WO2009156532A1 - Thermus thermophilus thermophilic esterase - Google Patents

Abstract

The invention relates to a thermophilic protein with esterase activity. Said protein, of the Thermus thermophilus organism, is expressed in recombinant yeasts and bacteria, in particular E.coli, K.lactis and S.cerevisiae. The esterase is expressed in fusion protein form with a signal sequence that enables secretion to the culture medium and, in addition, is operably linked to an inducible promoter. The recombinant strains are advantageous in that: (i) the cultivation thereof is much easier than that of the thermophilic organism that produces the enzyme, and (ii) they can be used to obtain a larger number of enzyme units per litre of culture than the original thermophilic strains.

Description

 Thermus thermophilic ester ester Thermophilus

TECHNICAL FIELD OF THE INVENTION

The present invention relates to thermophilic enzymes, in particular thermophilic esterases, of Thermus thermophilus that can be expressed in mesophilic organisms.

BACKGROUND OF THE INVENTION

Enzymes are very potent, selective and very specific catalysts. The term esterase is a general term to denote a hydrolase-like enzyme that hydrolyzes ester bonds to produce alcohols and carboxylic acids.

The applications of hydrolases, and in particular esterases, in today's industry are many, for example, ibuprofen, a non-steroidal anti-inflammatory agent has been synthesized by a stereo-selective hydrolysis of its methyl ester through the use of a carboxylesterase. The major industrial application of hydrolases, in particular esterases, includes the detergent industry where they are used to break down fat into easily removable hydrophilic substances.

The greatest requirement for a commercial enzyme is its thermal stability because thermal denaturation is a common cause of enzymatic inactivation. Additionally, by increasing the enzymatic thermostability, enzymatic reactions could be carried out at a higher temperature, which could help increase conversion rates, substrate solubility and reduce the possibility of microbial growth and viscosity in the reaction medium. . In food processing, from a hygiene point of view, enzymatic reactions at high temperatures are less dangerous for contamination by bacteria. In the decomposition of fats and oils present methods in which the fats and oils are contacted with steam at temperatures of about 250 ⁰ C and pressures of about 50 atmospheres are used, which implies the need to provide adequate facilities for carry out such reactions. Fats such as palmitic acid or stearic acid are solid at room temperature and have points melting point of about 65 ⁰ C. Therefore, hydrolysis reactions must be carried out at temperatures above said melting point.

The availability of thermophilic esterases would allow to operate at high temperatures, with the logical increase in the reaction rate and an easier solubilization of the substrates, an aspect that frequently constitutes a limiting factor in some applications of this type of enzymes (e.g. synthesis reactions in media with low water content).

Extremely sharp microorganisms have aroused great interest in recent years, due to the high thermal stability, resistance to chemical denaturants and extreme pHs of their enzymes, which make them especially suitable for use in biotransformation processes under extreme pH conditions, salinity and temperature Thermophilic microorganisms grow at temperatures above 45 ⁰ C, and in some cases (hyperthermophiles) even above 9O ⁰ C.

Although thermophilic organisms may be good candidates in producing thermostable enzymes, it is often impractical due to the low yield and high temperature fermentation equipment that would be necessary to use.

The production of enzymes of biotechnological interest from extreme thermophilic microorganisms is usually carried out by cloning and expression at a high level in an easily manipulated model bacterium or yeast, such as Escherichia coli or Saccharomyces cerevisiae. However, there are a large number of thermophilic enzymes whose synthesis complexity makes it impossible to obtain them actively in these model organisms (Hidalgo, A. and cois., 2004. Appl. Environ. Microbiol. VoI. 70 (7): 3839 -3844). This is the frequent case of hetero-oligomeric enzymes or that of those enzymes that require the incorporation of a cofactor in the synthesis process, which usually requires the help of specific chaperones that keep the enzyme structure in an open position for the entry of the mentioned cofactor. In such cases, which may constitute up to 50% of the enzymes encoded in any thermo edge, the obvious alternative is to use in the production of a thermophilic microorganism evolutionarily related to that from which the enzyme of interest comes from.

The bacteria of the genus Thermus spp. They are characterized by their ability to grow at high temperatures and by their ease of genetic manipulation, unique among extreme thermophilic organisms. The bacteria of the genus Thermus spp. they have great potential for use as systems for obtaining thermostable enzymes through functional selection at high temperatures, there are some examples of this in the scientific literature (Fridjonsson et al., 2002. J Bacteriol. VoI. 184: 3385-3391; Brouns and cois ., 2005. J Biol Chem. VoI. 280: 11422-31). However, the application of these methods depends on the generation of a strain of Thermus spp. Mutant that requires the functional activity of the enzyme to be thermostabilized for its growth, currently existing methodologies for obtaining such mutants are very limited.

International patent application WO9725058 describes the isolation, characterization and purification of esterases from Thermus sp. T351 (ATCC 31674). Said enzymes can be expressed in mesophilic organisms such as Escherichia coli from the infection of said cells with expression vector encoding said enzyme, such as lambda phage. On the other hand, WO2006082059 describes the expression of an enzyme with Alicyclobacillus acidocaldarius esterase activity in expression vectors comprising the nucleotide sequence encoding said enzyme in the form of a fusion protein. Said esterase is particularly useful as a marker of protein activity in cell-free translation systems (cell-free translation system) or in vivo systems, such as E. coli or yeast cells.

On the other hand, Fuciños P., et al (J Biotechnology, (2005) 117; 233-241) describe the identification of extracellular enzymes with lipase / esterase activity produced in Thermus thermophilus HB27 cultures. These authors describe the partial purification and preliminary biochemical characterization of them at the level of activity and stability at high temperatures (80-90 ⁰ C). However, the degree of Purification of said enzymes is very low (purification factor of 4 with respect to cell homogenate) and on the other hand, said authors do not describe the amino acid sequence of said proteins.

Therefore, there is a need to find new thermo-row enzymes with esterase activity that exhibit high temperature stability and that can be expressed in mesophilic organisms.

SUMMARY OF THE INVENTION In a first aspect, the invention relates to an isolated polypeptide with esterase activity comprising the amino acid sequence identified in SEQ ID NO: 1 or a functionally equivalent variant thereof.

In a second aspect, the invention relates to a fusion protein comprising i) a polypeptide according to the invention; and ii) a heterologous peptide.

In a third aspect, the invention relates to a nucleotide sequence encoding a polypeptide or a fusion protein according to the invention.

In another aspect, the invention also relates to a gene construct comprising a nucleotide sequence according to the invention.

In a further aspect, the invention relates to an expression vector comprising a nucleotide sequence or a gene construct according to the invention.

In another aspect, the invention relates to a cell comprising a nucleotide sequence, a gene construct or an expression vector according to the invention. In another aspect, the invention relates to a composition comprising a polypeptide according to the invention or a fusion protein according to the invention.

In another aspect, the invention also relates to a process for obtaining a polypeptide with esterase activity comprising culturing a cell according to the invention under conditions that allow the production of said polypeptide and, if desired, recovering said polypeptide.

In a further aspect, the invention relates to the use of a polypeptide according to the invention or a fusion protein according to the invention in the preparation of a detergent composition.

Finally, the invention relates to a method for the modification of fats or oils comprising contacting a polypeptide or a fusion protein according to the invention with a fat or oil under conditions where said polypeptide or fusion protein can hydrolyze said fat or oil.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows curves of esterase and biomass activity in the culture medium for the polypeptide fragment of the invention that lacks amino acids 1-16 of the N-terminal end (Figure IA) and the polypeptide fragment of the invention that lacks of amino acids 1-26 of the N-terminal end (Figure IB), both expressed in the yeast Kluyveromyces lactis NRRL-Yl 140 together with the secretion signal alpha-mating factor of K. lactis.

Figure 2 shows curves of esterase and biomass activity in the culture medium for the fragment of the polypeptide of the invention that lacks amino acids 1-16 of the N-terminal end expressed in the yeast Saccharomyces cerevisiae BJ3505 together with the sequence of the secretion signal of the alpha-mating factor of S. cerevisiae. Figure 3 shows an SDS-PAGE gel electrophoresis of the post-incubated culture medium (lane 2), concentrated by ultrafiltration (lane 3) and the eluted from affinity chromatography (lane 4). Lane 1 is the marker of Pm (Da). Treatment with EndoH glycosidase results in a single band (lane 5).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to an isolated polypeptide, hereinafter polypeptide of the invention, with esterase activity comprising the amino acid sequence identified in SEQ ID NO: 1 or a functionally equivalent variant thereof.

The term "esterase", as used herein refers to a hydrolase type enzyme that hydrolyzes ester bonds.

As used herein, the term "functionally equivalent variant" of an amino acid sequence refers to an amino acid sequence that (i) is substantially homologous to said amino acid sequence and (ii) exerts the same function (eg that has activity esterase).

An amino acid sequence is substantially homologous to a given amino acid sequence when it has a degree of identity of at least 70%, advantageously of at least 75%, typically at least 80%, preferably of at least 85%, more preferably of at least 90%, even more preferably of at least 95%, 97%, 98% or 99%, with respect to said determined amino acid sequence. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as, for example, BLAST (Altschul SF et al. Basic local alignment search tool J Mol Biol. 1990 Oct 5; 215 (3): 403-10). The person skilled in the art will understand that the amino acid sequences referred to in this description can be chemically modified, for example, by chemical modifications that are physiologically relevant, such as phosphorylations, acetylations, etc. Thus, the term "functionally equivalent variant", as used herein, means that the polypeptide or protein in question maintains at least one of the functions of the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, preferably, at least, a function related to hydrolysis, in particular, which maintains esterase activity. The esterase activity of the polypeptide of the invention can be determined by the use of conventional methods known to those skilled in the art, for example, by way of illustration only, the esterase activity of said polypeptide can be determined by methods such as the assays described in Examples 1-3 where it is described that for the measurement of lipolytic activity, the method described by Fuciños et al (2005) (J. Biotechnol. 117: 233-241) is employed.

In a particular embodiment, the polypeptide of the invention is a variant that has one or more insertions, deletions and / or modifications of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1, and maintains the esterase capacity.

In another particular embodiment, said variant is a fragment of the polypeptide of the invention. The term "fragment" as used herein refers to a polypeptide comprising a portion of said polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, that is, a contiguous amino acid sequence comprised within of said SEQ ID NO: 1. For use in the present invention said fragment must be functionally equivalent to said polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, that is, it must have esterase activity. In a particular embodiment of the invention, said esterase is a thermophilic esterase. In a specific embodiment, said fragment comprises the amino acid sequence shown in SEQ

ID NO: 1 in which the 16 or 26 amino acids have been removed from the N-terminal end, that is, the putative secretion signal (amino acids 1-16) and transmembrane helix

(amino acids 6-26), with respect to the amino acid sequence mentioned above. The term "thermophilic" as used herein refers to an enzyme, in particular, an esterase that can withstand relatively high temperature conditions, that is, it is stable and capable of carrying out its activity under elevated temperature conditions, in in particular at a temperature equal to or greater than 50 ⁰ C, more preferably at a temperature between 60 and 90 ⁰ C. In a particular embodiment, said enzyme comes from a thermophilic organism, in particular from Thermus thermophilus HB 27.

Additionally, the polypeptide of the invention can be part of a fusion protein. In this sense, by way of illustration, not limitation, said fusion protein may contain a region A consisting of a first polypeptide comprising the polypeptide of the invention linked to a region B comprising a second peptide. Said second peptide may be any suitable peptide, for example, an extracellular secretion signal peptide.

Said region B may be attached to the amino-terminal region of said region A, or alternatively, said region B may be linked to the carboxyl-terminal region of said region A. Both regions A and B may be linked directly or through of a spacer peptide (linker) between said regions A and B. The fusion protein can be obtained by conventional methods known to those skilled in the art, for example, by gene expression of the nucleotide sequence encoding said fusion protein in appropriate host cells.

Therefore, in another aspect, the invention relates to a fusion protein comprising i) a polypeptide according to the invention; and ii) a heterologous peptide.

In a particular embodiment of the invention, said heterologous peptide is an extracellular secretion signal peptide, that is, a sequence that allows the secretion of said fusion protein into the extracellular medium. By "extracellular secretion signal peptide," as used in the context of the present invention, is meant all peptide comprising or consisting of a signal sequence that is naturally associated with a protein other than the polypeptide according to the invention to be expressed. Thus, the present invention contemplates the use of signal sequences derived from any secreted peptide, including both those signal sequences belonging to the same organism whose polypeptide we want to express but whose function is to promote secretion to the medium of proteins other than the polypeptide of the invention, as well as of signal sequences that derive from other microorganisms and that are responsible for promoting secretion into the environment of any enzyme. Thus, illustrative but non-limiting examples of signal sequences that can be used in the context of the present invention include polypeptides that contain or consist of the signal sequence of the preproofactor α of S.cerevisiae and other species of the genus Kluyveromyces, Pichia, and Hansenula, the killer toxin signal sequence of K. lactis, the glucoamylase II signal sequence of S. diastaticus, the glucoamylase signal sequence of C. albicans, the phosphatase signal sequence of S. cerevisiae, the sequence 128 kDa killer toxin signal of S. cerevisiae, the invertase signal sequence of S. cerevisiae, as well as random sequences that are known for their ability to functionally replace native signal sequences of E. coli, as they have been described by Kaiser, C. et al. (Science, 1987, 235: 312-317). Thus, illustrative, non-limiting examples of E. coli signal sequences include the MBP protein (maltose-binding protein), the ribose-binding protein RBP (ribose-binding protein), the alkaline phosphatase and the OmpA signal peptide as well as other signal sequences that can be identified using methods known in the art (for example by Gallicioti, G. et al. J. Membrane Biology, 183: 175-182). In a preferred embodiment, when the cell to be transformed is S. cerevisiae, the fusion protein of the invention comprises a sequence coding for the alpha-mating signal sequence of S. cerevisiae factor fused through its 3 'end and in the same reading frame with the sequence encoding the polypeptide of the invention. In another preferred embodiment, when the cell to be transformed is K. lactis, the fusion protein of the invention comprises a sequence encoding the alpha-mating signal domain sequence of K. lactis fused through its 3 'end and in the same reading frame with the sequence encoding the polypeptide of the invention. The polypeptide of the invention or the fusion protein of the invention may further include an amino acid sequence useful for the isolation or purification of the fusion protein of the invention. Said sequence will be located in a region of the fusion protein of the invention that does not adversely affect the functionality of the polypeptide of the invention. Virtually any amino acid sequence that can be used to isolate or purify a fusion protein (generically referred to as "tag" or "tag" peptides) may be present in said fusion protein of the invention. By way of illustration, not limitation, said amino acid sequence useful for isolating or purifying a fusion protein can be, for example, an arginine tail (Arg-tag), a histidine tail (His-tag), FLAG-tag, Strep-tag, an epitope capable of being recognized by an antibody, such as c-myc-tag, SBP-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, chitin-binding domain, glutathione S -transferase-tag, maltose binding protein, NusA, TrxA, DsbA, Avi-tag, etc. (Terpe K., Appl. Microbiol. Biotechnol. (2003), 60: 523-525), β-galactosidase, VSV-glycoprotein, etc. In a preferred embodiment of the invention, said purification peptide is selected from a Flag peptide or a polyhistidine tail.

In another aspect, the invention relates to a nucleotide sequence, hereinafter nucleotide sequence of the invention, which encodes a polypeptide or a fusion protein according to the invention. For simplicity, the nucleotide sequence encoding the polypeptide of the invention is included under the name "nucleotide sequence of the invention", that is, the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, as well as a nucleotide sequence encoding a variant or a functionally equivalent fragment of said polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1.

In another aspect, the invention relates to a gene construct comprising a nucleotide sequence according to the invention. In a further aspect, the invention relates to an expression vector comprising a nucleotide sequence or a gene construct according to claim. The term "expression vector" refers to a replicative DNA construct used to express DNA that encodes the polypeptide of the invention or the fusion protein of the invention and that includes a transcriptional unit comprising an assembly of (1) element / s genetic / s that have a regulatory role in gene expression, for example, promoters, operators or enhancers

(enhancers), operably linked to (2) a DNA sequence encoding the polypeptide or fusion protein of the invention that is transcribed to messenger RNA and translated into protein and (3) appropriate transcription initiation and termination sequences and translation.

Preferably, the invention contemplates the use of vectors that can be propagated in both bacteria and yeast.

In a particular embodiment of the invention, when the cell is a prokaryotic cell, such as a bacterium, suitable vectors according to the invention are, for example, vectors pUC18, pUC19, pUC118, pUC119 (Messing, 1983. Meth. In Enzymology 101 : 20-77; Vieira and Messing, 1982. Gene 19: 259-268), Bluescript (Stratagene, La Jolla, California) and its derivatives, mpl8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, pNH8A, pNHlβa, pNH18a. In a particular embodiment of the invention, when said bacterium is E. coli, said vector is plasmid pET, in particular, plasmid pET21d. The genes cloned in said plasmid are transcribed under the control of the bacteriophage T7 promoter, when T7 RNA polymerase is activated in the host cell. The expression is induced with IPTG, which removes the repressor from the operator so that the transcription is carried out and the expression of the protein of interest is promoted.

Additionally, illustrative examples of vectors suitable for the present invention when said cell to be transformed is a yeast cell are the following:

Multicopy autonomous plasmids: These plasmids contain sequences that allow the generation of multiple copies of said vectors. These sequences can be called 2μ, such as the one that appears in episomal plasmids (YEp or "yeast episomal plasmids") or ARS-like sequences, such as those that appear in plasmids of replication (YRps or "yeast replication plasmids"). Examples of vectors based on this type of plasmids are p426GPD, p416GPD, p426TEF, p423GPD, p425GPD, p424GPD or p426GAL, YEp24 and YEplac.

Single-copy autonomous plasmids: Plasmids containing the autonomous ARSl replication sequence and a centromeric sequence (CEN4). This type of plasmids includes centromeric plasmids (YCps or "yeast centromere plasmids").

Integration plasmids: Plasmids that are capable of integrating into the genome of the cell that hosts them. This type of plasmids includes integration plasmids (YIPs or "yeast integrating plasmids"). Examples of vectors based on this type of plasmids are pRS303, pRS304, pRS305 or pRS306 and the like.

In a particular embodiment of the invention, when the transformed cell is a yeast cell, in particular Kluyveromyces lactis, the expression vector is plasmid pKLAC 1.

The choice of a promoter and other regulatory element / s generally varies depending on the host cell used. Suitable promoters in the context of the present invention include constitutive promoters that consistently promote the expression of the sequences associated with them and inducible promoters, which require an external stimulus to promote the transcription of the sequences associated with them.

Promoters useful for carrying out the present invention include:

Constitutive promoters such as the alcohol dehydrogenase (ADHl) promoter, the elongation factor 1 alpha (TEF) promoter and the promoter of the gene encoding the triose phosphate isomerase

(TPI), the glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter and the 3-phosphoglycerate kinase (GPK) promoter, MRP7 promoter and alcohol oxidase (AOXl) promoter.

Inducible promoters such as the metallothionein promoter (CUPl) whose expression is regulated by adding copper to the culture medium, the promoter of the gene encoding the FUSl gene or the FUS2 gene, whose expression is activated in the presence of pheromones (the factor α) as described in US5063154, the TET promoter whose expression is regulated in the presence of tetracyclines, the GALl-10, GALL, GALS promoters that are activated in the presence of galactose, the estrogen-inducible VP 16-ER promoter, and the phosphatase promoter (PH05) whose expression is activated in the presence of phosphate and the HSP 150 thermal shock protein promoter, whose expression is activated at elevated temperature.

- Suppressive promoters such as the promoter of the enolasa (ENO-I) gene of S.cerevisiae whose expression can be repressed when the microorganism is grown in a non-fermentable carbon source as well as promoters whose expression is subject to repression by glucose, so that the expression will be repressed when part of the lactose has been hydrolyzed and begins to increase the concentration of glucose in the medium, the promoter of glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) from S.cerevisiae and the galacto kinase promoter (GALl). Preferably, the promoter that is repressible by glucose is the ADH2 gene promoter.

In a particular embodiment, when the cell to be transformed with said vector or construct of the invention is a bacterium, said promoter is the beta-lactamase and lactose promoter system (Chang et al., Nature 275: 615, 1978), the T7 promoter RNA polymerase (Studier et al., Meth. Enzymol. 185: 60-89, 1990), the lambda promoter (Elvin et al., Gene 87: 123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. in Enzymology 101: 155, 1983) and the tac promoter (Russell et al, Gene 20: 231, 1982). In a more particular embodiment of the invention, said promoter is an inducible promoter. In a more particular embodiment, said promoter is a promoter inducible by galactose, lactose or by IPTG. In a preferred embodiment, said promoter is the beta-galactosidase promoter so that enzymes are secreted into the culture medium when the transformed cells grow in the presence of an inducer such as galactose or lactose. In another particular embodiment of the invention, said promoter is a promoter that is repressed in the presence of glucose.

The invention also contemplates the use of expression enhancers. In a particular embodiment, said expression enhancers are specific yeast enhancers (so-called UAS or "upstream activating sequences") in order to regulate the expression of the fusion polypeptide or protein of the invention.

Alternatively, hybrid synthetic promoters that result from the union of a UAS with the transcription activation region of another promoter can be used. Examples of hybrid promoters include the ADH gene regulatory region linked to the GAP gene promoter activation region, as well as promoters comprising the promoter regulatory sequences of ADH2, GAL4, GALIO, and

PHO5 combined with the transcription activation regions of genes encoding glycolytic enzymes, such as GAP or pyruvate kinase. Additionally, when the cell to be transformed is a yeast cell, the yeast promoters may contain promoters from other sources that have the ability to bind to the yeast RNA and initiate transcription.

In another embodiment, the expression vector of the invention incorporates a transcriptional terminator in the 3 'direction of the sequence encoding the polypeptide or fusion protein of the invention. Preferred transcriptional terminators that can be incorporated include terminators found in the S.cerevisiae enolasa gene, in the S.Crevisiae CYC l gene and in the glyceraldehyde-3-phosphate dehydrogenase gene.

In general, all vectors mentioned in Sikorski ("Extrachromsomoal cloning vectors of Saccharomyces cerevisiae", in Plasmid, A Practical Approach, Ed. K. G. Hardy, IRL Press, 1993) and in Ausubel et al. ("Yeast Cloning Vectors and Genes" Current Protocols in Molecular Biology, Section II, Unit 13.4, 1994) are useful in the context of the present invention.

Vectors that can be used in the context of the present invention typically include an origin of replication, an antibiotic resistance gene, an origin of replication in bacteria (necessary for propagation in bacteria), multiple cloning sites, and a genetic marker The genetic marker is usually a gene that confers resistance to an antibiotic or, alternatively, an autotrophic marker. Thus, marker genes useful in the context of the present invention include, for example, the neomycin resistance gene, which confers resistance to G418 aminoglycoside, the hygromycin phosphotransferase gene that confers hygromycin resistance, the ODC gene, which confers resistance to Ornithine decarboxylase (2- (difluoromethyl) -DL-ornithine (DFMO) inhibitor, the dihydrofolate gene reduced which confers resistance to metrotexate, the puromycin-N-acetyl transferase gene, which confers puromycin resistance, the gene that confers resistance to zeocin, the adenosine deaminase gene that confers resistance to 9-beta-D-xylofuranosyl adenine, the cytosine deaminase gene, which allows cells to grow in the presence of JV- (phosphonacetyl) -L- aspartate, thymidine kinase, which allows cells to grow in the presence of aminopterin or the Xanthine-guanine phosphoribosyltransferase gene, which allows cells to grow in the presence of xanthine and absence guanine Other autotrophic markers that can be used according to the present invention are the TRP1, URA3, LEU2, HIS3 or LYS2 genes, which complement genetic defects in the cells that carry them, which allows said cells to grow in the absence of tryptophan, uracil, leucine, histidine and usine, respectively.

The vector of the invention can be used to transform, transfect or infect cells capable of being transformed, transfected or infected by said vector. Said cells can be prokaryotic or eukaryotic. Therefore, in another aspect, the invention relates to a cell comprising a vector of the invention, for which said cell has been able to be transformed, transfected or infected with a vector provided by this invention. Suitable host cells include: cells prokaryotes or eukaryotic cells. Preferred host cells are prokaryotic cells, in particular, bacteria, or eukaryotic cells, in particular, yeast.

Thus, illustrative, non-limiting examples of bacteria according to the present invention include Gram-negative bacteria, for example, a strain of Escherichia spp. (e.g., E. coli, etc.), a strain of Salmonella spp. (e.g., S. tiphymurium, etc.), a strain of Pseudomonas spp. (e.g., P. aeruginosa, P. putida, etc.), etc., which can be transformed with a gene construct of the invention or with a vector of the invention, such as an expression vector provided by this invention. In a preferred embodiment, said bacterium is Escherichia coli. Illustrative examples of E. coli strains that can be used according to the invention are strains BL21 (DE3),

DHlOB and DH5α.

By yeast is meant any eukaryotic organism belonging to the type of ascomycetes that includes organisms generally known as yeasts as well as those generally known as filamentous fungi. Yeasts and filamentous fungi include Pichia sp (P. pastoris, P.finlandica, P .trehalophila, P.koclamae, P.membranaefaciens, P. minuta, P. opuntiae, P .thermotolerans, P. salictaria, P.guercuum, P.pijperi, P.stiptis, P.methanolica), Saccharomyces (S. cerevisiae), Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K thernotolerans, and K. marxianus; K. yarrowia), Trichoderma reesia, Neurospora crassa, Schwanniomyces, Schwanniomyces occidentalis, Penicillium, Totypocladium, Aspergillus (A.nidulans, A.niger, A.oryzae), Hansenula polymorpha, Candida, Kloe, Kloe, Kloe Torulopsis, and Rhodotorula, Hansenula, Kluyveromyces sp. (for example, Kluyveromyces lactis), Candida albicans, Aspergillus sp (for example, Aspergillus nidulans, Aspergillum niger, Aspergillus oryzae), Trichoderma reesei, Chrysosporium luchiowense, Fusarium sp. (for example, Fusarium gramineum, Fusarium venenatwή), Physcomitrella patens. Therefore, in another aspect, the invention relates to a cell comprising a nucleotide sequence or a gene construct or an expression vector according to the invention. In another preferred embodiment of the invention, said yeast is selected from Saccharomyces cerevisiae and Kluyveromyces lactis. The polypeptide or fusion protein of the invention can be obtained by various methods known to those skilled in the art, for example, by the use of recombinant DNA techniques. In fact, the nucleotide sequence or vector of the invention can be used to produce the polypeptide or fusion protein of the invention. Thus, by way of illustration, not limitation, a method of producing said polypeptide or said fusion protein of the invention comprises growing a cell provided by this invention under conditions that allow the production of said polypeptide or fusion protein. The conditions for optimizing the culture of said cell will depend on the cell used. If desired, the polypeptide or fusion protein of the invention can be isolated and, optionally, purified, by conventional methods.

Therefore, in another aspect, the invention relates to a process for obtaining a polypeptide with esterase activity comprising culturing a cell according to the invention under conditions that allow the production of said polypeptide and, if desired, recovering said polypeptide. Thus, the polypeptide of the invention, if desired, is isolated and, optionally, purified.

Virtually any method known in the art can be used for the recovery of the protein of interest from the cell interior or from the culture medium. If the protein is produced inside the cell, it is necessary to lyse the cells to release the proteins of interest.

Thus, in the case that said cell is a bacterium, said cells can be lysed by different methods including treatment with alkalis or heat, ionic or non-ionic detergents and organic solvents. The choice of the method of extraction will depend on the bacterial species in question so that the treatment must be modified according to the host strain (bacterial species or strain, due to the different composition of the cell wall of different microorganisms). Conventionally, when the cell is a yeast cell, they are lysed by hypotonic lysis of spheroplasts previously formed by glucanase treatment, by sonication or by agitation in the presence of glass balls.

Once the protein of interest is found in the medium, either by releasing the cellular interior or because the protein is secreted by the cell's own secretion machinery, conventional methods are used for the purification of said protein, including, without being limited, chromatography (for example, ion exchange, affinity, hydrophobic interaction, gel filtration, HPLC), electrophoretic methods (preparatory isoelectric focusing, preparative electrophoresis in polyacrylamide-SDS gels), differential solubility (precipitation with ammonium sulfate) , preparative ultracentrifugation in sucrose gradient. Once the desired degree of purity has been reached, which may require more than one graphic chromate step, it is often necessary to concentrate the protein or remove salts and ions that may be detrimental for its subsequent use. In that case, known techniques are used, such as lyophilization or ultrafiltration.

The determination of the degree of purity of said polypeptide can be estimated by the value of the specific enzyme activity that is calculated by dividing the number of units of enzyme activity by the amount of mg of protein in a given volume. Preferably, the enzymatic activity is determined by the method of measuring lipolytic activity described according to Fuciños et al (2005) (J. Biotechnol. 117: 233-241).

In another aspect, the invention relates to a composition comprising a polypeptide or a fusion protein according to the invention.

Esterases are enzymes capable of hydrolyzing ester type bonds of fats. Therefore, in another aspect, the invention relates to the use of a polypeptide or a fusion protein according to the invention in the preparation of a detergent composition. In another aspect, the invention relates to a method for the modification of fats or oils comprising contacting a polypeptide or a fusion protein according to the invention with a fat or oil in conditions where said polypeptide or fusion protein can hydrolyze said fat or oil

The invention is described below by means of the following examples that should be considered as merely illustrative and not limiting thereof.

EXAMPLE 1

Mesophilic strains of bacteria and yeasts modified to produce a thermophilic esterase of heterologous origin.

1. Cloning of the nucleotide sequence of the gene encoding the heterologous enzyme and production of recombinant strains of Escherichia coli

The nucleotide sequence of the gene encoding the heterologous enzyme with esterase activity (complete sequence encoding a 329 amino acid protein) of T. thermophilus HB27 (DE3) was amplified by PCR from T. thermophilus genomic DNA with the following primers comprising BamHI (F) and HindIII sites

(R):

PUREST-F: CGGGATCCGAatgaagcggcttatcgcgct (SEQ ID NO: 2)

PUREST-R: CCCAAGCTTaggccgcacccgggggggcg (SEQ ID NO: 3)

The amplification product was cloned into the corresponding restriction sites of the pET21d vector (NOVAGEN).

Escherichia coli BL21 (DE3) bacteria (NOVAGEN) was transformed with the sequence cloned into plasmid pET21d so that the protein is synthesized fused to a C-terminal tail of 6 histidines after induction with IPTG (Isopropyl β-D-thiogalactopyranoside) . Bacteria transformed with the recombinant plasmid, were grown in LBA liquid medium (1% Bacto-Triptone, 0.5% Bacto-Yeast-Extract, 0.5% Sodium Chloride, 0.1% Glucose, Ampicillin 40 mg / mL) at 37 ⁰ C in orbital shaking and OD at 600nm reached 0.6 was performed IPTG induction.

The measurement of lipolytic activity and definition of the enzyme unit of the recombinant enzyme was performed according to Fuciños et al (2005) (J. Biotechnol. 117: 233-241)

2. Cloning of the nucleotide sequence of the gene encoding the heterologous enzyme and production of recombinant strains of Kluweromvces lactis

On the other hand, strains of the yeast Kluyveromyces lactis were transformed

NRRL-Yl 140 with sequence variants integrated in multicopy in the genome fused to a secretion signal and expressed under the promoter of beta-galactosidase. In this way, enzymes are secreted into the culture medium when yeasts grow in the presence of an inducer such as galactose or lactose.

The sequence variants used consist of eliminating 16 or 26 amino acids from the N-terminal end, that is, the putative secretion signals

(amino acids 1-16) and transmembrane helix (amino acids 6-26) of the native protein and correspond to the sequences identified as SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

The sequences were amplified by PCR from genomic DNA of T. thermophilus HB27 with the following primers to clone them between the Xhol and Kpnl sites of plasmid pKLACl (New England Biolabs) in reading pattern with the secretion signal (K. lactis alfa -mating factor domain).

Starting at amino acid 27

ESTlKF (Xhol) TTTCTCGAGAAAAGAgaggtgcccggtggggtctgc (SEQ ID NO: 6) EST 1 KR (Kpnl) TTTGGT ACCtcaaggccgcacccgggggggcgt (SEQ ID NO: 7) Starting at amino acid 17

EST1KF2 (Xhol) TTTCTCGAGAAAAGAcagggcctcgaggccttctgg (SEQ ID NO: 8) ESTlKR (Kpnl) TTTGGT ACCtcaaggccgcacccgggggggcgt (SEQ ID NO: 7)

With the linearized plasmids by enzymatic digestion with SacII, the yeast K. lactis NRRL-Yl 140 was transformed and the transformants were selected by growth in solid medium with 5 mM acetamide as a nitrogen source, and it was found that the construction had been integrated into Multicopy in the genome. Yeasts were transformed according to the lithium acetate method described in Ito et al. (1983) (J. Bacteriol, 153: 163-168.).

The transformed strains were grown in IL flasks with 200 mL of YPgal medium (Yeast extract: 10 g / L, Bacto-peptone: 20 g / L, Lactose: 20 g / L) at 30 ° C in orbital shaking (100 rpm), obtaining an approximate esterase activity in the culture medium at 49 hours of 500 U / L in the case of the longer variant and 200 U / L in the case of the shorter variant, as can be seen in the Graphs of Figure 1. The specific activity (U / mg prot) in the cell-free post-incubated was 0.09 and 0.04 U / mg protein, respectively. The method of measuring lipolytic activity used is that described by Fuciños et al (2005) (J. Biotechnol. 117: 233-241).

3. Cloning of the nucleotide sequence of the gene encoding the heterologous enzyme and production of recombinant strains of Saccharomvces cerevisiae

The yeast Saccharomyces cerevisiae BJ3505 (Eastman Kodak) was transformed

Company) with the heterologous protein nucleotide sequence variant without the region corresponding to the 16 N-terminal amino acids cloned in yEpFlagl (Eastman Kodak Company) in reading pattern with the secretion signal sequence (from alpha-mating S. cerevisiae factor) and the Flag peptide. The plasmid carries a promoter that is repressed by glucose. Enzymes are secreted into the culture medium, when glucose has been depleted, fused to the Flag peptide by the N-terminal end.

The sequence was amplified by PCR from genomic DNA of T. thermophilus HB27 with the following primers having tails homologous to the MCS vector (multiple cloning site).

PFLAG-EST F: AAAAGAGACT AC AAGGATGACGATGAC AAGcagggcctcgaggccttctgg (SEQ ID NO: 9)

A: TGGGACGCTCGACGGATCAGCGGCCGCTTAaggccgcacccgggggggcgt (SEQ ID NO: 10)

With the PCR product and the vector linearized by digestion at the multiple cloning site with EcoRI and Salí, the S. cerevisiae BJ3505 yeast was transformed by selecting the transformants (containing the recombinant plasmid that has been recirculated by recombination) by growth in medium CM-trp. The CM medium without the corresponding amino acid (tryptophan) used as an auxotrophic marker was prepared according to (Zitomer and Hall, 1976, J. Biol. Chem., 251: 6320-6326). Yeasts were transformed according to the lithium acetate method of Ito et al. (1983) (J. Bacteriol, 153: 163-168).

The transformed strains were grown in a medium containing 1% glucose, 3% glycerol, 1% yeast extract and 8% peptone. A 100 ml culture is performed in a 500 ml flask, the culture is started with an initial inoculum at 0.1 of

DOβoo and incubated at 30 ° with continuous orbital agitation (250 rpm), obtaining a lipase activity approximately 72 hours in the culture medium of 1000 EU / L as can be seen in the graph as an example. The specific activity at that time was 3.79 U mg ^"1. The method of measuring lipolytic activity and defining the enzymatic unit according to Fuciños et al (2005) J. Biotechnol. 117: 233-241. 4. Purification of the heterologous protein

The protein produced extracellularly by the yeast was purified

Saccharomyces cerevisiae BJ3505 transformed with the sequence variant without the region corresponding to the 16 N-terminal amino acids cloned in yEpFlagl in reading pattern with the sequence of the secretion signal (from the alpha-mating factor of S. cerevisiae) and the peptide Flag

The cell-free post-incubated medium (72 hours of culture) was concentrated by tangential ultrafiltration (10,000 Da cut) and from the concentrate the esterase was purified by affinity chromatography with AntiFlag M2 agarose (Sigma) following the supplier's instructions. The specific activity (average of three experiments) increased from 7.30 U / mg in the concentrated post-incubation to 220.6 U / mg in the eluted affinity chromatography (Purification Factor = 30.2).

Figure 2 shows an SDS-PAGE gel electrophoresis of the post-incubated culture medium (lane 2), concentrated by ultrafiltration (lane 3) and the eluted from affinity chromatography (lane 4). Lane 1 is the marker of Pm (Da). After affinity chromatography, three major bands corresponding to the esterase are obtained (all three are revealed in the western blot with anti-Flag antibodies) but in different forms of glycosylation as treatment with the EndoH glycosidase results in a single band (lane 5) .

Claims

1. An isolated polypeptide with esterase activity comprising the amino acid sequence identified in SEQ ID NO: 1 or a functionally equivalent variant thereof. 2. Polypeptide according to claim 1, wherein said esterase is a thermophilic esterase. 3. The polypeptide of claim 2, wherein said thermophilic esterase is stable at a temperature of between 60 and 90 ⁰ C. 4. A fusion protein comprising i) a polypeptide according to any one of claims 1 to 3; and ii) a heterologous peptide. 5. Fusion protein according to claim 4, wherein said heterologous peptide is an extracellular secretion signal peptide. 6. A polypeptide or fusion protein according to any one of claims 1 to 5, wherein said fusion protein further comprises a purification peptide. 7. A polypeptide or fusion protein according to claim 6, wherein said purification peptide is selected from a Flag peptide or a polyhistidine tail. 8. A nucleotide sequence encoding a polypeptide according to any one of claims 1 to 3 or a fusion protein according to any one of claims 4 to 7. 9. A gene construct comprising a nucleotide sequence according to claim 8. 10. An expression vector comprising a nucleotide sequence according to claim 8 or a gene construct according to claim 9. 11. Expression vector according to claim 10, further comprising the nucleotide sequence of a promoter. 12. Expression vector according to claim 11, wherein said promoter is an inducible or repressible promoter. 13. Expression vector according to claim 12, wherein said promoter is a galactose or lactose inducible promoter. 14. Expression vector according to claim 12, wherein said promoter is an IPTG inducible promoter. 15. Expression vector according to claim 12, wherein said promoter is a promoter that is repressed in the presence of glucose. 16. A cell comprising a nucleotide sequence according to claim 8 or a gene construct according to claim 9 or an expression vector according to any of claims 10 to 15. 17. Cell according to claim 16, wherein said cell is selected from a bacterium and a yeast. 18. Cell according to claim 17, wherein said bacterium is Escherichia coli. 19. Cell according to claim 17, wherein said yeast is selected from Saccharomyces cerevisiae and Kluyveromyces lactis. 20. A composition comprising a polypeptide according to any one of claims 1 to 3 or a fusion protein according to any one of claims 4 to 7. 21. A process for obtaining a polypeptide with esterase activity or a functionally equivalent variant thereof comprising culturing a cell according to any of claims 16 to 19 under conditions that allow the production of said polypeptide and, if desired, recovering said polypeptide. 22. Method according to claim 21, wherein when said esterase activity polypeptide comprises the amino acid sequence of an extracellular secretion peptide, the recovery of said polypeptide is carried out from the culture medium. 23. Use of a polypeptide according to any one of claims 1 to 3 or a fusion protein according to any one of claims 4 to 7 in the preparation of a detergent composition or to obtain an enantiopide product. 24. Method for the modification of fats or oils comprising contacting a polypeptide according to any one of claims 1 to 3 or a fusion protein according to any one of claims 4 to 7 with a fat or oil under conditions where said polypeptide or fusion protein can hydrolyze said fat or oil.