KR101061412B1 - Cloning of Isoeugenol Monooxygenase and Uses thereof - Google Patents

Abstract

The present invention relates to an isoeugenol monooxygenase consisting of a novel amino acid sequence, a nucleic acid molecule encoding the same, and a method for synthesizing an enzymatic vanillin derivative using the same. Since the vanillin derivative synthesis process of the present invention uses only the enzyme itself, it is possible to produce vanillin derivatives more economically and efficiently outside the cell without the addition of coenzymes and / or cofactors, without converting vanillin to a substance such as vanillic acid.

Vanillin, Eugenol, Isoeugenol, Isoeugenol Monooxygenase, Pseudomonas Nitroreducence Jin1

Description

Cloning of Isoeugenol Monooxygenase and Uses}

The present invention relates to isoeugenol monooxygenase, nucleic acid molecules encoding the same and uses thereof.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the most commonly used spice substance used in foods, beverages, perfumes, and medicines. It is the main substance of vanilla flavour, which is widely known and produced tens of thousands of tons worldwide. What is obtained is about 120 tons. Biological vanilla scent is derived from Vanilla planifolia , a plant grown only in tropical climates such as Madagascar or Indonesia. It can now be chemically produced from the benzene derivative quaiacol, and the synthetic vanillin is priced at about $ 12 per kg. On the other hand, bio-based natural vanillin prices can be traded at $ 4,000 / kg.

In the process of producing natural vanillin from plants, vanilla beans can be obtained when vanilla flanifolia flowers harvest vanilla beans after about 8 months after several minutes and ripen for about 6 months. However, the amount of vanillin that can be obtained from vanilla beans is about 2% of the weight of vanilla beans, resulting in low productivity. Also, in the real market, there is an imbalance between undersupply and oversupply due to consumers' preference for "natural", "healthy" or "well-being" related products. For example, in 1990, 70% of all food spices in Germany were derived from natural substances (ie biological origins), and as health concerns grew, the preference for natural substances increased. For the same reason, the price of natural vanillin in the market differs significantly from that of synthetic vanillin.

However, there are several problems in relying only on plants for producing stable natural vanillin. As mentioned earlier, vanilla planifolia can only be grown in the tropics, and it takes more than a year to obtain vanillin, and productivity is affected by various environmental factors such as plant diseases and climate. Fertilizers or pesticides used to improve productivity also cause chronic environmental pollution. Therefore, methods for producing natural vanillin from renewable materials using microorganisms need to have more stable productivity than natural vanillin production derived from plants.

In the prior art, various methods have been proposed for producing natural vanillin using microorganisms. U.S. Pat.No.5017388 discloses the production of vanillin using microorganisms which bioconvert the isoeugenol and eugenol to vanillin. International Application No. PCT / US99 / 21323 discloses a process for making vanillic acid from a carbon source and converting vanillic acid to vanillin using a specific enzyme. International Patent No. PCT / EP99 / 07952 discloses a natural vanillin production method using a mutant strain in which vanillin dehydrogenase that degrades vanillin is inactivated so that vanillin is not degraded and converted to vanillic acid. EP 0542348 discloses a method for the bioconversion of isoeugenol or eugenol to vanillin using lipooxygenase (EC 1.13.11.12). U.S. Pat. No. 6,652,831 discloses a technique for cloning genes related to the metabolic process of producing vanillin from eugenol and genes that are expected to be involved in decoding lignostilbene deoxygenase.

To date, several microorganisms capable of converting isoeugenol into vanillin have been reported in the literature (Furukawa, H. et al. J. Biosci. Bioeng. (2003) 96: 401-3., Hua, D.et J. Biotechnol. (2007) 130: 463-70., Kasana, R C et al. Curr. Microbiol. (2007) 54: 457-61., Priefert, H. et. al. Appl.Microbiol.Biotechnol (2001) 56: 296-314., Shimoni, E. et. Al. J. Biotechnol. (2003) 105: 61-70., Shimoni, E. et. Al. J. Biotechnol. (2000) 78: 1-9., Unno, T. et. Al. J. Agric. Food.Chem. (2007) 55: 8556-61., Yamada, M. et. Al. Appl. Microbiol.Biotechnol. (2007) 73: 1025-30., Zhang, Y. et.al. Appl.Microbiol.Biotechnol. (2006) 73: 771-9., Zhao, L.-Q. et.al.Process Biochem. (2006) 41: 1673- 6., Zhao, LQ et. Al. Biotechnol. Lett. (2005) 27: 1505-9.). In many cases, however, microorganisms use isoeugenol as a carbon source or energy source, so vanillin is rapidly converted to a material such as vanillic acid, which increases the vanillin concentration in the culture drug and then decreases over time. The microbiological toxicity of vanillin itself also interferes with increasing vanillin concentration in the culture.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors tried to find a more economical and efficient method for synthesizing vanillin and synthesizing enzymes without converting vanillin to a substance such as vanillic acid using only the enzyme itself, unlike the conventional method for preparing vanillin using a microorganism. As part of such efforts, the present invention has been accomplished by securing novel isoeugenol monooxygenase related to vanillin synthesis and identifying nucleic acid molecules encoding the enzymes.

Accordingly, it is an object of the present invention to provide isoeugenol monooxygenase and nucleic acid molecules encoding the same, consisting of novel amino acid sequences.

Another object of the present invention is to provide a method for preparing a vanillin derivative using the isoeugenol monooxygenase.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the present invention provides an isoeugenol monooxygenase consisting of the amino acid sequence of SEQ ID NO: 2 sequence.

The present inventors tried to find a more economical and efficient method for synthesizing vanillin and synthesizing enzymes without converting vanillin to a substance such as vanillic acid using only the enzyme itself, unlike the conventional method for preparing vanillin using a microorganism. As a result, a novel isoeugenol monooxygenase enzyme was discovered and it was confirmed that vanillin can be produced more efficiently and at a lower production cost.

As used herein, the term “isoeugenol monooxygenase” refers to an oxygenase enzyme involved in the oxidation of eugenol or isoeugenol. On the other hand, isoeugenol monooxygenase of the present invention can induce oxidation reaction of eugenol or various derivatives of isoeugenol. Preferably the isoeugenol monooxygenase of the present invention catalyzes the bioconversion of isoeugenol or eugenol to vanillin derivatives.

According to a preferred embodiment of the invention, the isoeugenol monooxygenase is derived from a microorganism. More preferably, the isoeugenol monooxygenase is derived from Pseudomonas genus microorganisms, and most preferably from Pseudomonas nitroreducence Jin1 (KCTC 11023BP).

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the isoeugenol monooxygenase.

Most preferably, the nucleic acid molecule of the present invention comprises the nucleotide sequence represented by SEQ ID NO: 1.

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites as well as natural nucleotides. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

In the range having the ability to convert the carbon source, which is the most characteristic feature of the isoeugenol monooxygenase of the present invention, to vanillin derivatives, the isoeugenol monooxygenase of the present invention may be selected from the amino acid sequence It is apparent to those skilled in the art that the present invention is not limited to nucleotide sequences.

Variations in nucleotides do not result in changes in proteins. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (eg, by degeneracy of the codon, there are six codons for arginine or serine), or codons that encode biologically equivalent amino acids. And nucleic acid molecules.

In addition, variations in nucleotides may result in changes in the isoeugenol monooxygenase itself. Even in the case of mutations that bring about a change in the amino acid of isoeugenol monooxygenase, it can be obtained that exhibit almost the same activity as the isoeugenol monooxygenase of the present invention.

It is apparent to those skilled in the art that the biological function equivalents that may be included in the isoeugenol monooxygenase of the present invention will be limited to variations in amino acid sequences that exert biological activity equivalent to the isoeugenol monooxygenase of the present invention. .

Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing mutations, hydropathic idex of amino acids may be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference within the range of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

Considering the above-described variations with biologically equivalent activity, the isoeugenol monooxygenase of the present invention or nucleic acid molecule encoding the same is construed to include a sequence that exhibits substantial identity with the sequences listed in the Sequence Listing. . Such substantial identity may, for example, be at least 99% when the sequences of the present invention are aligned with each other as closely as possible and the aligned sequences are analyzed using algorithms commonly used in the art. It means a sequence showing homology of. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparison methods using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to another aspect of the invention, the invention provides a vector comprising the nucleic acid molecule of the invention described above which encodes isoeugenol monooxygenase.

The vector system of the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell as a host. Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a tac promoter, a lac promoter, a lac UV5 promoter, an lpp promoter, a p _L ^λ promoter) capable of promoting transcription , p _R ^λ promoters, rac 5 promoters, amp promoters, recA promoters, SP6 promoters, trp promoters and T7 promoters, etc.), ribosome binding sites for initiation of translation and transcription / detox termination sequences. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p) _L ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1 which are often used in the art). And M13, etc.) or viruses (eg, SV40, etc.).

Vectors of the present invention may be fused with other sequences to facilitate purification of the isoeugenol monooxygenase expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6x His (hexahistidine; Quiagen, USA), and most preferably Is 6 × His. Because of the additional sequence for this purification, the protein expressed in the host is purified quickly and easily through affinity chromatography.

According to a preferred embodiment of the present invention, the fusion protein expressed by the vector containing the fusion sequence is purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of this enzyme, can be used, and when 6x His is used, a desired iso isoform using a Ni-NTA His-binding resin column (Novagen, USA). Eugenol monooxygenase can be obtained quickly and easily.

Meanwhile, the vector of the present invention includes an antibiotic resistance gene commonly used in the art as an optional marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.

According to another aspect of the present invention, the present invention provides a transformant comprising the vector of the present invention described above.

Host cells capable of stable and continuous cloning and expression of the vectors of the present invention are known in the art and can be used with any host cell, for example E. coli JM109, E. coli BL21 (DE3), E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.

The method of carrying the vector of the present invention into a host cell is performed by CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol. , 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res. , 16: 6127-6145 (1988)) and the like.

Vectors injected into host cells can be expressed in host cells, in which case a large amount of isoeugenol monooxygenase is obtained. For example, when the expression vector contains a lac promoter, the host cell may be treated with IPTG to induce gene expression.

According to another aspect of the invention, the invention provides a process for the preparation of vanillin derivatives comprising the following steps:

(a) preparing the isoeugenol monooxygenase of the present invention as described above;

(b) contacting the isoeugenol monooxygenase with a compound of formula 1 or 2; And

(c) obtaining a vanillin derivative of formula (3).

Formula 1

In the above formula, R ₁ is -CH = CHCH ₃ or -CH ₂ CH = CH ₂ , R ₂ -R ₄ are each independently of each other hydrogen, C _1-5 alkyl group, hydroxyl group or C _1-5 alkoxy group to be.

Formula 2

In the above formula, R ₁ -R ₆ are each independently hydrogen, C _1-5 alkyl group, hydroxyl group or C _1-5 alkoxy group.

Formula 3

In the above formula, R ₁ -R ₃ are each independently of each other hydrogen, a C _1-5 alkyl group, a hydroxyl group or a C _1-5 alkoxy group.

The present inventors have attempted to find enzymes having vanillin biosynthesis and bacterial strains producing such enzymes, and have tried to develop various vanillin derivatives using the enzymes or strains. As a result, Pseudomonas genus-derived isoeugenol monooxygenase which can oxidize vanillin differently from the conventionally known synthetic method was discovered, and the production of vanillin derivative was confirmed from this.

The isoeugenol monooxygenase used in the present invention for preparing vanillin derivatives is preferably derived from microorganisms. More preferably, the isoeugenol monooxygenase is derived from Pseudomonas genus microorganisms, and most preferably from Pseudomonas nitroreducence Jin1 (KCTC 11023BP).

In the method of the present invention, isoeugenol monooxygenase to be used as a reaction enzyme comprises (i) isoeugenol monooxygenase in purified form, and (ii) isoeugenol monooxygenase-encoding gene. It may contain a microorganism expressing this enzyme in the cell, or (iii) a lysate or cell extract of the microorganism.

The compound used as a substrate of isoeugenol monooxygenase in the method of the present invention is a compound of Formula 1 or Formula 2.

According to a preferred embodiment of the invention, in formula 1 R ₂ is hydrogen, R ₃ is a hydroxyl group and R ₄ is a methoxy group. According to a preferred embodiment of the present invention, R ₁ -R ₆ in formula (2) are each independently hydrogen, methyl group, hydroxyl group or methoxy group.

In the formulas defining the above compounds, the term “C _1-5 alkyl group” means a straight or pulverized saturated hydrocarbon group of 1-5 carbon atoms, for example methyl, ethyl, n -propyl, isopropyl, isobutyl, n − Butyl, t -butyl and n -pentyl. The term “alkoxy” refers to an —Oalkyl group and includes, for example, methoxy and ethoxy.

According to a preferred embodiment of the invention, step (c) is carried out in the absence of coenzymes, cofactors or both.

One of the greatest features of the present invention is that the isoeugenol monooxygenase does not require coenzymes and cofactors to catalyze the oxidation reaction. Oxygenases, or enzymes that play oxygen, are known to require coenzymes such as NADH or metal cofactors such as iron ions to activate oxygen. It is known that iron ions are required for the reaction of carotenoid oxygenase and lignostilbene deoxygenase having high amino acid sequence similarity to isoeugenol monooxygenase of Pseudomonas nitroreducens Jin. However, in the case of the isoeugenol monooxygenase of the present invention, it is possible to bioconvert isoeugenol to vanillin regardless of conditions with and without NADH, and particularly, a metal cofactor such as iron ion in the reaction solution. Since the reaction occurs even without addition, it can be seen that the oxidase reacts independently of the coenzyme and the cofactor in performing the oxidation reaction in which oxygen is added to the reactants (see FIG. 5).

According to a preferred embodiment of the present invention, the isoeugenol monooxygenase is present outside the cell.

As isoeugenol monooxygenase does not require coenzymes and cofactors as described above, the reaction may occur when isoeugenol monooxygenase is present in the cell as well as outside the cell, in which case a substrate, such as Isoyuenol can save the energy of the cells consumed to enter the cell. Thus, not only the use of a microorganism containing an isoeugenol monooxygenase-encoding gene containing the enzyme in a cell, but also a lysate of the microorganism, a cell extract obtained from the lysate, or a purified form of isoeugenol Monooxygenase can be used to produce vanillin derivatives extracellularly without the addition of coenzymes and / or cofactors.

Contacting the isoeugenol monooxygenase with the compound of Formula 1 or 2 may be carried out under conventional enzymatic reaction conditions.

The features and advantages of the present invention are summarized as follows.

(Iii) The present invention provides an isoeugenol monooxygenase consisting of a novel amino acid sequence and a nucleic acid molecule encoding the same.

(Ii) The present invention provides a method for synthesizing a vanillin derivative enzymatically using only isoeugenol monooxygenase, unlike conventionally known synthetic methods.

(Iii) The method of the present invention does not convert vanillin to a substance such as vanillic acid using only the enzyme itself and can produce vanillin derivatives more economically and efficiently outside the cell without the addition of coenzyme and / or cofactors.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Pseudomonas nitro red sense  Genome library construction of Jin1 strain and screening method of colonies with isoeugenol degrading activity

In the strain Pseudomonas nitrolidusense Jin1 (Accession No: KCTC 11023BP), in which the present inventors bioconvert the isoeugenol previously invented (Korean Patent No. 10-0830691) to vanillin, the strain was used to find a gene involved in bioconversion. Jin1's genomic DNA library was constructed. BAC (Bacterial artificial chromosomes) plasmid pIndigoBAC536 (Caltech, Calififornia, USA) restriction enzyme Hind Ⅲ after treatment with the same restriction enzymes was treated parts of the genomic DNA of strain Jin1 combined (ligation) reaction with Escherichia coli (E. coil ) DH10B (Life Technologies, France) was transformed by electroporation. About 1,100 colonies with genomic DNA of strain Jin1 on the order of 50-100 kb were obtained.

The following method was performed to find colonies capable of decomposing isoeugenol in 1,100 colonies obtained. Dispense 1 ml Luria-Broth (LB, 10 g tryptone, 10 g NaCl, 5 g yeast extract, pH 7.5) with the addition of antibiotic chloramphanicol at a concentration of 12.5 μg / ml in a 24-well culture vessel. After inoculation with colonies, the cells were cultured at 37 ° C. for at least 12 hours. Eight different colonies were inoculated in a 20 ml LB containing 200 μl of antibiotics in a plastic culture vessel, and the incubation was performed using a Teflon-coated rubber stopper in a 125 ml serum bottle. In a vessel, 500 μM isoeugenol was added to the culture for the expression of isoeugenol degradation related genes. After incubation at 30 ° C. for 12 hours or more, the culture solution was centrifuged to collect E. coli, and then suspended in bacteria and centrifuged again in a minimal medium (see Reference Example). For the decomposition experiment of isoeugenol, 1 ml of a bacterial suspension and 50 μM of isoeugenol were added to a 15 ml serum bottle and reacted at 30 ° C. for 20 hours. 1 ml of ethyl acetate was added to the reaction solution, vigorously mixed for 30 seconds, and then centrifuged with a desktop centrifuge. The ethyl acetate layer and the aqueous solution layer were separated by centrifugation, and only the ethyl acetate layer was transferred to a 2 ml glass container for HPLC analysis, and the amount of isoeugenol or vanillin produced by gas chromatography was analyzed by the peak area of each substance. It was.

Reference Example 1 Stanier's basal minimal salts buffer

1) A solution: 69.5 g of Na ₂ HPO _{4 and} 69.5 g of KH ₂ PO ₄ were dissolved in water to a final volume of 1 L.

2) Solution B: Nitrilotriacetic acid 20.0 g, MgSO ₄ 28.90 g, CaCl 5.04 g, (NH ₄ ) ₆ MO ₇ O ₂₄ 4H ₂ O 18.50 mg, FeSO ₄ 7H ₂ O 198.0 mg, Nicotinic 100 mg of acid was dissolved in water, and then mixed with a solution containing 100 ml of metal ions to make a final volume of 1 L.

Composition of Metal Ion Solution (per 100 ml) compound content Disodium salt (EDTA) 0.25 g ZnSO ₄ ㆍ 7H ₂ O 1.1 g FeSO _{4 7} H ₂ O 0.5 g MnSO ₄ H ₂ O 0.154 g CuSO ₄ ㆍ 5H ₂ O 0.04 g Co (NO ₃ ) ₂ ㆍ 6H ₂ O 0.025 g Na ₂ B ₄ O ₇ 10 H ₂ O 0.018 g

3) C solution: Dissolve 20.0 g of (NH ₄ ) ₂ SO ₄ in 100 mL of water.

4) 4 ml of A solution, 2 ml of B solution and 0.5 ml of C solution were added per 1 L of water to make a minimum medium.

In the same manner as above, one colony group having a reduced peak area of isoeugenol was obtained on gas chromatography, and eight colonies included in the group were inoculated in 20 ml LB containing antibiotic chloramphenicol at a concentration of 12.5 μg / ml. Isoeugenol was added by the method and incubated. Among the 8 colonies, the colonies capable of decomposing isoeugenol were named 611A and the plasmids of the colonies were named p611A.

Example 2: inserted into plasmid p611A Pseudomonas nitro red sense  Jin1 genomic DNA sequencing and gene analysis

Analysis of the DNA sequence of the genomic DNA portion of the strain Jin1 possessed by the plasmid p611A confirmed that about 140 kb of genomic DNA was inserted. Based on the DNA base sequence, the amino acid sequence of the DNA portion estimated to be a gene having a nucleotide sequence of 400 bp or more based on ATG or GTG, a code that starts transcription of the gene, and TGA TAA TAG, a code that ends transcription. Compared to the NCBI Protein Blast search ( http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome ) database and contains 22 predicted genes Confirmed. The results are shown in Table 2 below. The predicted genes in the 29 kb DNA fragment are expected to be metabolic genes involved in eugenol and isoeugenol metabolism (ORF1, 3, 4, 6-10, 15-21) and transcriptional regulators. . the gene (ORF2, 5, 22) and Pseudomonas footage is IE27 (Pseudomona putida IE27) the mono iso-eugenol oxide cyclooxygenase (isoeugenol monooxygenase isolated from; Yamada, M. et al Arch Microbiol (2007) 187...: 511-7) ORF12 with similarity to the 81.4% amino acid sequence with the gene and ORF11 gene with 47% amino acid sequence similarity with the predicted gene expression regulator reported in the same paper. Figure 1 is a diagram showing the direction of the predicted gene of 29 kb DNA, Table 2 summarizes the gene annotation for each gene number. 30,813 bp of a part of the DNA sequence obtained in the present invention showed that 30,784 bp of the 32,679 bp DNA sequence (BLAST DNA sequence Accession No: A92090) presented in the existing patent (EP 0845532) was 98.4% similar. Existing patents reveal the functions of genes involved in the metabolic process of vanillic acid via eugenol conniperyl alcohol, conilferyl aldehyde, ferulic acid, vanillin, etc. Was done. ORF12 (1437 bp) shows 98% amino acid sequence similarity with the expected gene annotated with lignostilbene dioxygenase, 1518 bp of the existing patent. However, the existing patents are merely annotations, and the present invention reports the function of the expected gene (ORF12) for the first time.

Gene Numbers and Gene Annotations of Genes in 29kb DNA Fragments Gene number Gene annotation ORF1 Coniferyl alcohol dehydrogenase ( calA ) ORF2 Putative transcriptional activator ( eugR ) ORF3 Transcriptional regulator ORF4 Coniferyl aldehyde dehydrogenase ( calB ) ORF5 Transcriptional regulator, MarR family ORF6 Methyl-accepting chemotaxis protein ORF7 Beta-ketothiolase ( aat ) ORF8 Feruloyl-CoA-synthetase (fcs) ORF9 Vanillin dehydrogenase ( vdh ) ORF10 Enoyl-CoA-hydratase / aldolase ( ech ) ORF11 Putative transcriptional regulator ORF12 Isoeugenol monooxygenase ORF13 NADPH: quinone reductase and related Zn-dependent oxidoreductases ORF14 Predicted nucleoside-diphosphate-sugar epimerase ORF15 Eugenol hydroxylase flavoprotein subunit ( EhyB ) ORF16 Hypothetical protein ORF17 Eugenol hydroxylase flavoprotein subunit ( EhyA ) ORF18 Putative gamma-glutamylcysteine synthetase ( gcs ) ORF19 Putative formaldehyde dehydrogenase ( fdh ) ORF20 Vanillate O-demethylase oxidoreductase (v anB ) ORF21 Vanillate O-demethylase oxidoreductase ( vanA ) ORF22 Transcriptional regulator ( vanR )

Example 3: Comparison of amino acid sequences of isoeugenol monooxygenase and other enzymes

Isoeugenol monooxygenase has an amino acid sequence 81.4% identical to the isoeugenol monooxygenase (ISM) of Pseudomonas putida IE27 mentioned above. Pseudomonas Posey Mobi-less (Pseudomonas paucimobilis) TMY1009 lignoceric stilbene dioxygenase dehydratase (lignostilbene dioxygenase, LSD) than 41.6%, Novos ping hump moth Aromatikkushida time signal lance (Novosphingobium aromaticivorans) carotenoids oxide cyclooxygenase the DSM 12444 strain (carotenoid oxygenase, CRTO) has 39.2% amino acid sequence similarity. Figure 2 is arranged based on the amino acid sequence of each gene.

Example 4 Cloning of the Isoeugenol Monooxygenase Gene

1. Gene Cloning in Plasmid pUC19

The following primers were designed based on the DNA sequences obtained to determine if the isoeugenol monooxygenase gene can actually convert isoeugenol to vanillin.

5'-primer: 5'- ACA ACA AAG GAG ACC GTG CC -3 '

3'-primer: 5'- TTA AGG TCT GGG TAC CCA GC -3 '

After PCR polymerization using the genomic DNA obtained from the primer and strain Jin1 as a template, the obtained PCR band was inserted into the pGEMTeasy vector (Invitrogen, USA). Plasmid pGEM :: IEM was cut with restriction enzyme EcoR I and inserted into pUC19 and pUC19 :: IEM was transformed into E. coli. After incubating the transformed Escherichia coli for at least 12 hours at 30 ° C., the bacteria were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.0), and centrifuged again, and the absorbance was 10 at 600 nm. The bacteria were suspended in the same buffer. 2 ml suspension, 1 mM glucose, and 3 mM isoeugenol were added to a 15 ml serum bottle, followed by reaction at 30 ° C. for 2 hours. The reaction time of 0 hours and 2 hours was extracted with 4 mL ethyl acetate and the extract was analyzed by gas chromatography. As a result, when the reaction time was 2 hours, the peak area of isoeugenol was reduced compared to the reaction time of 0, but vanillin increased the peak area. This isoisogenol was confirmed that the bioconversion to vanillin by the isoeugenol monooxygenase gene.

2. Cloning Genes in Plasmid pET21-a

The isoeugenol monooxygenase gene was amplified by PCR polymerization with the following primers. The base sequence of the primer is as follows. Each primer artificially inserted a specific restriction enzyme.

5'-primer: AAAA CATATG GCG AGA CTC AAC CGC AAC

3'-primer: AAAA CTCGAG TTA AGG TCT GGG TAC CCA GC

(Bold italics means Nde I and Xho I)

The PCR band obtained after the PCR reaction was first inserted into the pGEMTeasy vector, DNA sequencing analysis was performed, and the plasmids were extracted, treated with restriction enzymes Nde I and Xho I, and then treated with the same enzyme, pET21-a ( Novagen, USA) was used to make pETIEM. Plasmid pETIEM was designed to contain a transcription stop code immediately before the Xho I recognition sequence of the 3'-primer so that no tag was expressed. The made plasmid E. coli BL21 (DE3) (Studier, FW et. Al, Methods Enzymol. (1990) 185: 6089) were transformed.

Example 5: HPLC Analysis Method

HPLC was analyzed by the following method. Reversed-phase C ₁₈ (5 μm particle size, 4.6 mm × 25 cm, Milford, Mass.) Was mounted on Varian ProStar HPLC (Varian, Walnut Creek, Calif.) And 20 μl of samples analyzed. H ₂ O and acetonitrile containing 0.1% formic acid were used as solvents. The solvent ratio started with 10% acetonitrile, 20% at 5 minutes, 40% at 12 minutes, and 90% acetonitrile at 17 minutes. The ratio was fixed up to and the flow rate was set to 1 ml per minute. The absorbance was measured with a UV detector at 270 nm using a Photo Diode Array (Varian, Walnut Creek, Calif.) To obtain an HPLC chromatogram. The retention times of vanillin and isoeugenol were 12.9 and 18.2 minutes, respectively.

Example 6 Experimental Method for Enzymatic Reaction of Isoeugenol Monooxygenase Overexpressed in Escherichia Coli

In the transformed E. coli BL21 (DE3) / pETIEM prepared in Example 4 to obtain a water-soluble isoeugenol monooxygenase under the following conditions. After inoculating the bacteria in 600 ml LB, the temperature of the incubator was changed to 20 ° C. when the absorbance reached 0.2 at 600 nm after growing at 37 ° C. and 100 rpm. When the absorbance was 0.4-0.6, IPTG was added to the concentration of 0.1 mM to induce gene expression. After incubation for 12 hours, the bacteria were collected by centrifugation, suspended in 20 mM Tris HCl buffer (Tris-HCl, pH8.0), and centrifuged again. Centrifuged bacteria were stored at -80 ° C.

20 ml of 20 mM hydrochloric acid tris buffer solution (pH 8.0) added with 10% glycerol and 1 mM ditriodiol (DTT) to 10 g of the centrifuged bacteria (based on the wet sample weight) to obtain a cell extract. The bacterial membrane was broken by using an ultrasonic crusher for 20 min at 70% intensity, and the supernatant was taken after centrifugation at 4 ° C. for 30 minutes at a speed of 18,000 g.

Enzyme reaction was carried out in the following manner. The reaction was started by adding the cell extract to 100 mM phosphate buffer and 10% ethanol and adding 7 mM isoeugenol. The total reaction solution was 1 ml, the reaction was carried out at 30 rpm for 30-30 minutes at 150 rpm and then strongly mixed for 10 seconds after the addition of 1 ml methanol. The supernatant was analyzed by HPLC after centrifugation.

Figure 3 is a reaction of the cell extract of E. coli BL21 (DE3) / pETIEM for 10 minutes (C), 20 minutes (B), 30 minutes (A) and the reaction without reacting for 30 minutes under the same conditions (D) HPLC chromatogram of the solution. X axis shows analysis time and Y axis shows absorbance at 270 nm. As the reaction time increases, the amount of isoeugenol decreases and the amount of vanillin increases. Based on these results, it can be seen that isoeugenol monooxygenase has an enzymatic function of bioconversion of isoeugenol to vanillin as shown in FIG. 4.

Example 7: Coenzyme and Cofactor Independence of Isoeugenol Monooxygenase

In the case of an oxidase, an enzyme that plays a role in oxygen, it is known that coenzymes such as NADH or metal cofactors such as iron ions and metals are required to activate oxygen. ISM isolated from Pseudomonas putida IE27 has not been significantly affected by iron ions or various coenzymes even though oxygen is required. In contrast, carotenoid oxygenase and lignostilbene deoxygenase, which have high amino acid sequence similarity to IEM of strain Jin1, are known to require iron ions for reaction. Therefore, in the present invention, the cell extract was prepared by the following method in order to test the dependency of coenzyme or cofactor of IEM. In the case of IEM obtained in this study, it was confirmed that the enzyme reaction occurs even in the absence of NADH. Coenzymes such as NADH have a problem of low economical efficiency in enzymatic process because their price is quite expensive. If enzymatic reaction occurs without NADH, it can be applied economically in various ways.

Figure 5 shows the condition (A) with or without coenzyme NADH using the cell extract of E. coli, which induced the expression of the isoeugenol monooxygenase gene after transforming pETIEM into Escherichia coli BL21 (DE3). HPLC analysis of the production of vanillin at The X axis shows the analysis time and the Y axis shows the absorbance of 270 nm. As shown by HPLC analysis, isoeugenol monooxygenase can convert isoeugenol to vanillin regardless of conditions with and without NADH, and especially without adding metal cofactors such as iron ions to the reaction solution. Since the reaction occurs, it can be seen that the oxidase reacts independently of the coenzyme and the cofactor in performing the oxidation reaction in which oxygen is added to the reactants. These results indicate that the reaction can occur when isoeugenol monooxygenase is present in the cell as well as outside the cell, for example by expressing a gene in the cell membrane to save energy of the cell that is consumed to enter the isoyugenol into the cell. It is expected that it can be used for more efficient vanillin production.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

FIG. 1 is a genetic map of approximately 29 kb DNA fragments in which genes related to eugenol and isoeugenol metabolism of some genomic DNAs of Pseudomonas nitrolidusense Jin1 inserted into plasmid p611A have been collected.

Figure 2 is an alignment of isoeugenol monooxygenase (IEM) of Pseudomonas nitroreducens Jin1 with high amino acid sequence similarity. ISM (iso eugenol mono oxide cyclooxygenase, Pseudomonas footage is IE27), LSD (lignoceric stilbene dioxygenase dehydratase, Pseudomonas Posey tombstones less TMY1009), CRTO (carotenoids oxide cyclooxygenase, Novos ping hump moth Maroni Marti time signal pullulans DSM 12444 ) Is aligned.

Figure 3 is transformed into E. coli BL21 (DE3) pETIEM transformed into E. coli induces the expression of E. coli cells extract (cell extract) 10 minutes (C), 20 minutes (B), It is the HPLC chromatogram of reaction liquid which reacted for 30 minutes (A) and the reaction liquid (D) for 30 minutes, without adding a cell extract.

Figure 4 shows the metabolic process of Pseudomonas nitroreducens Jin1 isoeugenol vanillin by isoeugenol monooxygenase.

Figure 5 shows the condition (A) with or without coenzyme NADH using the cell extract of E. coli, which induced the expression of the isoeugenol monooxygenase gene after transforming pETIEM into Escherichia coli BL21 (DE3). HPLC analysis of the production of vanillin at

<110> Gwangju Institute of Science and Technology <120> Cloning of Isoeugenol Monooxygenase and Uses <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1437 <212> DNA <213> Pseudomonas nitroreducens <220> <221> CDS (222) (1) .. (1434) <400> 1 atg gcg aga ctc aac cgc aac gac ccg caa tta gta gga aca ctt ctc 48 Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu   1 5 10 15 ccc acc cgt atc gag gca gac ttg ttc gat cta gag gtt gac ggc gaa 96 Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu              20 25 30 atc cca aaa tca ata aat gga acg ttc tac cgt aat acg cca gaa cct 144 Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro          35 40 45 caa gtt acc ccg caa aaa ttc cac acc ttc ata gat gga gat gga atg 192 Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met      50 55 60 gcc tct gcc ttc cac ttc gaa gat ggt cat gtc gac ttc atc agt cgc 240 Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg  65 70 75 80 tgg gtt aaa acc gct cga ttc acg gcc gaa cga cta gcg cga aaa tcg 288 Trp Val Lys Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Lys Ser                  85 90 95 cta ttt ggc atg tac aga aac ccc tat acc gac gac acc agt gta aaa 336 Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys             100 105 110 gga cta gac cgc acc gtt gcc aat aca agc atc att agc cat cac ggc 384 Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly         115 120 125 aag gtg ctg gcg gtg aag gaa gac ggc cta ccg tac gaa ctg gat cct 432 Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro     130 135 140 cgt aca ctt gaa act cgc gga cgc ttc gac tac gac ggc caa gtt acc 480 Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr 145 150 155 160 agc caa acc cac acc gcc cat cca aaa tat gac ccg gaa acg ggt gac 528 Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp                 165 170 175 ttg ttg ttc ttc ggt tcg gca gct aag ggc gaa gca act cca gac atg 576 Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met             180 185 190 gcc tat tac att gtc gac aag cac ggc aag gtg aca cat gaa act tgg 624 Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp         195 200 205 ttt gag cag ccc tat ggc gca ttc atg cac gac ttt gcc att acc cga 672 Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg     210 215 220 aat tgg tcc att ttc cca att atg ccg gcc acc aac agc ctg tcc cgc 720 Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg 225 230 235 240 ctc aag gcg aaa cag cca att tat atg tgg gag ccg gaa ctg ggc agc 768 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser                 245 250 255 tac att ggc gta ctg ccg cgc cgc ggc cag ggc agt cag att cgc tgg 816 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp             260 265 270 ctc aag gca ccg gcg ctc tgg gta ttt cat gtt gtg aat gct tgg gaa 864 Leu Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu         275 280 285 gtc gga acc aag att tat atc gac ctt atg gaa agt gaa atc ctg ccg 912 Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro     290 295 300 ttc ccc ttc ccc aac tca caa aac caa ccc ttc gcc cct gag aaa gcc 960 Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala 305 310 315 320 gta cca cgc ctg act cgt tgg gaa att gac ctc gat agc agc agc gac 1008 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp                 325 330 335 gag atc aag cga acc cgg cta cac gat ttc ttt gcg gaa atg cca atc 1056 Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile             340 345 350 atg gat ttt cgc ttc gcc ctg caa tgc aac cgc tat ggc ttt atg ggg 1104 Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly         355 360 365 gtg gac gat cca cgc aaa cca ctt gcg cat cag cag gcc gag aag ata 1152 Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile     370 375 380 ttt gcg tac aac tca ctc ggc atc tgg gac aac cac cga ggt gac tac 1200 Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr 385 390 395 400 gac ctc tgg tac tcc gga gaa gcc tcg gcg gcc cag gag ccg gcc ttc 1248 Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe                 405 410 415 gtc cct aga agt ccg acc gcc gcc gaa ggt gat ggg tac ttg ctg acc 1296 Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr             420 425 430 gtg gtt ggt cgt ctc gat gaa aat cgc agt gat ctg gta att ctc gac 1344 Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp         435 440 445 act caa gac atc cag tct ggt ccc gtg gca acc atc aag ctg cca ttc 1392 Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe     450 455 460 cgg tta agg gcc gct ctc cat ggc tgc tgg gta ccc aga cct taa 1437 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro 465 470 475 <210> 2 <211> 478 <212> PRT <213> Pseudomonas nitroreducens <400> 2 Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu   1 5 10 15 Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu              20 25 30 Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro          35 40 45 Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met      50 55 60 Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg  65 70 75 80 Trp Val Lys Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Lys Ser                  85 90 95 Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys             100 105 110 Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly         115 120 125 Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro     130 135 140 Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr 145 150 155 160 Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp                 165 170 175 Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met             180 185 190 Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp         195 200 205 Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg     210 215 220 Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg 225 230 235 240 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser                 245 250 255 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp             260 265 270 Leu Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu         275 280 285 Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro     290 295 300 Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala 305 310 315 320 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp                 325 330 335 Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile             340 345 350 Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly         355 360 365 Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile     370 375 380 Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr 385 390 395 400 Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe                 405 410 415 Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr             420 425 430 Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp         435 440 445 Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe     450 455 460 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro 465 470 475  

Claims (10)

Isoeugenol monooxygenase consisting of the amino acid sequence of SEQ ID NO: 2 sequence. A nucleic acid molecule encoding the isoeugenol monooxygenase of claim 1. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 1. An isoeugenol monooxygenase expression vector comprising a nucleic acid molecule encoding the isoeugenol monooxygenase of claim 2 or 3. A transformant comprising the vector of claim 4. Method for preparing a vanillin derivative comprising the following steps: (a) preparing the isoeugenol monooxygenase of claim 1; (b) contacting the isoeugenol monooxygenase with a compound of formula 1 or 2; And (c) obtaining a vanillin derivative of formula (3). Formula 1

In the above formula, R ₁ is -CH = CHCH ₃ or -CH ₂ CH = CH ₂ , R ₂ -R ₄ are each independently of each other hydrogen, C _1-5 alkyl group, hydroxyl group or C _1-5 alkoxy group to be. Formula 2

In the above formula, R ₁ -R ₆ are each independently hydrogen, C _1-5 alkyl group, hydroxyl group or C _1-5 alkoxy group. Formula 3

In the above formula, R ₁ -R ₃ are each independently of each other hydrogen, a C _1-5 alkyl group, a hydroxyl group or a C _1-5 alkoxy group. The method of claim 6, wherein in Formula 1, R ₂ is hydrogen, R ₃ is a hydroxyl group and R ₄ is a methoxy group. The method according to claim 6, wherein in Formula 2 R ₁ -R ₆ are each independently hydrogen, a methyl group, a hydroxyl group or a methoxy group. 7. The method of claim 6, wherein step (c) is carried out in the absence of coenzymes, cofactors, or both. 7. The method of claim 6, wherein said isoeugenol monooxygenase is present outside the cell.

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