KR20140087798A - Process of biologically producing a styrene and derivatives thereof - Google Patents

Abstract

The present invention relates to a method for preparing styrene or a derivative thereof; and a method for preparing a polymer using the same. The styrene or the derivative thereof can be obtained in a biological manner which has higher specificity than a chemical process. Further, the styrene or the derivative thereof can be obtained in an environment friendly and economical manner in which a lignin degradation product is economically and efficiently obtained by using lignin as a raw material and then used as a substrate.

Description

TECHNICAL FIELD [0001] The present invention relates to a process for producing styrene and derivatives thereof by a biological method,

The present invention relates to a method for producing styrene or a derivative thereof by a biological method. The present invention also relates to a process for preparing lignin degradation products by chemical and biological conversion and for producing styrene or derivatives thereof therefrom by a biological method.

In recent years, the petrochemical industry is in a situation where global crude oil prices are continuously rising due to global crisis of oil supply and demand and exhaustion of oil resources. To overcome these difficulties, efforts to develop processes for producing compounds from renewable raw materials under the principle of sustainability have become a global goal. Therefore, as an alternative to the petroleum-based petrochemical industry, the recent global efforts to produce alternative biomass-derived alternative compounds using industrial biotechnology have led to the development of bioenergy, bioplastics, It is becoming visible in various fields.

Styrene is a colorless, viscous liquid with a melting point of -30.6 ° and a boiling point of 145.2 °. Insoluble in water, soluble in ethanol, ether, acetone and very soluble in benzene and petroleum ether. Styrene is a monomer used in the production of polystyrene (PS), acrylonitrile-butadiene-styrene copolymer (ABS), styrene-acrylonitrile resin (SAN), styrene-butadiene elastomer copolymer and unsaturated polyester resin. These polymers are used in plastics, elastomers, food packaging, and processed food industries.

Styrene is produced commercially by dehydrogenating ethylbenzene using high temperature steam at about 720 DEG C in the presence of a metal oxidation catalyst (WO199708034; EP905112). Ethylbenzene is produced by alkylation of benzene with ethylene. However, the production of benzene by hydrodealkylation of toluene requires a lot of energy, and cracker and high cost separation techniques are required for the production of ethylene which is produced by cracking hydrocarbons such as ethane.

Thus, in order to overcome these drawbacks and to develop an energy-saving process, a process using carbon dioxide (Prepr.Pap.-Am.Chem.Soc., Div. Fuel Chem. 2004, 49 (1), 114) . However, since this process also uses 525-575 degrees of steam, a large amount of energy is still required.

In order to overcome the disadvantages of the conventional process and to achieve the sustainability of the process, a biological process based styrene synthesis method has been attempted (Spekreijse et al., Simultaneous production of biobased styrene and acrylates using ethenolysis, Green Chem. ; Rebekah McKenna and David R. Nielsen, Styrene biosynthesis from glucose by engineered E. coli , Metab Eng. 2011, 13 (5), 544). However, these methods use amino acids and the like in the cells as a substrate, and glucose and the like are used as nutrients for culturing microorganisms. Therefore, they may be affected by unstable grain prices, which may adversely affect the economical efficiency of the process. Therefore, it is necessary to develop a method using low-cost biomass such as lignin which can be obtained in abundance in nature.

In the case of bioplastics produced using biomass, polylactic acid (PLA), commercialized by Cargill-Dow, in 2002, replaced polyethylene terephthalate (PET) and polystyrene Recently, the market is rapidly expanding. Also, polyhydroxyalkanoate (PHA) -based bioplastics commercialized by Metabolix in the United States has been moved from the existing high-priced medical polymer market to a factory for entering the general-purpose polymer market have. In addition, PBS (polybutylene succinate) using succinic acid has been commercialized and used. In the biochemical market, 1,3-propanediol commercialized by DuPont at the end of 2006 is a successful example of replacing existing petrochemical processes with biomass-based bioconversion.

In contrast to the examples listed above, styrene is a very rare example of complete bioprocessing, and a paper published by Nielsen's group at Arizona State University (Metab Eng. 2011 Sep; 13 (5): 544-54) And a patent filed by the United States Department of Agriculture (PCT / US2009 / 063219). In the case of the Nielsen group, phenylalanine ammonia-lyase and trans-cinnamic acid decarboxylase are used as a starting material, which is produced by intracellularly produced phenylalanine (L-phenylalanine) US Department of Agriculture patent is a way to use whole cells of fungi.

Therefore, there is a strong need for the development of new styrene or derivatives thereof in view of the social needs, process simplification, and economic improvement of recent eco-friendly process development.

The present invention provides a method for producing styrene or derivatives thereof by a biological method having high specificity compared to a chemical process.

The present invention also provides a method for producing styrene or a derivative thereof by an environmentally friendly biological method using lignin as a raw material, economically and efficiently obtaining a lignin degradation product, and using the lignin degradation product as a substrate.

One embodiment of the present invention is a method for producing a styrene derivative having a 4-hydroxy group, which comprises contacting a substrate comprising styrene having 4-hydroxy group or a derivative thereof represented by the formula (2) with a biocatalyst for removing the 4-hydroxy group, 1 < / RTI > or a derivative thereof,

[Chemical Formula 1]

(2)

In the general formulas (1) and (2), X and Y may be the same or different and independently represent hydrogen, hydroxy or a C1-C4 alkoxy group.

The present invention also relates to a process for preparing a styrene derivative having a 4-hydroxy group represented by the following formula (2), wherein X and Y are not simultaneously hydrogen in the formula (2) At least one substituent selected from the group consisting of a hydroxyl group and a C1-C4 alkoxy group at the 3-position, the 5-position or the 3-position and the 5-position of the aromatic carboxylic acid of the above formula (2) Further comprising the step of contacting the biocatalyst to remove the biocatalyst.

The substrate comprising the 4-hydroxy group-containing styrene or derivative thereof represented by the above-mentioned formula (2) can be produced by reacting a substrate containing an aromatic carboxylic acid having a 4-hydroxy group represented by the following formula (3) And then contacting the biocatalyst with the catalyst.

(3)

When X and Y are not hydrogen at the same time in Formula 3, the catalyst containing the aromatic carboxylic acid having a 4-hydroxy group represented by Formula 3 may be treated with a biocatalyst to remove the carboxyl group, A biocatalyst that removes at least one substituent selected from the group consisting of a hydroxyl group and a C1-C4 alkoxy group at the 3-position, 5-position, or 3-position and 5-position of the aromatic carboxylic acid of Formula 3 Further comprising a step of contacting.

The present invention relates to a method for producing a polymer by polymerizing the styrene or derivatives thereof, wherein the polymer is a homopolymer using one monomer or, in addition to the styrene or derivative monomers thereof, acrylonitrile, Butadiene, acrylic acid, and methacrylic acid may be copolymerized with one or more comonomers selected from the group consisting of acrylic acid and methacrylic acid.

The present invention relates to a process for obtaining aromatic monomers using chemical and / or biological conversion and for producing styrene or derivatives thereof by biological methods, which is capable of producing styrene or derivatives thereof from renewable lithines in an environmentally friendly, highly specific and economical manner . The present invention focuses on completely eliminating the dependence on petrochemical raw materials and using biomass as a raw material as a raw material, and it is a process for producing aromatic monomers produced through chemical or biological decomposition step process using low cost lignin as a raw material, , Styrene or a derivative thereof can be obtained.

Hereinafter, the present invention will be described in more detail.

One embodiment of the present invention is a method for preparing a biodegradable biodegradable polymer, which comprises contacting a substrate comprising styrene having a 4-hydroxy group or a derivative thereof represented by the general formula (2) with a biocatalyst which removes a 4-hydroxy group, The present invention relates to a method for producing styrene or a derivative thereof,

[Chemical Formula 1]

(2)

In the general formulas (1) and (2), X and Y may be the same or different and independently represent hydrogen, hydroxy or a C1-C4 alkoxy group. The C1-C4 alkoxy group may be linear or branched, preferably methoxy or ethoxy.

In still another embodiment of the present invention, there is provided a process for producing styrene or a derivative thereof, comprising contacting a biocatalyst for removing the 4-hydroxy group, wherein X and Y are not simultaneously hydrogen, The method for producing a styrene derivative of the formula (2), which comprises treating a substrate comprising styrene having a 4-hydroxy group or a derivative thereof represented by the formula (2) with a biocatalyst for removing the 4-hydroxy group, And a biocatalyst that removes at least one substituent selected from the group consisting of a hydroxyl group and a C1-C4 alkoxy group at the 3-position, the 5-position or the 3-position and the 5-position of the acid.

The styrene having the formula (1) or a derivative thereof obtained according to the present invention preferably includes styrene, 3-hydroxystyrene, 3,5-dihydroxystyrene, 3-methoxystyrene and 3,5-dimethoxystyrene do.

The styrene having 4-hydroxy group or the derivative thereof represented by the above-mentioned formula (2) may be obtained by reacting 4-hydroxystyrene, 3,4-dihydroxystyrene, 3-methoxy-4-hydroxystyrene, 3,5-dimethoxy- It can be produced by petrochemical processes or biological processes. Preferably, the styrene having 4-hydroxy group of Formula 2 or a derivative thereof is a step of contacting a substrate containing an aromatic carboxylic acid having a 4-hydroxy group represented by the following Formula 3 with a biocatalyst for removing a carboxyl group . ≪ / RTI >

(3)

In Formula 3, X and Y may be the same or different and independently represent hydrogen, hydroxy or a C1-C4 alkoxy group. The C1-C4 alkoxy group may be linear or branched, preferably methoxy or ethoxy.

In another embodiment of the present invention, when X and Y are not hydrogen at the same time in the above formula (3), the aromatic carboxylate having a 4-hydroxy group represented by the above formula (3) At least one substituent selected from the group consisting of a hydroxyl group and a C1-C4 alkoxy group at the 3-position, the 5-position or the 3-position and the 5-position of the aromatic carboxylic acid of the above formula (3) Further comprising the step of contacting the biocatalyst.

Aromatic carboxylic acids having a 4-hydroxy group of the formula (3), i.e., 4-hydroxycinnamic acid or derivatives thereof, a petrochemical process or a biological process.

Chemically, p-hydroxybenzaldehyde and malonic acid can be obtained by reaction with aniline catalyst (Xu et al., Chemical Intermediates, 2006-10; Wang et al., Chemical Reagents, 2009-2; Vassilev et al., Comptes rendus de l'Academie bulgare des Sciences, Tome 34, No. 7, 1981, pp. 125-28). Or the compound of formula (3) can be obtained by separating from plants or lysing lignin. Or from recombinant microorganisms including phenylalanine hydroxylase (PAL) or tyrosine ammonia lyase (TAL) activity (R. Benrief et al. Phytochemistry 47: 825-832 (1998), US20020187207, US20030079255).

Specific examples of the compound of Formula 3 include 4-hydroxycinnamic acid, 3,4-dihydroxynnamic acid, 3,4,5-trihydroxycinnamic acid, 3-methoxy-4-hydroxycinnamic acid (Ferulic acid ), 3,5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid).

For example, the 4-hydroxycinnamic acid or a derivative thereof represented by the formula (3) can be obtained as a lignin degradation product obtained by chemically and / or biologically degrading lignin.

The lignin of the present invention includes lignin, lignin derivatives, lignin fragments, and lignin-containing materials. The lignin derivative means that the lignin is modified by a chemical reaction such as phenolation or acetylation, and the lignin fragment means that the lignin or lignin derivative is chemically or biologically decomposed.

Lignin can be obtained after separating cellulose and hemicelluloses from biorefineries or pulp processes. Examples include kraft lignin (alkaline lignin), dealkaline lignin, hydrolytic lignin, organosolv lignin and sodium lignin sulfonate And lignin, a by-product of the lignocellulosic bioethanol process, is also applicable. The lignin present in the biomass is an aromatic polymer that surrounds the microfibers and contains phenylpropanoids such as coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Is a material composed of a dendritic structure which is complicatedly polymerized with carbon-carbon bonds or carbon-oxygen bonds as constituent units.

The lignin degradation product may be selected from the group consisting of vanillin, syringaldehyde, p-hydroxybenzaldehyde, p-Coumaryl aldehyde, Sinapaldehyde, conipheryl aldehyde Monolignol aromatic alcohols such as aromatic aldehyde such as aliphatic aldehyde, coniferyl aldehyde and cinnamic aldehyde, cinnamic alcohol, coniferyl alcohol and p-coumaryl alcohol, A mixture of aromatic monomers including aromatic carboxylic acids such as cinnamic acid, 4-hydroxycinnamic acid, ferulic acid and sinapinic acid, (3) of the invention.

The decomposition of lignin may be carried out at high temperatures such as pyrolysis, gasification, hydrogenolysis, acidolysis, base decomposition, chemical oxidation, hydrolysis under supercritical condition, A method selected from the group consisting of enzymatic degradation can be performed.

In one example of the present invention, 0.1 to 5% (w / v) H ₂ SO ₄ , HCl or HNO ₃ , or high concentration (0.5 to 2.0 mol / L) of NaOH, or KOH aqueous solution or the like. The acid treatment and the base treatment are preferably carried out at about 80 to 350 DEG C for 5 to 120 minutes.

The decomposition of lignin by pyrolysis can be performed by decomposing lignin by pyrolysis at a high temperature of 350 to 650 ° C. by using a high pressure reactor and by using a catalyst for decomposing nitrobenzene, KMnO ₄ , H ₂ O ₂ and zeolite It is possible to increase the decomposition efficiency. Also, various physico-chemical decomposition methods such as hydrogenolysis and hydrolysis under supercritical condition can be used.

The lignin decomposition reaction is preferably carried out under an oxygen pressure of 2 to 20 bar in order to optimize the decomposition reaction. The reaction time of the above decomposition methods is preferably 200 minutes or less, but can be appropriately adjusted. As the biocatalyst having the activity of removing the carboxyl group of the formula (3) by contacting with the substrate containing the aromatic carboxylic acid having the 4-hydroxy group of the formula (3) of the present invention, the enzyme capable of removing the carboxyl group of the formula . The enzyme is not particularly limited, but specific examples of the biocatalyst for removing the carboxyl group include p-coumaric acid decarboxylase (EC 4.1.1.-), caffeic acid decarboxylase, EC 4.1.1.-), ferulic acid decarboxylase (EC 4.1.1.-.), Trans-cinnamic acid decaroboxylase (EC 4.1.1.-.) Phenolic acid decarboxylase (EC 4.1.1.-), 4-hydroxybenzoate decarboxylase (EC 4.1.1.61), aminobenzoate decarboxylase (EC 4.1.1.61), aminobenzoate decarboxylase 4.1.1.24), protocatechuate decarboxylase (EC 4.1.1.63), Orsellinate decarboxylase (EC 4.1.1.58), Gallate decarboxylase (EC 4.1. 1.59), 4,5-dihydroxyphthalate Dihydroxyphthalate decarboxylase (EC 4.1.1.55), 3,4-dihydroxyphthalate decarboxylase (EC 4.1.1.69), 3-hydroxy-2- (3-hydroxy-2-methylpyridine-4,5-dicarboxylate 4-decarboxylase, EC 4.1.1.51) and phenylphosphate decarboxylase .4.1.1-), the microorganism containing the enzyme, the microorganism lysate, or an extract of the microorganism lysate.

As an example of the enzyme, the phenolic acid decarboxylase (EC 4.1.1.-) may be a protein comprising the amino acid sequence of SEQ ID NO: 2. (Or GO No.) GO: 0016831, GO: 0018799, GO: 0047662, GO: 0050223, GO: 0050159, GO: 0018798, GO: 0018796, GO: 0047556, GO: 0047431.

Enzyme name Gene name Genbank accession No. microbe coumaric acid decarboxylase pdcA AAK85433.1. Lactobacillus sakei 과산산 decarboxylase FDC1 AAB64981.1. Saccharomyces cerevisiae 감산산 padC AAC46254.1. Bacillus subtilis 4-hydroxybenzoate decarboxylase ubiD AEJ42740.1. Alicyclobacillus acidocaldarius 개 aroY BAH20873.1. Klebsiella pneumoniae 4,5-dihydroxyphthalate decarboxylase phtD BAA03974.1. Comamonas testosteroni 3,4-dihydroxyphthalate decarboxylase Asphe3_40730 ADX75142.1. Arthrobacter phenanthrenivorans

The biocatalyst which removes at least one substituent selected from the group consisting of a hydroxyl group and a C1-C4 alkoxy group in the 3-position, the 5-position, or the 3-position and the 5-position of the compound of Formula 2 or the aromatic carboxylic acid of Formula 3, (EC 4.1.3.27)), aminodeoxychorismate lyase (EC 4.1.3.38), chorismate lyase (EC 4.1.3.40), 3-dehydrocholase Dehydroquinone hydro-lyase (EC 4.2.1.10)), 3-dehydroshikimate hydro-lyase (EC 4.2.1.118), prephenate hydro-lyase 5-O- (1-carboxyvinyl) -3-phosphoshikimate phosphate-lyase (EC 4.2.1.51), 5-O- (1-carboxyvinyl) -3-phosphosilicate phosphate-lyase EC 4.2.3.5)), isochorismate lyase (EC 4.2.99.21), and hydroxides May be a phenyl pyruvate synthase (hydroxyphenylpyruvate synthase (EC 5.4.99.5)) microorganism, lysate or extract of the microorganism microorganism debris which produces the enzyme, the enzyme is selected from the group consisting of one or more.

For example, the amino deoxycholyase (ADC Lyase) may be an enzyme comprising the amino acid sequence (ADC lyase) of SEQ ID NO: 4. Specifically, examples of the enzyme of the present invention include Gene Ontology Number (GO No.) GO: 004049, GO: 005950, GO: 004107, GO: 008813, GO: 0046565, GO: 0043904, GO: 008696, GO: 004664, GO : 003855, GO: 004106, and the like. Examples of the enzyme and the gene encoding the same include the following examples:

Enzyme name Gene name Genbank No . microbe Anthranilic acid-producing enzyme TRP2 AAA35175.1 Saccharomyces cerevisiae Aminodoxycholyase lyase ABZ2 DAA10190.1 Saccharomyces cerevisiae Chrismate lyase ubiC CAA47181.1 Escherichia coli 3-dehydroquinate hydro-lyase aroD ACR61804.1 Escherichia coli 3-dehydroximate hydro-lyase quiC AAC37159.1 Acinetobacter sp. (strain ADP1) Prefenate Hydro-Lyase pheA AAA22507.1 Bacillus subtilis 5-O- (1-carboxyvinyl) -3-phosphoshimate phosphate-lyase aroC AAA23487.1 Escherichia coli Isocyclymate lyase entB AAA16102.1 Escherichia coli Hydroxyphenylpyruvate-producing enzyme aroH ADE84133.1 Rhodobacter capsulatus

As the biocatalyst having the activity of removing the hydroxyl group of the styrene having 4-hydroxy group of the formula (2) of the present invention or its derivative, any enzyme which can remove the 4-hydroxy group is included. Such enzymes include, but are not limited to, bile acid 7-alpha-dehydroxylase (EC 1.17.99.5), 4-hydroxybenzoyl-CoA reductase (EC 1.3.7.9)), 3-dehydroquinate hydro-lyase (EC 4.2.1.10), aldos-2-ulose dehydratase EC 4.2.1.110), Biochim Biophys Acta. 2005, 1723 (1-3): 63-73), o-succinylbenzoate synthase (EC 4.2.1.113) (EC 4.2.1.118), prephenate hydro-lyase (EC 4.2.1.51), arogenate dehydratase (EC 4.2.1.51), 3-dehydroshikimate hydro-lyase (EC 4.2.1.94)), and 16α-hydroxyprogesterone hydro-lyase (EC 4.2.1.94), scytallone 7,8-hydro-lyase 1.98), J Steroid Bioch em Mol Biol. 1991, 38 (2): 257-63).

For example, the prefenate hydrolase may be an enzyme comprising the amino acid sequence of SEQ ID NO: 6 (PHA2). Specifically, as an example of the enzyme of the present invention, Gene Ontology Number (GO No.) GO: 0047769, GO: 0004664, GO: 0046565, GO: 0003855, GO: 0030411, GO: 0033991, GO: 0043748, GO: 0047455, GO : 0018525, and GO: 0033792. Examples of the enzyme and the gene encoding the enzyme include the following examples.

Enzyme name Gene name Genbank No. microbe Gt; 7-dehydroxylase < / RTI > baiA2 AAB61150.1 Eubacterium sp. (strain VPI 12708) 4-hydroxybenzoyl-CoA reductase hcrA CAA05038.1 Thauera aromatica 3-dehydroquinate hydrolase aroD ACR61804.1 Escherichia coli o-succinylbenzoate synthase menC AAA71917.1 Escherichia coli 3-dehydroximate hydrolase qa-4 CAA32750.1 Neurospora crassa Prefenate Hydro-Lyase PHA2 DAA10245.1 Saccharomyces cerevisiae Arogenate dehydratase Bphy ACC72194.1 Burkholderia phymatum Scythalone 7,8-hydro-lyase SDH1 BAA34046.1 Magnaporthe oryzae

In general, enzymes can exhibit activity against a variety of similar substrates and can also exhibit activity against substrates that are not known substrates. In addition, enzymes show differences in activity from substrate to substrate, which may induce enzyme mutation, directed evolution, etc., to increase enzyme activity or substrate specificity. As an example of the present invention, the enzyme of the present invention may be mutated or evolved to increase the substrate specificity or enzymatic activity of the compound represented by the general formula (2) or (3) to increase the production of the product of the present invention.

The biocatalyst of the present invention includes an enzyme, a microorganism containing the enzyme, a microorganism lysate thereof, or an extract of a microorganism lysate. Contacting the substrate with the biocatalyst can be carried out by contacting the enzyme, the microorganism comprising the enzyme, the microbial debris or the microbial debris with an extract, in contact with or contacting the substrate, under conditions sufficient to produce a product from each substrate, And culturing the microorganism in a medium.

The biological reaction of each step of the present invention may be a method of contacting the microorganism or the like containing an enzyme or an enzyme or culturing the microorganism in a medium containing the substrate. The enzyme reaction may be carried out at a pH ranging from pH 5.0 to pH 10.0, and the optimum pH may vary depending on the characteristics of each enzyme within the above ranges. Also, the enzyme reaction may be performed at a temperature ranging from 25 ° C to 50 ° C, and the optimum temperature may vary depending on the characteristics of each enzyme. In one example of the present invention, the conversion temperature may be from 30 캜 to 37 캜.

In an embodiment of the present invention, the microorganism performing each step of the present invention may be a recombinant or wild-type, and the recombinant microorganism is one in which the gene encoding the enzyme is introduced into the host cell by recombinant method .

In a specific example of the present invention, when a recombinant enzyme is used, 1) a step of preparing an expression vector comprising a gene encoding the enzyme; 2) transforming the expression vector into a host cell and culturing it; 3) a step of producing an enzyme from the host cell; And 4) reacting the enzyme with the substrate. The enzyme that reacts with the substrate may be purified or unpurified.

As the expression vector which can be used for production of styrene and its derivatives in the production of the recombinant expression vector, any vector used in the genetic recombination method can be used. Specifically, the host cell transformed with the recombinant expression vector may be Escherichia coli. However, if the strain is capable of producing an enzyme protein that expresses a desired gene and is active with a recombinant expression vector, it may be a bacterial strain, a fungus, or a yeast Either can be used.

The obtained styrene or derivatives thereof according to the present invention can be used alone or in combination with other comonomers to produce polymers to be used in plastics, elastomers, food packaging and processed food industries.

According to another embodiment of the present invention, there is provided a process for preparing styrene or derivatives thereof, comprising: preparing styrene or derivatives thereof represented by the following formula (1) from a substrate comprising 4-hydroxystyrene or a derivative thereof represented by the following formula And a method of polymerizing the styrene or a derivative thereof to prepare a polymer. The definitions of the formulas (1) and (2) are the same as those described above. (A) a biocatalyst for removing the 4-hydroxy group of Formula 2, and (b) optionally a hydroxyl group at the 3-position, 5-position or 3-position and 5-position of Formula 2, and C1 -C4 alkoxy group, and the resulting styrene or derivative thereof may be polymerized with a monomer to prepare a polymer.

The polymerization may be carried out by polymerizing styrene or a derivative thereof with a monomer to produce a homopolymer or by copolymerizing the styrene or derivative monomer with at least one selected from the group consisting of acrylonitrile, butadiene, acrylic acid and methacrylic acid Copolymers can also be prepared by polymerizing the comonomers.

The conditions for the step of polymerizing the styrene or derivatives thereof are not particularly limited as long as they can be applied under ordinary polymerization conditions.

The method for producing styrene or a derivative thereof according to the present invention and the method for producing a polymer using the same can obtain styrene or a derivative thereof by a biological method having high specificity compared with a chemical process, Styrene or derivatives thereof can be obtained in an environmentally friendly and economical manner by obtaining lignin degradation products efficiently as a substrate.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

≪ Analysis of aromatic monomers >

Quantitative analysis of lignin-derived phenolic monomers and styrene derivatives was performed using a Waters e2695 HPLC and a Waters 2489 UV / Vis (254 nm, 280 nm) detector. Columns were Xbridge C18 (4.6 x 150 mm, 5 μm), column temperature was 35 ° C and flow rate of mobile phase was 1 mL / min. (0% B) and 9.5 min (20% B) in a gradient condition, using a 5% acetonitrile aqueous solution containing 0.1% formic acid and a 50% acetonitrile aqueous solution containing 0.1% formic acid. ), 16.5 min (60% B), 21.5 min (76% B), 25 min (100% B) and 33 min Respectively.

Concentrations of lignin-derived phenolic monomers including 4-hydroxycinnamic acid, ferulic acid, cinnamic acid, and the like were confirmed using HPLC and ESI-MS / MS (Waters TQD). HPLC conditions were the same as the above HPLC conditions, and mass spectrometry conditions were optimized using a standard solution. The ionization method used anionization method and the capillary voltage (3 kV), cone voltage (25 V), source temperature (120 ℃), desolvation temperature (300 ℃), desolvation gas flow (600 L / Cone gas flow (60 L / hr, N2) was used. Compared with the results of the standard solutions, the peaks generated in HPLC-ESI-MS / MS were identified by the generation of specific fragment ions when given molecular ion and collision energy in scan mode (50-200, m / z) .

Example  One: Dehydroxylation  Production of styrene by reaction

1-1: Prefenate Heathrow - Lyase  A recombinant expression vector containing the gene and a transgenic microorganism preparation

To prepare prephenate hydrolyase (PHA2), the PHA2 gene derived from S. cerevisiae strain was cloned. First, genomic DNA was extracted from S. cerevisiae ATCC 204508. Based on the nucleotide sequence (Genbank Accession Number; CAA86380.1) encoding the PHA2 gene, the following primers were devised, respectively.

(Forward primer 1): 5'-AAACATATG AAAATAAAAATTTTAGTAGA -3 '(SEQ ID NO: 7)

(Reverse primer 1): 5'-AAACTCGAG TTTGTGATAATATCTCTCAT -3 '(SEQ ID NO: 8)

The primer was used to amplify the PHA2 gene by performing PCR (PCR) on S. cerevisiae ATCC 204508 genome.

For PCR, 100 ng of template, 10 pmol of each primer, 2 mM dNTPs, 1X PCR buffer and 2U Taq polymerase were used, and the total volume of reaction solution was 50 μl. The DNA amplification reaction conditions of the PCR were pre-denaturation at 95 ° C for 5 minutes followed by 95 ° C, denaturation 1 minute; 54 占 폚 annealing 30 seconds; 72 ° C elongation 2 minutes was made 25 cycles and post-polymerization was carried out at 72 ° C for 5 minutes for final elongation.

The amplified genes were simultaneously digested with NdeI / XhoI restriction enzymes and inserted into a plasmid vector pET28a (Novagen) digested with NdeI / XhoI restriction enzyme using T4 DNA ligase to prepare pET28a / PHA2 vector. PCR and cloning results were determined by 1.5% agarose electrophoresis.

The recombinant expression vector thus obtained was transformed into Escherichia coli BL21 (DE3) by a conventional transformation method. In addition, the transformed microorganism was stored in an ultra-low temperature freezer after addition of 15% glycerol solution so that it could be cultured for enzyme production later.

1-2: Prefenate Heathrow - Lyase  Production of enzymes

For mass production of prephenate hydrolase, recombinant E. coli BL21 (DE3) strain of cryopreserved Stage 1 was inoculated into a test tube containing 5 ml of LB medium and when the absorbance at 600 nm was 2.0 Lt; RTI ID = 0.0 > 37 C < / RTI > Then, the culture medium of the seed culture was added to a 300 ml flask containing 100 ml of LB medium, and the culture was carried out. When the absorbance at 600 nm was 0.6, 1 mM IPTG was added to induce a large-scale expression of the enzyme. The agitation speed was 250 rpm, the incubation temperature was kept at 30 ° C, and IPTG was added, followed by culturing for 10 hours under the same conditions.

The culture of the transformed strain was centrifuged at 4,000 × g for 30 minutes at 4 ° C., washed twice with PBS buffer, and then added with 50 mM Tris-HCl buffer (pH 7.5) The solution was disrupted with an ultrasonic disrupter. The cell lysate was centrifuged again at 13,000 x g for 20 minutes at 4 ° C, and the cell pellet was removed. The cell supernatant was removed and the enzyme was purified by Ni-NTA His-Tag chromatography. The purified enzyme was exchanged with a 50 mM Tris-HCl buffer solution (pH 7.5) using a centrifugal filter (10 kDa), concentrated, and the concentration was measured by protein assay (Bradford assay) mL, and used for the enzyme reaction. Proteins were analyzed by electrophoresis using 12% polyacrylamide gel.

1-3: Production of styrene and its derivatives

50 μL of a 5 mg / L prefenate hydro-lyase purified in Example 1-2 and 10 μL of 20 mM 4-hydroxystyrene (Sigma) were added to 140 μL of a 10 mM PBS buffer solution to prepare a reaction mixture 0.2 mL was reacted in a 37 ° C thermostat for 5 hours. After completion of the reaction, 1.8 mL of methanol was added to the sample, and impurities were removed using a syringe filter (0.22 μm) to prepare an analytical sample. The reduction of the substrate 4-hydroxystyrene and the concentration of styrene were analyzed by HPLC (Table 4).

Further, instead of 4-hydroxystyrene as a substrate, 4-hydroxy-3-methoxystyrene was used as a substrate to carry out the enzymatic reaction under the above conditions (Table 4).

division Kinds Concentration before enzyme reaction (mM) Enzyme Reaction Concentration (mM) temperament p-hydroxystyrene 1.05 0.27 product Styrene 0.00 0.62 temperament 4-hydroxy-3-methoxystyrene 1.13 0.36 product 3-methoxystyrene 0.00 0.56

As seen from the above, it can be seen that p-hydroxystyrene is dehydroxylated by prephenate hydro-lyase and 4-hydroxy-3-methoxy styrene is dehydroxylated by 3-methoxystyrene.

Example  2:

2-1. ADC Lyase  A recombinant expression vector containing the gene and a transgenic microorganism preparation

In order to prepare aminodeoxychorismate lyase (ADC lyase), ADC lyase gene derived from S. cerevisiae strain was cloned. First, genomic DNA was extracted from S. cerevisiae KCCM 50712. Based on the nucleotide sequence (Genbank Accession Number; DAA10190.1) coding for the ADC lyase of S. cerevisiae KCCM 50712, the following primers were devised, respectively.

(Forward primer 2)

5'-AAACATATGTCACTAATGGACAATTGGAA-3 '(SEQ ID NO: 9)

(Reverse primer 2)

5'-AAACTCGAGATATTTTTCTTCACTGTTC-3 '(SEQ ID NO: 10)

PCR was performed on S. cerevisiae KCCM 50712 genome using the above primers to amplify the base sequence of the ADC lyase gene.

For PCR, 100 ng of template, 10 pmol of each primer, 2.5 mM dNTPs, 1X PCR buffer, and 2.5 U Taq polymerase were used, and the total volume of the reaction solution was 50 μl. The DNA amplification reaction conditions of the PCR were 95 ° C pre-denaturation at 94 ° C followed by 94 ° C, denaturation 1 min; 55 占 폚 annealing 30 seconds; Elongation at 72 ° C for 3 minutes was 30 cycles and post-polymerization was performed at 72 ° C for 5 minutes for final elongation.

The amplified genes were digested with NdeI / XhoI restriction enzymes and inserted into a plasmid vector pET28a (Novagen) digested with the same restriction enzymes using T4 DNA ligase to construct a pET28a / ADCL vector. PCR and cloning results were determined by 1.2% agarose electrophoresis.

The recombinant expression vector thus obtained was transformed into Escherichia coli BL21 (DE3) by a conventional transformation method. In addition, the transformed microorganism was added with a 15% glycerol solution and stored in a freezer for subsequent cultivation for enzyme production.

2-2: ADC lyase Manufacturing

In order to mass-produce ADC lyase, the cryopreserved step 1 recombinant E. coli BL21 (DE3) strain was inoculated into a test tube containing 5 ml of LB medium and incubated at 37 ° C until the absorbance at 2.5 The seed culture was carried out with a shaking incubator. Then, the culture medium of the seed culture was added to a 300 ml flask containing 100 ml of LB medium, and the culture was carried out. Also, when the absorbance at 600 nm was 0.6, 0.5 mM IPTG was added to induce the large-scale expression of the enzyme. The agitation speed was 250 rpm and the incubation temperature was maintained at 33 ° C. After adding IPTG, the cells were incubated for 6 hours under the same conditions.

The culture of the transformed strain was centrifuged at 4,000 x g for 20 minutes at 4 ° C, washed twice with PBS buffer, and then added with 50 mM Tris-HCl buffer (pH 7.5) The solution was disrupted with an ultrasonic disrupter. The cell lysate was centrifuged again at 13,000 x g for 20 minutes at 4 ° C, and the cell pellet was removed. The cell supernatant was removed and the enzyme was purified by Ni-NTA His-Tag chromatography. The purified enzyme was exchanged with a 50 mM Tris-HCl buffer solution (pH 7.5) using a centrifugal filter (10 kDa), concentrated, and the concentration was measured by protein assay (Bradford assay) mL and used for the reaction.

2-3: Preparation of styrene by multistage enzymatic reaction

In order to remove the methoxy group and the hydroxyl group of 4-hydroxy-3-methoxystyrene, dehydroxylation and demethoxylation reaction were carried out. As the dehydroxylase, the prephenate hydrolyzate (ADC lyase) prepared in Example 1-2 and the aminodeoxychorismate lyase (ADC lyase) prepared in Example 2-2 were used as the demethoxylase, Enzyme was used. The recombinant enzyme was used for the reaction, and finally purified enzyme having a concentration of 5 mg / L was used for the reaction using the enzyme expression method in E. coli described in Example 1-2 and Example 2-2.

50 μL of 50 μL of 4-hydroxy-3-methoxystyrene, 1 mM of EDTA, and 20 mM of 2-mercaptoethanol were added to 0.5 mL of the reaction mixture containing 50 μL and 50 mM of the prefenate hydrolase and the amino deoxycholate ligase, Were reacted in a 37 ° C thermostatic chamber for 5 hours. After completion of the reaction, 4.5 mL of methanol was added to the sample to dilute the sample, and then the impurity was removed using a syringe filter (0.22 μm) to prepare an analytical sample. The reduction of the substrate 4-hydroxy-3-methoxystyrene and the concentration of styrene were determined by HPLC (Table 5).

Before enzyme reaction (mM) After the enzyme reaction (mM) 4-hydroxy-3-methoxystyrene 5.07 0.92 4-hydroxystyrene 0.00 1.14 3-methoxystyrene 0.00 0.54 Styrene 0.00 2.32

As can be seen from the above, it can be seen that 4-hydroxy-3-methoxystyrene is converted to styrene by the presence of prephenate hydrolase and aminodeoxycholymitlase.

Example  3: Production of styrene

3-1: Phenolic acid Decarboxylase  A recombinant expression vector containing the gene and a transgenic microorganism preparation

In order to prepare phenolic acid decarboxylase (PADC), the padC gene derived from Bacillus subtilis strain 168 was cloned. First, genomic DNA was extracted from B. subtilis ATCC 6051. The following primers were devised on the basis of the base sequence coding for the pad C gene (Genbank Accession Number; AAC46254.1.).

(Forward primer 3):

5'-AAACATATGGAAAACTTTATCGGAAGCCA-3 '(SEQ ID NO: 11)

(Reverse primer 3):

5'-AAACTCGAGTAATCTTCCCGCGCGAATAT-3 '(SEQ ID NO: 12)

The primer was used to amplify the pad C gene by PCR (PCR) from B. subtilis genomic DNA.

For PCR, 100 ng of template, 10 pmol of each primer, 2.5 mM dNTPs, 1X PCR buffer, and 2.5 U Taq polymerase were used, and the total volume of the reaction solution was 50 μl. The DNA amplification reaction conditions of the PCR were 95 ° C pre-denaturation at 94 ° C followed by 94 ° C, denaturation 1 min; 55 占 폚 annealing 30 seconds; 72 ° C elongation for 1 minute was 30 cycles and post-polymerization was carried out at 72 ° C for 5 minutes for final elongation.

The amplified genes were digested with NdeI / XhoI restriction enzymes and inserted into plasmid vector pET28a (Novagen), which was digested with the same restriction enzymes using T4 DNA ligase, to construct a pET28a / PADC vector. PCR and cloning results were determined by 1.2% agarose electrophoresis.

The recombinant expression vector thus obtained was transformed into Escherichia coli BL21 (DE3) by a conventional transformation method. In addition, the transformed microorganism was added with a 15% glycerol solution and stored in a freezer for subsequent cultivation for enzyme production.

3-2: Phenolic acid Decarboxylase  production

In order to mass-produce the phenolic acid decarboxylase enzyme, the frozen recombinant Escherichia coli BL21 (DE3) strain was inoculated into a test tube containing 5 ml of LB medium and incubated at 37 째 C until the absorbance was 2.5 at 600 nm Lt; / RTI > culture medium. Then, the culture medium of the seed culture was added to a 300 ml flask containing 100 ml of LB medium, and the culture was carried out. Also, when the absorbance at 600 nm was 0.6, 0.5 mM IPTG was added to induce the large-scale expression of the enzyme. The agitation speed was 250 rpm and the incubation temperature was maintained at 30 ° C. After adding IPTG, the cells were incubated for 6 hours under the same conditions.

The culture of the transformed strain was centrifuged at 4,000 rpm for 20 minutes at 4 ° C, washed twice with PBS buffer, and then added with 50 mM Tris-HCl buffer (pH 7.5) Was crushed by an ultrasonic crusher. The cell lysate was further centrifuged at 13,000 rpm for 20 minutes at 4 ° C. Cell pellet was removed, and the cell supernatant was obtained, and the enzyme was purified by Ni-NTA His-Tag chromatography. The purified enzyme was exchanged with a 50 mM Tris-HCl buffer solution (pH 7.5) using a centrifugal filter (10 kDa), concentrated, and the concentration was measured by protein assay (Bradford assay) mL, and used for the enzyme reaction.

3-3: 4- Hydroxystyrene  Production of

20 μL of phenolic acid decarboxylase purified in Example 3-2 at a concentration of 5 mg / L and 10 μL of 4-hydroxycinnamic acid (Sigma) of 20 mM were mixed in 170 μL of a 10 mM PBS buffer solution, For 3 hours. After completion of the reaction, 9-fold volume of methanol was added to the sample for extraction, and impurities were removed using a syringe filter (0.22 micrometer) to prepare analytical samples. The production and concentration of 4-hydroxystyrene were confirmed by HPLC (Table 6).

In addition, enzyme reaction was carried out for ferulic acid (Sigma) instead of 4-hydroxycinnamic acid, which is a substrate, and the results of the analysis are shown in Table 6.

division Kinds Concentration before enzyme reaction (mM) Enzyme Reaction Concentration (mM) temperament 4-Hydroxycinnamic acid 1.08 0.11 product 4-hydroxystyrene 0.00 0.74 temperament Ferulic acid 1.01 0.27 product 3-methoxy-4-hydroxystyrene 0.00 0.69

As can be seen from the above, PADC shows that 4-hydroxycinnamic acid is converted to 4-hydroxystyrene and ferulic acid is converted to 3-methoxy-4-hydroxystyrene.

Example  4: From Lee Green  Styrene manufacture

4-1: Alkali oxidation decomposition of lignin

The lignin decomposition reaction was carried out using a laboratory high-pressure reactor (450 mL, Parr 4562). 10.0 g of kraft lignin was added to a previously prepared 200 mL of 4M NaOH aqueous solution to prepare a reaction product having an initial lignin content of 5.0% (w / v). 1 g of H ₂ O ₂ was added as a catalyst. The reaction was packed in a stainless steel high pressure reactor having an internal volume of 450 mL, sealed, and stirred at 500 rpm. The reactor was filled with oxygen gas at about 7 bar for 2 minutes through a sampling line connected to the inside of the reactor, heating of the reactor was started, and the reaction was continued for 120 minutes from the time when the internal temperature of the reactor reached 170 ° C. The reaction temperature was controlled by a PID controller through a cooling water tube. After 120 minutes from the start of the present reaction, the sample was sampled through the sampling line and the reaction was terminated.

6 ml of HCl was added to 2 ml of the alkaline decomposition product of the lignin thus obtained to obtain a pH of 2.0. The diluted sample was diluted with distilled water to a final volume of 10 mL, and the lignin was removed with a filter (10 kDa MWCO). Methanol in 9 times volume was added to 1 mL of the sample from which the re-greens were removed, and the impurity was removed using a syringe filter (0.22 O) W to purify the lignin degradation product. Then, it was analyzed by HPLC and the composition as shown in Table 7 was obtained.

Ingredients Concentration (mM) 4-Hydroxycinnamic acid 10.36 Ferulic acid 1.80 Cinnamic acid 1.78 p-hydroxybenzoic acid 0 p-Hydroxybenzaldehyde 0.54 Vanillic acid 1.03 vanillin 1.15 Silinjisan 0.11 Cyhalide 0.07 p-coumarylaldehyde 0.06 Coniferyl aldehyde 0.07 Shoshin aldehyde 0.06 Shin Nam Aldehyde 0.10

4-2: From lignin Hydroxystyrene  production

A small amount of 10 M HCl was added to the lignin degradation product obtained in Example 4-1, and the solution was titrated to pH 7.5. The solution was filtered with a 10 kDa MWCO filter, and the filtrate was prepared as a reaction product. 100 μL each of the enzyme solution containing the phenolic acid decarboxylase and amino deoxycholymitlia obtained in Example 3 and Example 2 and 200 μL of the lignin degradation product were mixed and reacted at 37 ° C. for 5 hours in a thermostat. After completion of the reaction, the reaction mixture was centrifuged at 13,000 g for 20 minutes at 4 ^° C, and the supernatant was obtained. After diluting with 360 μL of methanol, impurities were removed using a syringe filter (0.22 μm) Respectively. The production concentration of 4-hydroxystyrene was analyzed by HPLC, and the results of analysis of the sample before and after the reaction are shown in Table 8.

matter Before reaction (mM) After the reaction (mM) 4-Hydroxycinnamic acid 10.36 3.2 Ferulic acid 1.80 0.9 Cinnamic acid 1.78 0.7 2-methoxy-4-vinylphenol 0.0 1.2 2,6-dimethoxy-4-vinylphenol 0.0 0.5 4-hydroxystyrene 0.0 2.4

Lignin degradation products containing 4-hydroxycinnamic acid, ferulic acid, and cinnamic acid were removed by enzymatic reaction using phenolic acid decarboxylase and aminodeoxycholyzmatease to remove methionine from ferulic acid and cinnamic acid to form 4- Hydroxycinnamic acid and the carboxyl group was removed by phenolic acid decarboxylase, resulting in the production of 2.4 mM 4-hydroxystyrene.

4-3: From hydroxystyrene  Production of styrene

50 μL of the 5 mg / L prefenate hydrolyzate purified in Example 1-2 was added to 250 μL of the 4-hydroxystyrene-containing enzyme conversion product obtained in Example 4-2, and the mixture was incubated at 37 ° C. in a thermostatic chamber And reacted for 5 hours. After completion of the reaction, 2.7 mL of methanol was added to the sample, and impurities were removed using a syringe filter (0.22 μm) to prepare an analytical sample. The decrease of 4-hydroxystyrene and the concentration of styrene were analyzed by HPLC (Table 9).

matter Before enzyme reaction (mM) After the enzyme reaction (mM) 4-hydroxystyrene 2.4 1.12 Styrene 0.00 0.92

As shown above, it was confirmed that styrene was synthesized from 4-hydroxystyrene produced through enzyme conversion of lignin degradation product by further dehydroxylation enzyme reaction.

<110> SAMSUNG PETROCHEMICAL CO., LTD. <120> PROCESS OF BIOLOGICALLY PRODUCING A STYRENE AND DERIVATIVES          THEREOF <130> dpp20129170kr <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 486 <212> DNA <213> Artificial Sequence <220> <223> phenolic acid decarboxylase <400> 1 atggaaaact ttatcggaag ccacatgatt tatacgtatg aaaacggatg ggaatacgag 60 atttatatta aaaacgacca tacaattgat tatagaattc atagcggaat ggttgccgga 120 cgctgggttc gagatcagga agtgaatatt gtcaaactga cagaaggcgt atataaagtg 180 tcttggacag agccgactgg cacggatgtt tcattaaact ttatgccaaa tgaaaaacgc 240 atgcatggca ttattttctt cccgaaatgg gtgcatgaac atcctgaaat tacggtttgc 300 taccaaaatg accacattga tttgatgaaa gaatcccgcg aaaaatatga aacgtatcca 360 aaatacgttg tacctgaatt tgcggaaatt acatttctga aaaatgaagg agtcgacaac 420 gaagaagtga tttcgaaggc tccttatgag ggaatgacag acgatattcg cgcgggaaga 480 ttataa 486 <210> 2 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> phenolic acid decarboxylase <400> 2 Met Glu Asn Phe Ile Gly Ser His Met Ile Tyr Thr Tyr Glu Asn Gly   1 5 10 15 Trp Glu Tyr Glu Ile Tyr Ile Lys Asn Asp His Thr Ile Asp Tyr Arg              20 25 30 Ile His Ser Gly Met Val Ala Gly Arg Trp Val Arg Asp Gln Glu Val          35 40 45 Asn Ile Val Lys Leu Thr Glu Gly Val Tyr Lys Val Ser Trp Thr Glu      50 55 60 Pro Thr Gly Thr Asp Val Ser Leu Asn Phe Met Pro Asn Glu Lys Arg  65 70 75 80 Met His Gly Ile Ile Phe Phe Pro Lys Trp Val His Glu His Pro Glu                  85 90 95 Ile Thr Val Cys Tyr Gln Asn Asp His Ile Asp Leu Met Lys Glu Ser             100 105 110 Arg Glu Lys Tyr Glu Thr Tyr Pro Lys Tyr Val Val Pro Glu Phe Ala         115 120 125 Glu Ile Thr Phe Leu Lys Asn Glu Gly Val Asp Asn Glu Glu Val Ile     130 135 140 Ser Lys Ala Pro Tyr Glu Gly Met Thr Asp Asp Ile Arg Ala Gly Arg 145 150 155 160 Leu     <210> 3 <211> 1125 <212> DNA <213> Artificial Sequence <220> <223> aminodeoxychorismate lyase, ADC lyase, DAA10190.1 <400> 3 atgtcactaa tggacaattg gaagactgat atggaaagtt acgatgaagg aggcctagtt 60 gctaatccga acttcgaggt tctggccact ttcaggtacg accctggttt tgcacgccag 120 tcagcgtcaa agaaagagat ctttgaaact ccagaccctc gattaggttt gagagacgaa 180 gatattaggc agcagataat taatgaggat tactcaagtt atttacgagt aagggaggtt 240 aattccggcg gtgaccttct cgaaaatatt cagcatcctg atgcttggaa gcatgattgc 300 aagaccattg tgtgccagcg tgtagaagat atgctacaag tcatttatga acgatttttt 360 ttattagatg aacaatacca aagaataaga atagcattat catactttaa aattgacttc 420 agcacgtctc tgaatgattt attgaagtta ttggttgaaa acttgattaa ttgtaaagaa 480 ggaaattcag agtatcacga aaaaattcaa aaaatgatca acgaaaggca atgctataaa 540 atgcgggtac ttgtctctaa gacaggagat atacgaattg aggcaattcc aatgcctatg 600 gagcctatcc taaaattaac aaccgattat gacagtgttt ccacatactt catcaaaacg 660 atgctcaatg gatttttaat tgatagcaca ataaattggg atgttgttgt ttcatctgaa 720 ccattgaacg catcagcttt caccagtttt aaaaccactt caagagatca ttacgctagg 780 gcgagagttc gcatgcaaac tgctataaat aacttaagag gttcagaacc tacttcttct 840 gtctcgcaat gcgaaatttt attttccaac aaatctggcc tgctgatgga aggttcaata 900 acaaacgtgg ctgtaattca aaaagatcct aacggttcta aaaagtatgt gacaccaaga 960 ttagcaactg gatgtttgtg cggaacaatg cgtcattatt tattgcggct cggccttatt 1020 gaagagggag atatagatat aggaagcctt accgttggca acgaagtttt gcttttcaat 1080 ggcgtcatgg gatgcataaa gggaacagtg aagacaaaat attga 1125 <210> 4 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> aminodeoxychorismate lyase, ADC lyase, DAA10190.1 <400> 4 Met Ser Leu Met Asp Asn Trp Lys Thr Asp Met Glu Ser Tyr Asp Glu   1 5 10 15 Gly Gly Leu Val Ala Asn Pro Asn Phe Glu Val Leu Ala Thr Phe Arg              20 25 30 Tyr Asp Pro Gly Phe Ala Arg Gln Ser Ala Ser Lys Lys Glu Ile Phe          35 40 45 Glu Thr Pro Asp Pro Arg Leu Gly Leu Arg Asp Glu Asp Ile Arg Gln      50 55 60 Gln Ile Ile Asn Glu Asp Tyr Ser Ser Tyr Leu Arg Val Val Glu Val  65 70 75 80 Asn Ser Gly Gly Asp Leu Leu Glu Asn Ile Gln His Pro Asp Ala Trp                  85 90 95 Lys His Asp Cys Lys Thr Ile Val Cys Gln Arg Val Glu Asp Met Leu             100 105 110 Gln Val Ile Tyr Glu Arg Phe Leu Leu Asp Glu Gln Tyr Gln Arg         115 120 125 Ile Arg Ile Ala Leu Ser Tyr Phe Lys Ile Asp Phe Ser Thr Ser Leu     130 135 140 Asn Asp Leu Leu Lys Leu Leu Val Glu Asn Leu Ile Asn Cys Lys Glu 145 150 155 160 Gly Asn Ser Glu Tyr His Glu Lys Ile Gln Lys Met Ile Asn Glu Arg                 165 170 175 Gln Cys Tyr Lys Met Arg Val Leu Val Ser Lys Thr Gly Asp Ile Arg             180 185 190 Ile Glu Ala Ile Pro Met Pro Met Glu Pro Ile Leu Lys Leu Thr Thr         195 200 205 Asp Tyr Asp Ser Val Ser Thr Tyr Phe Ile Lys Thr Met Leu Asn Gly     210 215 220 Phe Leu Ile Asp Ser Thr Ile Asn Trp Asp Val Val Val Ser Ser Glu 225 230 235 240 Pro Leu Asn Ala Ser Ala Phe Thr Ser Phe Lys Thr Thr Ser Arg Asp                 245 250 255 His Tyr Ala Arg Ala Arg Val Arg Met Gln Thr Ala Ile Asn Asn Leu             260 265 270 Arg Gly Ser Glu Pro Thr Ser Ser Val Ser Gln Cys Glu Ile Leu Phe         275 280 285 Ser Asn Lys Ser Gly Leu Leu Met Glu Gly Ser Ile Thr Asn Val Ala     290 295 300 Val Ile Gln Lys Asp Pro Asn Gly Ser Lys Lys Tyr Val Thr Pro Arg 305 310 315 320 Leu Ala Thr Gly Cys Leu Cys Gly Thr Met Arg His Tyr Leu Leu Arg                 325 330 335 Leu Gly Leu Ile Glu Glu Gly Asp Ile Asp Ile Gly Ser Leu Thr Val             340 345 350 Gly Asn Glu Val Leu Leu Phe Asn Gly Val Met Gly Cys Ile Lys Gly         355 360 365 Thr Val Lys Thr Lys Tyr     370 <210> 5 <211> 1107 <212> DNA <213> Artificial Sequence <220> <223> Prephenate hydro-lyase, PHA2, CAA86380.1 <400> 5 atgaaaataa aaattttagt agatgaatat aatacgcaga aagaacaggc taaaaaagta 60 cgaaaagcaa cttcattata tttccgcatt catccttcaa ttatggccag caagactttg 120 agggttcttt ttctgggtcc caaaggtacg tattcccatc aagctgcatt acaacaattt 180 caatcaacat ctgatgttga gtacctccca gcagcctcta tcccccaatg ttttaaccaa 240 ttggagaacg acactagtat agattattca gtggtaccgt tggaaaattc caccaatgga 300 caagtagttt tttcctatga tctcttgcgt gataggatga tcaaaaaagc cctatcctta 360 cctgctccag cagatactaa tagaattaca ccagatatag aagttatagc ggagcaatat 420 gtacccatta cccattgtct aatcagccca atccaactac caaatggtat tgcatccctt 480 ggaaattttg aagaagtcat aatacactca catccgcaag tatggggcca ggttgaatgt 540 tacttaaggt ccatggcaga aaaatttccg caggtcacct ttataagatt ggattgttct 600 tccacatctg aatcagtgaa ccaatgcatt cggtcatcaa cggccgattg cgacaacatt 660 ctgcatttag ccattgctag tgaaacagct gcccaattgc ataaggcgta catcattgaa 720 cattcgataa atgataagct aggaaataca acaagatttt tagtattgaa gagaagggag 780 aacgcaggcg acaatgaagt agaagacact ggattactac gggttaacct actcaccttt 840 actactcgtc aagatgaccc tggttctttg gtagatgttt tgaacatact aaaaatccat 900 tcactcaaca tgtgttctat aaactctaga ccattccatt tggacgaaca tgatagaaac 960 tggcgatatt tatttttcat tgaatattac accgagaaga ataccccaaa gaataaagaa 1020 aaattctatg aagatatcag cgacaaaagt aaacagtggt gcctgtgggg tacattcccc 1080 agaaatgaga gatattatca caaataa 1107 <210> 6 <211> 368 <212> PRT <213> Artificial Sequence <220> <223> Prephenate hydro-lyase, PHA2, CAA86380.1 <400> 6 Met Lys Ile Lys Ile Leu Val Asp Glu Tyr Asn Thr Gln Lys Glu Gln   1 5 10 15 Ala Lys Lys Val Arg Lys Ala Thr Ser Leu Tyr Phe Arg Ile His Pro              20 25 30 Ser Ile Met Ala Ser Lys Thr Leu Arg Val Leu Phe Leu Gly Pro Lys          35 40 45 Gly Thr Tyr Ser His Gln Ala Ala Leu Gln Gln Phe Gln Ser Thr Ser      50 55 60 Asp Val Glu Tyr Leu Pro Ala Ala Ser Ile Pro Gln Cys Phe Asn Gln  65 70 75 80 Leu Glu Asn Asp Thr Ser Ile Asp Tyr Ser Val Val Pro Leu Glu Asn                  85 90 95 Ser Thr Asn Gly Gln Val Val Phe Ser Tyr Asp Leu Leu Arg Asp Arg             100 105 110 Met Ile Lys Lys Ala Leu Ser Leu Pro Ala Pro Ala Asp Thr Asn Arg         115 120 125 Ile Thr Pro Asp Ile Glu Val Ile Ala Glu Gln Tyr Val Ile Thr     130 135 140 His Cys Leu Ile Ser Pro Ile Gln Leu Pro Asn Gly Ile Ala Ser Leu 145 150 155 160 Gly Asn Phe Glu Glu Val Ile Ile His Ser His Pro Gln Val Trp Gly                 165 170 175 Gln Val Glu Cys Tyr Leu Arg Ser Ser Ala Glu Lys Phe Pro Gln Val             180 185 190 Thr Phe Ile Arg Leu Asp Cys Ser Ser Thr Ser Glu Ser Val Asn Gln         195 200 205 Cys Ile Arg Ser Ser Thr Ala Asp Cys Asp Asn Ile Leu His Leu Ala     210 215 220 Ile Ala Ser Glu Thr Ala Ala Gln Leu His Lys Ala Tyr Ile Ile Glu 225 230 235 240 His Ser Ile Asn Asp Lys Leu Gly Asn Thr Thr Arg Phe Leu Val Leu                 245 250 255 Lys Arg Arg Glu Asn Ala Gly Asp Asn Glu Val Glu Asp Thr Gly Leu             260 265 270 Leu Arg Val Asn Leu Leu Thr Phe Thr Thr Arg Gln Asp Asp Pro Gly         275 280 285 Ser Leu Val Asp Val Leu Asn Ile Leu Lys Ile His Ser Leu Asn Met     290 295 300 Cys Ser Ile Asn Ser Arg Pro Phe His Leu Asp Glu His Asp Arg Asn 305 310 315 320 Trp Arg Tyr Leu Phe Phe Ile Glu Tyr Tyr Thr Glu Lys Asn Thr Pro                 325 330 335 Lys Asn Lys Glu Lys Phe Tyr Glu Asp Ile Ser Asp Lys Ser Lys Gln             340 345 350 Trp Cys Leu Trp Gly Thr Phe Pro Arg Asn Glu Arg Tyr Tyr His Lys         355 360 365 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer 1 <400> 7 aaacatatga aaataaaaat tttagtaga 29 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1 <400> 8 aaactcgagt ttgtgataat atctctcat 29 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer 2 <400> 9 aaacatatgt cactaatgga caattggaa 29 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 <400> 10 aaactcgaga tattttgtct tcactgttc 29 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer 3 <400> 11 aaacatatgg aaaactttat cggaagcca 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 <400> 12 aaactcgagt aatcttcccg cgcgaatat 29

Claims (15)

A step of contacting the substrate comprising styrene having a 4-hydroxy group or a derivative thereof represented by the general formula (2) with a biocatalyst for removing the 4-hydroxy group, by a biological method, styrene or a derivative thereof &Lt; / RTI &gt;
[Chemical Formula 1]

(2)

In the general formulas (1) and (2), X and Y may be the same or different and independently represent hydrogen, hydroxy or a C1-C4 alkoxy group. The method according to claim 1, wherein, when X and Y are not hydrogen at the same time in the formula (2), the 4-hydroxy group-removed styrene or derivative thereof having a 4-hydroxy group represented by the following formula At least one substituent selected from the group consisting of a hydroxyl group and a C1-C4 alkoxy group is removed at the 3-position, the 5-position, or the 3-position and the 5-position of the compound of Formula 2 Further comprising the step of contacting the catalyst. The substrate according to claim 1, wherein the substrate comprising the styrene having 4-hydroxy group or the derivative thereof represented by Formula 2 is a substrate having an aromatic carboxylic acid having 4-hydroxy group, represented by Formula 3, Contacting the biocatalyst to remove the biocatalyst.
(3)

In Formula 3, X and Y may be the same or different and independently represent hydrogen, hydroxy or a C1-C4 alkoxy group. 4. The method according to claim 3, wherein in the case where X and Y are not hydrogen at the same time in the formula (3), a catalyst for removing the carboxyl group is added to a substrate containing an aromatic carboxylic acid having a 4- At least one substituent selected from the group consisting of a hydroxyl group and a C1-C4 alkoxy group is removed at the 3-position, the 5-position or the 3-position and the 5-position of the aromatic carboxylic acid of Formula 3 Lt; RTI ID = 0.0 &gt; biocatalyst &lt; / RTI &gt; The method according to any one of claims 1 to 4, wherein the biocatalyst is an enzyme, a microorganism comprising the enzyme, a lysate of the microorganism, or an extract of a microbial lysate. 6. The method of claim 5, wherein the biocatalyst is a wild-type microorganism or a recombinant microorganism. The method of any one of claims 1 to 4, wherein contacting the substrate with the biocatalyst comprises contacting the enzyme with an enzyme, an extract of microbial debris or microbial debris containing the enzyme, Wherein said microorganism is cultured in a medium. The biocatalyst for removing the 4-hydroxy group according to claim 1 or 2, wherein the biocatalyst is selected from the group consisting of acetic acid (1.17.99.5), 4-hydroxybenzoyl-CoA reductase (EC 1.3.7.9) 2-euros dehydratase (EC 4.2.1.110), o-succinylbenzoate synthase (EC 4.2.1.113), 3-dehydroquinone hydrolase (EC 4.2.1.118), prephenate hydro-lyase (EC 4.2.1.51), argonate dehydratase (EC 4.2.1.91), skiltalon 7,8-hydro-lyase EC 4.2.1.94) and 16? -Hydroxyprogesterone hydro-lyase (EC 4.2.1.98), a microorganism comprising said enzyme, a microorganism lysate thereof or a microbial lysate. The method of claim 3 or 4, wherein the biocatalyst for removing the carboxyl group is selected from the group consisting of coumaric acid decarboxylase (EC 4.1.1.-), caffeic acid decarboxylase (EC 4.1.1.-.), Ferulic acid decar (EC 4.1.1.-), trans-cinnamic acid decarboxylase (EC 4.1.1.-.), Phenolic acid decarboxylase (EC 4.1.1.-.), 4-hydroxybenzoic acid Decarboxylase (EC 4.1.1.61)), aminobenzoic acid decarboxylase (EC 4.1.1.24), protocatechate decarboxylase (EC 4.1.1.63)), oxidase decarboxylase (EC 4.1.1.58) Dihydroxyphthalate decarboxylase (EC 4.1.1.59), 3,4-dihydroxyphthalate decarboxylase (EC 4.1.1.59), 3,4-dihydroxyphthalate decarboxylase (EC 4.1.1.51) and phenylphosphate decarboxylase (EC 4.1.1.-), as well as the enzymes selected from the group consisting of: hydroxy-2-methylpyridine-4,5-dicarboxylate 4-dicarboxylic acid The Wherein the microorganism is an extract of a microorganism, a microorganism lysate thereof or a microbial lysate. The biocatalyst for removing at least one substituent selected from the group consisting of the hydroxyl group and the C1-C4 alkoxy group according to claim 2 or 4 is selected from the group consisting of an anthranilic acid-producing enzyme (EC 4.1.3.27), an amino deoxycholyase EC 4.1.3.38), cholesterol lyase (EC 4.1.3.40), 3-dehydroquinone hydro-lyase (EC 4.2.1.10), 3-dehydrochymate hydro-lyase (EC 4.2.1.118) (EC 4.2.1.51), 5-O- (1-carboxyvinyl) -3-phosphosiminate phosphate-lyase (EC 4.2.3.5), isochorismate lyase (EC 4.2.99.21) (EC 5.4.99.5), a microorganism producing the enzyme, a microorganism lysate thereof, or a microorganism lysate. The method according to claim 3 or 4, wherein the substrate containing an aromatic carboxylic acid having a 4-hydroxy group represented by the general formula (3) is a lignin degradation product obtained by decomposing lignin. 12. The method of claim 11, wherein the decomposition of the lignin is carried out at elevated temperatures such as pyrolysis, gasification, hydrogenolysis, acidolysis, base decomposition, chemical oxidation and hydrolysis under supercritical condition). &lt; / RTI &gt; A process according to any one of claims 1 to 4, which comprises the steps of: preparing styrene or a derivative thereof having the following formula (1); And
[Chemical Formula 1]

In the above formulas, X and Y, which may be the same or different, are independently hydrogen, a hydroxy or a C1-C4 alkoxy group,
Wherein the styrene or a derivative thereof is polymerized to produce a polymer. 14. The method of claim 13, wherein the polymerizing step comprises polymerizing the styrene or a derivative thereof with a monomer to produce a homopolymer. 14. The method according to claim 13, wherein the polymerization step comprises polymerizing the styrene or a derivative monomer thereof and at least one comonomer selected from the group consisting of acrylonitrile, butadiene, acrylic acid and methacrylic acid to produce a copolymer .