WO2017152297A1

WO2017152297A1 - Recombinant vanillyl alcohol oxidase protein (vaor)

Info

Publication number: WO2017152297A1
Application number: PCT/CL2017/050012
Authority: WO
Inventors: Virginia GARRETON; Nicole EHRENFELD; Rosario CALDERÓN; Paola ALVAREZ; Pedro RODRÍGUEZ; Valery FUENTES
Original assignee: Universidad Santo Tomas
Priority date: 2016-03-10
Filing date: 2017-03-10
Publication date: 2017-09-14
Also published as: CL2016000572A1

Abstract

The invention is directed to a recombinant vanillyl alcohol oxidase protein (VAOr) which results from the fusion of VAO and MBP (maltose binding protein), wherein the VAO used is the vaoA gene of the fungus Penicillium simplicissimum. This recombinant protein is expressed in E. coli, is functional, has a high yield, a high enzymatic activity, is stable in a dimeric configuration, the histidine tail making it easier to purify the resultant enzyme. This enzyme can be used in natural processes for producing vanilla on a large scale.

Description

PROTEIN VAINILLIL ALCOHOL OXIDASE RECOMBINANT Q / AOr)

DESCRIPTIVE MEMORY

BACKGROUND OF THE INVENTION

Vanillin is the most abundant component in vanilla aroma, and is therefore one of the most used molecules in the food industry. Additionally, this important molecule is also used as an intermediate compound in the chemical and pharmaceutical industry.

Vanillin is obtained naturally from tropical climbing orchid pods, mainly Vanilla planifolia, however, less than 1% of the vanillin currently consumed in the world is obtained from these natural sources, the other 99% is obtained from chemical or biotechnological synthesis of the molecule. However, in the United States and Europe the labeling "natural vanilla" includes both the extraction from plants and their obtaining by logical biotech processes such as fermentation with microorganisms, transformed cells or enzymatic digestion.

At the same time, consumers are increasingly concerned about the origin of the products, being more positive to the production of "natural" molecules, especially throughout the use of environmentally friendly alternatives, and unlike chemical synthesis.

The most widely described biotechnological technique for obtaining vanillin is all the use of microbiological culture, in which the modified organisms have been selected to make them resistant to the cytotoxicity of the preferred substrates, such as ferulic acid, eugenol and isoeugenol, as well as to reaction byproducts (Rabenhorst and Hopp, 1991, 2000; Lambert et al., 2014). To a much lesser extent, pure enzymes have also been proposed for the bioconversion process that leads to vanillin. Among these enzymes it is possible to find soy lipoxidase (Markus et al., 1994), lignostilbeno-a, β-dioxygenase from Pseudomonas sp. (Kamoda et al., 1989) and enzyme preparations containing beta-glucosidases, pectinases, xylanases and cellulases mainly to improve the purification of vanillin from orchid pods (Ruiz-Terán et al., 2001).

The use of the enzyme vanillyl alcohol oxidase (VAO, EC: 1.1.3.38) to produce vanillin (4-hydroxy-3-methoxybenzaldehyde) through the oxidation of vanillyl alcohol (alcohol 4-) has also been described in the state of the art hydroxy-3-methoxybenzyl), the oxidative demethylation of 2-methoxy-4- (methoxymethyl) phenol, and the oxidative discouragement of vanillylamine (4-hydroxy-3-methoxybenzylamine) (Fraaije et al., 1995; van den Heuvel et al., 2001).

This VAO enzyme exists in nature as a 520 KDa homo octameric enzyme capable of catalyzing a broad spectrum of oxidations (Fraaije et al., 1995). Apart from the octameric form of VAO, which is very difficult to keep active, the existence of a dimeric form of the enzyme has also been demonstrated, but it exists in a much smaller proportion than the octameric form (Tahallah et al., 2002) .

To catalyze substrate oxidation, VAO relies on a prosthetic group of flavin adenine dinucleotide (FAD), covalently bound to each subunit (de Jong et al., 1992; Fraaije et al, 1999). This enzyme is found naturally in the fungus Penicillium simplicissimum (de Jong et al., 1992), and has been cloned from this fungus and expressed heterologously in Aspergillus niger and Escherichia coli as a flavinated and catalytically active enzyme, however , with low yields (Benen et al., 1998).

Thus, obtaining an enzyme capable of producing vanillin efficiently and stably is a desirable goal in the state of the art.

To solve this technical problem, the inventors have developed a new recombinant VAO vanillyl alcohol oxidase enzyme, which fuses VAO and maltose binding protein (MBP), where this new enzyme is not derived from the prior art and surprisingly has a stability improved and is high performance, which allows to obtain vanillin in an efficient way.

In the prior art some cloned VAO proteins are known, for example in US2010028963 Al (MANE FILS V [FR]; 2010-02-04), they clone the VAO enzyme in yeasts for the production of vanilla, despite the cloned protein It is not chimeric nor has it been modified to improve the stability of the enzyme obtained, so this document does not affect the novelty of the invention.

On the other hand, document CN103820375 A (UNIV ANHUI NORMAL; 2014-05-28), discloses a strain transformed with a vector that expresses vanillyl alcohol oxidase associated with other enzymes for the production of feluric acid, the document does not propose the modification of this enzyme, so this document does not affect the novelty of the invention. Finally, in the scientific publication of Van den Heuvel RH, (2004), VAO mutants with modifications in an amino acid are studied, the document does not include fusion proteins. As can be seen, the recombinant VAO protein of the invention is not anticipated in the state of the art.

DESCRIPTION OF THE FIGURES

FIGURE 1. Construction and purification of VAO recombinants. (A) Representation of the transport vector constructed to express VAO with MBP. Plasmid pMGWA was used to express a recombinant VAO enzyme bound to MBP at the N-terminal end and a His-tag at the C-terminal end (Hisox). Theoretical molecular weight is also shown for each peptide and for the VAOr protein. (B) Coomassie blue staining of SDS-PAGE of total cell extracts expressing pMGWA BL21-VAO. Lane 1: Uninduced cells, lane 2: induced cells, lane 3: Proteins eluted after the amylose resin purification step and lane 4: MBP-VAO-His after a second nickel resin purification. In each lane 15 g of total protein were loaded.

FIGURE 2. Enzymatic activity curve of the VAOr enzyme of the invention. The catalytic activity was determined using vanillyl alcohol as a substrate. The test was performed at pH 9.0 in 50 mM of carbonate buffer at room temperature (23-25 ° C) using 0.5 g of purified VAO in 500 (corresponding to 8 nM of VAO) and ethanol no more than 0 ,5%. Each point represents the average of four independent trials.

FIGURE 3. Maintaining the catalytic activity of MBP-VAO-His under different conditions, using vanillyl alcohol as a substrate, the tests were performed at pH 9 in 50 mM carbonate buffer using 0.5 g of VAO purified in 500 (corresponding to 8 nM of VAO) and ethanol not more than 0.5%; after keeping the enzyme for 1 hour at room temperature (23-25 ° C; RT), after 48 hours at room temperature (23-25 ° C; RT), and after keeping the enzyme for 1 hour at 40, 50 and 60 ° C. Each bar represents the average of four independent trials. DETAILED DESCRIPTION OF THE INVENTION

In the present invention, a new recombinant form of the enzyme vanillyl alcohol oxidase (VAO) has been developed where this enzyme is fused with the maltose binding protein (MBP), at its N-terminal end.

Optionally, the VAO enzyme is also fused at its C-terminal end with a molecule that facilitates its purification, an affinity tail or tag, such as a histidine tail, AviTag, Calmoduling-tag, Myc-tag, Xpress-tag or Flag-tag, among others, in a preferred embodiment hexahistidine (His) is chosen.

The preferred embodiment of the invention, the MBP-VAO-Hise recombinant protein was expressed by the inventors in E. coli, finding that the resulting protein is characterized by being soluble, with high yields, with very similar enzyme kinetics and improved stability in comparison with the parameters already described for wild VAO. This VAOr protein of the invention forms dimeros in buffer solution, which are more stable than the majority octameric conformation of wild VAO, and therefore this greater stability of the quaternary structure allows a better efficiency of the enzyme of the invention in processes. Industrial

Thus the invention is directed to a recombinant vanillyl alcohol oxidase protein comprising the enzyme vanillyl alcohol oxidase (VAO) fused with maltose binding protein (MBP) at its N-terminal end, where the enzyme has at least 80% of Homology with SEQ ID No. 1 and where the vanillyl alcohol oxidase enzyme maintains its activity. Optionally, this fusion protein has a molecule at its C-terminus that facilitates its purification; such as a histidine polypeptide.

Where the VAOr of the invention is highly stable, it forms dimeros in solution, and has a high enzymatic activity.

In a second aspect the invention relates to an isolated polynucleotide, which: a. encodes the protein of SEQ ID No. 1; or

b. It has at least 80% homology with the sequence SEQ ID No. 2;

C. it has a sequence that is degenerated as a result of the degeneracy of the genetic code with respect to the sequences defined in (a) or (b); or

d. it is the complementary reverse sequence of the sequences defined in (a), (b) or (c); where the vanillyl alcohol oxidase protein encoded by this polynucleotide maintains its activity.

Optionally, a sequence encoding a molecule that facilitates the purification of said protein or a polypeptide that anchors the protein to the cell membrane is included at the end encoding the C-terminus of the protein. A polypeptide is especially preferred to facilitate purification.

In a third aspect the invention is also directed to an expression cassette comprising the isolated polynucleotide, as described in the previous point, where the polynucleotide sequence is operably linked to at least one regulatory sequence that allows the expression of the polynucleotide sequence in an expression cell or organism, where this expression cassette can be inserted into an expression vector.

The fourth aspect also protects the transformed cell with the cassette and / or vector described in the previous paragraph; where preferably the transformed cell is a bacterium, especially Escherichia coli.

Finally, in a fifth aspect, the invention relates to the method of obtaining vanillin, in which vanillyl alcohol is contacted with the recombinant vanillyl alcohol oxidase enzyme of the invention or with a transformed cell expressing this enzyme of the invention.

The stability of the new recombinant enzyme of the invention is given by the fusion of the vanillyl alcohol oxidase (VAO) enzyme with the maltose binding protein (MBP), at its N-terminal end. The person skilled in the art knows that proteins can have modifications in their sequence that do not interfere with their activity, for this reason the invention comprises the protein as defined in SEQ ID No. 1, and all modifications in its sequence that do not modify the tertiary structure thereof, this includes all possible modifications, up to 70% homology with the sequence, secondly up to 80% homology is preferred, and if necessary only up to 90% homology, with the condition that the catalytic activity of the enzyme VAO is maintained.

At the same time, and optionally, but preferably, at the C-terminal end it is possible to include a tail or tag that facilitates the purification of the recombinant protein. It will be clear to the person skilled in the art that there are many molecules or peptides that fulfill that function, where the nature of this tail is not decisive for the protein of the invention, and any molecule that allows the purification of the protein of the invention can be used . Examples of these peptides are: poly histidine, AviTag, Calmoduling-tag, Myc-tag, Xpress-tag, Flag-tag, among others. The isolated polynucleotide of the invention is one that codes for the recombinant protein of the invention MBP-VAO, as we indicated, the VAO and MBP proteins can have modifications in their sequence that do not modify their activity or their conformation, all those modifications in the sequence they fall within the scope of the present invention, the fundamental parameter being that the VAO enzyme maintains its catalytic activity. Additionally, all nucleotide sequences encoding all possible sequences of the fused proteins, and their complementary reverse sequences are within the scope of the invention.

In order to obtain the VAOr protein of the invention, it must be cloned and expressed within a cell. For this, a polynucleotide that has at least 80% homology with the sequence SEQ ID No. 2 must first be operatively inserted into a plasmid or expression vector. This vector must have a regulatory sequence that allows the expression of said polynucleotide sequence in a host cell. In the state of the art there are many expression vectors, in this invention any of those available may be used, since the nature of the vector is not relevant for the realization of the invention. Notwithstanding the foregoing, the vector comprising a polynucleotide that has at least 80% homology with the sequence SEQ ID No. 2 is considered one of the products of the invention and is itself protected.

Once the vector expressing the VAOr protein of the invention has been obtained, it must be transfected into a host cell by any transformation method available in the art. The transformed cell is also a product of the present invention. Once the transformed cell is obtained, it is incubated and if necessary the expression of the protein of the invention is stimulated according to the conditions required by the regulatory sequence of the vector. Subsequently, the cells are broken and the VAOr protein of the invention is recovered from the soluble fraction of the supernatant.

In a preferred embodiment the protein is expressed within a bacterium, especially in E. coli, however, the person skilled in the art knows that proteins can be expressed in any cloning model available in the art, that is, yeasts, microalgae. , plants, plant cells, insect cells, animal cells or in vitro expression systems, developing in each case an expression vector and a nucleotide sequence suitable to the chosen host cell. If necessary, it is also possible that the tail of the C-terminal end is a transmembrane liposoluble protein or polypeptide, in which case the protein could be expressed in the host cell membrane, and the whole cell can be used in the process of obtaining the vanillin In the Preferred embodiment of the invention the enzyme is purified to be used in the production of vanillin.

Penicillium simplicissimum naturally produces an intracellular VAO when grown with veratryl alcohol as the sole source of carbon and energy, producing 14.7 mg / L of enzyme every two days (de Jong et al., 1992). Prior to this invention, recombinant VAOs had been produced in Aspergillus niger, where yields were not clearly reported, and E. coli with yields below 5 mg / L, even after optimization of the cloned sequence (Benen et al., 1998 ). The inventors previously tried to obtain VAO as a Spanish protein, but found difficulties in expressing and purifying this recombinant protein. In contrast, the MBP-VAO protein of the invention was expressed and easily purified from the soluble fraction of all E. coli extract with high yields (from 40-80 mg / 1) and purity (90%). No fermenter or optimization of culture conditions were required to achieve these enzyme production levels, suggesting that productivity can be easily increased even above these values.

To express MBP-VAO-His, we have cloned exactly the same cDNA described previously (Benen et al., 1998). However we have been able to produce and purify 10-15 times more protein.

While the use of MBP in recombinant proteins has been reported, in order to improve the solubility of the protein, it has also been demonstrated that the ability of MBP to promote the solubility of chimeric proteins can result in the induction of incorrect protein folding to which it is attached (Raran-Kurussi and Waugh, 2012). So it was not obvious that the binding of MBP and VAO resulted in an active VAO, and that is the contribution of this invention, where it is demonstrated that the recombinant protein is active and stable and solves a technical problem in the use of this enzyme in the industry.

When studying the kinetic parameters of the VAOr enzyme of the invention, a Km of 239 μΜ was obtained and the Kcat was 2.4 s-1, while for the wild VAO enzyme, Van den Heuvel and his collaborators (2004) reported a Km of 189 μΜ, while Kcat was 2.4 s-1 using vanillyl alcohol as a substrate at pH 10.0, while Fraaije and his collaborators (1995) estimated a Km value of 290 μΜ and Kcat of 5.4 s-1 at pH 10.0. That is, the enzyme of the invention has kinetics similar to that of the native enzyme, so binding to MBP did not affect its activity. The invention may be better understood in the light of the examples included below, which are illustrative and not limiting of one of the embodiments thereof.

Example 1.

VAO cloning

The P. simplicissimum vaoA gene was chosen, the gene sequence was obtained from the GenBank database (accession number Y15627) and was synthesized by GeneScript Inc (USA). The CACC sequence was added just before the 5'ATG of the original gene, to allow a correct insertion of the ORF vaoA into the pENTR / D-TOPO vector. After cloning into the pENTR vector, vaoA-pENTR was amplified and subsequently cloned, using clone reaction LR, into pMGWA where maltose binding protein was added to the N-terminal end and a 6 Histidine tail at the end C-terminal of the wild type vaoA. The correct insertion and reading frame of the vaoA gene cloned into the new pMGWA vector was verified by sequencing (Macrogen USA). As Figure 1A shows, the addition of these peptides increased the molecular weight of VAO by 52%. However, as will be seen later, these modifications did not affect the enzyme in a negative way.

VAOr expression and purification

E coli BL21 (DE3) pLysS transformed with the plasmid obtained in the previous stage, which carries pMGWAvaoA, was grown at 37 ° C in 1 L of Terrific broth supplemented with 100 mg of ampicillin until it reached DOeoo of 0.8. The expression of the enzyme of the invention was subsequently induced with 0.5 mM isopropyl-l-thio-D-galactopyranoside (IPTG) overnight at 18 ° C.

The cells were collected by centrifugation at 4 ° C and resuspended in 40 ml of 20 mM Tris buffer, pH 7.4, 0.2 M NaCl, 1 mM EDTA, and 1 protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Germany). The cells were broken by sonication at 4 ° C, and subjected to centrifugation for 30 min at 15000g and 4 ° C, to remove cell debris. The crude extracts were loaded in 15 ml of a column of amylose (New England Biolabs, United Kingdom) prebalanced in the same rupture buffer. Next, the column was washed with 150 ml of the same loading buffer and eluted with 50 ml of 20 mM Tris buffer, pH 7.4, 0.2 M NaCl, 1 mM EDTA and 10 mM maltose. The protein of the invention is found in this eluate. In an additional purification step, the preparation obtained from the elution with amylose was chromatographed on a pre-equilibrated HisPur Ni-NTA resin with a 20 mM Tris buffer, pH 7.4, 0.2 M NaCl, 1 mM EDTA . Subsequently, the enzyme eluted from the Ni-NTA column was dialyzed 3 times against 10 mM potassium phosphate buffer, pH 8.0, 0.2 M NaCl. Finally, the eluted fractions containing VAOr were pooled and stored. in 50% glycerol at -20 ° C.

The purity of the enzyme was verified by 12% SDS-PAGE, where Coomassie Blue R-250 was used for protein staining. The pure recombinant VAO Protein of the invention was obtained active, with a high yield, between 40 to 80 mg / L, with a purity of at least 90% (Figure IB, lanes 3 and 4). These results are significantly superior to those reported in the prior art, where Benen (Benen, et al, 1998. Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase. J. Biol. Chem. 273, 7865-72. Doi: 10.1074 / jbc.273.14.7865) reported the first cloning of VAO cDNA in an E. coli system with a final yield of less than 5 mg / L, ie the expression of The protein of the invention has yields 10 to 15 times higher than previously reported.

Example 2

VAOr characterization

A representative reaction of VAO activity is the synthesis of vanillin from its vanillyl alcohol precursor, and since it is also the most important commercially, the enzyme evaluation was studied in the catalysis of this reaction. .

The enzyme activity of the invention obtained in example 1, MBP-VAO-His, was estimated spectrophotometrically at 340 nm since the reaction product, vanillin, can be detected at that wavelength. The VAOr protein of the invention, as obtained in example 1, was diluted to a final concentration of 0.001 mg / ml (approximately 8 nM), from a stock solution at 0.37 mg / ml. The reaction was performed by adding vanillyl alcohol at different concentrations. The tests were performed at room temperature (RT), at pH 9.0, in 50 mM carbonate buffer and in triplicate. The reaction was monitored on an Optizen Pop UV / VIS spectrophotometer. To determine the Km of the reaction the substrate concentration was varied gradually (0, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 6 and 8 mM), while the enzyme concentration is kept constant at 0.001 mg / mL. The experimental curve was adjusted to the Michaelis-Menten equation and the non-linear regression was performed using the Sigmaplot 11.0 program (Systat Software, Inc)

Figure 2 shows the Michaelis-Menten kinetics of the enzyme of the invention under the conditions described, where the VAOr of the invention reaches its Vmax at 12.8 U / mg enzyme, corresponding to about 4 mM of vanillyl alcohol (8 nM VAO concentration). On the other hand, the Km obtained was 239 μΜ and the Kcat was 2.4 s-1.

Under our conditions, the VAO fusion protein showed similar kinetic parameters to those reported for the native enzyme (de Jong et al., 1992; Fraaije et al., 1995; Van den Heuvel et al, 2004).

Example 3

VAO Stability

The enzyme activity of the enzyme of the invention was evaluated using vanillyl alcohol as a substrate, the tests were performed at pH 9 in 50 mM carbonate buffer using 0.5 g of purified VAO in 500 (corresponding to 8 nM of VAO) and ethanol no more than 0.5%; after keeping the enzyme for 1 hour at room temperature (23-25 ° C; RT), after 48 hours at room temperature (23-25 ° C; RT), and after keeping the enzyme for 1 hour at 40, 50 and 60 ° C. The results are expressed in relative activity with respect to the 1 hour condition at room temperature and are shown in Figure 3, where each bar represents the average of 4 independent studies. It is observed that the activity of MBP-VAO -His treated at 40 ° C or 50 ° C for 1 hour remained almost the same as the sample that was kept at room temperature for 1 hour, decreasing the activity only after treatment at 60 ° C for one hour (Figure 3). Surprisingly, the recombinant enzyme MBP-VAO-His maintained its almost complete activity after 2 days at room temperature without any protection (Figure 3). This demonstrates the stability of the enzyme of the invention.

Benen, JA, Sánchez-Torres, P., Wagemaker, MJM, Fraaije, MW, van Berkel, WJH, Visser, J., 1998. Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl -alcohol oxidize. J. Biol. Chem. 273, 7865-72. doi: 10.1074 / jbc.273.14.7865 De Jong, E., van Berkel, WJH, van der Zwan, RP, de Bont, JAM, 1992. Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD. Eur. J. Biochem. 208, 651-7. doi: 10.1111 / j. l432-1033.1992.tbl7231.x

Fraaije, M.W., Veeger, C, van Berkel, W.J.H., 1995. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum. Evidence for the production of 4- hydroxycinnamyl alcohols from 4-allylphenols. Eur. J. Biochem. 234, 271-7. doi: 10.1111 / j.1432-1033.1995.271_c.x

Fraaije, M.W., van den Heuvel, R.H.H., van Berkel, W.J.H., Mattevi, A., 1999. Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase. J. Biol. Chem. 274, 35514-20. doi: 10.1074 / jbc.274.50.35514

Kamoda, S., Habu, N., Samejima, M., Yoshimoto, T., 1989. Purification and some properties of lignostilbene-a, -dioxygenase responsible for the C _a -C _{| ¾} cleavage of a diarylpropane type lignin model compound from Pseudomonas sp. TMY1009. Agrie Biol. Chem. 53, 2757-2761.

Lambert, F., Zueca, J., Ness, F., Aigle, M., 2014. Production of ferulic acid and coniferyl alcohol by conversion of eugenol using a recombinant strain of Saccharomyces cerevisiae. Flavor Fragr. J. 29, 14-21. doi: 10.1002 / ffj.3173

Markus, P.H., Peters, A.L., Roos, R., 1994. Process for the preparation of phenylaldehydes. US Pat. 5,358,861.

Rabenhorst, J., Hopp, R., 1991. Process for the preparation of vanillin. US Pat. 5,017,388.

Rabenhorst, J., Hopp, R., 2000. Process for the preparation of vanillin and microorganisms suitable therefor. US Pat. 6, 133,003.

Raran-Kurussi, S., Waugh, D.S., 2012. The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose-binding protein but their folding is either spontaneous or chaperone-mediated. PLoS One 7, e49589. doi: 10.1371 / journal.pone.0049589

Ruiz-Terán, F., Pérez-Amador, I., López-Munguia, A., 2001. Enzymatic extraction and transformation of glucovanillin to vanillin from vanilla green pods. J. Agrie. Food Chem. 49, 5207- 5209. doi: 10.1021 / jf010723h

Tahallah, N., van den Heuvel, R.H.H., van den Berg, W.A.M., Maier, C.S., van Berkel, W.J.H., Heck, A.J.R., 2002. Cofactor-dependent assembly of the flavoenzyme vanillyl-alcohol oxidase. J. Biol. Chem. 277, 36425-32. doi: 10.1074 / jbc.M205841200

Van den Heuvel, R.H.H., Fraaije, M.W., Laane, C, van Berkel, W.J.H., 2001. Enzymatic synthesis of vanillin. J. Agrie. Food Chem. 49, 2954-8. doi: 10.1021 / jf010093j

Van den Heuvel, R.H.H., van den Berg, W.A.M., Rovida, S., van Berkel, W.J.H., 2004. Laboratory- evolved vanillyl-alcohol oxidase produces natural vanillin. J. Biol. Chem. 279, 33492-500. doi: 10.1074 / jbc.M312968200

Claims

CLAIMS.

1. Recombinant vanillin alcohol oxidase protein CHARACTERIZED because it comprises vanillyl alcohol oxidase (VAO) enzyme fused with maltose binding protein (MBP) at its N-terminal end, where the enzyme has at least 80% homology with SEQ ID No. 1 and where the vanillyl alcohol oxidase enzyme maintains its activity.

2. Recombinant vanillyl alcohol oxidase protein according to claim 1 CHARACTERIZED because it has a molecule at its C-terminal end that facilitates its purification.

3. Recombinant vanillyl alcohol oxidase protein according to claim 2 CHARACTERIZED because the molecule that facilitates its purification is a histidine polypeptide.

4. Recombinant vanillyl alcohol oxidase protein, according to any of the preceding claims CHARACTERIZED because the VAOr is highly stable, forming dimers in solution, and has a high enzymatic activity.

5. Isolated polynucleotide CHARACTERIZED because:

to. encodes the protein of SEQ ID No. 1; or

b. It has at least 80% homology with the sequence SEQ ID No. 2;

6. Isolated polynucleotide according to claim 5 CHARACTERIZED in that at the end encoding the C-terminus of the protein encodes a molecule that facilitates the purification of said protein.

7. Isolated polynucleotide according to claim 5 CHARACTERIZED in that at the end encoding C-terminus of the protein encodes histidine polypeptide.

8. CHARACTERIZED vector because it contains the polynucleotide of claim 4.

9. Vector according to claim 8 CHARACTERIZED because it is an expression vector where the polynucleotide sequence is operably linked to at least one regulatory sequence that allows the expression of the polynucleotide sequence in a host cell.

10. Transformed cell CHARACTERIZED because it comprises the vector according to claims 8 or 9.

11. Transformed cell according to claim 10 CHARACTERIZED because it is a bacterium.

12. Transformed cell according to claim 11 CHARACTERIZED because it is Escherichia coli.

13. Method for obtaining CHARACTERIZED vanilla because vanillin alcohol is contacted with the recombinant vanillyl alcohol oxidase enzyme of any of claims 1 to 4, or with the transformed cell expressing this enzyme, according to any of claims 10 to 12.