WO2010139837A1 - Covalent complexes of lipases with proteins, dna probes, cofactors or other biomolecules - Google Patents

Abstract

The invention relates to complexes that include lipases covalently bonded, by means of regions not involved in the active centre thereof, to biomolecules of interest (proteins such as enzymes, antibodies or proteins A or G, cofactors or DNA probes). The complexes can be immobilised via the intact active centre of the lipases on all types of hydrophobic media, without requiring any modifications or activations thereof. The bonding of the lipase to the hydrophobic media is reversible, allowing the reversible immobilisation of the complexes. The invention provides two methods, chemical and biological, for preparing said complexes, as well as a method for immobilising said complexes. Said immobilised complexes can be used in industry, in laboratories or in diagnostics, given the potential use thereof as biocatalysts or as biosensors. The chemical method includes adsorbing the lipase to a porous hydrophobic medium, combining the lipase thus adsorbed with the biomolecule, and desorbing said complex from the medium using detergents. The biological method includes inserting the gene of the biomolecule in a plasmid containing the gene of the lipase, in a microorganism, in order for the latter to express the lipase fused with the biomolecule. The reversible immobilisation of the complexes generated using either of the two methods can be carried out on non-porous hydrophobic media.

Description

 COVALENT COMPLEXES OF LIPASES WITH PROTEINS, DNA PROBES, COFACTORS OR OTHER BIOMOLECULES

The present invention falls within the field of biochemistry and molecular biology and refers to complexes comprising covalently linked lipases, through regions not involved in their active center, to biomolecules of interest (proteins or other biomolecules). The complexes can be subsequently immobilized through the intact active center of the lipases on all types of hydrophobic supports. It also refers to two methods, chemical and biological, of preparing these complexes and a method of immobilization thereof. These immobilized complexes can have applications in the industry, in the laboratory or in diagnosis, due to their potential use as biocatalysts or as biosensors.

STATE OF THE PREVIOUS TECHNIQUE

The immobilization of biomolecules of industrial or biomedical interest is, in many cases, a necessary stage in the development of biocatalysts and biosensors. Most of the supports available for this purpose are hydrophobic in nature, such as, for example, most commercial magnetic particles (such as ferrite cores coated with polystyrene), carbon nanotubes, multiwell plates, some plates for preparing arrays , etc. However, the immobilization of proteins and other biomolecules on these hydrophobic surfaces generates a series of problems, such as the inactivation of the biomachromolecule (Mateo, C, et al., 2002, Biotechnology Progress, 18: 3, 629-634). In addition, these types of supports could adsorb analytes in a non-specific way, which makes it difficult to use them as biosensors. Some of these nanostructures are derivatized with difficulty, even once derivatized they cannot be easily manipulated. This is the case of magnetic nanoparticles, which can be added in the presence of co-solvents, high ionic strength, polyfunctional reagents, etc. Sort out these inconveniences would imply the possibility of applying, in the industry or in diagnosis, these immobilized biomachromolecules.

On the other hand, the development of reversible immobilization techniques would provide a series of advantages in the immobilization of biomolecules. For example, when the support is very expensive or the reactor very complex, the possibility of reuse would be interesting (You, C, et al., 2009, Talanta, 78: 3, 705-710).

Methods of covalent immobilization of biomolecules on carriers and membranes have been developed in the presence of His-Tag, whose amino acid residues are directly involved in covalent binding. To increase the probability of immobilization, the hydrophobicity of the membrane can be increased (EP0991944 A1).

On the other hand, the non-specific binding of biomolecules to carbon nanotubes can be overcome by binding the target proteins to polyethylene oxide chains present in the nanotubes (Robert J. Chen, 2003, PNAS, 100: 9, 4984 ^ 1989).

Another approach is the coating of the nanoparticles with silica for the binding of active biomolecules, such as antibodies, enzymes or nucleotides, to mark cells, detect and / or isolate nucleic acid molecules that have specific nucleotide sequences, etc. (WO03089906 A2).

The regeneration of cofactors such as NAD, NADP, NADPH, ATP, UDP, etc., is a problem in the design of a multitude of biotransformations. A solution involves the immobilization of the cofactor in non-porous nanoparticles, using the enzymes involved in the process also in this type of support (Liu, W., et al., 2009, Journal of Biotechnology, 139: 1, 102-107) . However, it would be necessary to have reversible immobilized cofactors in particles, so as to simplify the recycling systems of these molecules. In the case of enzymes, in those that must act on insoluble substrates (such as cellulases or proteases), it would be interesting to use non-porous nanoparticles to immobilize large amounts of these enzymes on the surface of the support and to be able to perform this type of reactions. However, its small size makes its handling complicated. The use of magnetic particles would be a simplification, but given its high price it is a system of difficult use in the short term. In addition, most of the nanoparticles that are stable are hydrophobic, which causes their aggregation and the possible inactivation of the biomacromolecules immobilized on them.

Lipases have a great affinity for hydrophobic surfaces (Bastida, A., et al., 1998, Biotechnology and Bioengineering, 58: 5, 486-493) and exist in nature in two conformations. They have their active center in an extremely hydrophobic pocket and perform their functions at the interface of the drops of their substrates (oil drops). In homogeneous aqueous media, the majority form of lipases is that in which the active center is isolated from the medium by a polypeptide chain called "lid", being considered inactive. In the presence of hydrophobic surfaces, lipases undergo a conformational change, adsorbing them through the inner face of the lid (which is hydrophobic) and the hydrophobic areas surrounding the active center, which leads to strong adsorption. An example of this is that lipases can be exposed to concentrations of 30-40% organic cosolvents without being released into the environment. However, despite this great affinity for hydrophobic supports, they can be easily recovered without the need to induce modifications, but simply by washing them with high concentrations of detergents, which are capable of releasing lipases (Bastida, A., et al., 1998, Biotechnology and Bioengineering, 58: 5, 486-493).

The development of simple methods of immobilization of biomolecules on nanostructures and other hydrophobic supports is essential to carry out the applications of them. These methods must allow the selective union of the biomolecule of interest, that is, without there being nonspecific interactions with other molecules, since otherwise their applications as biocatalysts or as biosensors would be very limited. In addition, in some cases it would be interesting that these methods allow the reuse of the support once it has been used.

DESCRIPTION OF THE INVENTION

The present invention provides complexes comprising covalently linked lipases, through regions not involved in their active center, to biomolecules of interest (proteins or other biomolecules). The complexes can be subsequently immobilized through the intact active center of the lipases on all types of hydrophobic supports. It also refers to two methods, chemical and biological, of preparing these complexes and a method of immobilization thereof. These immobilized complexes can have applications in the industry, in the laboratory or in diagnosis, due to their potential use as biocatalysts or as biosensors.

Lipase acts as a transport protein and is immobilized by interfacial activation on hydrophobic magnetic particles or other hydrophobic supports without the need for any modification or prior activation of the supports. So, once the biomolecule is attached to the lipase, the immobilization of this complex on a hydrophobic support can be carried out avoiding the inactivation of the biomolecule, since the lipase is adsorbed by interfacial interaction on the hydrophobic nanoparticle. The immobilization of lipase is rapid, intense, reversible and can be performed under mild experimental conditions. Once the nanostructures or hydrophobic supports have been used, they can be recovered by desorbing the complexes with high concentrations of detergent, thus allowing the reuse of the supports for the adsorption of new complexes. The complexes are simple to prepare, both by chemical and biological methods. In addition, this immobilization of biomolecules on surfaces, even when they are small, can be quantified following the catalytic activity of the transporter lipase. The fact that the transport protein, that is, lipase, has a high catalytic activity towards synthetic substrates facilitates the quantification of very small amounts of biomolecules (of the order of micrograms) that are immobilized on the surfaces of the nanostructures.

The immobilization of biomolecules on hydrophobic supports by means of the method provided by the present invention reduces unwanted nonspecific interactions and could even allow the stabilization, by co-valent multiple-junction, of enzymes on rigid transporter lipases.

Therefore, a first aspect of the invention refers to a complex, hereinafter "complex of the invention", which comprises a lipase covalently linked to a biomolecule.

In this description "lipase" means the enzyme present in the lysosomes and released by the pancreas, responsible for the breakdown of fats or lipids into fatty acids. A "biomolecule" is any constituent organic molecule of living beings, such as, but not limited to, carbohydrates, lipids, proteins, cofactors or nucleic acids; In the present description, nucleic acid probes are also included within the biomolecules.

In a preferred embodiment of this aspect of the invention, the lipase comes from a thermophilic microorganism, and in a more preferred embodiment the thermophilic microorganism is Bacillus thermocatenulatus or Thermus thermophillus. Although any lipase capable of adsorbing strongly to hydrophobic surfaces could be used in the present invention, the more stable the lipase is, the greater the range of conditions in which it can be used and more variety of chemical modifications can be made on it to optimize The immobilization of the biochromolecule. In addition, the greater the stiffness of the lipase molecule, the greater the possibility of stabilizing the biomolecule to which it is joined by covalent multipoint union. Therefore, the lipases of this invention preferably are derived from thermophilic microorganisms, which are defined as those living microorganisms and develop under extreme temperature conditions, ie, above 45 ⁰ C, the optimum temperature is from 50 to 75 ⁰ C and its maximum temperature between 80 and 113 ⁰ C. Examples of thermophilic microorganisms could be, but not limited to, Pyrococcus furíosus, Thermus aquaticus, Thermus thermophilus, Chloroflexus aurantiacus, Thermococcus littorales, Pyrodictium abyssi (archaea), Bacillus stearothermophilus, Rhodothermusicus, Rhod obamensis, Marínithermus hydrothermalis, Deferríbacter desulfuricans, Thermodesulfobacterium hydrogeniphilum or Bacillus thermocatenulatus.

"Covalent bonding" means the bond in which the atoms of two nonmetallic elements are held together because they share two electrons. In the present invention, covalent bonds between lipases and biomolecules are made through regions not involved in the active center of lipase, thus keeping the active center intact to subsequently immobilize the complex on a hydrophobic support.

In another preferred embodiment of this aspect of the invention, the biomolecule of the complex of the invention is a protein. In a more preferred embodiment, the protein is an antibody. In another preferred embodiment the protein is: protein A or protein G. In another preferred embodiment the protein is an enzyme. By "protein or peptide" is meant any macromolecule formed by linear chains of amino acids linked by peptide bonds, whether it has a structural, regulatory, catalytic or enzymatic, transport, defensive or contractile function. Both simple proteins (consisting only of amino acids) and conjugates (consisting of amino acids and prosthetic groups) are included within this definition. Examples of proteins are, but are not limited to, hormones, antibodies, enzymes, receptors, etc.

In this description, "antibodies or immunoglobulins" means those glycoproteins produced by B cells in response to an antigen. The typical antibody consists of basic structural units, each with two large heavy chains and two smaller light chains. Although the general structure of all antibodies is very similar, a small region is extremely variable, which allows the existence of millions of antibodies, each with a slightly different end. This part of the protein, called hypervariable region, is that which gives the antibody the specificity of binding to its antigen.

The lipase binding to an antibody can be carried out through its glycosyl chain (after oxidation with periodate), by reaction with the amino groups of the lipase plants. This immobilization maintains the ability of the antibody to recognize its antigen.

Protein A is a 42,000 daltons polypeptide that is the usual constituent of the Staphilococcus aureus cell wall, has four antibody binding sites, however, only one can be used at a time. Protein A is bifunctional, allowing the formation of multimeric complexes. Any antibody has at least two binding sites to protein A. Due to these properties, protein A is capable of recognizing antibodies of different species, such as humans, donkey, rabbit, dog, pig or guinea pig, and with a lower affinity, but still useful, for mouse, cow or horse antibodies, between others, because of what has been used in many immunohistochemical methods. Protein A is purified from both natural sources (S. aureus) and from recombinant protein overexpression systems. Protein G is a polypeptide of between 30,000 and 35,000 daltons isolated from the cell wall of beta-hemolytic streptococcus of strains C or G. It has an affinity for antibodies different from that of protein A, which is complemented by it, being also useful for immunohistochemical methods. Proteins A and G bound to a lipase and immobilized on magnetic particles or hydrophobic nanotubes, or in general on any hydrophobic support, are useful both for the purification of antibodies (used in mixtures containing even suspended solids) and for the oriented immobilization of antibodies and their use as biosensors.

The "enzymes" are substances of a protein nature that catalyze chemical reactions, when it is thermodynamically possible, decreasing the level of the "activation energy" of the reactions. These enzymes can be, but not limited to, oxidoreductases, transferases, hydrolases, isomerases, Nasas or ligases. Examples of enzymes could be, but are not limited to, dehydrogenases, kinases, glucosidases, decarboxylases, synthetases, etc. There are enzymes that act on insoluble substrates or cofactors, examples of them are, without limitation, cellulases or proteases. The enzymes that are part of the complex of the invention are preferably enzymes that act on insoluble substrates or cofactors.

In another preferred embodiment, the biomolecule of the complex of the invention is a cofactor. In a more preferred embodiment, the cofactor is selected from the list comprising: NAD (P) ⁺ , NAD (P) H, ATP, ADP or FAD. In another preferred embodiment, the biomolecule is a DNA probe.

By "cofactor" is meant any non-protein, thermostable and low molecular weight component, necessary for the action of an enzyme, whether metal ions or organic molecules or coenzymes. Examples of cofactors are, but not limited to, Fe ²⁺ , Cu ²⁺ , K ⁺ , Mn ²⁺ , Mg ²⁺ , vitamins, NAD (P) ⁺ , NAD (P) H, ATP, ADP or FAD.

"DNA probe" means any fragment of fluorescently labeled DNA.

In another preferred embodiment, the complex of the invention also comprises an activatable polymer between the lipase and the biomolecule. In a more preferred embodiment the activatable polymer is dextran.

"Activable polymer" means any polymer that, when modified with any chemical group, facilitates the union between a lipase and another biomolecule. An example of an activatable polymer is, without limitation, dextran. "Dextran" is understood as a complex and branched polysaccharide formed by many glucose molecules linked in chains of variable length (from 10 to 150 kilodaltons), which is used as an antithrombotic because it reduces the viscosity of the blood. The chain consists of α1-6 glycosidic junctions, while branching begins at α1-4 junctions (in some cases also at α1-2 and α1-3 junctions). Dextran is synthesized from sucrose by certain lactic acid bacteria, of which the best known are, but not limited to, Leuconostoc mesenteroides and Streptococcus mutans. If a lipase is modified with dextran and this in turn joins a cofactor, it would be possible to have magnetic particles, or in general any hydrophobic support, with the cofactor immobilized reversibly, greatly simplifying the recycling systems of these molecules. In the same way, the union of lipase to dextran and this to a DNA probe allows obtaining the immobilized probe reversibly on magnetic particles, or in general on any hydrophobic support. Therefore, the activatable polymer, preferably dextran, is between the lipase and the biomolecule, preferably when the biomolecule is a cofactor or a DNA probe. In another preferred embodiment, the complex of the invention is immobilized on a non-porous hydrophobic support. In a more preferred embodiment, the hydrophobic support is selected from the list comprising: carbon nanotube, hydrophobic array, hydrophobic array, multiwell plate, hydrophobic magnetic nanoparticle or hydrophobic non-magnetic nanoparticle. In an even more preferred embodiment, the hydrophobic magnetic nanoparticle is a ferrite core coated with polystyrene.

"Non-porous hydrophobic support" is understood as any support, including nanostructures, which is repelled by water and lacking pores.

Examples of non-porous hydrophobic supports are, without limitation, carbon nanotube, hydrophobic array, hydrophobic array, multiwell plate, hydrophobic magnetic nanoparticle or hydrophobic non-magnetic nanoparticle.

An example of a hydrophobic magnetic nanoparticle is, without limitation, a polystyrene coated ferrite core. It is understood as "ferrite core" that which is formed by iron powder combined with other elements that give very good magnetic properties. It is understood as

"polystyrene" the thermoplastic polymer that is obtained from the polymerization of styrene.

Another aspect of the invention relates to a method of chemical preparation of the complex of the invention, hereafter referred to as the "first method of the invention", which comprises:

to. adsorb a lipase to a hydrophobic, porous and inert solid support, b. conjugate the lipase of step (a) with a biomolecule, and c. desorb the complex obtained in step (b) of the support.

In a preferred embodiment of this second aspect of the invention, the hydrophobic solid support of step (a) is: octyl agarose or glass coated with C4-C18 alkyl groups In another preferred embodiment, the solid support The hydrophobic of step (a) has a hydrophilic surface coated with hydrophobic nanostructures. In a more preferred embodiment, the hydrophobic nanostructures are selected from the list comprising: hydrophobic proteins, hydrophobic protein molecules, hydrophilic proteins modified with hydrophobic reagents, hydrophobic polymers or hydrophilic polymers modified with hydrophobic reagents.

In the present description, "hydrophobic, porous and inert solid support" means any support, including nanostructures, which is repelled by water and which has pores. Examples of hydrophobic supports of this type are, but are not limited to, octyl agarose or glass coated with alkyl groups. By "alkyl groups" is meant the organic functional groups formed by the removal of a hydrogen atom from a hydrocarbon that are always attached to another atom or group. The hydrophobic support used in step (a) of the first method of the invention can also have a hydrophilic surface, understanding as such any surface that exhibits affinity for water, modified by coating with hydrophobic nanostructures. In the present description, "nanostructure" means a structure with an intermediate size between the molecular and microscopic structures, of micrometric size. Examples of hydrophobic nanostructures could be, but are not limited to, hydrophobic proteins, hydrophobic protein molecules, hydrophilic proteins modified with hydrophobic reagents, hydrophobic polymers or hydrophilic polymers modified with hydrophobic reagents. An example of hydrophobic proteins could be, but not limited to, hydrophobins.

The complex of the invention is synthesized in solid phase, in a previous stage, in a porous hydrophobic support where intermolecular interactions between adsorbed biomolecules are impossible, which prevents their aggregation. This can be done thanks to the possibility of immobilizing lipases reversibly on hydrophobic supports. The lipase immobilized in the porous hydrophobic solid support is conjugated with the biomolecule of interest, as shown in the examples of the present invention. So that at the end of this stage of the process, the lipase is obtained together with the porous hydrophobic solid support and the biomolecule of interest. Subsequently, the porous support complex is separated by detergents, as shown in the examples of the present invention, to obtain the complex formed by the lipase and the biomolecule in its soluble form.

In another preferred embodiment, the lipase of step (a) is modified with dienophiles, with ionized amino groups coated with epoxy groups or glutaraldehyde groups or with activated polymers to chemically conjugate with biomolecules. In a more preferred embodiment, the activated polymers contain carboxyl groups, epoxy groups or amino groups.

"Dienophiles" means any substance that is attracted to dienes. "Amino groups" are functional groups derived from ammonia or some of its alkylated derivatives by elimination of one of its hydrogen atoms. "Ionization" means the chemical or physical process by which ions are produced. By "epoxy group" is meant the radical formed by an oxygen atom attached to two carbon atoms, which in turn are linked together by a single covalent bond. By "glutaraldehyde" is meant a simple five-carbon molecule with aldehyde groups at each end, of chemical formula OCH-CH ₂ -CH ₂ -CH ₂ -CHO. Examples of activated polymers to be conjugated with biomolecules with which the lipase modification could be carried out are, without limitation, dextran or polyethylene glycol. By "carboxyl group" is meant a group of organic formula COOH attached to an organic moiety, represented as R.

In the cases in which the stability of the biomolecule is much lower than that of the lipase, the chemical chimera through the multi-point covalent junction would have advantages. For this, all carboxyl groups of lipase can be transformed into amino groups by modification with ethylenediamine. These groups can then be modified with 1,4 butanediol diglycidyl. ether, to generate epoxide groups, or with glutaraldehyde. Even the biomolecule of interest could be adsorbed by ion exchange on the aminated lipase and subsequently treated with glutaraldehyde. All these techniques allow a great activation of lipase, which could allow a very intense multipoint covalent union of the biomolecule with lipase, increasing its stability.

Another aspect of the invention relates to a method of biological preparation of the complex of the invention, hereinafter "second method of the invention", which comprises inserting the gene of the biomolecule into a plasmid containing the lipase gene.

Preferably, the second method of the invention is carried out when the biomolecule of the complex of the invention is a protein. "Plasmid" means the extrachromosomal nucleic acid fragment found in the cytoplasm of some prokaryotes, of varying size but smaller than the main chromosome, with a linear, circular or super-rolled structure. To the thermophilic microorganisms containing the lipase gene in a plasmid, they can be inserted, by means of genetic engineering techniques known by any expert in the field, for example but without limiting ourselves, through the use of restriction enzymes, the gene of Ia biomolecule of interest, for example but not limited to, a protein. By cutting, with the same restriction enzyme, the lipase gene and the biomolecule gene of interest, compatible ends are generated for the binding of both genes in reading phase by, for example, but not limited to, a ligase . The microorganism that carries the plasmid containing this genetic construction expresses the lipase-biomolecule fusion protein. In this way the complex of the invention is obtained ready to be immobilized on a support.

Another aspect of the invention relates to the use of the complex of the invention for the immobilization of biomolecules in hydrophobic supports. Another aspect of the invention relates to a method of obtaining the complex of the invention immobilized in a non-porous hydrophobic support, hereinafter "third method of the invention", comprising the steps of the first method of the invention or the second method of the invention, and also:

d. adsorb the complex of the invention to a non-porous hydrophobic support.

Once the complex of the porous hydrophobic solid support where the conjugation with the biomolecule of interest is performed, or obtained the complex by means of the recombinant fusion of the lipase with the biomolecule, the next step is to adsorb it to a non-porous hydrophobic support or nanostructure without No type of activation or modification, by means of the procedure described in the examples of the present invention. So, after this step, the complex formed by the lipase linked to the biomolecule immobilized on a hydrophobic support is obtained, useful for use in industry, in the laboratory or in diagnosis.

In a preferred embodiment, the third method of the invention also comprises:

and. coating the non-porous hydrophobic support with other lipases prior to the adsorption of step (d).

The non-porous hydrophobic support can be coated with other unmodified and low molecular weight lipases, to completely cover the surface of the support with hydrophilic and inert protein structures. With this, a more inert surface is obtained, so that there is hardly any hydrophobic surface of the support exposed to the medium.

Once used, the nanostructures or non-porous hydrophobic supports of step (d) containing the complexes can be recovered by desorbing the complexes with high concentrations of detergents, and thus can be reused as supports for new complexes to immobilize biomolecules. In addition, the use of detergents allows complexes to be obtained in their soluble form. So that the immobilization of the complexes of the invention is reversible, thanks to the ability of the lipases to be desorbed from the supports through the use of detergents.

Therefore, in another preferred embodiment, the third method of the invention also comprises desorbing the non-porous hydrophobic support complex of step (d) by using a detergent. In a more preferred embodiment, the detergent is sucrose laurate.

In this description, "detergent" is understood as any substance capable of breaking the lipid barrier by solubilizing the proteins and interrupting the lipid-lipid, lipid-protein and protein-protein interaction.

Another aspect of the invention relates to the use of the complex of the invention as a biocatalyst.

In this description "biocatalyst" is understood as any catalyst for the biochemical reactions of living beings, which reduces the activation energy of a chemical reaction, making it faster. By immobilizing the complexes of the invention formed by lipases and, for example, but without limiting ourselves, proteins, preferably enzymes, or cofactors, biocatalysis of macromolecules or, in general, any biochemical reaction can be carried out. Therefore, the biomolecules that are part of the complex of the invention when it is used as a biocatalyst are preferably enzymes or cofactors.

Another aspect of the invention refers to the use of the complex of the invention as a biosensor. In this description, "biosensor" means a system for the measurement (quantitative or qualitative) of biological or chemical parameters, which usually combines two components of a biological nature or a component of a biological nature and another of a physical-chemical nature.

The biomolecules of the complex of the invention that can be used as biosensors, can be, without limitation, cofactors, DNA probes or proteins, preferably enzymes, antibodies or proteins A or G. Proteins have the ability to bind to other proteins, as they can being the antibodies, so having proteins immobilized on a support enables binding with antibodies of interest for their application, for example, but not limited to, in diagnosis. Therefore, in a preferred embodiment, the lipase bound biomolecule in the complex of the invention that is used as a biosensor is a protein, for use in the reversible immobilization of antibodies, and more preferably the protein is protein A or protein G In another preferred embodiment, the lipase bound biomolecule in the complex of the invention that is used as a biosensor is an antibody for the detection of antigens.

DNA probes can hybridize, in complementarity, with nucleic acid fragments. Therefore, in another preferred embodiment, the biomolecule bound to the lipase in the complex of the invention that is used as a biosensor is a DNA probe, for use in the detection of DNA fragments.

Another aspect of the invention relates to the use of the complex of the invention for the regeneration of immobilized cofactors. Due to the ability of the immobilized complexes of the invention to be desorbed from their supports through the use of detergents, as explained above, it is possible to recycle both the biomolecules and the supports once they are used. When the biomolecule is a cofactor, the immobilized complex can be used for, for example, but not limited to, catalyze redox processes, phosphorylation processes, etc. After which, thanks to the union of lipase to support is reversible, the lipase-cofactor complex can be desorbed and thus the cofactor can be reused for future use.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Represents the immobilization course of the BTL-DH chimera on unmodified magnetic nanoparticles. (O) BTL enzymatic activity in the supernatant; (^) BTL activity in suspension; (•) BTL activity in the soluble complex.

Fig. 2. Shows the lipase complexes with biomolecules that can be prepared by the methods of the invention.

Fig. 3. Shows the lipase complexes with biomolecules immobilized on hydrophobic magnetic nanoparticles.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which show the effectiveness of the complexes comprising lipases linked to biomolecules, as well as the methods of preparation and immobilization thereof. These specific examples that They provide serve to illustrate the nature of the present invention and are included for illustrative purposes only, so they are not to be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1. Lipase enzyme complex.

PREPARATION AND IMMOBILIZATION OF THE LIPASA- DEHYDROGENASE COMPLEX

A thermostable lipase from Bacillus thermocatenulatus (BTL) is used as a transporter lipase and as a biomolecule of industrial interest, the enzyme Pseudomonas sp. (DH).

2 mg of BTL are adsorbed per 1 g of agarose-octyl by hydrophobic adsorption at 25 ⁰ C in sodium phosphate 10 mM pH 7. The BTL adsorbed is enriched in amino groups by treatment with 1 M ethylenediamine at pH 4, 75 and 25 ⁰ C for 1.5 h by adding 10 mM carbodimide. This amination transforms all the carboxyl groups of lipase into amino groups, these groups can be modified with 1,4 butanediol diglycidyl ether to generate epoxide groups, or with glutaraldehyde. Even, the enzyme can be adsorbed by ion exchange on the aminated lipase and subsequently treated with glutaraldehyde. This modification of the lipase is carried out in order to establish a multi-point covalent union in cases where the stability of the enzyme is much lower than that of the lipase. Subsequently, Ia BTL aminated solid phase is incubated for 16 h at 25 ⁰ C in a solution of diglycidyl butanediol ether: 16% (v / v) in 100 mM sodium bicarbonate at pH 9 and 10% (v / v) acetone . Each gram of BTL immobilized and enriched in ionized amino groups coated by epoxy groups offers 0.9 mg dehydrogenase (DH) in 9 mL of sodium phosphate buffer 10 mM at pH 7. In this way 0.5 mg of DH is immobilized on 1 mg of BTL (100% of the DH offered and 25% of the maximum possible). Subsequently, the BTL-DH complex 16 has 25 ⁰ C and then incubated Ia entire DH is covalently joined to the BTL. Next, each gram of DH-BTL-agarose-octyl derivative is incubated in 10 mL in a 10 mM sodium phosphate solution at pH 7 with 0.5% (v / v) lauryl sucrose. It is thus possible to desorb more than 60% of the BTL-DH chimera. Subsequently, the detergent is removed by hydrolysis catalyzed by a covalently immobilized derivative of the lipase of Candida Antarctica B.

For the immobilization of the BTL-DH complex on a hydrophobic magnetic particle, a solution of the BTL-DH chimera dissolved in 25 mM sodium phosphate pH 7 at 25 ⁰ C is offered to a suspension of 100 mg of magnetic nanoparticles. The immobilization follows, seeing that both the lipase activity and the DH activity disappear from the supernatant and are incorporated into the support (see Figure 1). In this way, 67 mg of chimera are immobilized per 1 g of nanoparticle.

EXAMPLE 2. Lipase complex with DNA probe.

DNA probes immobilized on different supports are a tool for the development of biosensors, especially in the field of diagnosis. By joining the DNA probes, containing a thiol or disulfide group introduced at the 3 ^' or 5 ^' ends, to lipases containing highly reactive epoxide groups or disulfide groups, the lipases of several probe molecules can be coated. This allows the immobilization of DNA probes on all types of hydrophobic surfaces. If in addition the lipase is very stable and the lipase-surface junction is stable at high temperatures, a PCR of the DNA sample hybridized with the immobilized complementary probe can even be performed. In the presence of high concentrations of detergent, the hybridized DNA bound to the lipase can be desorbed and the support can be reused, with the economic advantage that this entails.

EXAMPLE 3. Lipase complex with cofactor.

PREPARATION AND IMMOBILIZATION OF THE LIPASA COFACTOR COMPLEX

A thermostable lipase from Bacillus thermocatenulatus (BTL) is used as a transporter lipase and as a cofactor the NAD + (nicotin adenine diphosphate) which is a key cofactor for enzymatic oxidation and reduction processes.

8 mg of BTL per 1 g of agarose-octyl is adsorbed by hydrophobic adsorption at 25 ⁰ C in 10 mM sodium phosphate at pH 7. The BTL adsorbed is partially aminated by treatment with 1 M of ethylenediamine at pH 4.75 and 25 ⁰ C for 1.5 h by adding 1 mM carbodimide.

The partially aminated BTL lipase is coated with dextran by the application of the following protocol: oxidized dextran aldehyde 100% molecular weight 6,000 daltons is prepared. It provides 1 g of agarose-octyl amino BTL- 9 mL of dextran aldehyde 33 mg / mL at pH 7.5, and the suspension is stirred gently at 25 ⁰ C for 1, 5h. Finally, the derivative is filtered, washed with distilled water and used immediately to obtain agarose-octyl-BTL-dextran-carboxyl. This unreduced agarose-octyl-BTL-dextran derivative is incubated in a solution of sodium aspartate: thus 1 g of agarose-octyl-BTL-dextran-aldehyde is incubated in 9 mL of 3M sodium aspartate at pH 8.5 in 150 mM trimethyl amino borane. The suspension was subjected to gentle shaking at 25 ⁰ C for 16 h. After this time it is reduced with borohydride by means of the suspension in 90 mL of 100 mM sodium bicarbonate at pH 10 containing the total volume 1 mg / mL of sodium borohydride for 2 h. Finally, the derivative is filtered and washed with 100 mM sodium phosphate at pH 7 and abundant distilled water. 2 g of agarose-octyl-BTL-dextran-carboxyl or agarose-octyl-BTL-carboxyl are incubated in an aqueous solution in a total volume of 10 ml_ containing 100 mM NAD ⁺ at pH 4.75. The carboxyl-amino coupling is activated by the addition of 1-ethyl-3- (3-dimethiamino-propyl) carbodiimide (EDCI) to a total concentration of 100 mM every 1.5 h for 6 h total reaction. The reaction mixture is subjected to gentle stirring at 25 ^° C. Finally, it is washed thoroughly with a saturated solution of sodium chloride, 500 mM sodium phosphate pH 7 and 10 mM sodium phosphate pH 7. The result is a modification with 0.05 μmol NAD ⁺ / mg of BTL. Being able to desorber 50% of the BTL-dextran-NAD ⁺ by means of the incubation of 1 g of derivative in 10 mL of total volume at 0.5% triton in 25 mM of sodium phosphate at 25 ⁰ C.

To immobilize the BTL-NAD ^{+ complex} on magnetic nanoparticles, an excess of BTL is offered over the maximum loading capacity of the nanoparticles, so that the amount of BTL-amino-epoxid present in 5 g of agarose-octyl is desorbed activated with 1.5 mg / g BTL with

0.5% triton in 25 mM sodium phosphate at pH 7, the supernatant of the desorption is offered 50 mg of nanoparticles, 25 ⁰ C by diluting the volume of immobilization sodium phosphate 10 mM pH 7 to Ia The concentration of triton is 0.005%, obtaining a kinetics of immobilization similar to the previous one, immobilizing 75% of the amount offered, which consists of a modification of up to 7.5 mg of BTL per 100 mg of nanoparticles (equivalent, per both at 0.35 μmoles of NAD ⁺ per 100 mg of nanoparticles).

Claims

1. Complex comprising a lipase covalently linked to a biomolecule. 2. Complex according to claim 1 wherein the lipase comes from a thermophilic microorganism. 3. Complex according to claim 2 wherein the thermophilic microorganism is: Bacillus thermocatenulatus or Thermus thermophillus. 4. Complex according to any of claims 1 to 3 wherein the biomolecule is a protein. 5. Complex according to any of claims 1 to 4 wherein the protein is an antibody. 6. Complex according to any of claims 1 to 4 wherein the protein is: protein A or protein G. 7. Complex according to any of claims 1 to 4 wherein the protein is an enzyme. 8. Complex according to any one of claims 1 to 3 wherein the biomolecule is a cofactor. 9. Complex according to claim 8 wherein the cofactor is selected from the list comprising: NAD (P) ⁺ , NAD (P) H, ATP, ADP or FAD. 10. Complex according to any of claims 1 to 3 wherein the biomolecule is a DNA probe. 11. A complex according to any of claims 8 to 10, which further comprises an activatable polymer between the lipase and the biomolecule. 12. Complex according to claim 11 wherein the activatable polymer is dextran. 13. Complex according to any one of claims 1 to 12 which, in addition, is immobilized on a non-porous hydrophobic support. 14. Complex according to claim 13 wherein the hydrophobic support is selected from the list comprising: carbon nanotube, hydrophobic array, hydrophobic array, multiwell plate, hydrophobic magnetic nanoparticle or hydrophobic non-magnetic nanoparticle. 15. Complex according to claim 14 wherein the hydrophobic magnetic nanoparticle is a ferrite core coated with polystyrene. 16. Method of chemical preparation of the complex according to any of claims 1 to 15, comprising: to. adsorb a lipase to a hydrophobic, porous and inert solid support, b. conjugate the lipase of step (a) with a biomolecule, c. desorb the complex obtained in step (b) of the support. 17. Method of chemical preparation of the complex according to claim 16, wherein the hydrophobic solid support of step (a) is: octyl agarose or glass coated with alkyl groups of C ₄ -CiS- 18. Method of chemical preparation of the complex according to claim 16, wherein the hydrophobic solid support of step (a) has a hydrophilic surface coated with hydrophobic nanostructures. 19. Method of chemical preparation of the complex according to claim 18, wherein the hydrophobic nanostructures are selected from the list comprising: hydrophobic proteins, hydrophobic protein molecules, hydrophilic proteins modified with hydrophobic reagents, hydrophobic polymers or hydrophilic polymers modified with hydrophobic reagents. 20. Method of chemical preparation of the complex according to any of claims 16 to 19 wherein the lipase of step (a) is modified with dienophiles, with ionized amino groups coated with epoxy groups or glutaraldehyde groups or with activated polymers to chemically conjugate with biomolecules . 21. Method of chemical preparation of the complex according to claim 20, wherein the activated polymers contain carboxyl groups, epoxide groups or amino groups. 22. Method of biological preparation of the complex according to any one of claims 1 to 7 which comprises inserting the gene of the biomolecule into a plasmid containing the lipase gene. 23. Use of the complex according to any of claims 1 to 15 for the immobilization of biomolecules on hydrophobic supports. 24. Method of obtaining the complex according to any of claims 13 to 15, comprising the steps of the method according to any of claims 16 to 21 or of the method according to claim 22, and further: d. adsorbing the complex of any one of claims 1 to 12 to a non-porous hydrophobic support. 25. Method of obtaining the complex according to claim 24, which further comprises: and. coating the non-porous hydrophobic support with other lipases prior to the adsorption of step (d). 26. Use of the complex according to any of claims 1 to 4, 7 to 9 or 11 to 15 as a biocatalyst. 27. Use of the complex according to any of claims 1 to 15 as a biosensor. 28. Use of the complex according to any of claims 8 or 9 or 11 to 15 for the regeneration of immobilized cofactors.