KR102439135B1 - Method for biocatalytic production of biogenic amines with in situ product removal - Google Patents

Abstract

Disclosed herein is a biological production method of a biogenic amine using an amino acid as a substrate, an amino acid dicarboxylase, an open reactive group, and a cation exchange resin. When an amino acid produced by fermentation is used as a substrate, the biogenic amine produced by the present invention may be treated as a naturally-derived compound, unlike a chemically synthesized compound. In addition, the production method of the present invention removes the enzyme reaction product at the same time as the reaction through vaporization and ion exchange resin adsorption, thereby alleviating strong product inhibition of amino acid dicarboxylase, thereby producing biogenic amines with high production efficiency. . In another aspect, the present invention enables easy product recovery by separating the biogenic amine from the reaction solution simultaneously with the enzymatic reaction.

Description

Method for biological production of biogenic amines through simultaneous product separation {Method for biocatalytic production of biogenic amines with in situ product removal}

The present invention relates to a biological process for producing biogenic amines using aromatic amino acid decarboxylase, and more particularly, carbon dioxide as a product in the decarboxylation reaction and It relates to a method for producing biogenic amines with high efficiency by isolating the amine products simultaneously with the reaction through vaporization and ion exchange resins, respectively.

Biogenic amine is a generic term for amines produced in the living body. In particular, it includes monoamine neurotransmitter and trace amine, which are biochemical information transmitters that act as neurotransmitters. It plays a pivotal role in function. For example, a decrease in the monoamine neurotransmitters serotonin, dopamine, and norepinephrine causes depression, and trace amines 2-phenylethylamine and tryptamine are known to be involved in mood and appetite regulation. In addition, biogenic amines are known to be importantly involved in intercellular communication in addition to neurotransmission functions. Due to such biological importance, the utilization of biogenic amines in pharmaceutical applications is attracting attention. Despite the recent increase in demand for natural products, biogenic amines have been produced by chemical synthesis until now, and studies on eco-friendly production methods based on biological processes have hardly been conducted. It can be obtained by extraction from natural products, but it is not realistic in terms of production cost because trace amounts exist. For example, the 2-phenylethylamine contained in chocolate is only about 3 mg per kg of chocolate.

Aromatic amino acid decarboxylase is an enzyme that performs decarboxylation of amino acids in vivo using pyridoxal-5'-phosphate (PLP) as a cofactor. For example, in the case of L-dopa dicarboxylase, the decarbonization reaction of L-3,4-dihydroxyphenylalanine is performed to produce dopamine and carbon dioxide. Due to these properties, aromatic amino acid dicarboxylase is very suitable for the biological synthesis of biogenic amines.

In a reaction process using a biocatalyst, substrate inhibition by a reactant and product inhibition by a product frequently occur depending on the characteristics of the enzyme. Since the inhibition of the enzyme activity lowers the reaction efficiency, it is essential to develop a customized process that can improve the reaction efficiency by alleviating the inhibition of the activity for industrial use.

There have been many reports of inhibition of activity by reactants and products in the decarbon dioxide reaction performed by dicarboxylase. In the prior literature (Biochimica et Biophysica Acta, 1986, 870, 31-40), Drosophila L-dopa dicarboxylase derived from melanogaster showed inhibition of enzymatic activity by reactants with respect to L-dopa (L-3,4-dihydroxyphenylalanine), and inhibition of activity by dopamine, a product, has also been reported. In addition, in the prior literature (Archives of Biochemistry and Biophysics, 1999, 365(1), 17-24), it has been reported that Pseudomonas stutzeri -derived oxaloacetate dicarboxylase inhibits enzyme activity by products for pyruvate and carbonate. .

However, despite the high industrial applicability of amino acid dicarboxylase, studies on a high-efficiency process capable of solving activity inhibition by its reactants and products have hardly been made. Patent Document 1 (US Patent, 6015698) describes a method for producing biogenic amines through decarbonization reaction from amino acids using Gibberella and Fusarium , Aspergillus , Pleurotus , and Nectria strains, but the reaction yield is very low within 22%. Prior Art Document 1 (Microbial Cell Factories, 2019, 18, 74) describes a method for producing L-dopa in E. coli through metabolic engineering, but a process design strategy for reactant and product inhibition has not been reported.

US Patent US 06015698A

Microbial Cell Factories, 2019, 18, 74

An object of the present invention is to provide a method for producing biogenic amines with high efficiency by separating the product from the reaction solution at the same time as the biocatalytic reaction and alleviating the inhibition of enzyme activity by the product of amino acid dicarboxylase.

In order to achieve the above object, the present invention includes a cation exchange resin (CER) in an amino acid dicarboxylase reaction solution and facilitates gas exchange by keeping the lid of the reactor open to increase the productivity of biogenic amines. provide a way

The biogenic amine production method presented in the present invention relieves the inhibition of enzyme activity by the product during the enzyme reaction by separating the reaction product from the reaction solution through cation exchange resin adsorption and vaporization at the same time as the amino acid decarboxylase reaction, thereby improving production efficiency and yield All of these have the advantage of being high. In another aspect, the present invention can purify the adsorbed biogenic amine product after separating only the cation exchange resin through filtering without additional treatment of the reaction solution after completion of the reaction because the desired amine product is adsorbed to the cation exchange resin. By providing a product separation method, the separation and purification of biogenic amines can be easily performed. In addition, biogenic amines produced by using L-aromatic amino acids produced by fermentation as a substrate can be treated as naturally-derived compounds, unlike chemically synthesized compounds.

1 is a diagram showing product inhibition of aromatic amino acid dicarboxylase (hereinafter, AADC-BA) derived from Bacillus atrophaeus .
2 is a diagram comparing the effects of closed and open reactors in 2-phenylethylamine production reaction by amino acid dicarboxylase (AADC-BA).
3 is a diagram showing the results of adsorption of L-phenylalanine or 2-phenylethylamine to a cation exchange resin (Dowex 50WX8/Na ⁺ ).
4 is a diagram comparing conversion rates with or without a cation exchange resin in the production of 2-phenylethylamine using an open reactor and amino acid dicarboxylase (AADC-BA).
5 is a diagram showing the production of serotonin through amino acid dicarboxylase (AADC-BA) in an open reactor including a cation exchange resin.

The present invention will be described in detail as follows.

The present invention relates to a method for producing a biogenic amine using an amino acid dicarboxylase using a cation exchange resin and an open reactor to separate and produce the product at the same time as the reaction, comprising the following steps.

(a) providing an amino acid as a substrate for an amino acid decarboxylase;

(b) adding a cation exchange resin to the reaction solution;

(c) reacting the amino acid dicarboxylase with the amino acid to produce a biogenic amine and carbon dioxide;

(d) adsorbing the biogenic amine to a cation exchange resin and separating it from the reaction solution;

(e) separating the carbon dioxide from the open system reactor through volatilization; and

(f) recovering the biogenic amine from the cation exchange resin;

A method for biocatalytic production of biogenic amines, characterized in that it comprises

[Scheme 1]

The amino acid is a compound containing an amine group and a carboxy group, preferably phenylalanine, tryptophan, tyrosine, aspartic acid, 3,4-dihydroxyphenylalanine (dopa), 5-hydroxytryptophan, homophenylalanine, m -fluorophenylalanine , p -fluorophenylalanine, p -chlorophenylalanine, p -bromophenylalanine, or p -nitrophenylalanine.

As shown in Scheme 1, the present invention can produce biogenic amines through decarbonization of amino acids provided as substrates by amino acid dicarboxylase. Under neutral reaction conditions (eg, pH 7), amino acids as substrates exist as electrically neutral zwitterions, and biogenic amines in products exist as cations. Also, the other product, carbon dioxide, is mostly gasified by equilibrium.

An ion exchange resin is a resin that can cause ion exchange, and is used to remove or separate ions from a solution. When a cation exchange resin is used, the biogenic amine present as a cation in Scheme 1 can be selectively adsorbed and separated from the reaction solution. Since the amino acid, which is a substrate, is electrically neutral, it is not adsorbed to the ion exchange resin, but exists in the reaction solution and is continuously consumed in the reaction.

Carbon dioxide produced through the decarbonization reaction may exist as carbonic acid, bicarbonate, or carbonate in an aqueous solution, but is mostly gasified by hydration equilibrium. Since the solubility of carbon dioxide in water decreases at high temperature and low pressure, carbon dioxide produced in the aqueous phase is generally present in an extremely low concentration under the enzyme reaction conditions. Therefore, in an open reactor, carbon dioxide removal through gasification can be promoted through continuous gas exchange.

Therefore, the amine and carbon dioxide are continuously separated from the reaction solution at the same time as they are generated by the ion exchange resin and vaporization, and are present at a low concentration in the reaction solution, so the problem of inhibition of enzyme activity by the product of amino acid dicarboxylase can be solved.

The biogenic amine changes from cation to neutral when changing from neutral to basic conditions. Therefore, the biogenic amine adsorbed to the cation exchange resin can be easily desorbed from the cation exchange resin by increasing the pH of the solution. Therefore, it is possible to selectively separate and purify only the biogenic amine, which is a desired product, from the cation exchange resin separated from the reaction solution using this.

The above-described cation exchange resin may be used by adding it to the reaction solution in the form of a resin. When the amino acid decarboxylase reaction is completed, the biogenic amine can be recovered from the cation exchange resin by separating the cation exchange resin to which the biogenic amine is adsorbed from the reaction solution and adding a basic solution such as NaOH solution. The base is selected from a basic solution of pH 12 or higher, and may preferably be 1N sodium hydroxide and 10% aqueous ammonia. The recovered solution is further subjected to extraction through an organic solvent after lowering the pH, so that the purification of biogenic amine is possible. The organic solvent is selected from hydrophobic organic solvents, and may preferably be n -hexane.

In addition, the ion exchange resin may be used in an enzyme reactor in the form of a column. In this case, the enzyme reactor is selected from a continuous flow reactor, and may preferably be a continuous flow reactor including a reaction vessel, a pump, and a cation exchange resin column. Since the continuous flow reactor is connected to the reactor in the form of a module, it can be separated and easily replaced even in the middle of the reaction. At this time, it is possible to recover the biogenic amine by directly flowing the basic solution to the separated cation exchange resin column. The base is selected from a basic solution with a pH of 12 or higher, and preferably 10% aqueous ammonia, sodium hydroxide (0.1 or 1 N).

The open reactor may be used together with a stirrer or an air pump. The stirrer may be a magnetic stirrer or an overhead stirrer. The air pump may be installed and used separately from the reactor and may be connected to the inside or outside of the reaction solution through tubing to promote gas circulation.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. Terms not otherwise defined herein have the meanings commonly used in the art to which the present invention pertains.

Example 1. Amino Acids of dicarboxylate cloning and tablets

Amino acid dicarboxylase was expressed in E. coli through cloning of Bacillus atrophaeus or Lactobacillus brevis , Methanococcus jannaschii strain. Each amino acid dicarboxylase is conveniently AADC-BA ( Bacillus atrophaeus ) or AADC-LB ( Lactobacillus brevis ), AADC-MJ ( Methanococcus ) jannaschii ). For this purpose, the primers shown in Table 1 were cloned into pET28a or pET26b vectors.

PCR primer sequence for cloning of amino acid dicarboxylase Primer sequences AADC-BA forward: 5′-AGGAGATATACCATGAAACAAGTGTCGGAAAAC-3′
reverse: 5′-GTGGTGGTGCTCGAGTTCAGCAACGCATGGATACG-3′ AADC-LB forward: 5′-AGGAGATATACCATGGAAAAAAGTAATCGCTCAC-3′
reverse: 5′-GTGGTGGTGCTCGAGAACATTTTCCTTTTGATTAACCG-3′ AADC-MJ forward: 5′-AGGAGATATACATATGCGGAACATGCAAGAA-3′
reverse: 5′-GGTGGTGGTGGCTCGAGGTCCCGCTTAATCGAATTCAG-3′

KTAprime plus FPLC (GE Healthcare, USA)를 사용해 정제하였다. 상기 세포추출액을 버퍼(20 mM 소듐포스페이트, 0.15 M 염화나트륨, 0.5 mM PLP, pH 7.6)로 충전된 HisTrap HP 컬럼에 흘려준 후, 이미다졸 농도를 0.05-0.5 M로 변화시키면서 아미노산 디카복실레이스를 용출시켰다. 이후 상기 용출액을 버퍼(50 mM 인산나트륨, 0.15 M 염화나트륨, 0.2 mM PLP, pH 7)로 충전된 HiTrap HP 컬럼 (GE Healthcare)에 흘려주어 잔류 이미다졸을 제거하였다. Escherichia transformed with the plasmid coli BL21(DE3)) cells were suspended at 37°C in 1 L of LB broth (trypton 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L) containing kanamycin (50 µg/mL). It was cultured up to an optical density (OD) of 0.4, at which time IPTG was added to a final concentration of 0.1 mM and further cultured at 30° C. for 12 hours to overexpress the protein. Thereafter, E. coli cells containing the overexpressed protein were obtained by centrifugation at 5,000×g and 4°C for 5 minutes. It was suspended in 15 mL buffer (50 mM Tris/HCl, 50 mM sodium chloride, 1 mM EDTA, 1 mM beta-mercaptoethanol, 0.1 mM PMSF, 0.5 mM PLP, pH 8), cooled on ice, and ultrasonicated in a cell disrupter. Cell disruption was performed using a sonicator. After centrifugation at 10,000×g, 4℃ for 1 hour to remove cell debris, the cell-free crude extract obtained was subjected to HisTrap HP column (GE Healthcare, USA) and

Purification was performed using KTAprime plus FPLC (GE Healthcare, USA). The cell extract was flowed through a HisTrap HP column filled with a buffer (20 mM sodium phosphate, 0.15 M sodium chloride, 0.5 mM PLP, pH 7.6), and then amino acid dicarboxylase was eluted while changing the imidazole concentration to 0.05-0.5 M. did it Thereafter, the eluate was flowed through a HiTrap HP column (GE Healthcare) filled with a buffer (50 mM sodium phosphate, 0.15 M sodium chloride, 0.2 mM PLP, pH 7) to remove residual imidazole.

The purified amino acid dicarboxylase was added to a solution containing a final concentration of 10 mM L-phenylalanine and 50 mM potassium phosphate buffer (pH 7) to perform an enzymatic reaction at 37 degrees, and 10 minutes after the start of the reaction, it was generated through HPLC analysis The obtained 2-phenylethylamine was quantified. The reactivity of the obtained enzyme is shown in Table 2 below. 1 U means the amount of enzyme capable of producing 1 μmol 2-phenylethylamine per minute under the above conditions.

Comparison of activity of three amino acid dicarboxylase Specific activity AADC-BA 0.28 U/mL/μM-enzyme AADC-LB 0.04 U/mL/μM-enzyme AADC-MJ 0.009 U/mL/μM-enzyme

Example 2. Amino acids of dicarboxylate Substrate specificity

In order to confirm the substrate specificity of amino acid dicarboxylase, an enzymatic reaction was performed at pH 7 (50 mM potassium phosphate), 37°C. The reaction was started by adding purified amino acid dicarboxylase (AADC-BA or AADC-MJ) to a substrate (final concentration 1 or 2, 5, 10 mM L-amino acid), and a portion of the reaction solution was reacted at 0-60 minutes. was used to quantify the amount of residual substrate through HPLC analysis. Among the substrates, the reaction was performed at 1 mM for L-homophenylalanine and 2 mM for L-tyrosine, and the reaction rate of L-phenylalanine was measured under the same substrate concentration condition to calculate the relative reaction rate. The reaction rate of the racemic mixture in the substrate was measured under the condition of 5 mM L-phenylalanine, and the relative reaction rate was calculated. The obtained substrate specificities are shown in Table 3 below. Both AADC-BA or AADC-MJ showed the highest activity against tryptophan. AADC-BA showed high reactivity to various aromatic amino acids, including phenylalanine substitution.

Substrate specificity of amino acid dicarboxylase temperament AADC - BA AADC -MJ L-Phenylalanine 100% 100% L-Tryptophan 590%

0%

0% L-Tyrosine 21% 407% L-Aspartate

0% 27% L-Glutamate

0%

0% L-DOPA 24% 532% 5-Hydroxy-L-tryptophan 71% None L-Homophenylalanine 3% L-Phenylglycine

0% m -Fluoro-DL-phenylalanine 180% p -Fluoro-DL-phenylalanine 290% p -Chloro-DL-phenylalanine 833% p -Bromo-DL-phenylalanine 300% p -Nitro-DL-phenylalanine 450%

Example 3. Amino acids of dicarboxylate Activity as a function of pH

To check the activity of amino acid dicarboxylase (AADC-BA) by pH, 50 mM buffer (pH 4-6, sodium citrate; pH 6-8, potassium phosphate; pH 8-10, sodium borate; pH 7, HEPES), the enzymatic reaction was performed at 37 °C. The reaction was started by adding purified amino acid dicarboxylase (AADC-BA) to the substrate (final concentration 10 mM L-phenylalanine), and the amount of the product was quantified through HPLC analysis 20 minutes after the start of the reaction. The results are shown in Table 4 below. pH 7-8 was found to be the most suitable pH condition for enzyme activity.

Activity change according to pH of AADC-BA Buffer pH AADC - BA's activation
(mM/min/mM-enzyme) sodium citrate 4 <10 5 20±5 6 190±10 Potassium phosphate 6 100±10 7 270±20 8 280±20 Sodium borate 8 200±10 9 30±20 10 <10 HEPES 7 140±10

Example 4. Amino acids of dicarboxylate Activity and stability with temperature

To check the activity of amino acid dicarboxylase (AADC-BA) by temperature, an enzymatic reaction was performed in 50 mM buffer (pH 7, potassium phosphate), 30-65°C. The reaction was started by adding purified amino acid dicarboxylase (AADC-BA) to the substrate (final concentration 30 mM L-phenylalanine), and the amount of the product was quantified through HPLC analysis 20 minutes after the start of the reaction. The results are shown in Table 5 below. 37-45 degrees was found to be the most suitable temperature condition for enzyme activity.

AADC-BA activity change according to temperature Temperature AADC-BA activity
(mM/min/mM-enzyme) 30℃ 290±10 37℃ 370±40 45℃ 380±20 55℃ 240±2 65℃ 35±5

To confirm the stability of amino acid dicarboxylase (AADC-BA) by temperature, purified amino acid dicarboxylase (AADC-BA) was added to a solution containing 50 mM buffer (pH 7, sodium phosphate) and 150 mM sodium chloride. After 15 hours of storage at 30-63 ℃, the residual activity was confirmed. The reaction was started by adding the amino acid dicarboxylase stored at each temperature to the substrate mixture (final concentration 10 mM L-phenylalanine, 50 mM potassium phosphate buffer pH 7), and 20 minutes after the start of the reaction, the amount of the product was analyzed by HPLC. was quantified through The results are shown in Table 6 below. The stability of the enzyme was relatively high up to 55°C, but the stability of the enzyme rapidly decreased above that temperature.

Temperature-dependent stability of AADC-BA Temperature 15 hours incubation after
residual activity 30℃ 106±8% 45℃ 93±2% 55℃ 85±13% 63℃ 32±1%

Example 5. Amino Acid by Product of dicarboxylate active inhibition

In order to confirm the activity inhibition by the product of amino acid dicarboxylase (AADC-BA), the enzymatic reaction was performed at 37°C. In order to confirm activity inhibition by 2-phenylethylamine, including substrate (final concentration 10 mM L-phenylalanine), product (0-200 mM 2-phenylethylamine), and buffer (50 mM potassium phosphate, pH 7) Purified amino acid dicarboxylase (AADC-BA) was added to the solution to react, and the amount of residual substrate was quantified 10 or 60 minutes after the start of the reaction through HPLC analysis. To confirm the inhibition of enzymatic activity by carbon dioxide, purified amino acid dika in a solution containing substrate (final concentration 10 mM L-phenylalanine), carbon dioxide product (0-100 mM bicarbonate), and buffer (5 mM potassium phosphate, pH 7) Voxylase was added to react, and the amount of the product was quantified through HPLC analysis 30 minutes after the start of the reaction. The results are shown in FIG. 1 . AADC-BA was observed to inhibit 71 or 73% activity in 100 mM bicarbonate or 2-phenylethylamine, respectively.

Example 6. In closed or open reactors biogenic amine Productivity Comparison

In order to confirm the improvement of the reaction yield according to the vaporization and removal of carbon dioxide products in the decarbonization reaction of amino acid dicarboxylase, biogenic amine generation reactions were performed in closed and open reactors. In a 1.8 mL container, add 1 mL of a reaction solution containing substrate (50 mM L-phenylalanine), buffer (50 mM potassium phosphate, pH 7), and AADC-BA overexpressing cells (0.83 U/mL), and then place the container in a closed condition ( The reaction was carried out under closed reactor) and unsealed conditions (open reactor). The reaction was mixed through a magnetic stirrer at 37°C. A portion of the reaction solution was sampled according to the reaction time, and the product was analyzed by HPLC. The results are shown in FIG. 2 . When vaporization and removal of carbon dioxide product was promoted using an open reactor, the reaction yield was increased by 20% (38 reaction time) compared to a closed reactor.

Example 7. Adsorption of Enzyme Reaction Composition on Cation Exchange Resin

Adsorption of reactants and products in amino acid decarboxylase reaction was observed using a cation exchange resin (CER). As a cation exchange resin, Dowex 50WX8 (100-200 mesh) was used as a representative example. Dowex 50WX8/H ⁺ (Sigma Aldrich Cat#217506), sold to prepare the Na ⁺ ion form of Dowex 50WX8, was treated with sodium hydroxide solution. Specifically, 10 mL of 1 N sodium hydroxide solution was added to Dowex 50WX8/H ⁺ 1 g and stirred at 37° C. for 1 hour (200 rpm), and when the pH reached 6-8 to remove residual sodium hydroxide The cation exchange resin was washed with ultrapure water until

The adsorption of the enzyme reaction composition to the cation exchange resin was examined using the representative substrate (L-phenylalanine) and its product (2-phenylethylamine) of amino acid dicarboxylase (AADC-BA). A 1 mL solution in which 0-21 mg/mL cation exchange resin was added to a solution containing a substrate or product (final concentration 10 mM) was stirred (200 rpm) at 37° C. for 30 minutes. After centrifugation at 13,000 rpm for 1 minute, each substrate/product was quantified by measuring the absorbance of the supernatant using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). This result is shown in FIG. 3 . The product, 2-phenylethylamine, showed 92% adsorption on the cation exchange resin at a concentration of 21 mg/mL, but the substrate L-phenylalanine showed 4% adsorption at the same concentration of the cation exchange resin, so it was virtually not adsorbed.

Amino acid dicarboxylase (AADC-BA) was added at a final concentration of 10 μM to a cation exchange resin (final concentration 0-21 mg/mL cation exchange resin) to check whether amino acid dicarboxylase was adsorbed on the cation exchange resin. The 1 mL solution was stirred (200 rpm) at 37°C for 30 minutes. This was centrifuged at 13,000 rpm for 1 minute, and the concentration of residual amino acid dicarboxylase in the supernatant was confirmed by measuring absorbance at 280 nm using Nanodrop2000. The results are shown in Table 7. No adsorption of amino acid dicarboxylase on the cation exchange resin was observed.

Measurement of adsorption amount of cation exchange resin of AADC-BA Dowex 50WX8 /Na ⁺ density Enzyme Residue in Solution 2.1 mg/mL 100% 6.3 mg/mL 100% 21 mg/mL 100%

Example 8. Variety of open reactors biogenic amine Produce

Various biogenic amine production reactions were performed using amino acid dicarboxylase in an open reactor. The reaction was started by adding the final 1 mL reaction solution (10 mM L-amino acid, 50 mM potassium phosphate pH 7) and AADC-BA overexpressing E. coli (0.13 U/mL) to a 1.8 mL reaction vessel, and a magnetic stirrer at 37°C. was used to stir the reaction solution. The results are shown in Table 8 below. All five biogenic amines could be produced through an enzymatic reaction with a conversion rate of over 90%.

Production of various biogenic amines in an open reactor product reaction time conversion rate 2-Phenylethylamine 3 h 96.9% Dopamine 6.5 h 90.5% Serotonin 6.5 h 96.9% Tryptamine 0.3 h >99% Tyramine 44.5 h 95.1%

Example 9. Production of 2-phenylethylamine in an open reactor with added cation exchange resin

The reaction efficiency according to the presence or absence of a cation exchange resin in an open reactor was compared using L-phenylalanine, a representative substrate of AADC-BA. Cation exchange resin (0 or 400 mg/mL) and purified AADC-BA (1.4 U/mL) overexpressing E. coli (0.13) in the final 1 mL reaction solution (150 mM L-phenylalanine, 50 mM HEPES pH 7) in a 1.8 mL container U/mL) was added to initiate the reaction. The reaction solution was mixed through a magnetic stirrer at 37°C to accelerate the removal of the reaction product. The results are shown in FIG. 4 . When the cation exchange resin was additionally included in the open reactor, the reaction efficiency increased by 37% (78% conversion) after 1 hour of reaction. In addition, it was observed that the concentration of 2-phenylethylamine in the reaction solution was kept low at the level of 3.1 mM.

Example 10. Production of serotonin on a small scale in an open reactor with added cation exchange resin

In order to show the utility of the present invention, a biocatalytic production of serotonin was performed. The reaction was carried out in a 100 mL jacketed reactor with 40 mL reaction solution (405 mg 5-hydroxy-L-tryptophan, 50 mM potassium phosphate pH 7) and cation exchange resin (100 mg/mL Dowex 50WX8/Na ⁺ ), purified AADC- BA (4 U/mL) was added and proceeded at 37°C. The reaction solution was stirred (150 rpm) through a magnetic stirrer, and the substrate and product were analyzed by HPLC depending on the reaction time. The results are shown in FIG. 5 . After 40 minutes of reaction, >99% conversion was shown, and it was observed that most of the produced serotonin was adsorbed to the cation exchange resin and was present at a low concentration (1.7 mM after 40 minutes of reaction) in the reaction solution.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Method for biocatalytic production of biogenic amines with in in situ product removal <130> PD20-119 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 480 <212> PRT <213> amino acid decarboxylase <400> 1 Met Ser Glu Asn Leu Gln Leu Ser Ala Glu Glu Met Arg Gln Leu Gly 1 5 10 15 Tyr Gln Ala Val Asp Leu Ile Ile Asp His Met Asn His Leu Lys Ser 20 25 30 Lys Pro Val Ser Glu Thr Ile Asp Ser Asp Ile Leu Arg Asn Lys Leu 35 40 45 Thr Glu Ser Ile Pro Glu Asn Gly Ser Asp Pro Lys Glu Leu Leu His 50 55 60 Phe Leu Asn Arg Asn Val Phe Asn Gln Ile Thr His Val Asp His Pro 65 70 75 80 His Phe Leu Ala Phe Val Pro Gly Pro Asn Asn Tyr Val Gly Val Val 85 90 95 Ala Asp Phe Leu Ala Ser Gly Phe Asn Val Phe Pro Thr Ala Trp Ile 100 105 110 Ala Gly Ala Gly Ala Glu Gln Ile Glu Leu Thr Thr Ile Asn Trp Leu 115 120 125 Lys Ser Met Leu Gly Phe Pro Asp Ser Ala Glu Gly Leu Phe Val Ser 130 135 140 Gly Gly Ser Met Ala Asn Leu Thr Ala Leu Thr Val Ala Arg Gln Ala 145 150 155 160 Lys Leu Asn Asn Asp Ile Glu Asn Ala Val Val Tyr Phe Ser Asp Gln 165 170 175 Thr His Phe Ser Val Asp Arg Ala Leu Lys Val Leu Gly Phe Lys His 180 185 190 His Gln Ile Cys Arg Ile Glu Thr Asp Glu His Leu Arg Ile Ser Val 195 200 205 Ser Ala Leu Lys Lys Gln Ile Lys Glu Asp Arg Thr Lys Gly Lys Lys 210 215 220 Pro Phe Cys Val Ile Ala Asn Ala Gly Thr Thr Asn Cys Gly Ala Val 225 230 235 240 Asp Ser Leu Asn Glu Leu Ala Asp Leu Cys Asn Asp Glu Asp Val Trp 245 250 255 Leu His Ala Asp Gly Ser Tyr Gly Ala Pro Ala Ile Leu Ser Glu Lys 260 265 270 Gly Ser Ala Met Leu Gln Gly Ile His Arg Ala Asp Ser Leu Thr Leu 275 280 285 Asp Pro His Lys Trp Leu Phe Gln Pro Tyr Asp Val Gly Cys Val Leu 290 295 300 Ile Arg Asn Ser Gln Tyr Leu Ser Lys Thr Phe Arg Met Met Pro Glu 305 310 315 320 Tyr Ile Lys Asp Ser Glu Thr Asn Val Glu Gly Glu Ile Asn Phe Gly 325 330 335 Glu Cys Gly Ile Glu Leu Ser Arg Arg Phe Arg Ala Leu Lys Val Trp 340 345 350 Leu Ser Phe Lys Val Phe Gly Val Ala Ala Phe Arg Gln Ala Ile Asp 355 360 365 His Gly Ile Met Leu Ala Glu Gln Val Glu Ala Phe Leu Gly Lys Ala 370 375 380 Lys Asp Trp Glu Val Val Thr Pro Ala Gln Leu Gly Ile Val Thr Phe 385 390 395 400 Arg Tyr Ile Pro Ser Glu Leu Ala Ser Thr Asp Thr Ile Asn Glu Ile 405 410 415 Asn Lys Lys Leu Val Lys Glu Ile Thr His Arg Gly Phe Ala Met Leu 420 425 430 Ser Thr Thr Glu Leu Lys Glu Lys Val Val Ile Arg Leu Cys Ser Ile 435 440 445 Asn Pro Arg Thr Thr Thr Glu Glu Met Leu Gln Ile Met Met Lys Ile 450 455 460 Lys Ala Leu Ala Glu Glu Val Ser Ile Ser Tyr Pro Cys Val Ala Glu 465 470 475 480 <210> 2 <211> 396 <212> PRT <213> amino acid decarboxylase <400> 2 Met Arg Asn Met Gln Glu Lys Gly Val Ser Glu Lys Glu Ile Leu Glu 1 5 10 15 Glu Leu Lys Lys Tyr Arg Ser Leu Asp Leu Lys Tyr Glu Asp Gly Asn 20 25 30 Ile Phe Gly Ser Met Cys Ser Asn Val Leu Pro Ile Thr Arg Lys Ile 35 40 45 Val Asp Ile Phe Leu Glu Thr Asn Leu Gly Asp Pro Gly Leu Phe Lys 50 55 60 Gly Thr Lys Leu Leu Glu Glu Lys Ala Val Ala Leu Leu Gly Ser Leu 65 70 75 80 Leu Asn Asn Lys Asp Ala Tyr Gly His Ile Val Ser Gly Gly Thr Glu 85 90 95 Ala Asn Leu Met Ala Leu Arg Cys Ile Lys Asn Ile Trp Arg Glu Lys 100 105 110 Arg Arg Lys Gly Leu Ser Lys Asn Glu His Pro Lys Ile Ile Val Pro 115 120 125 Ile Thr Ala His Phe Ser Phe Glu Lys Gly Arg Glu Met Met Asp Leu 130 135 140 Glu Tyr Ile Tyr Ala Pro Ile Lys Glu Asp Tyr Thr Ile Asp Glu Lys 145 150 155 160 Phe Val Lys Asp Ala Val Glu Asp Tyr Asp Val Asp Gly Ile Ile Gly 165 170 175 Ile Ala Gly Thr Thr Glu Leu Gly Thr Ile Asp Asn Ile Glu Glu Leu 180 185 190 Ser Lys Ile Ala Lys Glu Asn Asn Ile Tyr Ile His Val Asp Ala Ala 195 200 205 Phe Gly Gly Leu Val Ile Pro Phe Leu Asp Asp Lys Tyr Lys Lys Lys 210 215 220 Gly Val Asn Tyr Lys Phe Asp Phe Ser Leu Gly Val Asp Ser Ile Thr 225 230 235 240 Ile Asp Pro His Lys Met Gly His Cys Pro Ile Pro Ser Gly Gly Ile 245 250 255 Leu Phe Lys Asp Ile Gly Tyr Lys Arg Tyr Leu Asp Val Asp Ala Pro 260 265 270 Tyr Leu Thr Glu Thr Arg Gln Ala Thr Ile Leu Gly Thr Arg Val Gly 275 280 285 Phe Gly Gly Ala Cys Thr Tyr Ala Val Leu Arg Tyr Leu Gly Arg Glu 290 295 300 Gly Gln Arg Lys Ile Val Asn Glu Cys Met Glu Asn Thr Leu Tyr Leu 305 310 315 320 Tyr Lys Lys Leu Lys Glu Asn Asn Phe Lys Pro Val Ile Glu Pro Ile 325 330 335 Leu Asn Ile Val Ala Ile Glu Asp Glu Asp Tyr Lys Glu Val Cys Lys 340 345 350 Lys Leu Arg Asp Arg Gly Ile Tyr Val Ser Val Cys Asn Cys Val Lys 355 360 365 Ala Leu Arg Ile Val Val Met Pro His Ile Lys Arg Glu His Ile Asp 370 375 380 Asn Phe Ile Glu Ile Leu Asn Ser Ile Lys Arg Asp 385 390 395

Claims (4)

(a) contacting an aromatic amino acid, an amino acid dicarboxylase and a cation exchange resin in a reaction solution;
(b) removing carbon dioxide generated in the reaction solution; and
(c) recovering the biogenic amine from the cation exchange resin; A method for biologically producing a biogenic amine comprising:
The amino acid dicarboxylase is an amino acid dicarboxylase derived from Bacillus atrophaeus or Methanococcus jannaschii ,
When the amino acid dicarboxylase is an amino acid dicarboxylase derived from Bacillus atrophaeus , the aromatic amino acid is at least one amino acid selected from the group consisting of phenylalanine, tryptophan and tyrosine, and at least one aromatic amino acid selected from the group consisting of derivatives thereof,
When the amino acid dicarboxylase is an amino acid dicarboxylase derived from Methanococcus jannaschii , the aromatic amino acid is at least one amino acid selected from the group consisting of phenylalanine, tyrosine and asphaltic acid, and at least one aromatic amino acid selected from the group consisting of derivatives thereof In, way.
According to claim 1,
When the amino acid dicarboxylase is an amino acid dicarboxylase derived from Bacillus atrophaeus , the aromatic amino acids are phenylalanine, tryptophan, tyrosine, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan, homophenylalanine, m-fluoro At least one aromatic amino acid selected from the group consisting of lophenylalanine, p-fluorophenylalanine, p-chlorophenylalanine, p-bromophenylalanine and p-nitrophenylalanine,
When the amino acid dicarboxylase is an amino acid dicarboxylase derived from Methanococcus jannaschii , the aromatic amino acid is at least one aromatic amino acid selected from the group consisting of phenylalanine, tyrosine, asphaltic acid and 3,4-dihydroxyphenylalanine (DOPA). , Way.
According to claim 1,
The reaction solution is present in an open reactor, and the step (c) is a method for biologically producing a biogenic amine, characterized in that the carbon dioxide is removed by vaporizing.
According to claim 1,
The cation exchange resin is a method for biologically producing a biogenic amine, characterized in that the sulfone group ion exchange group consisting of a polystyrene polymer.

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