KR20140087797A - Process of biologically producing a 4-hydroxystyrene and derivatives thereof - Google Patents

Abstract

The present invention relates to a method for preparing 4-hydroxystyrene or a derivative thereof; and a method for preparing a polymer using the same. The 4-hydroxystyrene or the derivative thereof can be obtained in a biological manner which has higher specificity than a chemical process. Further, the 4-hydroxystyrene 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 preparing 4-hydroxystyrene and derivatives thereof by a biological method,

The present invention relates to a process for preparing 4-hydroxystyrene 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 4-hydroxystyrene or derivatives thereof therefrom by biological methods.

In recent years, oil prices have been rising steadily in the petrochemical industry due to global uncertainty about 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.

4-hydroxystyrene can be used in various industrial applications, and 4-hydroxystyrene or 4-acetoxystyrene can be used as a monomer in the production of liquid crystalline polymers or in the production of resins, elastomers, .

A method of chemically or biologically producing 4-hydroxystyrene is known. An example of such a chemical method can be obtained from ethylbenzene or p-hydroxyacetophenol through several steps (US 4,503,271, US 5,523,378). Or 4-hydroxycinnamic acid is reacted with copper powder at a temperature of 225 ° C to produce 4-hydroxystyrene (Sovish J. Org. Chem. 24: 1345-1347 (1959)). US 5,324,804 discloses a process for preparing 4-hydroxystyrene by decarboxylating caffeic acid in dimethylformamide at 150 ° C. It is also known to decarboxylate substituted cinnamic acid derivatives in an aqueous solution and at a temperature of 100 ° C (Lebensmittel-Wissenschraft u. Technol. 10: Food Science and Technology: 145-147 (1977)). However, such a chemical method requires expensive raw materials and reagents, high reaction conditions and high energy. Also, unwanted impurities are generated in large quantities, requiring complex purification steps, which is expensive to manufacture.

In order to overcome the shortcomings of existing chemical processes and achieve process sustainability, a bio-process based 4-hydroxystyrene synthesis method has been attempted. In US20040018600, 4-hydroxystyrene was prepared using a recombinant strain from glucose. However, when a recombinant strain is used, 4-hydroxystyrene as a product inhibits the production of 4-hydroxystyrene by feedback, resulting in decreased productivity and microbial growth. In addition, the reaction using the amino acid in the cell as a substrate may be greatly influenced by the unstable grain price because glucose or the like is used as a nutrient for culturing the microorganism, 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.

Therefore, there is a strong demand for the development of new 4-hydroxystyrene or its manufacturing process from the viewpoint of social requirements, process simplification, and economic improvement in recent eco-friendly process development

The present invention provides a method for producing 4-hydroxystyrene or a derivative thereof by a biological method having high specificity compared to a chemical process.

The present invention also provides a method for producing 4-hydroxystyrene 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 it as a substrate.

The present invention relates to a process for the production of 4- (4-hydroxyphenyl) -4-hydroxy-4-methylphenylcarbamate having the following formula (1) by a biological method, comprising the step of contacting a substrate comprising an aromatic carboxylic acid having a 4- To a process for preparing hydroxystyrene and derivatives 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 compound of formula (2) wherein X and Y are not hydrogen at the same time, (B) a biocatalyst that removes at least one substituent selected from the group consisting of a hydroxy 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, Can be performed.

The present invention relates to a process for producing a polymer by polymerizing the 4-hydroxystyrene or a derivative thereof, wherein the polymer is a homopolymer using one monomer, or a polymer obtained by polymerizing 4-hydroxystyrene or a derivative thereof At least one comonomer selected from the group consisting of styrene, 2,5-diemthyl-2,4-hexadiene, and diallyl maleate as comonomers May be copolymerized.

The present invention relates to a process for obtaining aromatic monomers using chemical and / or biological conversion and for the production of 4-hydroxystyrene or derivatives thereof by biological methods, wherein 4-hydroxystyrene or a derivative thereof from renewable lignin is environmentally friendly , Can be produced economically with high specificity. 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, To obtain 4-hydroxystyrene or a derivative thereof.

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

The present invention relates to a process for the production of 4- (4-hydroxyphenyl) -4-hydroxy-4-methylphenylcarbamate having the following formula (1) by a biological method, comprising the step of contacting a substrate comprising an aromatic carboxylic acid having a 4- To a process for preparing hydroxystyrene and derivatives thereof.

[Chemical Formula 1]

(2)

In the above general formulas (1) and (2), X and Y may be the same or different and are independently a hydrogen, a hydroxy or a C 1 -C 4 alkoxy group, preferably a hydroxy or a C 1 -C 4 alkoxy group. The C1-C4 alkoxy group may be linear or branched, and is preferably methoxy or ethoxy.

The 4-hydroxystyrene or its derivative having the formula (1) obtained according to the present invention is preferably 4-hydroxystyrene, 3,4-dihydroxystyrene, 3-methoxy-4-hydroxystyrene, -Dimethoxy-4-hydroxystyrene.

Another embodiment of the present invention is a process for preparing a compound of formula (2), wherein when X and Y are not hydrogen at the same time, it is preferable that the substrate containing an aromatic carboxylic acid having 4-hydroxy group Removing 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 after the treatment with the catalyst, Hydroxystyrene or a derivative thereof represented by the general formula (1), which comprises the step of further contacting a biocatalyst.

Hydroxycinnamic acid or a derivative thereof, and specific examples thereof include 4-hydroxycinnamic acid, 3,4-dihydroxynnamic acid, 3,4, 5-trihydroxycinnamic acid, 3-methoxy-4-hydroxycinnamic acid (Ferulic acid) and 3,5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid).

The aromatic carboxylic acid represented by Formula 2 may be prepared by a chemical 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 (2) can be obtained by separating from plants or lysing lignin. Or from recombinant microorganisms that contain phenylalanine hydroxylase (PAL) or tyrosine ammonia lyase (TAL) activity (R. Benrief et al. Phytochemistry 47: 825-832 (1998), US2002 / 0187207, US20030079255 ).

For example, the 4-hydroxycinnamic acid or its derivative represented by the formula (2) 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 degradation products of lignin include 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, The compounds of formula (2) of the invention are included.

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 invention, 0.1 to 5% (w / v) H ₂ SO ₄ , HCl or HNO ₃ , or a 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 pyrolysis at a high temperature of 350 to 650 ° C using a high-pressure reactor, and decomposition of lignin can be carried out by using catalysts for decomposing nitrobenzene, KMnO ₄ , H ₂ O ₂ and zeolite The decomposition efficiency can be increased. 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 (2) by contacting with the substrate containing the aromatic carboxylic acid having the 4-hydroxy group of the formula (2) 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 aromatic carboxylic acid of Formula 2 is anthranilate synthase (EC 4.1.3.27)), aminodeoxychorismate lyase (EC 4.1.3.38), chorismate lyase (EC 4.1.3.40), 3-dehydroquinone hydrolase 3-dehydroquinate hydro-lyase (EC 4.2.1.10)), 3-dehydroshikimate hydro-lyase (EC 4.2.1.118), prephenate hydrolyase EC 4.2.1.51), 5-O- (1-carboxyvinyl) -3-phosphoximate phosphate-lyase (EC 4.2.3.5) ), Isochorismate lyase (EC 4.2.99.21), and hydroxyphenyl pyruvate generation enzyme (Hydroxyphenylpyruvate synthase (EC 5.4.99.5)), the microorganism producing the enzyme, the microorganism lysate or the microorganism lysate.

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

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. In one example of the present invention, the enzyme of the present invention may be mutated or evolved to increase the substrate specificity or enzyme activity of the compound of formula (2) 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 in the production of the 4-hydroxystyrene and the derivative thereof 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 4-hydroxystyrene or derivatives thereof according to the present invention can be used in various applications such as production of monomers, resins, elastomers and coatings in the production of liquid crystalline polymers, and can be used alone or in combination with other comonomers And then polymerized together to prepare a polymer.

According to another embodiment of the present invention, there is provided a process for preparing 4-hydroxystyrene or a derivative thereof, comprising: preparing 4-hydroxystyrene or a derivative thereof from a substrate comprising an aromatic carboxylic acid represented by the following formula 2: And a method of polymerizing the 4-hydroxystyrene or a derivative thereof to prepare a polymer. The definitions of the formulas (1) and (2) are the same as those described above. The 4-hydroxystyrene represented by the above formula (1) or its derivative is obtained by reacting (a) a biocatalyst which removes the carboxyl group and (b) a hydroxyl group at the 3-position, 5-position or 3-position and 5-position of the aromatic carboxylic acid, And a C1-C4 alkoxy group. The obtained 4-hydroxystyrene or its derivative may be polymerized with a monomer to prepare a polymer.

In the polymerization step, 4-hydroxystyrene or a derivative thereof may be polymerized with a monomer to prepare a homopolymer, and the 4-hydroxystyrene or derivative monomer thereof and styrene, 2,5-dimethyl A copolymer may be prepared by polymerizing at least one comonomer selected from the group consisting of 2,5-diemthyl-2,4-hexadiene and diallyl maleate. have.

The conditions for the step of polymerizing the 4-hydroxystyrene or derivative thereof are not particularly limited as long as they can be applied under ordinary polymerization conditions.

The method for producing 4-hydroxystyrene or derivative thereof according to the present invention and the method for producing a polymer using the same can obtain 4-hydroxystyrene or a derivative thereof by a biological method having a higher specificity than a chemical process, In addition, using lignin as a raw material, hydroxystyrene or a derivative thereof can be obtained in an economical and economical manner using the lignin degradation product economically and 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 4-hydroxystyrene were analyzed 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  1: 4- Hydroxystyrene  production

1-1: 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 1):

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

(Reverse primer 1):

5'-AAACTCGAGTAATCTTCCCGCGCGAATAT-3 '(SEQ ID NO: 6)

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.

1-2: 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 production of 4-hydroxystyrene.

1-3: Production of 4 -hydroxystyrene

20 μL of phenolic acid decarboxylase purified in Example 1-2 at a concentration of 5 mg / L and 10 μL of 20 mM 4-hydroxycinnamic acid (Sigma) were mixed with 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 3).

Also, ferulic acid (Sigma) was used instead of 4-hydroxycinnamic acid in the same manner as described above, and the results of the analysis are shown in Table 3.

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  2: 4- Hydroxystyrene  Produce

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 1)

5'-AAACATATGTCACTAATGGACAATTGGAA-3 '(SEQ ID NO: 7)

(Reverse primer 2)

5'-AAACTCGAGATATTTTTCTTCACTGTTC-3 '(SEQ ID NO: 8)

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; 72 ° C elongation 3 minutes 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 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: 4- Hydroxystyrene  Produce

In order to remove the carboxyl group of 3-methoxy-4-hydroxycinnamic acid (ferric acid), the phenolic acid decarboxylase of Example 1-2 was used, and in order to remove the methoxy group, 4-hydroxystyrene was prepared by carrying out an enzymatic reaction by performing an enzyme reaction using ADC Lyase. Each of the enzymes was used for the reaction with a purified enzyme having a concentration of 5 mg / L.

As a reaction for removing the methoxy group, 0.5 mL of a reaction mixture containing 50 μL of ADC Lyase and 5 mM 3-methoxy-4-hydroxycinnamic acid (ferulic acid), 1 mM EDTA and 20 mM 2-mercaptoethanol was added at 37 ° C. And allowed to react in a thermostat for 5 hours. The decrease of the substrate 3-methoxy-4-hydroxycinnamic acid and the concentration of 4-hydroxycinnamic acid were analyzed by HPLC (Table 4).

In order to prepare 4-hydroxystyrene through a decarboxylation enzyme reaction, 10 μL of 5 mg / L phenolic acid decarboxylase was added to the obtained reaction solution, and the mixture was allowed to react for 3 hours at 37 ° C. in a thermostat. After completion of each enzyme reaction, 100 μL samples were taken, diluted with 900 μL of methanol, and then analyzed using a syringe filter (0.22 μm) to remove impurities. The results of the analysis are shown in Table 4.

Before enzyme reaction (mM) After ADC Lyase treatment (mM) After decarboxylase treatment (mM) 3-Methoxy-4-hydroxycinnamic acid 4.99 2.18 0.32 4-Hydroxycinnamic acid 0.0 2.45 0.38 4-hydroxystyrene 0.00 0.00 1.98

As shown above, after 2-methoxy-4-hydroxycinnamic acid is converted to 4-hydroxycinnamic acid by aminodeoxycholyzmatease, it is finally converted to 4-hydroxystyrene by phenolic acid decarboxylase Is produced.

Example  3: From Lee Green  production

3-1: 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. After analysis by HPLC, the composition as shown in Table 5 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

3-2: 4- Hydroxystyrene  production

A small amount of 10 M HCl was added to the lignin decomposition product obtained in Example 3-1, 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 phenolic acid decarboxylase and amino deoxycholymitlia obtained in Examples 1-2 and 2-2 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. Then, 360 μL of methanol was added to dilute the sample, and then the impurities were removed using a syringe filter (0.22 μm) . 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 6.

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

4-hydroxycinnamic acid, ferulic acid, and cinnamic acid were degraded to remove the methoxy groups of ferulic acid and cinnamic acid through an enzyme reaction using phenolic acid decarboxylase and aminodeoxycholyzmatease, Hydroxycinnamic acid, and then the carboxyl group was removed by phenolic acid decarboxylase, resulting in the production of 2.4 mM 4-hydroxystyrene.

<110> SAMSUNG PETROCHEMICAL CO., LTD. <120> PROCESS OF BIOLOGICALLY PRODUCING A 4-HYDROXYSTYRENE AND          DERIVATIVES THEREOF <130> DPP20129169KR <160> 8 <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> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer 1 <400> 5 aaacatatgg aaaactttat cggaagcca 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1 <400> 6 aaactcgagt aatcttcccg cgcgaatat 29 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer 2 <400> 7 aaacatatgt cactaatgga caattggaa 29 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 <400> 8 aaactcgaga tattttgtct tcactgttc 29

Claims (12)

Comprising contacting a substrate comprising an aromatic carboxylic acid having a 4-hydroxy group represented by the formula (2) with a biocatalyst which removes the carboxyl group, by a biological method, 4-hydroxystyrene And derivatives 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. 2. The method according to claim 1, wherein when X and Y are not hydrogen at the same time in the formula (2), a catalyst for removing the carboxyl group is added to a substrate containing an aromatic carboxylic acid having a 4-hydroxy group represented by the formula A biocatalyst which 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 aromatic carboxylic acid, Further comprising the step of: The method according to claim 1, wherein the biocatalyst is an enzyme, a microorganism containing the enzyme, a lysate of the microorganism, or an extract of microorganism lysate. 4. The method of claim 3, wherein the biocatalyst is a wild-type microorganism or a recombinant microorganism. The method according to claim 1, wherein the step of contacting the substrate with the biocatalyst comprises contacting the enzyme, an enzyme-containing microorganism, a microorganism disruptor or an extract of microbial debris with the substrate, or contacting the microorganism with the substrate, Lt; / RTI &gt; The method according to claim 1, 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 decarboxylase 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)), oxidized decarboxylase (EC 4.1.1.58), gallate decar (EC 4.1.1.59), 4,5-dihydroxyphthalate decarboxylase (EC 4.1.1.55), 3,4-dihydroxyphthalate decarboxylase (EC 4.1.1.69), 3-hydroxy- An enzyme selected from the group consisting of 2-methylpyridine-4,5-dicarboxylate 4-dicarboxylic acid (EC 4.1.1.51) and phenyl phosphate decarboxylase (EC 4.1.1.-) Ha Is an extract of a microorganism, microorganism lysate or microbial lysate thereof. The biocatalyst for removing at least one substituent selected from the group consisting of the hydroxyl group and the C1-C4 alkoxy group is at least one selected from the group consisting of an anthranilic acid-producing enzyme (EC 4.1.3.27), aminodoxychrismate lyase (EC 4.1.3.38 ), Chrysomate lyase (EC 4.1.3.40), 3-dehydroquinone hydro-lyase (EC 4.2.1.10), 3-dehydrochymate hydro-lyase (EC 4.2.1.118), prefenate hydro- (EC 4.2.1.51), 5-O- (1-carboxyvinyl) -3-phosphosiminate phosphate-lyase (EC 4.2.3.5), and isochorismate lyase (EC 4.2.99.21) Wherein the enzyme is selected from the group consisting of a bait-forming enzyme (EC 5.4.99.5), a microorganism producing the enzyme, a microorganism lysate thereof, or a microbial lysate. The method according to claim 1, wherein the substrate containing an aromatic carboxylic acid having a 4-hydroxy group represented by the formula (2) is a lignin degradation product obtained by decomposing lignin. 9. The method of claim 8, wherein the decomposition of lignin is carried out at elevated temperatures such as pyrolysis, gasification, hydrogenolysis, acidolysis, base decomposition, chemical oxidation, and hydrolysis under supercritical condition. &lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; Preparing a 4-hydroxystyrene or a derivative thereof having the following formula (1) by the process according to claim 1 or 2; And
[Chemical Formula 1]

(2)

Wherein X and Y may be the same or different and independently represent hydrogen,
Wherein the 4-hydroxystyrene or the derivative thereof is polymerized to prepare a polymer. 11. The method according to claim 10, wherein the polymerization step comprises polymerizing the 4-hydroxystyrene or a derivative thereof with a monomer to produce a homopolymer. 11. The method of claim 10, wherein the polymerizing step comprises mixing the 4-hydroxystyrene or a derivative monomer thereof with at least one or more selected from the group consisting of styrene, 2,5-dimethyl-2,4-hexadiene and diallyl maleate A method for producing a copolymer by polymerizing a monomer.