ES2352779A1 - Covalent complexes of lipases with proteins, dna probes, cofactors or other biomolecules - Google Patents

Abstract

Covalent complexes of lipases with proteins, DNA probes, cofactors, or other biomolecules. Complexes that comprise lipases covalently linked, through regions not involved in their active center, to biomolecules of interest (proteins, such as enzymes, antibodies or proteins A or G, cofactors or DNA probes). The complexes can be immobilized through the intact active center of the lipases on all types of hydrophobic supports without the need to carry out modifications or activations on them. The binding of lipase to hydrophobic scaffolds is reversible, allowing reversible immobilization of the complexes. The invention provides two methods, chemical and biological, of preparing these complexes and a method of immobilizing them. These immobilized complexes may have applications in industry, in the laboratory, or in diagnosis, due to their potential use as biocatalysts or biosensors. The chemical method comprises the adsorption of the lipase to a porous hydrophobic support, the conjugation of the thus adsorbed lipase with the biomolecule and desorbing this complex from the support by using detergents. The biological method comprises inserting the biomolecule gene into a plasmid containing the lipase gene, in a microorganism, so that it expresses the lipase fused to the biomolecule. Reversible immobilization of the complexes created from either of the two methods can be performed on non-porous hydrophobic supports.

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, across regions not involved in its active center, to biomolecules of interest (proteins or other biomolecules). The complexes can be subsequently immobilize through the intact active center of the lipases on all types of hydrophobic supports. Also refers to two methods, chemical and biological, of preparing these complexes and a method of immobilization thereof. These Fixed assets may have applications in the industry, in the laboratory or in diagnosis, due to its potential use as biocatalysts or as biosensors.

Prior art

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 most commercial magnetic particles (such as polystyrene-coated ferrite cores), carbon nanotubes, multiwell plates, some arrays , etc. However, the immobilization of proteins and other biomolecules on these hydrophobic surfaces generates a number of problems, such as the inactivation of the biomachromolecule (Mateo, C., et al ., 2002, Biotechnology Progress , 18: 3, 629-634). In addition, this type 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. Solving 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, 4984-4989).

Another approach is the coating of nanoparticles with silica for the binding of active biomolecules, 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. One solution involves 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 should 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 be able to perform this type of reactions. However, their Small size makes handling difficult. The use of magnetic particles would be a simplification, but given its high Price is a system of difficult use in the short term. Besides, the most nanoparticles that are stable are hydrophobic, which which causes its aggregation and the possible inactivation of biomachromolecules 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 others hydrophobic supports is essential to carry out the applications thereof. These methods should allow union selective of the biomolecule of interest, that is, without existing nonspecific interactions with other molecules, since other way its applications as biocatalysts or as biosensors are They would find very limited. In addition, in some cases it would be interesting that these methods allowed the reuse of Support once it has been used.

Description of the invention

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

Lipase acts as a transporter protein and it is immobilized by interfacial activation on magnetic particles hydrophobic or other hydrophobic supports without the need to perform No modification or previous activation of the supports. From so that once the biomolecule is attached to the lipase, it can be carry out the immobilization of this complex on a support hydrophobic avoiding the inactivation of the biomolecule, being the lipase which is adsorbed by interfacial interaction on the hydrophobic nanoparticle Lipase immobilization is rapid, Intense, reversible and can be performed in conditions Soft experimental Once the nanostructures have been used or hydrophobic supports, can be recovered by desorbing the complexes with high concentrations of detergent, thus allowing reuse of the supports for the adsorption of new complex.

The complexes are simple to prepare, both by chemical methods as biological. In addition, this immobilization of biomolecules on surfaces, even when they are small, can be quantified following the catalytic activity of The transport lipase. The fact that the protein transporter, that is, lipase, has a high activity catalytic 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 supports hydrophobic by the method provided herein invention reduces unwanted unspecific interactions and it could even allow stabilization, by covalent bonding multi-point, of enzymes on rigid lipases Conveyors

Therefore, a first aspect of the invention is refers to a complex, from now on "complex of the invention ", which comprises a lipase covalently linked to a biomolecule

This description means "lipase" the enzyme present in lysosomes and released by the pancreas, responsible for the breakdown of fats or lipids in fatty acids. A "biomolecule" is any organic molecule constituent of living beings, as for example, but without limit ourselves, carbohydrates, lipids, proteins, cofactors or acids nucleic; in this description they are also included within biomolecules nucleic acid probes.

In a preferred embodiment of this aspect of the invention, the lipase is derived 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, the greater the range of conditions under 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 higher the stiffness of the lipase molecule, the greater the possibility of stabilizing the biomolecule to which it is joined by covalent multipoint binding. Therefore, the lipases of the present invention preferably come from thermophilic microorganisms, which are defined as those microorganisms that live and develop under extreme temperature conditions, that is, above 45 ° C, whose optimum temperature is between 50 and 75 ° C and its maximum temperature between 80 and 113ºC. Examples of thermophilic microorganisms could be but is not limited thereto, Pyrococcus furiosus, Thermus aquaticus, Thermus thermophilus, Chloroflexus aurantiacus, coastal Thermococcus, Pyrodictium abyssi (archaea), Bacillus stearothermophilus, Rhodotermus obamensis, Marinithermus hydrothermalis, Deferribacter desulfuricans, Thermodesulfobacterium hydrogeniphilum or Bacillus thermocatenulatus.

"Covalent union" means the link in which the atoms of two non-metallic elements remain united because they share two electrons. At the moment invention, covalent junctions between lipases and Biomolecules are made through regions not involved in the lipase active center, thus keeping the active center intact to subsequently immobilize the complex on a support hydrophobic.

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 a antibody. In another preferred embodiment the protein is: protein A or protein G. In another preferred embodiment the protein is a enzyme.

It is understood as "protein or peptide" any macromolecule formed by linear chains of amino acids joined by peptide bonds, already have a structural function, regulatory, catalytic or enzymatic, transporter, defensive or contractible. This definition includes both simple proteins (consisting only of amino acids) such as conjugates (consisting of amino acids and prosthetic groups). Examples of proteins are, but not limited to, hormones, antibodies, enzymes, receptors, etc.

This description means "antibodies or immunoglobulins" those glycoproteins produced by B lymphocytes in response to an antigen. He typical antibody consists of structural units basic, each with two large heavy chains and two light chains of smaller size. 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 what gives the antibody the specificity of binding to its antigen.

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

Protein A is a 42,000 Dalton polypeptide that is a common 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, among others, so it 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 so it is complemented with 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.

"Enzymes" are the substances of protein nature that catalyze chemical reactions, when it is thermodynamically possible, decreasing the level of "energy of activation "own reactions. These enzymes can be, but not limited to, oxidoreductases, transferases, hydrolases, isomerases, nasas or ligases. Examples of enzymes could be, but not limited to, dehydrogenases, kinases, glucosidases, decarboxylases, synthetases, etc. There are enzymes that act on insoluble substrates or cofactors, examples of them are, without limit ourselves, 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 one more embodiment Preferred 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 probe of DNA

"Cofactor" means 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 without limitation, Fe 2+, Cu 2+, K +, Mn 2+, Mg 2+, vitamins, NAD (P) +, NAD (P) H, ATP, ADP or FAD.

"DNA probe" means any DNA fragment labeled with fluorescence.

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

"Activable polymer" means any polymer that, when modified with a 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 the branches begin at α1-4 junctions (in some cases also 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 to obtain 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.

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In another preferred embodiment, the complex of the invention is immobilized on a hydrophobic support not porous. In a more preferred embodiment the hydrophobic support is select from the list comprising: carbon nanotube, array hydrophobic, hydrophobic array, multiwell plate, nanoparticle hydrophobic magnetic or hydrophobic non-magnetic nanoparticle. In an even more preferred embodiment, the magnetic nanoparticle Hydrophobic is a ferrite core coated with polystyrene.

It is understood as "hydrophobic support not porous "any support, including nanostructures, which is repelled by water and lacking pores. Media examples Non-porous hydrophobic are, without limitation, carbon nanotube, hydrophobic array, hydrophobic array, multiwell plate, hydrophobic magnetic nanoparticle or non-magnetic nanoparticle hydrophobic An example of a hydrophobic magnetic nanoparticle is, without limiting ourselves, a ferrite core coated with polystyrene. Be understood as "ferrite core" that which is formed by iron powder combined with other elements that give it very good magnetic properties It is understood as "polystyrene" the thermoplastic polymer that is obtained from the polymerization of the styrene

Another aspect of the invention relates to a chemical preparation method of the complex of the invention, now hereinafter "first method of the invention", comprising:

to.

adsorb a lipase to a solid support hydrophobic, porous and inert,

b.

conjugate the lipase from step (a) with a biomolecule, and

C.

desorb the complex obtained in the step (b) of the support.

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

In this description you understand by "hydrophobic, porous and inert solid support" any support, including nanostructures, which is repelled by water and that presents pores. Examples of hydrophobic supports of this type they are, but not limited to, octyl agarose or glass coated with alkyl groups. It is understood by "alkyl groups" organic functional groups formed by the elimination of a hydrogen atom of a hydrocarbon that are always found attached to another atom or group. The hydrophobic support used in the step (a) of the first method of the invention may also have a hydrophilic surface, understanding any surface as such that has an affinity for water, modified by coating with hydrophobic nanostructures. At the moment description means "nanostructure" a structure with an intermediate size between molecular structures and Microscopic, micrometric size. Examples of nanostructures hydrophobic could be, but not limited to, proteins hydrophobic, hydrophobic protein molecules, proteins hydrophilic modified with hydrophobic reagents, polymers hydrophobic or hydrophilic polymers modified with reagents hydrophobic An example of hydrophobic proteins could be, but without limiting ourselves, hydrophobins.

The complex of the invention is synthesized in phase solid, in a previous stage, in a porous hydrophobic support where intermolecular interactions between adsorbed biomolecules they are impossible, which prevents their aggregation. This can be done thanks to the possibility of immobilizing lipases in a way reversible on hydrophobic supports. 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 at the end of this stage of the process you get the lipase bound to 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 present invention, to obtain the complex formed by 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, epoxide groups or amino groups.

"Dienophiles" means any substance that is attracted to dienes. The "amino groups" they are functional groups derived from ammonia or any of its alkylated derivatives by elimination of one of its atoms of hydrogen. "Ionization" means the chemical process or physical by which ions are produced. It is understood by "group epoxide "the radical formed by an oxygen atom attached to two carbon atoms, which in turn are linked together by a Only covalent bond. "Glutaraldehyde" means a simple five-carbon molecule with aldehyde groups in each extreme, chemical formula OCH-CH 2 -CH 2 -CH 2 -CHO.  Examples of activated polymers to conjugate with biomolecules with which the lipase modification could be carried out they are, without limitation, dextran or polyethylene glycol. It is understood by "carboxyl group" a group of organic formula COOH attached to a organic residue, represented as R.

In cases where the stability of the biomolecule is much smaller than that of lipase, the chemical chimera to Through the multipunual covalent union would have advantages. For this can transform all the carboxyl groups of the lipase in amino groups by modification with ethylenediamine. Then, these groups can 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 treat it with glutaraldehyde All these techniques allow a great activation of the lipase, which could allow a covalent bond Very intense multi-point biomolecule with lipase, increasing its stability

Another aspect of the invention relates to a method of biological preparation of the complex of the invention, of hereinafter "second method of the invention", which comprises inserting the biomolecule gene 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" is understood as the extrachromosomal nucleic acid fragment found in the cytoplasm of some prokaryotes, of varying size although smaller that the main chromosome, with linear, circular or supercoiled. To thermophilic microorganisms containing the gene of the lipase in a plasmid, they can be inserted, by genetic engineering techniques known to any expert in the matter, for example but without limiting ourselves, by using restriction enzymes, the biomolecule gene of interest, by example but without limiting ourselves, a protein. By cutting, with the same restriction enzyme, the lipase gene and the gene of the biomolecule of interest, compatible extremes are generated for the linkage of both genes in reading phase by, for example, but without limiting ourselves, a ligase. The microorganism that carries the plasmid containing this genetic construct expresses the protein Lipase-biomolecule fusion. This way it obtains the complex of the invention ready to be immobilized in 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, hereafter "third method of the invention ", which comprises the steps of the first method of the invention or the second method of the invention, and in addition:

d.

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

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

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

and.

coat the hydrophobic support no porous with other lipases prior to adsorption of the passage (d).

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The hydrophobic support can not be coated porous with other unmodified and low molecular weight lipases, to fully cover the surface of the support with structures hydrophilic and inert proteins. With this, you get a more inert surface, so that hardly any surface remains hydrophobic 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 complexes with high detergent concentrations, and so they can be reused as supports of new complexes to immobilize biomolecules. In addition, the use of detergents allows to obtain complexes in their soluble form. So that the immobilization of the complexes of the invention is reversible, thanks to the ability of lipases to be desorbed from the supports through the use of detergents.

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

This description means "detergent" any substance capable of breaking the barrier lipid solubilizing proteins and disrupting the interaction lipid-lipid, lipid-protein and protein-protein

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

This description means "biocatalyst" any catalyst of reactions biochemicals of living things, which reduces activation energy of a chemical reaction, making it faster. Through the immobilization of the complexes of the invention formed by lipases and, for example, but not limited to proteins, preferably enzymes, or cofactors, the biocatalysis of macromolecules or, in general, of any reaction biochemistry. Therefore, the biomolecules that are part of the complex of the invention when it is used as a biocatalyst They are preferably enzymes or cofactors.

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

This description means "biosensor" a system for measurement (quantitative or qualitative) of biological or chemical parameters, which usually combine two components of a biological nature or a component of biological nature and other nature physical chemistry.

The biomolecules of the complex of the invention which can be used as biosensors, can be without limit ourselves, cofactors, DNA probes or proteins, preferably enzymes, antibodies or proteins A or G. Proteins have ability to bind to other proteins, such as antibodies, so you have immobilized proteins in a support enables binding with antibodies of interest to your application, for example, but not limited, in diagnosis. By this, 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 biomolecule bound to the lipase in the complex of the invention that is used as a biosensor is an antibody for the detection of antigens.

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

Another aspect of the invention relates to the use of the complex of the invention for cofactor regeneration immobilized Due to the capacity of the complexes of the invention immobilized from being desorbed from its supports by The use of detergents, as explained above, is possible to recycle both biomolecules and supports once They are used. When the biomolecule is a cofactor, the immobilized complex can be used for, for example, but without limit ourselves, catalyze redox processes, phosphorylation processes, etc. After which, thanks to the lipase binding to the support is reversible, the complex can be desorbed lipasa-cofactor and thus the cofactor be reused For future use.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of 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 . (?) Enzymatic activity of BTL in the supernatant; (\ ding {117}) 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 .

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Examples

The invention will be illustrated below through tests carried out by the inventors, who put manifested the effectiveness of complexes comprising lipases bound to biomolecules, as well as the methods of preparation and immobilization thereof. These specific examples that provide serve to illustrate the nature of the present invention and are included for illustrative purposes only, so they should not be construed as limitations to the invention that here it is claimed. Therefore, the examples described below illustrate the invention without limiting the scope of the same.

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Example 1 Lipase enzyme complex Preparation and immobilization of the complex lipase dehydrogenase

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 per 1 g of agarose-octyl are adsorbed by hydrophobic adsorption at 25 ° C in 10 mM sodium phosphate at pH 7. The adsorbed BTL is enriched in amino groups by treatment with 1 M of ethylenediamine at pH 4.75 and 25 ° C for 1.5 h by adding 10 mM carbodimide. This amination transforms all the carboxyl groups of the lipase into amino groups, these groups can be modified with 1,4 butanediol diglycidyl ether to generate epoxide groups, or with glutaraldehyde. The enzyme can even be adsorbed by ion exchange on the aminated lipase and subsequently treated with glutaraldehyde. This modification of the lipase is carried out to establish a multi-point covalent bond in cases where the stability of the enzyme is much lower than that of the lipase. Subsequently, the solid phase aminated BTL is incubated for 16 h at 25 ° C in a solution of butanediol diglycidyl 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 with epoxy groups is offered 0.9 mg dehydrogenase (DH) in 9 mL of 10 mM sodium phosphate buffer at pH 7. Thus 0.5 mg of DH are immobilized on 1 mg of BTL (100% of the DH offered and 25% of the maximum possible). Subsequently, the BTL-DH complex is incubated 16 h at 25 ° C and then all DH is covalently bound 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 Antarctic Candida lipase B.

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

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Example 2 Lipase complex with DNA probe

DNA probes immobilized on different supports constitute a tool for the development of biosensors, especially in the field of diagnosis. Joining the DNA probes, containing a thiol or disulfide group introduced into 3 'or 5' ends, to lipases containing epoxy groups or groups very reactive disulfide, several lipases can be coated probe molecules. This allows the immobilization of probes of DNA on all types of hydrophobic surfaces. If lipase also It is very stable and the lipase-surface junction is stable at high temperatures, you can even perform a PCR of the DNA sample hybridized with the complementary probe immobilized 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 it entails

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Example 3 Lipase complex with cofactor Preparation and immobilization of the cofactor lipase complex

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

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

BTL, partially aminated lipase is coated with dextran by applying the following protocol: prepares 100% molecular weight oxidized dextran aldehyde 6,000 daltons. It is offered at 1 g of agarose-octyl-BTL-amino  9 mL of dextran-aldehyde 33 mg / mL at pH 7.5, and the suspension is allowed to stir gently at 25 ° C for 1.5 h. Finally the derivative is filtered, washed with distilled water and use immediately to obtain agarose-octyl-BTL-dextran-carboxyl. This derivative agarose-octyl-BTL-dextran  without reducing it is incubated in a solution of sodium aspartate: so 1 g of agarose-octyl-BTL-dextran-aldehyde Incubate in 9 mL of 3M sodium aspartate at pH 8.5 in 150 mM of trimethyl amino borane. The suspension is subjected to gentle agitation to 25 ° C for 16 h. After this time it is reduced with borohydride by suspending 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 phosphate of 100 mM sodium at pH 7 and abundant distilled water.

2 g of are incubated agarose-octyl-BTL-dextran-carboxyl or of agarose-octyl-BTL-carboxyl  in an aqueous solution in a total volume of 10 mL containing 100 mM NAD + at pH 4.75. The coupling carboxyl-amino is activated by the addition of 1-ethyl-3- (3-dimethiamino-propyl) carbodiimide (EDCl) up 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, wash thoroughly with a saturated solution of sodium chloride, 500 mM sodium phosphate pH 7 and with 10 mM sodium phosphate pH 7. The result is a modification with 0.05 µm NAD + / mg BTL. Rotting desorber 50% of the BTL-dextran-NAD + through the incubation of 1 g of derivative in 10 mL of total volume at 0.5% triton in 25 mM sodium phosphate at 25 ° C.

To immobilize the complex BTL-NAD + on magnetic nanoparticles, is offers an excess of BTL over the maximum load capacity of the nanoparticles, so that the amount of BTL-amino-epoxidada present in 5 g of activated agarose-octyl with 1.5 mg / g BTL with 0.5% triton in 25 mM sodium phosphate at pH 7, the supernatant of desorption is offered at 50 mg of nanoparticles, at 25 ° C diluting the volume of immobilization with 10 mM sodium phosphate at pH 7 up to the concentration of triton is 0.005%, obtaining a immobilization kinetics 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 (28)

1. Complex comprising a bound lipase covalently 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 the claims 1 to 3 wherein the biomolecule is a protein. 5. Complex according to any of the claims 1 to 4 wherein the protein is an antibody. 6. Complex according to any of the claims 1 to 4 wherein the protein is: protein A or protein G. 7. Complex according to any of the claims 1 to 4 wherein the protein is an enzyme. 8. Complex according to any of the 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 the claims 1 to 3 wherein the biomolecule is a DNA probe. 11. Complex according to any of the 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 of the claims 1 to 12 which, in addition, is immobilized in 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, plate multi-well, hydrophobic magnetic nanoparticle or non-nanoparticle hydrophobic magnetic 15. Complex according to claim 14 wherein the hydrophobic magnetic nanoparticle is a ferrite core polystyrene coated. 16. Method of chemical preparation of the complex according to any of claims 1 to 15, comprising:

to.

adsorb a lipase to a solid support hydrophobic, porous and inert,

b.

conjugate the lipase from step (a) with a biomolecule,

C.

desorb the complex obtained in the step (b) of the support.

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17. Method of chemical preparation of the complex according to claim 16, wherein the hydrophobic solid support of the step (a) is: octyl agarose or glass coated with alkyl groups of between C 4 -C 18. 18. Method of chemical preparation of the complex according to claim 16, wherein the hydrophobic solid support of the 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 the 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.

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22. Method of biological preparation of the complex according to any one of claims 1 to 7 comprising insert the biomolecule gene into a plasmid containing the gene of the lipase. 23. Use of the complex according to any of the claims 1 to 15 for immobilization of biomolecules in 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.

adsorb the complex of any of claims 1 to 12 to a hydrophobic support not porous.

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25. Method of obtaining the complex according to claim 24 which further comprises:

and.

coat the hydrophobic support no porous with other lipases prior to adsorption of the passage (d).

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26. Use of the complex according to any of the claims 1 to 4, 7 to 9 or 11 to 15 as a biocatalyst. 27. Use of the complex according to any of the claims 1 to 15 as biosensor. 28. Use of the complex according to any of the claims 8 or 9 or 11 to 15 for the regeneration of cofactors immobilized