WO2021009550A1

WO2021009550A1 - Preparation of immobilized enzymes

Info

Publication number: WO2021009550A1
Application number: PCT/IB2019/056159
Authority: WO
Inventors: Laura GODOY; Carlos Fischer
Original assignee: Rhodia Brasil Ltda
Priority date: 2019-07-18
Filing date: 2019-07-18
Publication date: 2021-01-21

Abstract

A process for the preparation of a catalytic system comprising an enzyme covalently bound to the surface of a precipitated silica carrier, the catalytic system obtained therefrom and its uses. The catalytic system may be advantageously used for the transesterification reaction of fatty acid feedstock with short chain alcohols to obtain fatty acid esters of short chain alcohols.

Description

PREPARATION OF IMMOBILIZED ENZYMES

Technical Field

[0001] The present invention relates to a catalyst system comprising an enzyme covalently immobilized on a precipitated silica support and to the process for its preparation. Depending on the nature of the enzyme, the system can be used in a number of different catalytic processes.

Background Art

[0002] Enzyme-based catalysts systems are known. In order to ensure economic use of such catalysts, some conditions have to be satisfied: the catalyst has to be active for a sufficiently long time under the reaction conditions, it should be readily removable after the end of the reaction and it should be reusable as often as possible. Ideally, these requirements should be satisfied for a very wide range of reaction conditions (for example temperature range, type of solvents, pressures, etc.), in order to provide a catalyst as universal as possible.

[0003] It is also known to immobilize enzymes on non-soluble supports.

Frequently, enzymes are non-covalently immobilized on carriers. The carriers used are typically ion exchange resins or polymer particles.

Examples of such systems are for instance Novozym 435, Lipozym RM IM or Lipozym TL IM commercially available from Novozymes A/S, Denmark.

[0004] A disadvantage of the use of such non-covalently immobilized enzymes is the desorption of the enzyme from the support which occurs depending on the reaction system used. In addition, the use of polymeric supports for immobilization purposes (such as acrylic resins), may show limitations regarding the chemical stability of the catalytic system in organic mediums, which are extensively used as reaction medium.

[0005] There is therefore still a need for methods of enzyme immobilization which overcome the disadvantages of the prior art, in order to implement enzyme-based catalytic processes which have not been realizable to date. [0006] It is therefore an object of the present invention to provide catalytic systems which do not have one or more of the disadvantages of the prior art systems. In particular, catalytic systems which have a high stability with respect to desorption of the enzyme from the carrier and also possess industrially meaningful specific activities. A further object of the present invention is to provide catalytic systems which can be removed from the reaction system in a simple manner and that can be reused.

[0007] The present invention provides an enzyme-based catalytic system which comprise an enzyme immobilized on an inert carrier, and their use as catalyst systems in industrial applications.

[0008] The present invention also provides a process for preparing the inventive catalytic system, which comprise enzymes immobilized on an inert carrier, which is precipitated silica.

[0009] The inventive catalyst system also has the advantage that the selection of the carrier material and of the associated particle size distribution allows the particle size to be adjusted such that simple removal of the catalyst system from the reaction and, hence, also the reuse of the catalyst is possible.

Summary of invention

[0010] The first object of the invention is a catalyst system comprising enzyme molecules bound to a precipitated silica carrier via spacer molecules, and at least one polyfunctional molecule bound to at least two enzyme molecules.

[0011] In the inventive catalyst system, free amino groups are first introduced at the surface of the precipitated silica carrier by means of spacer molecules. The spacer molecules form covalent bonds with the free silanol groups on the precipitated silica surface. Enzyme molecules are then bound to the amino groups of the spacer molecules. Without being bound by theory it is believed that covalent bonds are formed between the amino group of the spacer molecule and the lateral chains of the aminoacids glutamine, asparagine, glutamic and aspartic acids, that are present in most enzymes. [0012] Polyfunctional molecules capable of reacting with functional groups present on the enzyme molecule, notably amino, guanidine, amide, hydroxyl, thiol, benzyl, phenyl and indole functional groups, as well as with itself, further secure the enzyme molecules around the precipitated silica carrier. Said polyfunctional molecules form crosslinks binding the enzyme molecules together, thus providing stability to the catalyst system.

[0013] The precipitated silica carrier

[0014] By "precipitated silica" it is meant a silica that is prepared by precipitation from a solution containing silicate salts (such as sodium silicate), with an acid (such as sulfuric acid). The expression“precipitated silica carrier” is used herein to refer to the bulk of precipitated silica particles that serve as the carrier for the enzyme molecules.

[0015] Precipitated silica used in the invention may be prepared according to methods known in the art , such as anyone of the methods described in EP396450A, EP520862A, EP670813A, EP670814A, EP762992A, EP762993A, EP917519A, EP1355856A, W003/016215A,

W02009/112458A, W02011/117400A, WO2013/110659A,

WO2013/139934A, W02008/000761A.

[0016] The precipitated silica may be in any form suitable to match the shape and/or size of reactor conditions. The precipitated silica may be in any physical form such as beads, fibers, or plates.

[0017] In a preferred embodiment, the precipitated silica used in system of the present invention is in the form of beads. The term“beads” is used herein to refer to solid particles having a median particle size in the range of from 100 pm to 900 pm. The particle size is preferably at least 200 pm, more preferably at least 400 pm. The particle size is preferably not more than 800 pm, more preferably not more than 700 pm. An advantageous range of particle size is from 400 to 700 pm, preferably 450 to 650 pm.

[0018] The median particle size is typically determined by laser diffraction.

[0019] The precipitated silica is characterized by a BET specific surface area of from 80 to 650 m²/g.

[0020] The precipitated silica typically has a BET specific surface of at least 90 m²/g, in particular of at least 100 m²/g, even of at least 120 m²/g. The BET specific surface generally is at most 240 m²/g, in particular at most 200 m²/g.

[0021] The BET specific surface is determined according to the Brunauer- Emmett -Teller method described in The Journal of the American

Chemical Society, Vol. 60, page 309, February 1938, and corresponding to the standard NF ISO 5794-1 , Appendix E (June 2010).

[0022] In general, the precipitated silica has a CTAB specific surface of between 100 and 625 m²/g. The precipitated silica typically has a CTAB specific surface of at least 90 m²/g, in particular of at least 100 m²/g, even of at least 120 m²/g. The CTAB specific surface generally is at most 240 m²/g, in particular at most 200 m²/g. The CTAB specific surface is the external surface, which can be determined according to the standard NF ISO 5794- 1 , Appendix G (June 2010).

[0023] The precipitated silica is characterised by silanol density, that is the

number of OFI groups per surface area, expressed as number of OFI/nm², which is equal to or greater than 2 OFI/nm², even greater than 2 OFI/nm². The number of OFI groups per surface area typically does not exceed 40 OFI/nm², more typically it does not exceed 20 OFI/nm².

[0024] Notable, non-limiting examples of precipitated silica, which could be used as the carrier in the present invention, are for instance Tixosil^® 38X, Tixosil^® 68, Tixosil^® 38A, Zeosil^® 1165MP, Zeosil^® HRS1200MP, or Zeosil^® Premium 200MP, all commercially available from Solvay.

[0025] The spacer molecules

[0026] The terms“spacer molecule” or“spacer” are interchangeably used herein to refer to a low molecular weight molecule which is covalently bound to both the precipitated silica support and the enzyme molecule. The spacer acts as an immobilizing group for binding the enzyme molecule to the precipitated silica carrier.

[0027] The spacer can be an optionally substituted bivalent compound. The

spacer generally is 1 to 30 atoms, preferably, 1 to 20 atoms, and, more preferably, 2 to 12 atoms in length, measured from the catalytic species to the carrier surface. [0028] In an embodiment of the invention the spacer comprises a linear alkyl chain of formula -(Chhjm- wherein m is an integer from 1 to 20, preferably from 2 to 14, more preferably from 2 to 12, even more preferably from 2 to 10.

[0029] The expression“spacer (molecule) precursor” is used herein to refer to a bifunctional compound which, once reacted with the precipitated silica carrier and the enzyme molecule, generates the spacer. Spacer precursors comprise at least one functional group capable of reacting with silanol groups on the precipitated silica surface and at least one functional group capable of reacting with functional groups in the lateral amino acid chains of the enzyme molecule. Notable examples of said amino acid’s functional groups are amino, guanidine, amide, hydroxyl, thiol, benzyl, phenyl and indole groups.

[0030] In a preferred embodiment of the invention the spacer precursor is

provided with one amino functional group and with a functional group capable of reacting with the silanol groups on the silica surface.

[0031] Typically, the functional group capable of reacting with the silanol groups on the silica surface is an alkoxysilane group of formula -SiR3-_n(OR’)_n wherein n is an integer from 1 to 3, preferably 3, and R and R’ are both independently selected from alkyl groups having from 1 to 20 carbon atoms, preferably from alkyl groups having from 1 to 5 carbon atoms, more preferably from alkyl groups having from 1 to 3 carbon atoms.

[0032] In a particularly preferred embodiment the spacer precursor is 3- aminopropyltriethoxysilane and the spacer is -(Chh^-.

[0033] The polyfunctional molecule

[0034] The expression“polyfunctional molecule precursor” is used herein to refer to a molecule comprising at least two functional groups which are capable of reacting with functional groups present on the enzyme molecule as well as with themselves. Notable, non-limiting examples of said groups are for instance functional groups deriving from the carboxy group, notably - COOH, -COOR” or -CHO groups.

[0035] Notable non-limiting examples of suitable polyfunctional molecule

precursors are aldehydes such as glutaraldehyde, glyoxal, malondialdehyde, succinaldehyde, adipaldehyde and phthalaldehyde, as well as polyaldehydes including, but not limited to oxidized sugars, oxidized polysaccharides, dialdehyde starch, oxidized cellulose, oxidized gum arabic and oxidized guar.

[0036] In a preferred embodiment of the invention the polyfunctional molecule precursor is glutaraldehyde.

[0037] Without being bound by theory it is believed that the polyfunctional

molecule precursor reacts both with functional groups on the enzyme (e.g. amino, guanidine, amide, hydroxyl, thiol, benzyl, phenyl and indole groups) as well with other polyfunctional molecule precursors to form a “net” further securing the enzyme to the carrier. Once the reaction with the enzyme molecules and/or other polyfunctional molecule precursor has taken place, the polyfunctional molecule precursor becomes a

polyfunctional molecule.

[0038] The polyfunctional molecule precursor is different from the spacer

molecule precursor.

[0039] The enzyme

[0040] Suitable enzymes include, but are not limited to, enzymes selected from the enzymatic families including oxidoreductases, transferases,

hydrolases, lyases, isomerases, ligases and esterases. Exemplary enzymes from the oxidoreductase family, include, but are not limited to, reductases, peroxidases, hydrogenases, dehydrogenases and catalyses. Exemplary enzymes from the transferase family include, but are not limited to, glycosyltransferases and mannosyltransferases. Exemplary enzymes from the hydrolase family include, but are not limited to, esterases, glucoamylases, transcarbamylases, nucleases, ribonucleases, ATPases, peptidases, proteases and phosphodiesterases. Exemplary enzymes from the lyase family include, but are not limited to, polysaccharide lyases. Exemplary enzymes from the isomerase family include, but are not limited to, topoisomerases. Exemplary enzymes from the ligase family include, but are not limited to, snyntheteases. [0041] Preferably the enzyme is selected from the group consisting of the hydrolases and more preferably from the esterases. In a particularly preferred embodiment of the invention the enzyme is a lipase.

[0042] The lipase may be derived from a strain of the genus Humicola (also

known as Thermomyces ), Pseudomonas, Candida, or Rhizomucor, preferably the species H. lanuginosa, C. antarctica or R. miehei.

[0043] Advantageously, the catalytic system comprises from 20 to 50% by weight of enzyme with respect to the total weight of the precipitated silica carrier and the enzyme, preferably from 30 to 45%, more preferably from 35 to 40 % by weight.

[0044] Process for the preparation of the catalytic system

[0045] A second object of the invention is a process for the preparation of the catalytic system, said process comprising:

- providing a precipitated silica carrier;

- reacting said precipitated silica carrier with at least one spacer precursor to provide a modified silica carrier comprising at least one spacer molecule attached to the surface of the silica carrier;

- reacting the modified silica carrier with enzyme molecules to obtain enzyme molecules bound to the silica carrier via spacer molecules; and

- providing at least one polyfunctional molecule precursor and allow it to react with the enzyme molecules bound to the silica carrier.

[0046] In the first step of the process, a modified silica carrier is prepared by

reacting the precipitated silica carrier with a spacer (molecule) precursor having at least two functional groups, in a manner sufficient to attach at least one functional group of the spacer precursor molecule to least one surface of the precipitated silica.

[0047] In a preferred embodiment of the invention, the spacer precursor

comprises at least one alkoxysilane group of formula -SiR3-_n(OR’)_n wherein n is an integer from 1 to 3, preferably 3, and R and R’ are both independently selected from alkyl groups having from 1 to 20 carbon atoms, preferably from alkyl groups having from 1 to 5 carbon atoms, more preferably from alkyl groups having from 1 to 3 carbon atoms. [0048] The reaction of the spacer molecule with the precipitated silica carrier is performed under reflux conditions in an appropriate solvent, as known to one skilled in the art.

[0049] The amount of spacer precursor used in the first step of the process is sufficient to ensure full conversion of the silanol groups on the surface of the silica carrier. The amount of spacer precursor to be used can be determined by the person skilled in the art based on the nature of the precursor and on the silanol density of the silica carrier being used.

[0050] When the spacer precursor molecule is 3-aminopropyltriethoxysilane the amount is generally from 5 to 15% by weight of spacer precursor per weight of silica, and, preferably, from 10 to 12% by weight.

[0051] At the end of the reaction a modified a silica carrier comprising at least one spacer molecule attached to at least one surface of the silica carrier is obtained.

[0052] In a subsequent step of the process, a sufficient amount of the selected enzyme molecules is reacted with the modified silica carrier. Enzyme molecules are reacted with the free functional group of the spacer molecule now covalently attached to the silica carrier such that the enzyme is covalently bound to the surface of the carrier.

[0053] The reaction is performed under conditions suitable to promote the

reaction of the functional group of the spacer precursor molecule, typically an amino group, with the functional groups of the enzyme molecule.

Typically the reaction involves functional groups in the lateral amino acid chains of the enzyme molecule, such as amino, guanidine, amide, hydroxyl, thiol, benzyl, phenyl and indole groups.

[0054] In the last step of the process, the polyfunctional precursor molecules are reacted with the enzyme molecules which are bound to the silica carrier. In a particularly preferred embodiment of the invention, the polyfunctional molecule is glutaraldehyde. In such a case the amount of glutaraldehyde is typically from 50 to 250 grams per kilogram of solid enzyme, preferably from 190 to 220 grams per kilogram of solid enzyme.

[0055] The parameters of each step of the process can be adjusted based upon the precipitated silica carrier, the enzyme, the spacer precursor molecule and the polyfunctional precursor molecule as known by the person skilled in the art.

[0056] Use of the catalytic system

[0057] The inventive catalyst system may be used to catalyze reactions generally known in the art to be catalyzed by the corresponding free enzyme. For example, the catalyst system may be used to catalyze reactions, including, but not limited to, oxidation/reduction reactions; the transfer of groups of atoms, e.g. amino, acetyl, phosphoryl, and glycosyl groups; hydrolytic cleavage of bonds; non-hydrolytic cleavage of, for example, C-C, C-0 or C-N bonds; isomerization and transfer reactions; and the like.

[0058] A major advantage of the catalyst system of the invention is the ease with which it can be recovered after reaction of reactants in a mixture to obtain the desired product. Catalytic species which are soluble in the reaction medium, such as enzymes, are typically very difficult to separate and recover. However, catalyst systems in accordance with the present invention are easily separated by conventional solid-liquid separation techniques, such as, for example, filtration or centrifugation, for recovery and re-use.

[0059] Catalyst systems in accordance with the present invention may be used in conventional reactors such as, for example a fixed (column) or fluidized bed reactors. The catalyst system may be used in a continuous or batch mode.

[0060] A further object of the present invention is a process wherein a reaction is catalysed by the inventive catalyst system.

[0061] In particular a further object of the invention is a process of catalyzing a reaction comprising:

(a) providing a reaction mixture comprising reactant molecules capable of reacting to produce a desired product; and

(b) contacting the reaction mixture with a catalyst system in an amount and for a time sufficient to catalyze the reaction of the reactant molecules in the reaction mixture to provide the desired product.

[0062] The process typically further comprises the step of removing the catalyst system from the reaction mixture. [0063] In an advantageous embodiment of the inventive process, the catalyst system comprises a lipase enzyme and the reaction to be catalysed is the transesterification reaction of fatty acid feedstock with short chain alcohols, in particular methanol, ethanol or propanol, to obtain fatty acid esters of short chain alcohols. Said esters are commonly known under the name of biodiesel as they are used as additives to or as replacement of fossil diesel.

[0064] Thus, in said embodiment, the reaction mixture comprises a fatty acid

feedstock and at least one short chain alcohol.

[0065] The expression’’short chain alcohol” refers to a branched or linear alcohol having 1 to 5 carbon atoms and mixtures thereof. Preferred short chain alcohols are methanol, ethanol, propanol and mixtures thereof.

[0066] The alcohol content in the mixture is preferably less than 4.0, less than

3.0, even less than 2.0, even 1.5 or 1.0 molar equivalents to the amount of fatty acids in the reactant mixture (free and glyceride bound fatty acids). The alcohol may be added stepwise and/or continuously to the reaction mixture over the length of or a part of the reaction period.

[0067] The term "fatty acid feedstock" is defined herein as a fatty acid feedstock substrate comprising triglyceride. The substrate may furthermore comprise fatty acid alkyl esters, diglyceride, monoglyceride, free fatty acid or any combination thereof. Any oils and fats of vegetable or animal origin comprising fatty acids may be used as substrate for producing fatty acid alkyl esters in the process of the invention.

[0068] The fatty acid feedstock may be oil selected from the group consisting of: algae oil, canola oil, coconut oil, castor oil, coconut oil (copra oil), corn oil, cottonseed oil, flax oil, fish oil, grape seed oil, hemp oil, jojoba oil, mustard oil, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, and oil from halophytes, pennycress oil, camelina oil, jojoba oil, coriander seed oil, seashore mallow oil, microbial oils or any combination thereof.

[0069] The fatty acid feedstock may be fat selected from the group consisting of: animal fat, including tallow from pigs, beef and sheep, lard, chicken fat, fish oil, yellow grease, brown grease, or any combination thereof. [0070] The invention will be now described with reference to the following examples, whose purpose is merely illustrative and not intended to limit scope of the invention.

[0071] Should the disclosure of any patents, patent applications, and publications which are incorporated herein by reference conflict with the description of the present application to the extent that it may render a term unclear, the present description shall take precedence.

[0072] EXPERIMENTAL

[0073] Particle size

[0074] The median particle size may be determined by laser diffraction using a MALVERN (MasterSizer 2000) particle sizer, employing the Fraunhofer theory. The analysis protocol includes a first full deagglomeration of the precipitated silica sample to be carried out before the laser diffraction determination.

[0075] The full deagglomeration of the precipitated silica sample is carried out directly in the sample dispersion unit of the MasterSizer 2000 by setting the following parameters, until median particle size variation between two consecutive analyses is inferior to 5%: Hydro 2000G sample dispersion unit; Stirring conditions : 500 rpm; Pump conditions : 1250 rpm; Ultrasonic probe : 100%;

[0076] Measurement parameters: Obscuration range : 8-15%; Background

measurement duration : 10 s; Measurement duration: 10 s; Delay between measurements: 1 s.

[0077] Time to reach a stable median particle size with such protocol is typically around one hundred seconds.

[0078] Silanol density determination

[0079] The samples were analyzed using ATD-ATG technique on Mettler's

LF1100 thermobalance and a Tensor 27 Bruker spectrometer equipped with a gas cell, with the following program: Temperature rise from 25°C to 1100°C at 10°C/min, under air (60 mL/min), in AI203 crucible of 150 pL. The silanol density is directly related to the loss of mass between 200°C and 800°C. The loss of mass (%) between 200°C and 800°C is identified as AW% this value. The silanol ratio (mmol/g) is defined by:

TSiOH = AW^*2^*1000 / (18.015^*100) = 1.11^*AW

Silanol density (OH/nm²) is calculated by :

D = TSiOhPNa / 1 021 *S_BET = TSiOH^*602.2/ SBET

wherein Na : Avogadro’s number and SBET is the BET surface area.

[0080] EXAMPLE 1

[0081] Precipitated silica particles (Tixosil^® 68, Solvay) were sieved in order to isolate beads having an average particle size in the range of 450-650 pm.

[0082] 1 g of the beads was suspended in 20 mL of anhydrous ethanol. 0.12 g of 3-aminopropyltriethoxysilane (3-APTES; CAS: 919-30-2) were added to the mixture and the reaction was performed at 40°C for 1.5 h.

[0083] The reaction medium was filtered using stainless steel membrane (pore 45 pm) and solids were washed with 10 mL of anhydrous ethanol. The surface modified silica carrier beads were left to dry at room temperature.

[0084] The yield of the silica carrier modification reaction was 94%, calculated based on the amount of 3-APTES remained in the reaction medium by potentiometric acid-base titration (5 g of the reaction supernatant was titrated using KOH solution with concentration 0.05 mol/L previously standardized using potassium hydrogen phthalate in a potentiometric titrator with combined pH glass electrode).

[0085] 1 g of dry surface-modified silica carrier was added to a medium

containing 1.65 g of water, 0.75 g of glycerol and 0.6 g of free lipase enzyme (CALB L from Novozymes). The reaction was conducted at 40°C for 1 h.

[0086] The reaction medium was filtered using stainless steel membrane (pore 45 pm) and solids were washed with 10 mL of deionized water.

[0087] The yield was 38% w/w enzyme/support, quantified by UV

spectrophotometry (Bradford assay He, F. (2011). Bradford Protein Assay. Bio-protocol Bio101 : e45. DOI: 10.21769/BioProtoc.45). The overall yield considering the available enzyme in the medium was 83 by weight.

[0088] The enzyme/precipitated silica carrier particles were directly used in the next step. [0089] 1 g of the enzyme/precipitated silica carrier particles were added to a medium containing 5 g of water and 0.146 g of glutaraldehyde (50% w/w aqueous solution). The mixture was kept at 40°C during 1 h. The

crosslinking yield was 99.2% based on the glutaraldehyde consumption, quantified by hydroxylamine method using potentiometric titration.

[0090] The catalytic system (SILICA_MVP), after washing and drying, was directly used in the next step.

[0091] The sugar esterification trials were performed according to Vescovi et al.

(Process Biochemistry, 51 (12), pp. 2055-2066).

[0092] In these trials, the purpose was to produce fructose oleate in nine

sequential batches, quantifying the fructose conversion in each batch to determine the stability of the enzyme activity over repeated use. The experimental conditions were 55°C, 5 ml_ of tert-butyl alcohol, using a fructose/oleic acid molar ratio of 1 :5 (fructose concentration of 130 mM), 20% (w/v) molecular sieve (type 3 A) and enzyme load of 8.40 U/mL of solvent, under stirring in a Marconi incubator (MA 430/1) equipped with 360°-stirring carrousel (Marconi®, Piracicaba, Brazil). At the end of each batch (6 h each), the catalytic system and the molecular sieve were recovered by filtration at room temperature in a stainless steel membrane (pore 45 pm), and reused in a new batch. Esterification conversions were evaluated by oleic acid consumption determination using acid-base potentiometric titration (a desirable amount of sample was titrated using isopropanol as solvent and NaOH with concentration of 0.4 mol.L·¹ previously standardized using potassium hydrogen phthalate in a potentiometric titrator with combined pH glass electrode).

[0093] The inventive system SILICA_MVP was evaluated in parallel with

Novozym 435 (a solid catalyst preparation comprising lipase form C.

Antarctica immobilised on polyacrylate beads from Novozymes), in triplicate for each cycle, measuring the fructose conversion after each batch with duration of 6 h. The results are compiled in Figure 1 which shows the conversion of oleic acid to fructose oleate for each batch.

[0094] As shown in Figure 1 the inventive catalytic system exhibits better stability, conserving more of the initial activity if compared to Novozym 435 system. [0095] As overall result, the inventive catalytic system achieved 89% activity preservation after 9 cycles of 6 h (calculated as the fructose conversion in the ninth cycle divided by the fructose conversion in the first cycle), while Novozym 435 preserved only 57% of its initial activity.

[0096] Microscopy analysis of the two systems was performed comparing initial and final particles (at the end of the 9^th batch) of the two catalytic system.

[0097] At the end of the nine catalytic cycles, the particles of Novozym 435 were shrunk with respect to their initial size with subsequent pores clogging or collapse, exposing liquid enzymes to leaching due to medium interactions. The particle dimensions were around 30% smaller than the initial diameter.

[0098] At the end of the nine catalytic cycles, the particles of the inventive catalyst system (SILICA_MVP) preserved their morphology. The particles presented dimensions around 5% smaller than the initial particles.

Claims

1. A catalyst system comprising enzyme molecules bound to a precipitated silica carrier via spacer molecules, and at least one polyfunctional molecule bound to at least two enzyme molecules.

2. The catalyst system of claim 1 wherein the enzyme is selected from the group consisting of hydrolases, esterases and lipases.

3. The catalyst system of claim 1 or 2 wherein the enzyme is a lipase.

4. The catalyst system according to anyone of the preceding claims wherein the precipitated silica carrier is in the form of particles having a median particle size in the range of from 400 to 700 pm.

5. A process for the preparation of the catalytic system of anyone of claims 1 to 4, said process comprising:

- providing a precipitated silica carrier;

- reacting said precipitated silica carrier with at least one spacer molecule precursor to provide a modified silica carrier comprising at least one spacer molecule attached to at least one surface of the silica carrier;

- reacting the modified silica carrier with enzyme molecules to obtain enzyme molecules bound to the silica carrier via spacers; and

6. The process according to claim 5 wherein the spacer molecule precursor is 3- aminopropyltriethoxysilane.

7. The process according to anyone of claims 5 or 6 wherein the polyfunctional molecule precursor is selected from the group consisting of glutaraldehyde, glyoxal, malondialdehyde, succinaldehyde, adipaldehyde, phthalaldehyde, oxidized sugars, oxidized polysaccharides, dialdehyde starch, oxidized cellulose, oxidized gum arabic and oxidized guar.

8. The process according to claim 7 wherein the polyfunctional molecule

precursor is glutaraldehyde.

9. A process of catalyzing a reaction comprising:

(a) providing a reaction mixture comprising reactant molecules capable of reacting to produce a desired product; and (b) contacting the reaction mixture with a catalyst system of anyone of claims 1 to 4 in an amount and for a time sufficient to catalyze the reaction of the reactant molecules in the reaction mixture to provide the desired product.

10. The process of claim 9 wherein the catalyst system is the catalyst system of claim 3 and the reaction mixture comprises a fatty acid feedstock and at least one short chain alcohol.