WO2016193516A1 - General method for obtaining biocatalysts which comprises enzyme immobilisation during the synthesis of metallo-organic materials - Google Patents

Abstract

The present invention relates to a general method for obtaining biocatalysts which comprises immobilising enzymes in situ in the intercrystalline mesoporosity formed by nanocrystal aggregation during the synthesis of a metallo-organic material (MOF). The method of the invention allows the obtainment of a high enzyme content on the support , the minimisation of the reduction of catalytic efficiency in relation to that of the free enzyme, and the reduction of losses as a result of leaching, thereby resolving the limitations identified in the known methods for obtaining biocatalysts, which are based on in-situ enzyme immobilisation. The invention also relates to the biocatalysts directly obtained by the method.

Description

 GENERAL PROCEDURE FOR OBTAINING BIOCATALIZERS UNDERSTANDING THE IMMOBILIZATION OF ENZYMES DURING THE SYNTHESIS OF METAL-ORGANIC MATERIALS DESCRIPTION

SECTOR OF THE INVENTION

The present invention falls within the scope of the development of catalysts that can be applied in very different fields, such as chemical, pharmaceutical, agricultural, energy and biotechnology. Specifically, it refers to a procedure for obtaining biocatalysts that is general, and that allows the immobilization of any enzyme during the synthesis of nanocrystalline metallo-organic materials (MOFs), taking advantage of intercrystalline mesoporosity, generated by agglomeration or aggregation of nanocrystals.

STATE OF THE TECHNIQUE

The immobilization on solid supports of enzymes allows their heterogeneization, that is, having the biomolecule supported in solid phase. The obtaining of solid biocatalysts favors, among other aspects, the separation of said enzymes from the reaction medium and their reuse in successive reaction cycles.

The chemical nature and textural properties of the materials used as supports give different properties to the final catalyst. The binding of an enzyme on a preexisting support (hereinafter post-synthesis immobilization) can be carried out through covalent or non-covalent bonds, and in any case a high chemical affinity between both species is convenient. To accommodate high loads of an enzyme, the support must offer a high specific surface area, so materials that have a high porosity with a pore diameter greater than the molecular dimensions of the enzyme to be immobilized are desirable.

These two characteristics (chemical nature and textural properties) restrict the possibilities of finding a general or universal method to immobilize any enzyme, regardless of its characteristics, and makes a design necessary " measure "according to the chemical composition of the enzyme surface and its molecular dimensions.

As an alternative to post-synthesis immobilization, the possibility of entrapment of the enzyme during the support synthesis process, hereinafter in-situ immobilization (thus the synthesis of siliceous materials in the presence of enzymes is uniquely known) has been explored by several tracks.

Among the porous materials with more emergent projection, stand out the metal-organic materials (MOFs). In the context of a possible application in the immobilization of enzymes, these materials comply with the two main premises indicated above (specific surface area and pore diameter). Thus, on the one hand, its structural versatility is such that, unlike other conventional microporous materials, some of its structures have pores that go deep into the mesopore range (Deng et al., Science, 2012, 336, 1018 ). On the other hand, its versatility of composition, which covers almost all of the elements of the Periodic Table or of the known organic functionalities, guarantees the possibility of designing MOFs, which if necessary are chemically related to each enzyme through the control of hydrophobicity or the presence / concentration of electrostatic charges and / or pre-designed functional groups in the porous environment.

Within the patent literature, methods for obtaining biocatalysts comprising post-synthesis immobilization of enzymes in MOFs are known. Thus, the document WO2012174402A2 which refers to the mixture of a mesoporous MOF such as Tb-mesoMOF, with a solution containing the enzyme microperoxidase-11, and which is incubated for approximately 10 to 150 hours at an approximate temperature of 37 degrees Celsius, achieving the immobilization of the enzyme in the nanoscopic cells of the MOF. For its part, document US2014342429A1 deals with a method for immobilizing any type of biomolecule, including enzymes, on porous materials such as MOFs, and singularly on MOF MIL-53 (AI).

On the other hand, in the non-patent literature, the obtaining of biocatalysts comprising MOFs on which they are subsequently incorporated is also disclosed. small-sized enzymes with a diameter of less than 3.3 nm (Deng et al., Science, 2012, 336, 1018; Lykourinou et al., J. Am. Chem. Soc. 201 1, 133, 10382; Chen et al. ., J. Am. Chem. Soc. 2012, 134, 13188). However, post-synthesis immobilization in MOFs has several limitations. In the first place, the vast majority of MOF materials are microporous so they do not have pores large enough to immobilize enzymes inside, and those that possess them require a great effort in their preparation, because they are constituted by non-commercial organic ligands , expensive and difficult to synthesize, necessarily in steps prior to the formation of MOF itself (Deng et al., Science, 2012, 336, 1018). Second, the pores offered by these MOFs are not large enough for many biomolecules to diffuse through them, and therefore, the method is far from being considered universal. As regards the in-situ immobilization of enzymes on MOFs, only the document of Shieh et al. (J. Am. Chem. Soc. 2015, 137, 4276) which disseminates a method for synthesizing MOFs in the presence of an enzyme at room temperature and in aqueous conditions, and which consists of mixing an aqueous solution of zinc nitrate with another solution aqueous containing imidazolate-2-carboxaldehyde (ICA), catalase and a buffering agent. However, the catalyst thus obtained is characterized by being formed by microcrystals; a very reduced encapsulation efficiency that is 5% by weight of the enzyme (which would correspond approximately to a percentage of incorporation of the enzyme below 64%, considering a yield below 100% and a complete formation of the MOF ); and also a very reduced catalytic efficiency of the encapsulated enzyme (33 times lower than that of the free enzyme itself).

Based on the above, a general, simple and quick procedure is considered of interest, which based on the in-situ immobilization of enzymes, during the formation of MOFs, allows biocatalysts to be obtained with high enzymatic load, with a high retention capacity of enzyme and with a catalytic efficiency that is not excessively impaired with respect to the activity of the biomolecule without immobilization.

EXPLANATION OF THE INVENTION In a first aspect, the invention relates to a process for obtaining a biocatalyst because it comprises: a) partially or totally synthesizing a metallo-organic material (MOF) in the presence of at least one enzyme, until obtaining a solid and, b) isolating the solid synthesized according to step a), preferably by centrifugation or filtration, and where the synthesized MOF immobilizes the enzyme in the intercrystalline mesoporosity formed between crystals or between agglomerated or aggregated domains of homogeneous sized nanocrystalline particles.

Preferably, the intercrystalline mesoporosity comprises at least one, and preferably more than one, hollow volume between 2 and 50 nm.

The process of the invention is characterized in that in step (a), the synthesis of the MOF comprises contacting two solutions in the presence of at least one enzyme, where said solutions can be aqueous, organic or an aqueous-organic mixture, and where preferably the first solution is a solution of a metallic source and the second, an organic ligand solution.

The second organic ligand solution is chosen from an aqueous solution of an organic ligand salt or an aqueous solution of protonated organic ligand in the presence of a deprotonating agent.

Preferably the process of the invention is carried out at temperatures between 4 and 70 ° C and more preferably between 20 and 30 ° C. Preferably the enzymes that are used are β-glucosidase and lipase.

In a second aspect, the invention relates to the biocatalyst directly obtained by the process of the first aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION The technical problem that solves the present invention is the development of a general or universal, simple, fast and economical procedure, which can be used extensively to obtain biocatalysts comprising an MOF support and any enzyme that is immobilized therein during its synthesis, and that solves the problems detected in the state of the art for other biocatalysts that are based on in-situ immobilization, singularly the reduced capacity of enzymatic load, the limited retention capacity of the immobilized enzymes, and an excessive reduction of the catalytic efficiency with respect to that of the immobilized biomolecule.

The present invention is based on a method of in-situ immobilization of the enzyme Aspergillus niger β-glucosidase (β-Glu) and Candida Antarctic lipase (CalB) on different MOFs, which allows to obtain biocatalysts characterized by comprising polycrystalline particles formed by nanocrystals (about 40 nm) of homogeneous size, which in their aggregation generate hollow, stable and ordered mesopore volumes (between 2 and 50 nm in diameter), and that allow them to immobilize a significant amount of the enzyme inside, which reaches at least 15% by weight of the enzyme and more than 86% of the enzyme exposed in the reaction medium see Examples 1 to 5 and 7) and additionally, reduces the leachate losses thereof (see Example 6 ).

On the other hand, the reaction conditions used in this invention to obtain the biocatalysts, favor the maintenance of the catalytic activity of the enzymes conferring them a high catalytic efficiency (see Examples 1 to 5 and 7), obtaining values much higher than indicated by Shieh et al. (J. Am. Chem. Soc. 2015, 137, 4276) who recorded an efficiency 33 times lower in the biocatalyst with respect to the free enzyme, while following the procedure included in the present invention the fall is only about 6-12 times tested for the enzyme β-glucosidase in aqueous medium (Examples 1-3), even considering that the activity of the free enzyme has been measured under optimized conditions of pH, dilution, concentration of buffers, while the activity of the Biocatalyst has been measured by directly taking an aliquot (suspension) of the synthesis system (and, therefore, in the presence of a base, without pH control, without the presence of buffers, etc.). In the case of lipase (Example 7), the biocatalyst obtained in the present invention it maintains without any fall the catalytic efficiency, which is only 3% lower than the activity of the free enzyme. It is noteworthy that also in this case the soluble enzyme is tested only in the presence of a pH buffer optimized to enhance its activity, while the aliquot of the biocatalyst is tested by taking it directly from the reaction medium and therefore in the presence of all the components of the same.

Finally, the procedure for obtaining biocatalysts that is included in the invention, allows the stabilization of the structure and activity of the enzyme for at least 48 hours in a priori means not very favorable for it, such as Ν, Ν- dimethylformamide, where the free enzyme is completely inactivated after a few minutes (see Examples 4 and 5).

Certain embodiments of this invention are described in detail herein (see Examples 1 to 5 and 7). However, the present invention can be carried out in a wide range of embodiments beyond those explicitly described.

In a first aspect, the invention relates to a method of obtaining a biocatalyst, hereinafter method of obtaining the invention, comprising the following steps: a) partially or totally synthesizing a metallo-organic material (MOF) in the presence of at least one enzyme, until a solid is obtained, and b) isolate the synthesized solid according to step a), and where the synthesized MOF immobilizes the enzyme, in the intercrystalline mesoporosity formed between nanocrystals or between agglomerated or aggregated nanodominiums in micrometric particles. It is precisely the fact that immobilization occurs in this intercrystalline mesoporosity, that is, in hollow, stable and orderly mesoporous volumes, delimited by the agglomeration of nanocrystals, unlike the (micro) porosity intrinsic to the crystal structure of an MOF , which gives the procedure for obtaining the invention its considerable technical advantages. The document by Sánchez-Sánchez et al. (Oreen Chem. 2015, 17, 1500) of the inventors themselves, claims a process for preparing MOFs, which are prepared in water and at room temperature using salts as a source of organic ligands or, failing that, in the protonated form of the corresponding organic ligands previously deprotonated by the action of a base. These MOFs, which are those that are used as the basis in the present invention, are characterized by lacking intercrystalline pores large enough to encapsulate biomolecules the size of enzymes. In addition, the crystals that form these materials are nanocrystalline with a great tendency to agglomerate or aggregate with each other in the reaction medium, giving rise to particles compact enough to be considered non-disintegrable in their crystals or crystalline domains, and containing mesopores between the crystals, which potentially covers the sizes required to house a wide range of enzymes inside. In the present invention, "intercrystalline mesoporosity" means the presence in the MOF of at least one, and preferably more than one, intercrystalline hollow volume, stable, orderly and in the range of mesopores, which is established between particles formed by agglomeration or aggregation of crystals or domains of nanoporous particles of homogeneous size, compact enough to be considered non-disintegrable in their crystals or crystalline domains.

According to the classification of the IUPAC and in the context of this patent, mesoporo is understood as any hollow volume in a material whose diameter is between 2 and 50 nm, which preferably will have a pore distribution that conforms to the dimensions of the enzyme to be immobilized (in the case of β-glucosidase with values close to 10 nm and in the case of 5 nm lipase). Pores below 2 nm are called micropores.

The intercrystalline mesoporosity in solid particles, unlike the intrinsic porosity to the crystalline structure, is that generated by the agglomeration or aggregation of crystals. Therefore, it has a much wider pore distribution than the intrinsic one and, more importantly, it is under experimental control because it depends on the modification of the MOF preparation conditions, and therefore, it can cover all the sizes required for harbor any type of enzyme inside. By "biocatalyst" is meant the solid that is synthesized in stage a) and that is isolated in stage b) of the process for obtaining the invention, and which is a composite material comprising, a MOF support and at least one enzyme , which is immobilized in the intercrystalline mesoporosity of the MOF during its total or partial synthesis.

By "MOF or metallo-organic material" is meant an organic-inorganic hybrid material in which metal atoms or metalloids or clusters of those atoms are linked to each other through organic ligands, at least bidentate, to give rise to three-dimensional crystalline networks porous The method is applicable to any MOF prepared with organic ligands that preferably contain carboxylic groups through which metals are coordinated. Preferably, the MOF materials used are MIL-53 (AI), its structural counterpart NH ₂ -MIL-53 (AI) and Mg-MOF-74, as well as MOF Fe-BTC, all of them of small particle size . In the first two cases, the metal atoms that form in MOF are Al (like Al ³⁺ ) and the organic ligands are terephthalate or 2-amino-terephthalate, respectively. In the case of Mg-MOF-74, the metal is Mg (with formal charge 2+) and the ligand is 2,5-dihydroxyterephthalate. The MOF Fe-BTC material, marketed as Basolite F300, is composed of clusters of three Fe ³⁺ atoms linked together through benzene-1, 3,5-tricarboxylates. Unlike the MOF MIL-100 (Fe), formed by the same metal, the same clusters and the same organic ligand, the Fe-BTC material is semi-morph, although it has a high specific surface area (approximately 1000 m ² / g) for containing some cavities and pores similar to those of MIL-100 (Fe).

By "immobilization" is meant, in this document, the attachment of at least one enzyme, and preferably more than one, preferably in the intercrystalline mesoporosity of the MOF material, and which is favored over others such as embedding. By "embedding" is meant the immobilization of the enzyme when trapped by MOF crystals in the crystalline growth process, so that in the final biocatalyst it is accessible only through the intrinsic (micro) pores of the MOF.

Preferably, the MOF synthesis of step a) of the process for obtaining the invention comprises mixing, in the presence of at least one enzyme, of two solutions, a first solution or solution of a metallic source, which preferably has an acidic pH, and another second solution or solution of the organic ligand. In the scope of the invention, "first solution or solution of metallic source" means a solution of a metal salt, preferably in a polar solvent such as water. Examples of metal source solution that are included in the scope of the invention are an aqueous solution of an aluminum salt, or a solution of a magnesium salt in Ν, dime-dimethylformamide, and preferably solutions of aluminum nitrate nonahydrate or Mg acetate tetrahydrate or Fe (lll) chloride hexahydrate.

By "second solution or solution of organic ligand" is meant a solution of the source of organic ligand, either a salt or the acid, in a solvent preferably in a polar solvent such as water. If the acid is used as an organic source in water, a deprotonant agent is needed. Examples of organic ligand solution that are included in the scope of the invention are aqueous solution of organic ligands containing at least two groups of carboxylic acids per molecule and preferably 2-aminoterephthalic acid or 2,5-dihydroxyterephthalic acid.

The process for obtaining the invention can be carried out using aqueous solutions, organic solutions or solutions that are aqueous-organic mixtures. Preferably, the second solution is selected from an aqueous solution of an organic ligand salt or a protonated organic ligand in the presence of a deprotonation agent, with a pH (preferably neutral or basic) that at least guarantees the deprotonation or activation of two functional groups. which have to be subsequently coordinated to metals to form MOF materials.

On the other hand, the deprotonant agent is preferably selected from a strong base, a medium base or a weak base. Examples of strong base that are included in the scope of the invention are alkali hydroxides and preferably sodium hydroxide. Middle-base examples that are included in the scope of the invention are the amines, preferably triethylamine. Example of weak base that are included in the scope of the invention is ammonium hydroxide or ammonia.

By "enzyme" is meant any molecule of a protein nature that catalyzes chemical reactions. It can be immobilized, and the biocatalyst can be used in the field of industry (chemical, pharmaceutical or food, among others), preferably with respect to the soluble enzyme. Examples of enzymes that can be used in the invention are any of the enzymes existing in nature, with very diverse catalytic activities. Preferably, glucosidases, peroxidases, lacases, amylases, or lipases, and more preferably β-glucosidases and lipases.

The process of the invention prefers temperature between 4 and 70 ° C, and preferably between 20 and 30 ° C, that is to say those demanded by enzymes to preserve their structure and, therefore, their catalytic activity.

The enzyme is added in the course of the synthesis of the MOF according to step a) of the process for obtaining the invention, once the two solutions have been mixed, to avoid conditions that may be adverse. However, it is also possible to add the enzyme on either of the two solutions, taking care that the viability of the enzyme (especially catalytic activity) suffers as little as possible, that is, preferably, the enzyme is added on the solution whose pH is more close to enzymatic stability, which will suffer less (abrupt) changes in pH or that do not contain chemical species that may favor the denaturation of the enzyme or the inactivation of its active centers. The enzyme is added in any of the formats known in the state of the art, and preferably as an aqueous solution from an extract, which may comprise other chemical substances. These chemicals or the enzyme itself can affect different aspects of MOF synthesis, such as particle size (and, therefore, intercrystalline pores), the kinetics of the process or the presence or abundance of impurities formed, or catalytic activity of the immobilized enzyme.

Preferably, one of the solutions used for the synthesis of the MOF according to step a) of the process for obtaining the invention is added dropwise onto the other, keeping the system under stirring. This addition entails the formation of a solid either during the course of the addition or after a time that can reach from 1 second to 350 h. The solid formed is the biocatalyst, which comprises part or all of the enzyme, and is separated from the reaction mixture in step b) of the process for obtaining the invention preferably by centrifugation or by filtration after a time, between 1 second and 200 hours after finishing the addition of one solution over the other.

The process for obtaining the invention includes the possibility of simultaneous use of different enzymes. Thus, for example, it is feasible to employ more than one different class of enzyme than those mentioned herein.

Alternatively, the process for obtaining the invention comprises an additional stage or stage c) comprising drying the isolated solid according to stage b) under conditions compatible with the preservation of the structure and properties of the enzyme including the catalytic activity.

In a particular embodiment of the process for obtaining the invention, the solution of the metallic source is an aqueous solution of aluminum nitrate nonahydrate, the solution of the organic ligand is an aqueous solution of 2- aminoterephthalic acid, the deprotonant agent is selected from triethylamine , sodium hydroxide and ammonia, the enzyme is Aspergillus niger β-glucosidase which is added on the organic ligand solution and the reaction temperature is 25 ° C.

In another particular embodiment of the process for obtaining the invention, the solution of the metal source is a solution of Mg acetate tetrahydrate on Ν, dime-dimethylformamide, the solution of the organic ligand is a solution of 2,5-dihydroxyterephthalic acid on Ν , Ν-dimethylformamide, the enzyme is Aspergillus niger β-glucosidase which is added on the solution of the metal source and the reaction temperature is 25 ° C. In this embodiment, the stabilization of the enzyme structure and the maintenance of its catalytic activity is achieved for 48 hours, while the same enzyme in the free state in the same reagent is completely inactive after a few minutes.

In another particular embodiment of the process of obtaining, the solution of the metallic source is preferably iron chloride (lll) hexahydrate (FeCI ₃ -6H ₂ 0), the Organic ligand solution is a solution of benzene-1,3,5-tricarboxylic acid (BTC) in the presence of a deprotonating agent, preferably sodium hydroxide, the enzyme is Candida Antarctic Lipase B which is preferably added over the organic ligand solution and the reaction temperature is 25 ° C. The addition of one solution over the other, preferably of the metallic solution over that of the organic ligand, produces the immediate formation of an orange-brown precipitate, which after a reaction time of between 10 minutes and 22 hours, is separated by centrifugation processes or filtration The biocatalysts, far from being stable in the reaction medium during the time of synthesis, evolve in terms of the enzyme content and the catalytic activity in the test reaction studied, getting as active biocatalysts as the free enzyme itself.

Additionally, the process for obtaining the invention may comprise another stage in which the solid isolated according to stage b) or dried according to stage c) is again exposed to at least one enzyme, to increase the enzyme load thereof using a method of post-synthesis immobilization, since in the process of immobilization in-situ the enzymes could act as a mesoporgenic agent, creating larger mesopores. The technical effect achieved in the biocatalysts obtained by the method of obtaining the invention is evident through the techniques of physical-chemical and biochemical characterization that are collected in Examples 1 to 7, particularly in relation to the high amount of immobilized enzyme, at the high retention capacity of the enzyme and the reduction of the catalytic efficiency reduction with respect to that of the immobilized biomolecule. The high efficiency of enzymatic immobilization is based on the fact that the formation of the MOF material is preferably due to the instantaneous formation of a colloidal solution when the metallic solution and the organic ligand solution are mixed, as a consequence of the large number of MOF nuclei. that precipitate or crystallize when the metal cation and organic anion are found. Precisely because nucleation is so favored, the nuclei are abundant and barely grow because the metal and the organic ligand are depleted in the formation of the rest of the nuclei, thus forming suspended nanoparticles with a colloid appearance. The subsequent isolation of the solid from the solution, preferably by centrifugation, causes the enzyme to also recover in the solid, which together with the affinity between the biomolecule and the MOF material can give high loads of immobilized biomolecules, up to 100% of the enzyme present in the medium.

In a second and final aspect, the invention relates to the biocatalyst directly obtained by the process of the invention.

BRIEF DESCRIPTION OF THE CONTENT OF THE FIGURES

Figure 1. Standardized powder X-ray diffractograms of the sample of NH ₂ - MIL-53 (AI) (gray) and of the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -NaOH-24h (black).

Figure 2. Thermograms (solid lines) and their derivatives (dashed lines) of the β-Glu soluble enzyme extract (light gray), of MOF NH ₂ -MIL-53 (AI) material prepared in the absence of enzyme (gray), and of the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) - NaOH-24h (black).

Figure 3. Standardized powder X-ray diffractograms of the sample of NH ₂ - MIL-53 (AI) (gray) and the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -TEA-48h (black). Figure 4. Thermograms (solid lines) and their derivatives (broken lines) of the soluble enzyme extract of β-Glu (gray) and of the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) - TEA-48h (black) .

Figure 5. Standardized powder X-ray diffractograms of β-Glu @ NH ₂ -MIL-53 (AI) -NH ₃ -1 h (gray) and -24h (black) biocatalysts.

Figure 6. Thermograms (solid lines) and their derivatives (dashed lines) of the soluble enzyme extract of β-Glu (gray) and of the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) - NH ₃ -24h (black ).

Figure 7. Standardized powder X-ray diffractograms of the Mg-MOF-74 material (gray) and the p-Glu @ Mg-MOF-74-24h biocatalyst (black). Figure 8. Electrophoresis gel of: 1) high molecular weight reference; 2) soluble extract of the β-Glu enzyme; 3) p-Glu @ NH ₂ -MIL-53 (AI) -NaOH-1 h biocatalysts; 4) β- Glu @ NH ₂ -MIL-53 (AI) -NaOH-24h; 5) p-Glu @ NH ₂ -MIL-53 (AI) -TEA-2h; 6) p-Glu @ NH ₂ - MIL-53 (AI) -TEA-48h; 7) p-Glu @ Mg-MOF-74-2h; 8) p-Glu @ Mg-MOF-74-24h; 9) p- Glu @ NH ₂ -MIL-53 (AI) -NH ₃ -1 h; and 10) p-Glu @ NH ₂ -MIL-53 (AI) -NH ₃ -24h.

Figure 9. Electrophoresis gel of: 1) high molecular weight reference; 2) soluble extract of the enzyme p-Glu; 3) p-Glu @ Mg-MOF-74-5min biocatalyst; 4) p-Glu @ Mg- MOF-74-20h.

Figure 10. Scheme of the catalytic test reaction of pNPG hydrolysis in the presence of the enzyme p-glucosidase.

Figure 11. Schematic of the catalytic test reaction of a) hydrolysis of pNPA towards p-nitrophenol and b) hydrolysis of tributirin towards butyric acid in the presence of the enzyme lipase.

Figure 12. Thermograms (solid lines) and their derivatives (dashed lines) of the lipase soluble enzyme extract (light gray), of the MOF Fe (3) -BTC-NaOH material prepared in the absence of enzyme (gray), and of the biocatalyst Lip @ Fe (3) -BTC-NaOH- 1 h (black).

EMBODIMENTS OF THE INVENTION Example 1. Procedure for obtaining a p-Glu @ NH ₂ -MIL- 53 (AI) -NaOH-24h biocatalyst, comprising the synthesis of the NH ₂ -MIL-53 (AI) system using NaOH as a deprotonating agent and in the presence of the β-Glucosidase enzyme This example shows how to obtain a biocatalyst by immobilizing the β-glucosidase enzyme of Aspergillus niger (β-glu) during the formation of the MOF NH ₂ - MIL-53 (AI) system in which sodium hydroxide (NaOH) was used as the base. The Aspergillus niger β-Glucosidase enzyme has approximate dimensions of -12.3 nm x -10.7 nm x -8.1 nm, a molecular weight -240 KDa, and an isoelectric point 4.2. The first solution was prepared with 2.005 g of aluminum nitrate nonahydrate (AI (N0 ₃ ) 3-9H ₂ 0) (metal source) and 6.012 g of deionized water, giving a pH of 2.0. On the other hand, the second solution was prepared with 0.483 g of the organic ligand 2-aminoterephthalic acid NH ₂ -H ₂ BDC, 5.206 g of 1 M NaOH solution and 10.462 g of deionized water, giving a clear solution in a few minutes with a pH 6, 1. Then, 2.75 ml of Novozymes β-Glucosidase enzyme extract (EC 3.2.1.21) from Novozymes, supplied as a liquid enzyme preparation (Novozym 188), was added to the second solution with a concentration of the extract of β-Glucosidase measured by Bradford analysis of 14.54 mg / ml, which modified the pH to 5.6. Next, the first solution was added to the second solution, with stirring, which caused the formation of a yellowish solid in suspension practically immediately and at room temperature (25 ° C), giving a pH of 3.1, after a time ranging from 1 to 24 hours, different aliquots (suspension) were taken, from which the biocatalyst called p-Glu @ NH ₂ -MIL- 53 (AI) -NaOH-nh, was removed from the supernatant (solution) by centrifugation (13,400 rpm for 90 seconds).

The catalytic activity of both the suspension and the supernatant in hydrolysis of 10 mM para-nitrophenyl-beta-D-glucopyranoside (pNPG) dissolved in 0.1 M phosphoric acid / trisodium citrate buffer pH 5.0 was measured to give p -nitrophenol and beta-D-glucose. The catalytic activity was measured by spectrophotometry at 405 nm, with an array diode spectrophotometer (Agilent 8453 UV-Vis) provided with thermostatting and a magnetic stirring device to keep the samples in homogeneous suspension during the tests. The enzymatic activity was measured in any case by the increase of absorbance per minute at 405 nm produced by the release of p-nitrophenol due to the action of the enzyme β-glucosidase (Figure 10) at 35 ° C and at a pH of 5, 0. For this, on the test cuvettes containing 1.5 ml of buffer at pH 5.0 and 0.5 ml of 10 mM pNPG, 100 μΙ of soluble enzyme or 100 μ o of the suspension was added, or 100 μΙ of the supernatant or 100 μΙ of a resuspension of immobilized β-glucosidase. The molar extinction coefficient of p-nitrophenol measured under these conditions was 240 M ^"1 cnT ¹ . When the catalytic activity of the supernatants obtained from aliquots taken at different times remained constant or zero, the process of immobilization of the enzyme was terminated. The concentration of non-immobilized enzyme present in the supernatant as well as in the initial enzyme solution were determined by Bradford analysis.

After the immobilization monitoring was finished (after 24 hours), the remaining solid was filtered under vacuum using a Vidra FOC plate (666/1) of pore size 4 and 0.45 μηι HV Millipore filter paper. The biocatalyst obtained once dry, heavy and ground, was resuspended (10 mg in 5 ml_ of buffer) and its catalytic activity was tested in the same reaction indicated above.

Table 1 shows the percentage of immobilized β-Glu enzyme, determined by Bradford analysis by measuring the initial protein concentration in the enzyme solution and protein in the supernatant after 1 and 24 hours. After 1 h, 33% of the enzyme present in the medium was immobilized, while after 24 h 96% of the enzyme was immobilized. These percentages of enzymatic immobilization evolved in parallel with the presence of the NH ₂ -MIL-53 (AI) phase in the recovered solid, which over time grew progressively to the detriment of the phase corresponding to the protonated organic ligand (Sánchez-Sánchez et al., Green Chem. 2015, 17, 1500), which evidenced that it is the MOF phase and not the purely organic phase that contributes to the immobilization of the enzyme.

Table 1. Percentages of immobilization (by Bradford analysis); enzymatic load, catalytic activity and catalytic efficiency (by spectrophotometry); and sulfur content (by CHNS elemental chemical analysis) of the biocatalysts p-Glu @ NH ₂ -MIL- 53 (AI) -NaOH-1 h and -24h.

Sample ³ Immobilized enzyme (%) ^b / S ^d (%) Activity Efficiency

Enzymatic load (mg / g) ^c catalytic ⁶ catalytic '

1 h 33/28 _a 6 0.21

24 h 96/79 0.14 31 0.39 ³ The sample is designated as nh (number of hours of synthesis) of the β-Glu @ NH ₂ -MIL-53 (AI) -NaOH-nh biocatalyst.

b Percentage of enzyme immobilized in the solid versus total enzyme added. ^c mg of enzyme per g of biocatalyst.

d Percentage of S in the sample according to CHNS elementary analysis.

⁶ Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

f Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 3) in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

9 Not measured.

The enzyme load (Table 1) was determined from the milligrams of protein in the synthesis medium (calculated from Bradford analysis) and the grams of biocatalyst obtained after filtering the final synthesis solution. It is expressed as mg of protein / g of recovered biocatalyst. The presence of enzyme in the biocatalyst was also detected qualitatively by elementary chemical analysis CHNS (Table 1), which gives the contents of carbon, hydrogen, nitrogen and sulfur. These analyzes were carried out on a LECO CHNS-932 Elemental Analyzer device. The presence / absence of sulfur in the biocatalyst is particularly informative because it is part of the enzyme and not the MOF material studied. In good agreement with the estimate from the Bradford method, in the biocatalyst β-Glu @ NH ₂ -MIL-53 (AI) -NaOH-24h the sulfur content was 0.14%.

Additionally, Table 1 also shows the catalytic activity (expressed in U / g biocatalyst) and the catalytic efficiency (in U / mg protein) of the biocatalyst obtained after being resuspended and calculated from equations 2 and 3 below, respectively.

donde U son las unidades de actividad catalítica de la enzima definidas como transformación de 1 μηιοΙ de sustrato por minuto.

where U are the units of enzyme catalytic activity defined as transformation of 1 μηιοΙ of substrate per minute.

On the other hand, the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -NaOH-24h obtained, once dry, was characterized with various physical-chemical techniques, such as X-ray powder diffraction using a diffractometer X-ray Polycrystalline X ^' Pert Pro PANalytical, for structural identification. Figure 1 compares the powder X-ray diffractograms of the p-Glu @ NH ₂ -MIL-53 (AI) -NaOH-24h biocatalyst and the homologous MOF in the absence of enzyme. The diffractogram of the enzyme-free material is typical of a nanocrystalline NH ₂ -MIL-53 (AI) prepared at room temperature (Sánchez-Sánchez et al., Green Chem. 2015, 17, 1500). The addition of the enzyme extract caused some narrow and intense peaks to appear, which have been attributed to the protonated NH ₂ -H ₂ BDC ligand, in line with the drop in pH caused by this addition in the overall mixture. Nanocrystallinity is an indispensable condition for particles formed by their aggregation to contain intercrystalline porosity in the range of mesopores. Finally, the thermogram (TGA) was also recorded and its derivative (DTG) of the MOF NH ₂ -MIL-53 (AI) without enzyme, from the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -NaOH- was calculated 24h and of the β-Glu enzyme extract, in a Perkin-Elmer TGA7 device with a temperature sweep of 20-900 ° C at a heating rate of 20 ° C / min under a stream of dry air, and its derivative ( DTG) is presented in Figure 2.

There was 2.9% weight loss (190-271 ° C) in the enzyme extract assigned to the enzyme. In the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -NaOH-24h, an overall weight loss of 88% was detected; in the temperature range between 152-246 ° C the weight loss was 9.5%, while in the MOF without enzyme there was no appreciable weight loss in that range, which suggests the presence of enzyme in the biocatalyst. Example 2. Procedure for obtaining the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) - TEA-48h, which comprises the synthesis of the NH ₂ -MIL-53 (AI) system using ASD as a deprotonant agent and in the presence of the enzyme β-Glucosidase

This experiment shows how to obtain a biocatalyst by immobilizing the same Aspergillus niger β-Glucosidase enzyme used in Example 1, during the formation of a MOF NH ₂ -MIL-53 (AI) system in which triethylamine base is used ( TORCH).

The first solution was prepared with 2,000 g of aluminum nitrate nonahydrate (AI (N0 ₃ ) 3-9H ₂ 0) (metallic source) and 6.030 g of deionized water, giving a pH of 2.0. On the other hand, the second solution was prepared with 0.483 g of the organic ligand 2-aminoterephthalic acid NH ₂ -H ₂ BDC, 0.538 g of TEA and 13.246 g of deionized water, giving a clear solution in a few minutes with a pH of 6, 1. Then, 2.75 ml_ of β-Glucosidase enzyme extract (EC 3.2.1.21) from Novozymes, supplied as a liquid enzyme preparation (Novozym 188), with a concentration of β-Glucosidase extract was added to the second solution. measured by Bradford analysis of 14.54 mg / ml, which modified the pH of the mixture to 5.5. Then, the first solution was added dropwise on the second solution, under stirring, which caused the formation of a yellowish solid in suspension almost immediately and at room temperature (25 ° C), giving a pH of 3.1. After a time that ranged between 5 minutes and 48 hours, different aliquots (suspension) were taken, from which the biocatalyst called p-Glu @ NH ₂ -MIL-53 (AI) -TEA-nh, was removed from the supernatant (dissolution) by centrifugation (13,400 rpm for 90 seconds).

The catalytic activity was measured, following the same experimental steps described in example 1 above, except that the sampling times were 2 and 48 hours, respectively, for the biocatalysts p-Glu @ NH ₂ -MIL-53 (AI) -TEA-2h and - 48h. The use of ASD as a deprotonator agent for the organic MOF ligand also resulted in an efficient immobilization of the β-Glu enzyme (99% of the enzyme exposed after 48 hours has been encapsulated). The catalytic efficiency of the resulting biocatalyst (0.87 U / mg enzyme) (Table 2), was significantly higher than that obtained in the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -NaOH-24h (0, 39 U / mg enzyme, Table 1), which suggests that weaker bases are preferred to strong bases in deprotonation of the organic ligand of the MOF when the in-situ immobilization of the enzyme proceeds, presumably because of the negative repercussion that These bases may have the intrinsic activity of the enzyme.

In good agreement with the estimate from the Bradford method, the sulfur content in the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -TEA-48h was almost five times higher than that of its counterpart p-Glu @ NH ₂ -MIL-53 (AI) -TEA-2h (Table 2).

Table 2. Percentages of immobilization (by Bradford analysis); enzymatic load, catalytic activity and catalytic efficiency (by spectrophotometry); and sulfur content (by CHNS elementary chemical analysis) of the biocatalysts p-Glu @ NH ₂ -MIL- 53 (AI) -TEA-2h and p-Glu @ NH ₂ -MIL-53 (AI) -TEA-48h.

Sample ³ Immobilized enzyme (%) ^b / S ^d (%) Activity Efficiency

Enzymatic load (mg / g) ^c catalytic ⁶ catalytic '

2h 17/18 0.04 40 2.22

 48h 99/108 0.16 94 0.87

³ The sample is designated as nh (number of hours of synthesis) of the β-Glu @ NH ₂ -MIL-53 (AI) -TEA-nh biocatalyst.

b Percentage of enzyme immobilized in the solid versus total enzyme added. ^c mg of enzyme per g of biocatalyst.

d Percentage of S in the sample according to CHNS elementary analysis.

6 Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol ^f Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 3 of Example 1) in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol Figure 3 compares powder X-ray diffractograms of the β-Glu @ NH ₂ -MIL-53 (AI) -TEA-48h biocatalyst and the homologous MOF obtained in the absence of enzyme in the synthesis medium, NH ₂ -MIL-53 (AI). It can be seen that the presence of the β-Glu enzyme introduced changes in the crystalline nature of the MOF phase formed, so that the diffractogram obtained is typical of a nanocrystalline NH ₂ -MIL-53 (AI) (Sánchez-Sánchez et al., Green Chem. 2015, 17, 1500), with some impurity of protonated organic ligand. Since the diffractogram of the biocatalyst is very similar to that of the MOF material without enzyme, particularly in terms of bandwidth, its crystal size must also be very similar and, therefore, its intercrystalline mesoporosity.

Finally, the thermogram (TGA) and its derivative (DTG) of the p-Glu @ NH ₂ -MIL-53 (AI) -TEA-48h biocatalyst and the β-Glu extract, which is presented in the Figure, were also determined 4. In the TGA curve of the enzyme extract, two global weight losses can be distinguished: the first between 30 and 190 ° C (87.1%), which is attributed to water, and the second between 190 and 271 ° C ( 2.9%), which is attributed to the enzyme. In the thermogram of the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -TEA-48h, the weight loss that occurred between 166 and 242 ° C (7%) is detected, which by analogy with that of the extract Enzymatic, is assigned to the loss of the enzyme. Example 3. Procedure for obtaining the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) - NH ₃ -24h, comprising the synthesis of the NH ₂ -MIL-53 (AI) system using NH ₃ as a deprotonant agent and in presence of the enzyme β-Glucosidase

This experiment shows how to obtain a biocatalyst by immobilizing the same Aspergillus niger β-Glucosidase enzyme from the previous examples, during the formation of an MOF NH ₂ -MIL-53 (AI) system in which NH ₃ is used as the base.

The first solution was prepared with 2,041 g of aluminum nitrate nonahydrate (AI (N0 ₃ ) 3-9H ₂ 0) (metal source) and 6.023 g of deionized water. On the other hand, the The second solution was prepared with 0.482 g of the organic 2-aminoterephthalic acid NH ₂ -H ₂ BDC ligand, 0.362 g of 25% NH ₃ aqueous solution and 10.008 g of deionized water. The mixture at room temperature (25 ° C) took several hours to reach the solution, which was finally orange. Then 2.75 ml of enzyme extract (concentration = 14.54 mg enzyme / ml) was added on the second solution and then the first solution was added on the mixture of the second solution and the enzyme.

After one hour, a sample was taken, which was centrifuged at 13400 rpm for 90 seconds. The supernatant was separated to measure its enzymatic activity and protein concentration by Bradford analysis in the same manner as in the previous examples. The remaining solid was filtered under vacuum, obtaining the dry solid that was labeled as p-Glu @ NH ₂ -MIL-53 (AI) -NH ₃ -1 h, weighed, ground and resuspended to measure its enzymatic activity. After 24 h the rest of the sample was centrifuged and filtered under vacuum. The supernatant was removed, and the dry solid was labeled β-Glu® _NH2 -MI L-53 (AI) ₃ -NH 24h.

In this case the catalytic efficiency of the p-Glu @ NH ₂ -MIL-53 (AI) - NH ₃ -nh biocatalysts exceeded that of any other biocatalyst described in the previous examples, which again suggests that less strong bases (the Ammonia is less strong than TEA amine) affects the intrinsic activity of the enzyme less.

Table 3. Percentages of immobilization (by Bradford analysis); enzymatic load, catalytic activity and catalytic efficiency (by spectrophotometry); and sulfur content (by CHNS elementary chemical analysis) of the biocatalysts p-Glu @ NH ₂ -MIL- 53 (AI) -NH ₃ -1 h and -24h.

Sample ³ Immobilized enzyme (%) ^b / S ^d (%) Activity Efficiency

Enzymatic load (mg / g) ^c catalytic ⁶ catalytic '

1 h 5/7 _ to 18 2.57

24h 98/56 0.13 51 0.91 ^a The sample is designated as nh (number of synthesis hours) of the biocatalyst p- Glu @ NH ₂ -MIL-53 (AI) -NH ₃ -nh. ^b Percentage of enzyme immobilized in the solid versus total enzyme added. ^c mg of enzyme per g of biocatalyst.

d Percentage of S in the sample according to CHNS elementary analysis.

6 Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

f Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 3 of Example 1) in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

9 Not measured.

Figure 5 shows the X-ray diffractograms of the β-Glu @ NH ₂ -MIL-53 (AI) -NH ₃ -1 h and -24h biocatalysts. In the two diffractograms two phases are identified: the one corresponding to a nanocrystalline NH ₂ -MIL-53 (AI) with intercrystalline mesoporosity and the organic ligand NH ₂ -H ₂ BDC. As in the previous examples, the NH ₂ -MIL-53 (AI) phase was increasing to the detriment of NH ₂ -MIL-53 (AI) with the synthesis time.

For its part, Figure 6 shows the thermogravimetric (TG) profiles of the biocatalyst p-Glu @ NH ₂ -MIL-53 (AI) -NH ₃ -24h and the β-glucosidase enzyme extract. In the biocatalyst, there is an overall weight loss of 88%; In the temperature range between 187-278 ° C the weight loss is 18.2%.

The sequence of experimental examples 1, 2 and 3 shows how the change in the nature of the base used to deprotonate the organic ligand 2-amino-terphthalic acid (NH ₂ -H ₂ BDC), which forms the MOF NH ₂ -MOF-53 (AI) nanocrystalline at room temperature and in water by simple contact with an aqueous solution of Al, affects the immobilization efficiency of the Aspergillus niger β-Glucosidase enzyme. Not less important, it also affects the catalytic activity per immobilized enzyme unit, presumably as a consequence of the influence of these parameters on the physicochemical properties of the MOF support such as specific surface area, size and distribution of intercrystalline pores, or size of the agglomerates or aggregates of nanocrystals. Example 4. Method of obtaining the biocatalyst p-Glu @ Mg-MOF-74-24h, comprising the synthesis of the Mg-MOF-74 system using N, N-dimethylformamide as solvent and in the presence of the enzyme β-Glucosidase. In the present experiment it is shown how to obtain a biocatalyst by immobilizing the β-Glu enzyme during the formation of the nanocrystalline MOF-74 system prepared with Mg at room temperature (25 ° C) in Ν, Ν-dimethylformamide (DMF) as solvent, and that In the absence of enzyme it contains an acceptable ordered mesoporosity (Díaz-García et al., Cryst. Growth Des., 2014, 14, 2479).

To obtain the first solution, 0.561 g of Mg acetate tetrahydrate was added over 10.013 g of DMF, which dissolved in a few minutes. On the other hand, to obtain the second solution, 0.202 g of 2,5-dihydroxyterephthalic acid (dhtp) was dissolved in 10,021 g of DMF. On the first solution 0.5 ml of the same enzyme extract containing β-Glu was added in a concentration of 14.54 mg enzyme / ml of the previous examples, and immediately the second solution dropwise. A sample was taken at 2 hours and centrifuged for 15 seconds at 12,500 rpm thus obtaining the supernatant. The solid was filtered under vacuum and labeled as p-Glu @ Mg-MOF-74-2h. The reaction lasted for 24 hours, after which the solid biocatalyst p-Glu @ Mg-MOF-74-24h was recovered according to the same procedure.

In this case, no basis was added because the previous deprotonation of the organic ligand was not necessary for the formation of MOF-74 at room temperature (Díaz-García et al., Cryst. Growth Des., 2014, 14, 2479).

Table 4, in addition to ratifying the presence of the β-Glu enzyme in the p-Glu @ Mg-MOF-74 biocatalysts, indicates that this enzyme retained some catalytic activity despite having been in DMF, a medium that is very adverse , as evidenced in the total loss of the activity of the enzyme extract in DMF for 10 minutes. This suggests that the Mg-MOF-74 material, in this case, was not a simple immobilization of the β-Glu enzyme that immobilizes, but also in some way helped to preserve its enzymatic activity against the solvent inhibiting that activity, DMF On the other hand, the efficiency of enzymatic immobilization was very high from the first minutes, probably as a result of the mixture of solutions of metal and organic ligand caused the formation of a virtually colloidal suspension, which left the enzyme little chance not to be part of the solid, once the MOF material was recovered.

Table 4. Immobilization percentages (by Bradford analysis); enzymatic load, catalytic activity and catalytic efficiency (by spectrophotometry); and sulfur content (by CHNS elementary chemical analysis) of the enzyme extract after being subjected to a DMF solution for 10 minutes, and of the biocatalysts p-Glu @ Mg-MOF-74-2h and -24h.

Sample ³ Immobilized enzyme (%) ^b / S ^d (%) Activity Efficiency

Enzymatic load (mg / g) ^c catalytic ⁶ catalytic '

2h 86/112 _a 18 0.16

 24h 86/113 0.10 7 0.06

P-Glu ^h 0 0.00

³ The sample is designated as nh (number of hours of synthesis) of the biocatalyst β-Glu @ Mg-MOF-74-nh

b Percentage of enzyme immobilized in the solid versus total enzyme added. ^c mg of enzyme per g of biocatalyst.

d Percentage of S in the sample according to CHNS elementary analysis.

6 Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

f Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 3 of Example 1) in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

9 Not measured.

h Extract of the β-Glu enzyme after 10 minutes in DMF.

Figure 7 compares the diffractograms of a Mg-MOF-74 prepared at room temperature as published (Díaz-García et al., Cryst. Growth Des., 2014, 14, 2479), but containing 0.5 ml of water to match the added with the enzymatic extract, and of the biocatalyst p-Glu @ Mg-MOF-74-2h. The two diffractograms are typical of a MOF-74 structure of very small crystal size, and that contains an intercrystalline mesoporosity. Example 5. Method of obtaining the biocatalyst p-Glu @ Mg-MOF-74-20h, which comprises the synthesis of the Mg-MOF-74 system using N, N-dimethylformamide and in the presence of a greater amount of enzyme than Example 4 In this experiment the same conditions as in Example 4 are repeated, with the difference that a greater amount of the β-Glu enzyme is used. For the first solution, 0.566 g of Mg acetate tetrahydrate was added over 10.015 g of DMF, which dissolved in a few minutes. On the other hand, 0.211 g of 2,5-dihydroxyterephthalic acid (dhtp) was dissolved in 10,013 g of DMF to form the second solution. On the first solution, 0.5 ml of enzyme extract was added, containing β-Glu in a concentration of 24.76 mg enzyme / ml, and immediately the second solution dropwise.

A sample was taken at 5 minutes and centrifuged for 15 seconds at 12,500 rpm thus obtaining the supernatant. The solid was filtered under vacuum and labeled as β-Glu @ Mg-MOF-74-5min. The reaction lasted for 20 hours, after which the solid biocatalyst p-Glu @ Mg-MOF-74-20h was recovered according to the same procedure. Table 5 indicates that when there was more enzyme in the reaction medium (12.8 mg of enzyme versus 7.27 mg in Example 4), higher enzyme immobilization percentages (93% in Example 5 versus) were achieved. to 86% in Example 4), which indicates that the saturation of the system has not been reached, that is, the amount of enzyme offered in the medium could potentially continue to increase.

Table 5. Percentages of immobilization (by Bradford analysis); enzymatic load, catalytic activity and catalytic efficiency (by spectrophotometry); and content of Sulfur (by CHNS elemental chemical analysis) of the biocatalysts p-Glu @ Mg- MOF-74 -5min and -20h.

Sample ³ Immobilized enzyme (%) ^b / S ^d (%) Activity Efficiency

Enzymatic load (mg / g) ^c catalytic ⁶ catalytic '

5min 93/79 _a 2 0,02

20h 92/276 0.33 4 0.01 ³ The sample is designated as nmin or nh (number of minutes / hours of synthesis) of the biocatalyst p-Glu @ Mg-MOF-74-nmin -nh.

b Percentage of enzyme immobilized in the solid versus total enzyme added. ^c mg of enzyme per g of biocatalyst.

d Percentage of S in the sample according to CHNS elementary analysis.

6 Catalytic activity (expressed in units of activity U per g of biocatalyst and calculated according to equation 2 of Example 1), in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

f Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 3 of Example 1) in the hydrolysis of para-nitrophenyl-beta-D-glucopyranoside releasing paranitrophenol.

9 Not measured.

Table 5, in addition to ratifying the presence of the β-Glu enzyme in the p-Glu @ Mg-MOF-74 biocatalysts, indicates that this enzyme retained some catalytic activity despite having been in DMF just as it had been observed in the Example 4. Again, the Mg-MOF-74 material was not a simple host of the β-Glu enzyme that immobilizes, but also somehow helped to preserve its enzymatic activity against the solvent that inhibits that activity, DMF. Example 6. Electrophoresis in polyacrylamide gels of the biocatalysts obtained in Examples 1 to 5.

With all the biocatalysts obtained in Examples 1 to 5, electrophoresis was carried out in polyacrylamide gels (PAGE) with SDS (sodium dodecyl sulfate sulfate). Biocatalysts undergo the denaturing conditions of the technique (SDS, mercaptoethanol, 100 ° C, 5 min) that induces the complete loss of the tridimentional structure of the protein, which is reduced to the linear chain of amino acids and in this way should be easily released from the framework of the MOF. Supernatants undergo electrophoresis to investigate the presence of protein bands in the gel. Figures 8 and 9 show the electrophoresis corresponding to the biocatalysts of examples 1 to 5, all prepared with Beta-glu. The band corresponding to the enzyme was observed in the biocatalyst with the Mg-MOF-74 material, which certifies its presence. However, no protein band was observed in the NH ₂ -MIL-53 (AI) biocatalysts, which cannot mean the absence of protein, since this has been detected by the rest of the techniques used (determination of the enzymatic activity of the biocatalyst, Bradford analysis, CHNS elementary chemical analysis, as indicated in Tables 1 to 3, and by thermogravimetry (shown in Figures 2, 4 and 6)). What it can indicate is that the protein chain is more strongly chemically retained by interaction with the material, and more protected against leachate.

Example 7. Procedure for obtaining a solid lip @ Fe-BTC-NaOH-nh biocatalyst, comprising the synthesis of the MOF Fe-BTC system (similar to the MOF marketed as Basolite F300) using NaOH as a deprotonation agent and in the presence of the enzyme lipase

This example shows how to obtain a biocatalyst by immobilizing the enzyme Candida Lipase Antarctic B (CaLB) during the formation of the MOF Fe-BTC system in which sodium hydroxide (NaOH) was used as the base. The lipase enzyme of Candida Antarctica B has approximate dimensions of ~ 3 nm x ~ 4 nm x ~ 5 nm, a molecular weight -32 KDa, and an isoelectric point 6.0.

The first solution was prepared with 0.508 g of iron trichloride hexahydrate FeCI ₃ -6H ₂ 0 (metal source) and 10.0 g of deionized water, giving a pH of 1.36. On the other hand, the second solution was prepared with 0.263 g of the organic ligand benzene-1, 3,5-tricarboxylic acid, or trimesic acid (H ₃ BTC), 3.685 g of 1.06 M NaOH solution and 1 g of water deionized, giving a clear solution in a few minutes with a pH of 8.0. Then, 5 mL of Novozymes enzyme extract (EC 3.1.1.3) from Novozymes, supplied as a liquid enzyme preparation (Lipozyme Calb L), was added to the second solution, with a concentration of the extract of lipase measured by Bradford analysis of 5.9 mg / mL, which changed the pH to 7.83. Then, the first solution was added on the second solution, under stirring, which caused the formation of an orange brown solid in suspension practically immediately and at room temperature (25 ° C), giving a pH of 3.1, after a time that ranged between 1 min and 24 hours, different aliquots (suspension) were taken, from which the biocatalyst called Lip @ Fe (3) -BTC-NaOH-nh, was removed from the supernatant (solution) by centrifugation (13,400 rpm for 90 seconds) and filtration. The catalytic activity of both the suspension and the supernatant in the hydrolysis of the 0.4 mM para-nitrophenyl acetate (pNPA) substrate dissolved in phosphoric acid buffer (H ₃ PO4) / di-sodium hydrogen phosphate (NaHP0 ₄ -2H _{2) was} measured 0) 0.05 M pH 7.0, to give p-nitrophenol and acetic acid at 25 ° C (Figure 1 1a). To prepare the substrate solution, 80 μΙ_ of 200 mM pNPA in acetonitrile is added over 40 ml_ of 0.05 M sodium phosphate buffer pH 7.0, with a rapid mixing agitation rate. This solution is stored in an ice bath.

The catalytic activity was measured by spectrophotometry at a wavelength of 348 nm, with an array diode spectrophotometer (Agilent 8453 UV-Vis) provided with thermostatization and a magnetic stirring device to keep the samples in homogeneous suspension during the tests. On the test cuvettes, which contained 1.9 ml_ of substrate solution consisting of 0.4 mM pNPA in 0.05 M buffer at pH 7.0, 50 μΙ_ of soluble enzyme or 50 μΙ_ of the suspension was added or 50 μΙ_ of the supernatant. The molar extinction coefficient of p-nitrophenol measured under these conditions was 5150 M ^"1 cnT ¹ .

When the catalytic activity of the supernatants obtained from aliquots taken at different times remained constant or zero, the process of immobilization of the enzyme was terminated. The concentration of non-immobilized enzyme present in the supernatant as well as in the initial enzyme solution were determined by Bradford analysis.

After the immobilization monitoring was completed (after a maximum of 24 hours), the remaining solid was filtered under vacuum using a Vidra FOC (666/1) size plate. Pore 4 and filter paper Millipore 0.45 μηι HV. The biocatalyst obtained once dry, heavy and ground, was resuspended (between 5 and 20 mg) and its catalytic activity was tested in the hydrolysis reaction of tributirin or 1, 2,3-Glyceroltributirine (TB) (Figure 1 1 b) . This reaction is more specific than the previous one and is based on the release of butyric acid that is generated by contacting tributirin and lipase.

A Mettler Toledo DL-50 pHstate is used to monitor this reaction. The experimental procedure that has been carried out in each reaction has been: 48.5 mL of phosphoric acid buffer (H ₃ P0 ₄ ) / di-sodium hydrogen phosphate (NaHP0 ₄ -2H ₂ 0) 10 mM pH is stirred in a vessel 7.0 and 1.47 mL of 0.1 M tributyrin. Next, a known biocatalyst mass, mB (between 5-20 mg) is introduced. The recording of the rate of addition of 0.1 M soda, v _Na0H , to maintain the constant pH of 7.0 gives us a line whose slope corresponds to the rate of hydrolysis, and therefore, the activity of the enzyme. Tributirin units (U _T B) are calculated from Equation 4:

/ _ v _NaOH (mL / min) -100 ^ mol / mL) _{(Ec 4)}

^Υ ΤΒ \ ^Υ ΎΒ ') - ^~

m _B (g)

The catalytic efficiency of an immobilized biocatalyst is defined as the activity / load ratio, using Equation 5:

_^ _, U _TR I g catalyst CFr 'i

Catalytic Efficiency (U _TB / mg Lipase) = ->

 C immobilized arga {mg Lipase I g support) where U are the units of catalytic activity of the enzyme defined as transformation of 1 μηιοΙ of substrate per minute.

Table 6 shows the percentage of immobilized lipase enzyme, determined by Bradford analysis by measuring the initial protein concentration in the enzyme solution and protein in the supernatant after 10 min (0.17 h), 1 h, 4 h and 22 hours. After 10 min, 95% of the enzyme present in the medium was immobilized, while after 22 h 87% of the enzyme was immobilized. These percentages of enzymatic immobilization indicate that as soon as the metal source solutions are contacted with the organic ligand + enzyme solution mixture, practically all the enzyme present in the medium is instantly immobilized in the MOF. Enzymatic load (Table 6) was determined both by difference between the activity of the suspension and the activity of the supernatant measured spectrophotometrically in the p-NPA hydrolysis test (Figure 11 a), and from the milligrams of protein in the synthesis medium (calculated from analysis Bradford) and the grams of biocatalyst obtained after filtering the final synthesis solution.

Table 6. Lipase immobilization percentages (by Bradford analysis); enzymatic load, catalytic activity and catalytic efficiency (by spectrophotometry); and nitrogen and sulfur content (by CHNS elemental chemical analysis) of the Lip @ Fe-BTC-NaOH-nh biocatalysts.

³ The sample is designated as Lip @ Fe-BTC-NaOH-nh where n is the number of hours of synthesis of the biocatalyst.

 b Percentage of enzyme immobilized in the solid versus total enzyme added. c mg of enzyme immobilized per g of recovered biocatalyst.

 d Percentage of N and S in the sample according to CHNS elementary chemical analysis.

⁶ Catalytic activity (expressed in units of U _T B activity per g of biocatalyst and calculated according to equation 4), in the hydrolysis of tributirin releasing butyric acid. f Catalytic efficiency (expressed in units of activity U per mg of enzyme and calculated according to equation 5) in the hydrolysis of tributyrin releasing butyric acid.

The presence of enzyme in the biocatalyst was also detected qualitatively by elementary chemical analysis CHNS (Table 6), which gives the content of carbon, hydrogen, nitrogen and sulfur. These analyzes were carried out on a LECO CHNS-932 Elemental Analyzer device. In good agreement with the estimate from the Bradford method, the Fe-BTC-NaOH-nh biocatalysts have both nitrogen and sulfur, but the MOF material without enzyme in which the nitrogen and sulfur content was 0.0% . Additionally, Table 6 also shows the catalytic activity (expressed in U / g biocatalyst) and the catalytic efficiency (in U / mg protein) of the biocatalyst obtained after being resuspended and calculated from equations 4 and 5 above, respectively. It should be noted that catalytic activity and catalytic efficiency increase as immobilization time increases. The activity of the free enzyme is 370U / mg. Therefore, the lipase immobilized in the Lip @ Fe-BTC-NaOH-22h biocatalyst practically maintains the same activity as the native enzyme, whose activity is 370.23 U / mg.

On the other hand, the Lip @ Fe-BTC-NaOH-nh biocatalysts obtained, once dried, were characterized with various physicochemical techniques, such as thermogravimetric analysis. Figure 12 shows the thermograms (TGA) and their derivatives (DTG) of the MOF Fe-BTC-NaOH without enzyme, the Lip @ Fe-BTC-1 h biocatalyst and the Lipase enzyme extract (Figure 12). The thermograms were recorded on a Perkin-Elmer TGA7 device with a temperature scan of 20-900 ° C at a heating rate of 20 ° C / min under a dry air flow of 40 mL / min. In the thermogram of the Lip @ Fe-BTC-1h biocatalyst a double weight loss was detected in the temperature range between 130-342 ° C, with a pattern similar to that detected in the thermogram of the enzyme extract in temperature ranges similar while in the thermogram of the MOF without enzyme there was no appreciable weight loss in that interval, which evidences the presence of enzyme in the biocatalyst.

Claims

1. - Procedure for obtaining a biocatalyst, characterized in that it comprises the following steps: a) partially or totally synthesizing a metallo-organic material (MOF) in the presence of at least one enzyme until obtaining a solid and, b) isolating the solid synthesized according to step a), and where the enzyme is immobilized in-situ during the synthesis of the MOF in at least one of the hollow volumes of diameter between 2 and 50 nm of the intercrystalline mesoporosity formed between crystals or between agglomerated or aggregated domains of homogeneous size nanocrystalline particles 2. - Method according to claim 1, characterized in that in step (a), the synthesis of the MOF comprises contacting a first solution of a metal source and another second solution of organic ligand, in the presence of at least one enzyme. 3. - Method according to any of claims 1 and 2, characterized in that in step (a) the solutions are aqueous. 4. - Method according to any of claims 1 and 2, characterized in that in step (a) the solutions are organic or an aqueous-organic mixture. 5. - Method according to any of claims 1 to 4, characterized in that in step (a) the second organic ligand solution is chosen from an aqueous solution of an organic ligand salt or an aqueous solution of protonated organic ligand in the presence of a deprotonating agent. 6. - Method according to claim 5, characterized in that the deprotonant agent is selected from a strong base, a medium base or a weak base. 7. Method according to any of claims 1 to 6, characterized in that step (a) is carried out at a temperature between 4 and 70 ° C and preferably between 20 and 30 ° C. 8. Method according to any of claims 1 to 7, characterized in that in step (a) the enzyme is added on either of the two solutions, before the synthesis of MOF. 9. - Method according to any of claims 1 to 7, characterized in that in step (a) the enzyme is added on the mixture of the two solutions, before the synthesis of MOF. 10. - Method according to any of claims 1 to 9, characterized in that the enzyme is selected from β-glucosidases and lipases. 1. Method according to claim 10, characterized in that the enzyme is a β-glucosidase. 12. - Method according to claim 10, characterized in that the enzyme is a lipase. 13. - Method according to any of claims 1 to 12, characterized in that the isolation of the solid of step (b) is carried out by centrifugation or filtration. 14. Method according to any of claims 1 to 13, characterized in that it additionally comprises a stage (c) for drying the solid isolated in step (b). 15. Method according to any of claims 1 to 1 1 and 13-14, characterized in that the first solution of the metal source is an aqueous solution of nonahydrated aluminum nitrate, the second solution of the organic ligand is an aqueous solution of acid 2-aminoterephthalic and an agent Deprotonant, the reaction temperature 25 ° C and the enzyme is Aspergillus niger β-glucosidase which is added on the organic ligand solution. 16. - Method according to claim 15, characterized in that the deprotonant agent is selected from triethylamine, sodium hydroxide and ammonia. 17. - Method according to any of claims 1 to 1 1 and 13-14, characterized in that the first solution of the metal source is a solution of Mg acetate tetrahydrate on Ν, Ν-dimethylformamide, the second solution of the organic ligand is a solution of 2,5-dihydroxyterephthalic acid on N, N-dimethylformamide, the reaction temperature 25 ° C and the enzyme is Aspergillus niger β-glucosidase which is added on the solution of the metal source. 18. - Method according to any of claims 1 to 10 and 12, characterized in that the first solution of the metal source is a solution of iron chloride (lll) hexa hydrate or, the second solution of the organic ligand is an acid solution Benzene-1, 3,5-tricarboxylic and a deprotonant agent, the reaction temperature 25 ° C and the enzyme is Candida Antarctic B lipase which is added on the organic ligand solution. 19. - Process according to claim 18, characterized in that the deprotonant agent is sodium hydroxide. 20. - Biocatalyst obtained by the process according to any of claims 1 to 19.