellobiohydrolase, hemicellulase, mannanase, xylanase, pectinase, pectin esterase, pectin and pectate lyase, gamanase, esterase, laccase, and lipase, and mixtures thereof, the composite bead having a surface and pores containing the adsorption material, wherein the enzyme is immobilized by the absorption material on the surface of the bead or by the absorption material on the surface of the bead and within the pores, and wherein the bead has an average particle diameter from 200 pm to 1 cm; wherein the composite bead is obtainable by the process defined herein above.

For the purposes of the invention, the expressions “obtainable”, “obtained” and similar equivalent expressions are used interchangeably and, in any case, the expression “obtainable” encompasses the expression “obtained”.

All the embodiments disclosed herein for the composite bead and its preparation process apply also for the composite bead obtainable by this process.

As mentioned above, the porous water-insoluble immobilized enzyme-containing composite beads of the present disclosure can be used for the production of pulp for the manufacture of paper products.

The enzymes are typically used in combination, although this is not essential and pulp may only be treated with one type of enzyme or one class of enzyme such as cellulases. The enzyme dosage depends on the specific enzyme and the other treatment conditions, in particular pulp consistency and temperature. The effective amount of enzyme can be determined by a person skilled in the art and is such that results in increased fiber drainage of the pulp relative to non-enzyme treated pulp and/or which enhances paper sheet strength of the paper made using the enzyme treated pulp. Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

Polysulfone (ref: 428302; average Mw -35,000 by LS, average Mn -16,000 by MO) was provided by Sigma-Aldrich. Sodium alginate (ref: 177772500) was provided by Acros Organics. Activated carbon Norit (prepared from wood, ref: 97876) was provided by Sigma-Aldrich and activated carbon Fluka (decolorizing, ref: 161551) was supplied by Honeywell. The enzymes used for the immobilization experiments were Viscozyme proteins (Viscozyme® L, Sigma-Aldrich, ref: V2010; a multi-enzyme complex containing a wide range of carbohydrases, including arabanase, cellulase, p-glucanase, hemicellulase, and xylanase). N,N-Dimethylformamide (DMF, ref: D/3840/17) was provided by Fisher Scientific. Carboxymethyl cellulose sodium salt (CMC, ref: 21902) was provided by Sigma. Calcium chloride (ref: C1016) was provided by Sigma-Aldrich.

Instruments used

- Syringe pump- kdScientific Model KDS-410-CE (or equivalent);

- Syringe - Henke-Sass Wolf, Norm-Ject syringe, ref: 4100-00V0;

- Needle - B Braun, Sterican needle, ref: 4665643;

- UV-Vis spectrophotometer - Shimadzu Model UV-1800 (or equivalent);

- Hot plate magnetic stirrer - 1 KA™ model RCT basic with electronic contact thermometer ETS-D5 (or equivalent).

Test procedure

1. Syringe pump- kdScientific Model KDS-410-CE (or equivalent)

1.1. Turn on the kdScientific syringe pump.

1.2. Loosen the Knobs for syringe positioning and rotate the Pusher Block Knob until it snaps into place. This will disengage the Pusher Block and allow the user to position the block to accept the syringe.

1.3. Fill the syringe with polymeric solution and place the needle on the syringe. 1.4. Place the syringe in the holder, making sure the flange is resting against the Syringe Holder Block and the plunger is against the Pusher Block. Rotate the Adjustable Clamp until it rests on the Syringe barrel and tighten the Knobs. Engage the Pusher Block by pulling the Knob out and rotate the knob 90°clockwise.

1.5. Set the volume and the rate of injection on the display for 3 mL and 0.4 mL/min respectively. Then select the “Inf” mode on the display.

1.6. The equipment kdScientific Model KDS-410-CE is ready to use, so press the button “run”.

2. UV-Vis spectrophotometer - Shimadzu, Model UV-1800 (or equivalent)

2.1. Turn on the UV-Vis spectrophotometer.

2.2. Wait until the equipment will be ready (all parameters on the display must show OK)

2.3. On the display, press the button PC mode.

2.4. Load the desired method (/.e., Bradford method A _m ax = 595 nm, Reducing Sugar DNS method Amax ^— 540 nm).

2.5. Zero the instrument with two blank samples and then, replace the sample holder with subsequent standard samples to prepare the calibration curve. Afterwards measure absorbance of the samples.

3. Hot plate magnetic stirrer - 1 KA™ model RCT basic with electronic contact thermometer ETS-D5 (or equivalent)

3.1. Turn on the hot plate magnetic stirrer

3.2. Set the desired temperature on the electronic contact thermometer ETS-D5 (/.e., 50 °C)

3.3. On the display, set the desired stirring rate (/.e., =350 rpm)

Preparation of enzymatic beads (Examples 1-35 and Comparative Example 1)

The composition of polymeric solutions of Comparative Example 1 and of Examples 1 to 6 is summarized in Table 1 below.

Table 1. Composition of different polymeric solutions

Examples 1 , 2, 3, 4 and 5 were prepared using Activated carbon Norit a Activated carbon Fluka, decolorizing b Activated carbon Norit with enzymes immobilized

Example 1 - Enzyme immobilization on bead surface

In order to obtain beads a first solution was prepared by dissolving 20 g of polysulfone and 0.5 g of activated carbon Norit in 79.5 g of in DMF. The mixture was stirred at 500 rpm during 24 h at room temperature. Afterwards, the beads were obtained by dosing 3 mL of the polymeric solution into a coagulation bath containing 100 mL of distilled water as a non-solvent. The polymer solution was dropwise dosing using a syringe pump equipped with a syringe and a needle. The dosing rate of polymeric solution was 0.4 mL/min and the distance between the needle and the surface of water in the coagulation bath was 3.5 cm. Then, the prepared beads were filtered and left 1 h in a clean water bath under gentle stirring in order to extract the rest of DMF. Finally, the beads were filtrated and, in order to remove any potentially reminded solvent, they were subsequently dried employing a rotary evaporator. The size of bead was 2.5 mm. Afterwards, in order to immobilize enzymes on the bead, first the aqueous solution of enzyme (1 g _pro tein/L) was prepared using Viscozyme proteins. Then, 1 g of beads was immersed in 25 mL of the enzyme solution containing 25 mg of Viscozyme proteins and gently stirred using a magnetic stirrer for 24 h at room temperature. The immobilized amount of the enzymes was indirectly determined by analyzing the remaining amount of enzymes in the solutions by the standard Bradford method using UV-Vis spectrophotometer. The amount of immobilized enzymes in 1 g of beads was 0.8 mg.

Example 2

The procedure was analogous to Example 1 except that the amount of DMF was 79 g and the amount of activated carbon Norit was 1 g. The size of bead was 2.6 mm. The amount of immobilized enzymes in 1 g of beads was 0.9 mg.

Example 3 The procedure was analogous to Example 1 except that the amount of DMF was 78 g and the amount of activated carbon Norit was 2 g. The size of bead was 2.6 mm. The amount of immobilized enzymes in 1 g of beads was 1.0 mg.

Example 4

The procedure was analogous to Example 1 except that the amount of DMF was 75 g and the amount of activated carbon Norit was 5 g. The distance between the needle and the surface of water in the coagulation bath was 8,5 cm. The size of bead was 2.6 mm. The amount of immobilized enzyme in 1 g of beads was 1.4 mg.

Example 5

The procedure was similar to Example 2, except that activated carbon Fluka was used instead of activated carbon Norit. The size of bead was 2.6 mm. The amount of immobilized enzymes in 1 g of beads was 0.6 mg.

Example 6 - Enzyme immobilization on active carbon

First, enzyme immobilization was carried out by immersing 500 mg of activated carbon Norit in 250 mL of enzyme aqueous solution containing 250 mg of Viscozyme proteins. The obtained enzyme-immobilized activated carbon was collected on a paper 8-10 microns porous filter using a Buchner funnel and the material was dried at 40 ± 2 °C for 4h. Then, in order to obtain the beads a similar process as in Example 4 was carried out, except in that enzyme-immobilized activated carbon Norit was used instead of activated carbon Norit. The size of bead was 2.6 mm. The amount of immobilized enzymes in 1 g of carbon was 36.8 mg. After beads formation, enzymatic beads were dried under fume hood until constant weight instead of employing a rotary evaporator. Additionally, in order to evaluate if all enzymes immobilized on activated carbon were encapsulated, the amount of enzymes in the coagulation bath was analyzed by the standard Bradford method using UV-Vis spectrophotometer and no enzymes were found in the coagulation bath. The amount of immobilized enzymes in 1 g of prepared beads was 7.4 mg.

Comparative Example 1

The procedure was similar to Example 1 , except that the amount of DMF was 80 g and no activated carbon was used. The size of bead was 2.5 mm. The amount of immobilized enzymes in 1 g of beads was 0.6 mg.

In Examples 7 to 34 below, similar beads were prepared either by using other polymers instead of polysulfone or other adsorption materials instead of activated carbon. Example 7

The procedure was similar to Example 2, except polyethersulfone (PESLI) was used instead of polysulfone.

Example 8

The procedure was similar to Example 2, except polyvinylidene difluoride (PVDF) was used instead of polysulfone.

Example 9

The procedure was similar to Example 2, except polystyrene (PS) was used instead of polysulfone.

Example 10

The procedure was similar to Example 2, except polyacrylonitrile (PAN) was used instead of polysulfone.

Example 11

The procedure was similar to Example 2, except 1-methyl-2-pyrrolidone (NMP) was used instead of DMF.

Example 12

The procedure was similar to Example 11 , except PESLI was used instead of polysulfone.

Example 13

The procedure was similar to Example 11 , except PVDF was used instead of polysulfone.

Example 14

The procedure was similar to Example 11 , except PS was used instead of polysulfone.

Example 15

The procedure was similar to Example 11 , except PAN was used instead of polysulfone.

Example 16

The procedure was similar to Example 5, except PESLI was used instead of polysulfone. Example 17

The procedure was similar to Example 5, except PVDF was used instead of polysulfone.

Example 18

The procedure was similar to Example 5, except PS was used instead of polysulfone.

Example 19

The procedure was similar to Example 5, except PAN was used instead of polysulfone.

Example 20

The procedure was similar to Example 5, except NMP was used instead of DMF.

Example 21

The procedure was similar to Example 20, except PESLI was used instead of polysulfone.

Example 22

The procedure was similar to Example 20, except PVDF was used instead of polysulfone.

Example 23

The procedure was similar to Example 20, except PS was used instead of polysulfone.

Example 24

The procedure was similar to Example 20, except PAN was used instead of polysulfone.

Example 25

The procedure was similar to Example 2, except activated silica was used instead of activated carbon.

Example 26

The procedure was similar to Example 2, except celite was used instead of activated carbon.

Example 27

The procedure was similar to Example 2, except zeolite was used instead of activated carbon.

Example 28

The procedure was similar to Example 2, except ceramic was used instead of activated carbon.

Example 29

The procedure was similar to Example 2, except resin was used instead of activated carbon.

Example 30

The procedure was similar to Example 6, except activated silica was used instead of activated carbon.

Example 31

The procedure was similar to Example 6, except enzyme-immobilized celite was used instead of enzyme-immobilized activated carbon.

Example 32

The procedure was similar to Example 6, except enzyme-immobilized zeolite was used instead of enzyme-immobilized activated carbon.

Example 33

The procedure was similar to Example 6, except enzyme-immobilized ceramic was used instead of enzyme-immobilized activated carbon.

Example 34

The procedure was similar to Example 6, except enzyme-immobilized resin was used instead of activated carbon.

Example 35

In order to obtain alginate beads a first solution was prepared by dissolving 2 g of sodium alginate and 0.5 g of activated carbon Norit in 97.5 g of water. The mixture was stirred at 500 rpm during 24 h at room temperature. Afterwards, the beads are obtained by dosing 12 mL of the polymeric solution into a coacervation bath containing 100 mL of 2% calcium chloride in water. The polymer solution was dropwise dosing using a syringe pump equipped with a syringe and a needle. The dosing rate of polymeric solution was 0.4 mL/min and the distance between the needle and the surface of the coagulation bath was 8.5 cm. The size of bead was 2.7 mm. Then, the prepared beads were filtered.

Afterwards, the procedure of enzyme immobilization on the beads was performed analogously to Example 1 , except 0.3 g (dry weight) of beads was used instead of 1 g. The amount of immobilized enzymes was 45.2 mg/gbeads.

Enzymatic bead activity test (Examples 36-45 and Comparative Example 2)

Examples below were carried out to verify the activity of prepared enzymatic beads. Results with polysulfone beads of Example 4 (Examples 36 to 40) and with alginate beads of Example 35 (Examples 41-45) are shown in FIG. 5. In can be seen that the enzymatic activity of polysulfone beads is maintained without significantly decreasing from the second cycle of use onwards, and that the enzymatic activity of alginate beads is maintained without significantly decreasing from the fourth cycle of use onwards.

Example 36

First, 1 g/L of CMC in acetate buffer (0,05M, pH 5) was prepared. Then, 10 g of beads prepared in Example 4, equivalent to 13.6 mg of enzyme proteins, were immersed in 25 mL of the CMC solution and stirred using a hot plate magnetic stirrer under around 350 rpm for 24 h at 50 °C. Blank test with 25 mL of the CMC solution was also carried out. The amount of reducing sugars liberated during cellulose hydrolysis was determined according to Reducing Sugar DNS method using UV-Vis spectrophotometer. The amount of reducing sugars liberated during cellulose hydrolysis was 102 ± 5.7 mg/L.

Example 37

The 2 ^nd cycle experiment of beads reuse was analogous to Example 36, except beads recovered after 1 ^st activity test in Example 36 were used instead of beads prepared in Example 4. The amount of reducing sugars liberated during cellulose hydrolysis was 81 ± 2.9 mg/L.

Example 38

The 3 ^rd cycle experiment of beads reuse is analogous to Example 36, except beads recovered after 2 ^nd activity test in Example 37 were used instead of beads prepared in Example 4. The amount of reducing sugars liberated during cellulose hydrolysis is 78 ± 1.7 mg/L.

Example 39 The 4 ^th cycle experiment of beads reuse is analogous to Example 36, except beads recovered after 3 ^rd activity test in Example 38 were used instead of beads prepared in Example 4. The amount of reducing sugars liberated during cellulose hydrolysis is 79 ± 0.2 mg/L.

Example 40

The 5 ^th cycle experiment of beads reuse is analogous to Example 36, except beads recovered after 4 ^th activity test in Example 39 were used instead of beads prepared in Example 4. The amount of reducing sugars liberated during cellulose hydrolysis is 78 ± 0.8 mg/L.

Example 41

The procedure is analogous to Example 36, except 0,3 g (dry weight) of beads prepared in Example 35, equivalent to 13.6 mg of enzyme proteins, was used instead of 10 g of beads prepared in Example 4. The amount of reducing sugars liberated during cellulose hydrolysis is 373 ± 13.3 mg/L.

Example 42

The 2 ^nd cycle experiment of beads reuse is analogous to Example 41 , except beads recovered after 1 ^st activity test in Example 41 were used instead of beads prepared in Example 35. The amount of reducing sugars liberated during cellulose hydrolysis is 162 ± 0.3 mg/L.

Example 43

The 3 ^rd cycle experiment of beads reuse is analogous to Example 41 , except beads recovered after 2 ^nd activity test in Example 42 were used instead of beads prepared in Example 35. The amount of reducing sugars liberated during cellulose hydrolysis is 109 ± 2.8 mg/L.

Example 44

The 4 ^th cycle experiment of beads reuse is analogous to Example 41 , except beads recovered after 3 ^rd activity test in Example 43 were used instead of beads prepared in Example 35. The amount of reducing sugars liberated during cellulose hydrolysis is 77 ± 1.5 mg/L.

Example 45

The 5 ^th cycle experiment of beads reuse is analogous to Example 41 , except beads recovered after 4 ^th activity test in Example 44 were used instead of beads prepared in Example 36. The amount of reducing sugars liberated during cellulose hydrolysis is 75 ± 2.0 mg/L.

Comparative Example 2

The procedure is similar to Example 36, except 161 pL of pure non-immobilized enzyme (Viscozyme proteins), equivalent to 13.6 mg of enzyme proteins was used instead of 10 g of enzymatic beads. The amount of reducing sugars liberated during cellulose hydrolysis is 544 ± 26.3 mg/L.

Bead characterization

Both external and internal morphology of prepared beads were analyzed by Environmental Scanning Electron Microscope (ESEM, FEI Quanta 600). To analyze the internal morphology, micrographs of the cross-section of beads were taken. In average particle diameter order to obtain a cross-section without modifying the polymeric structure, the beads were cryogenically broken. For this procedure, a cryostat (Leica CM 1850) was employed. First of all, the beads were attached over a specimen disc with a freezing medium. It was used an embedding medium for frozen tissue specimens (Sakura Tissue). Once the beads were fixed over the specimen disc, the disc was immersed into liquid nitrogen. Then, the specimen disc was located in the cryochamber. After that, the sample was cut with thickness intervals of 1 pm. Finally, the beads were analyzed by ESEM. All the cross-section images were analyzed with the Imaged software (Imaged developed at the National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin)) in order to quantify the mean pore size ("pore diameter") and the macrovoid size of the beads.

External metrology of the beads was also observed by optical microscopy (TE2000-E, NIKON) in order to visualize carbon particles in their surface.

Additionally, the bead weight and size (diameter) were measured using an analytical balance (ED224S, Sartorius) and a digital calliper (Mitutoyo Digital ABS Caliper), respectively. The average was determined with at 10 measures.

Moreover, the overall bead porosity (E), defined as the volume of the pores divided by the total volume of the bead, was calculated using a method based on density measurements. For polysulfone bead without carbon, the overall bead porosity was determined from bulk and polysulfone densities by using the following equation: where p _c and p _psf (1.24 g/cm ³ ) correspond to bead and polysulfone density, respectively.

For the beads with activated carbon the equation is slightly different: where p _c is a bead density and p _pS f _+a c is a weighted average of the material density, considering POCNORIT = 0.4 g/cm ³ and POCFLUKA = 0.35 g/cm ³ .

Table 2 shows the characteristics of the prepared beads. As it is shown in Table 2, the diameter of beads did not significantly change by increasing the carbon content. On the other hand, the bead weight slightly increased by rising the carbon content. The porosity showed dependence on the carbon content; the higher the carbon content, the lower the porosity of beads. Pore size did not change significantly. However, the macrovoids were smaller for carbon beads as compared to pure polysulfone beads. As macrovoids have different dimensions within a single bead cross section, the corresponding standard deviation values are very high (Values are means ± SD, n = 10).

Table 2. Polysulfone beads characterization

Examples 1, 2, 3, and 4 were prepared using Activated carbon Norit a Activated carbon Fluka, decolorizing b Activated carbon Norit with enzymes immobilized Norit The influence of activated carbon on the polymeric bead was also analyzed in terms of internal and external morphology by ESEM as well as by optical microscope. The optical microscope was used in order to visualize the carbon particles in the polymeric bead surface, which were not possible to be observed in ESEM micrographs. As it is shown in FIG. 1 , the carbon-modified bead is darker due to the black carbon particles, which are clearly observed. The ESEM cross section micrographs demonstrated that prepared beads are matrix type spheres without shell (FIG. 2).

As can be seen in FIG. 3, as the carbon loading increases, the porous structure becomes closer and the size and number of macrovoids are reduced. On the other hand, there are no important changes in the microporous structure, as the mean pore size did not change significantly (Table 2).

In case of surface morphology (FIG. 4) no significant changes were observed for carbon content 2.4 wt% and 4.8 wt% as compared to 0 wt%. However, for carbon content 9.1 wt% the porous structure became closer and pore size seemed to be reduced, which clearly was observed for the highest concentration of carbon (20 wt%).

Enzyme immobilization on beads

Table 3 shows the results of enzymes immobilization on bead surface for Examples 1-5 and on activated carbon for Example 6. In case of Example 6, the enzymes were first immobilized on carbon and then the carbon-immobilized enzymes were used to prepare a polymeric solution, which further was used to form the beads. The leaching test demonstrated the absence of enzymes in the coagulation bath, which means that all enzymes immobilized on carbon were enclosed in the beads.

Table 3

Examples 1, 2, 3, and 4 were prepared using Activated carbon Norit a Activated carbon Fluka, decolorizing b Activated carbon Norit with enzymes immobilized Norit

Values are means ± SD, n = 3

As it is shown in Table 3, the higher the carbon content, the higher the amount of immobilized enzymes. The highest amount of immobilized enzymes was obtained in Example 6 (20 wt% of carbon based on polysulfone), where the enzymes were first immobilized on the activated carbon.

Comparing the results of different carbons (Example 2 and Example 5), it can be stated that carbon Norit gave better results than carbon Fluka.

Recovery and reapplication tests of the modified beads

To demonstrate the stability of enzymes immobilized on the beads surface or enclosed in the beads, recovery and replication tests were performed. Each test was carried out in a batch experiment where 1 g of enzymes immobilized beads was placed in an Erlenmeyer flask with 25 mL of distilled water for a blank test, or with 25 mL of water solution containing 0.1 g of CMC to simulate the preferable concentration ratio between enzymes and dried fiber for enzymatic treatment of pulp in the pulp mill or deink plant. These beads were left in the solution for 2 weeks. During this time, samples were periodically withdrawn from the solution in order to analyze the enzymes content in case of leaching (standard Bradford method, using UV-Vis spectrophotometer (UV-1800, Shimadzu)).

Results indicated that the enzymes were immobilized in the beads and leaching to the aqueous medium in which they were storage was not detected.

Example 46

In order to obtain beads a first solution was prepared by dissolving 18.75 g of polysulfone and 5 g of activated carbon Norit, and 1.25 g of iron magnetic microparticles (IOTHINKS- POWDER, particle size < 400 pm (< 35 mesh)) in 75 g of in DMF. The mixture was stirred with an overhead stirrer IKA at 500 rpm during 24 h at room temperature (i.e., at a temperature of 20-25 °C). Afterwards, the beads were obtained by dosing 3 mL of the polymeric solution into a coagulation bath containing 100 mL of distilled water as a nonsolvent. The polymer solution was dropwise dosing using a syringe pump equipped with a syringe and a needle. The dosing rate of polymeric solution was 0.4 mL/min and the distance between the needle and the surface of water in the coagulation bath was 3.5 cm. Then, the prepared beads were filtered and left 1 h in a clean water bath under gentle stirring in order to extract the rest of DMF. Magnetic properties of the beads are showed in FIG. 7.

Example 47

The procedure was analogous to Example 46 except that the amount of iron magnetic microparticles was 0.25 g and the amount of polysulfone was 19.75 g.

Example 48

The procedure was analogous to Example 46 except that the amount of iron magnetic microparticles was 2.5 g and the amount of polysulfone was 17.5 g.

Example 49

In order to obtain alginate/carbon/magnetic beads immobilized with enzyme, in the first step, maghemite (y-Fe2C>3) nanoparticles were synthesized according to the Massart’s method (Massart 1981, ibid.). This procedure involves alkaline co-precipitation performed by rapid addition of concentrated ammonium hydroxide (20%, 1L) in an aqueous mixture of 180 g of FeCl2 (0.9 mol) and 715 mL of FeC solution (1.5 mol) in acidic medium (HCI). The resulting dark black magnetite (FesC ) were then stirred in nitric acid (360 mL, 2M), oxidized into maghemite by a boiling solution of Fe(NOs)3 (323g, 1.3 mol) and, after washing, dispersed into water leading to a stable magnetic fluid called ferrofluid. The pH value of the resulting suspension is about 2. At this time, nanoparticles are positively charged with nitrate counterions. Then 2g of the alginate/carbon beads obtained in Example 35 were mixed with 100 mL of the diluted ferrofluid solution (prepared in the above described first step) in order to give them magnetic properties. Obtained magnetic alginate/carbon/iron beads were finally used for enzyme immobilization following the procedure described in Example 6.

Cited references

1. Massart R. “Preparation of aqueous magnetic liquids in alkaline and acidic media”, IEEE Trans Magn. 1981 ; 17(2):1247-8.

2. Bee A, Massart R, Neveu S. “Synthesis of very fine maghemite particles”, J Magn Magn Mater. 1995; 149; 6-9. For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

Clause 1. An enzymatic bead that is a porous water-insoluble immobilized enzymecontaining composite bead comprising:

- a polymer matrix;

- an adsorption material; and

- an enzyme selected from the group consisting of cellulase, endo-glucanase, cellobiohydrolase, hemicellulase, mannanase, xylanase, pectinase, pectin esterase, pectin and pectate lyase, gamanase, esterase, laccase, lipase, and mixtures thereof, the composite bead having a surface and pores containing the adsorption material, wherein the enzyme is immobilized by the absorption material on the surface of the bead or by the absorption material on the surface of the bead and within the pores, and wherein the bead has an average particle diameter from 200 pm to 1 cm.

Clause 2. The enzymatic bead of clause 1 , wherein the enzyme is adsorbed by the absorption material within the pores of the composite bead and on the surface of the bead.

Clause 3. The enzymatic bead of clause 1 , wherein the enzyme is adsorbed by the absorption material on the surface of the bead.

Clause 4. The enzymatic bead of any one of clauses 1 to 3, wherein the absorption material is selected from the group consisting of activated carbon, activated silica, celite, zeolite, cordierite ceramic, crosslinked phenol-formaldehyde polycondensate resin, and mixtures thereof.

Clause 5. The enzymatic bead of clause 4, wherein the absorption material is activated carbon.

Clause 6. The enzymatic bead of any one of clauses 1 to 5, wherein the weight amount of adsorption material is from 1 to 30 wt%, with respect the weight amount of polymer.

Clause 7. The enzymatic bead of any one of clauses 1 to 6, wherein the amount of enzyme is from 0.5 to 50 mg/gbeads, or from 0.5 to 20 mg/gbeads, or from 0.5 to 10 mg/gbeads.

Clause 8. The porous water-insoluble immobilized enzyme-containing composite bead of any one of clauses 1 to 7, wherein the polymer is selected from the group consisting of polysulfone, polyethersulfone, polyvinylidene difluoride, polystyrene, polyacrylonitrile, and mixtures thereof; alginate, carrageenan, gellan gum, carboxyl methyl cellulose, hyaluronic acid, and mixtures thereof.

Clause 9. The enzymatic bead of any one of clauses 1 to 8, wherein the polymer is polysulfone and the amount of enzyme is from 0.5 to 10 mg/gbeads.

Clause 10. The enzymatic bead of any one of clauses 1 to 8, wherein the polymer is alginate and the amount of enzyme is from 20 to 50 mg/gbeads, or from 30 to 45 mg/gbeads.

Clause 11. A composition comprising the enzymatic bead as defined in any of the clauses 1 to 10 and one or more excipients or carriers.

Clause 12. A process for the preparation of the enzymatic bead as defined in any one of clauses 1 to 10 comprising the following steps: a) providing a polymeric solution of a polymer in a suitable solvent; b) adding to the polymer solution an adsorption material in order to obtain a polymeric mixture; c) dropwise dosing the polymeric mixture into a non-solvent or into a solvent containing a chelating agent in order to obtain composite beads having an average diameter in the range from 200 pm to 1 cm; d) adding the composite beads to an enzyme aqueous solution in order to obtain composite beads comprising immobilized enzyme on the surface of the beads; e) isolating the immobilized enzyme-containing composite beads; or, alternatively, comprising the following steps: a) adding an adsorption material to an enzyme aqueous solution in order to obtain an enzyme immobilized on an adsorption material; b) providing a polymeric solution of a polymer in a suitable solvent and adding thereto the enzyme immobilized on an adsorption material in order to obtain a polymeric mixture; c) dropwise dosing the polymeric mixture into a non-solvent or into a solvent containing a chelating agent in order to obtain immobilized enzyme-containing composite beads having a diameter in the range from 200 pm to 1 cm; d) isolating the immobilized enzyme-containing composite beads.

Clause 13. The process for the preparation of the enzymatic bead according to clause 12, wherein dropwise dosing is carried out with a dropping device having an exit tube or a nozzle having an internal diameter from 0.2-1 .5 mm and at a rate from 0.05 to 1 mL/min, such as 0.4 mL/min.

Clause 14. The process for the preparation of the enzymatic bead according to clauses 12 or 13, wherein the distance between the dropping device and the surface of water in the coagulation bath is from 2 to 10 cm, such as 3.5 cm

Clause 15. Use of the enzymatic bead as defined in clauses 1 to 11 or of the composition as defined in claim 12 for the production of pulp for the manufacture of paper products.