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WO2021118137A1 - Method for enzymatically producing beta amino acid using novel ester hydrolase - Google Patents

Method for enzymatically producing beta amino acid using novel ester hydrolase Download PDF

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Publication number
WO2021118137A1
WO2021118137A1 PCT/KR2020/017233 KR2020017233W WO2021118137A1 WO 2021118137 A1 WO2021118137 A1 WO 2021118137A1 KR 2020017233 W KR2020017233 W KR 2020017233W WO 2021118137 A1 WO2021118137 A1 WO 2021118137A1
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beta
trifluorophenyl
formula
pseudomonas
lip
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French (fr)
Korean (ko)
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윤형돈
최인석
전현우
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CKD Bio Corp
University Industry Cooperation Corporation of Konkuk University
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CKD Bio Corp
University Industry Cooperation Corporation of Konkuk University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)

Definitions

  • the present invention relates to an enzymatic production method of beta amino acids using a novel ester hydrolase, and more particularly, to a novel ester hydrolase (lipase) in synthesizing beta-keto acid from beta-keto ester during the synthesis of beta amino acid.
  • a novel ester hydrolase lipase
  • / esterase relates to a method that can increase the productivity of beta amino acids by applying.
  • beta amino acids are recognized as important pharmacological compounds due to their unique pharmacological activity, and have been used importantly in the research of various peptide drugs because of their resistance to proteolytic enzymes under physiological conditions.
  • ester hydrolases such as lipase are essential enzymes for enzymatically synthesizing beta amino acids. While the previously reported lipase (see Patent Application No. 10-2014-0098351) is commercially available, it has a problem in that expression in E. coli is not easy and a cost problem because it is a lipase derived from a fungus, and also has a low activity of beta There was a problem in the synthesis process of amino acids.
  • the present invention provides a method for producing beta amino acids using a novel ester hydrolase (lipase / esterase).
  • the present invention also provides a composition for the production of beta amino acids from beta-keto esters comprising an ester hydrolase, a transaminase and an amino group donor derived from Pseudomonas sp. strain.
  • the present invention is ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate [ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate] Pseudomonas genus
  • 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid]
  • the 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] was used as a transaminase and an amino group donor
  • a method for producing (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid is provided.
  • the present invention is from ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate, including an ester hydrolase, a transaminase and an amino group donor derived from Pseudomonas sp. strain
  • ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate including an ester hydrolase, a transaminase and an amino group donor derived from Pseudomonas sp. strain
  • (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid is provided.
  • the present invention reacts a beta-keto ester represented by the following formula (1 ) with an ester hydrolase derived from a Pseudomonas sp . strain to synthesize a beta-keto acid represented by the following formula (2), and then converts the beta-keto acid to a transaminase And by reacting with an amino group donor, it provides a method for producing a beta amino acid represented by the following formula (3).
  • R is , , , , , and selected from the group consisting of
  • the Pseudomonas sp. strain may include one or more of Pseudomonas avellanae (Pseudomonas avellanae) or Pseudomonas stutzeri (Pseudomonas stutzeri).
  • the hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the transaminase may be an omega transaminase.
  • the reaction of the beta-keto ester with the ester hydrolase may be performed at 20 to 45° C. for 20 to 180 minutes.
  • the reaction of the beta-keto acid with the transaminase and the amino group donor may be performed at 20 to 45° C. for 12 to 60 hours.
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • ethanol in the reaction of the beta keto ester with an ester hydrolase and/or the beta keto acid with a transaminase and an amino group donor may further include any one organic solvent selected from the group consisting of methanol and acetone.
  • the amino group donor is L-alanine, beta-alanine, 3-aminopropionic acid (3-aminopropionic acid; 3-APA), isopropyl amine (iPrNH2), benzylamine (benzylamine; BnNH2) and (S) -Alpha methylbenzylamine ( ⁇ -methylbenzylamine; MBA) may include one or more selected from the group consisting of.
  • the concentration ratio of the ester hydrolase and the transaminase may be 1: 1 to 12.
  • the ester hydrolase and transaminase may be expressed in E. coli. More preferably, it may be co-expressed in the same E. coli.
  • the present invention also provides a composition for the production of beta amino acids represented by Formula 3 from the beta-keto ester represented by Formula 1, comprising an ester hydrolase, a transaminase, and an amino group donor derived from Pseudomonas sp. strain can provide
  • the hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate [ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate] Pseudomonas ) by reaction with an ester hydrolase derived from the genus strain, 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid ], and then 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] was synthesized by transaminase and A method for producing (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid by reaction with an amino group donor is provided.
  • the hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid may be a precursor of sitagliptin.
  • ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate containing an ester hydrolase, a transaminase and an amino group donor derived from Pseudomonas sp. strain
  • a composition for the production of (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid from The hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • beta amino acids in the enzymatic production process of beta-amino acids, by applying a novel ester hydrolase (lipase / esterase) that is more active than the conventional commercial lipase (Candida rugose), beta amino acids can be produced in high yield.
  • a novel ester hydrolase lipase / esterase
  • Candida rugose conventional commercial lipase
  • 1 shows a candidate group of 42 ester hydrolases (lipase/esterase) selected by relatively comparing the lengths of sequences among candidates selected through BLAST.
  • Figure 2 shows selected E. coli- derived ester hydrolase (lipase / esterase) and Pseudomonas ( Pseudomonas ) It is an SDS-PAGE picture of the derived ester hydrolase (lipase/esterase).
  • 3 is a graph showing the results of measuring the activity of the selected 8 lipase / esterase.
  • 5 is a graph analyzing the amount of decrease in activity according to the optimum temperature and time of Lip PA and Lip PS.
  • 6 is a graph analyzing the difference in the activity of Lip PA and Lip PS according to the concentration of DMSO.
  • Lip PA Pseudomonas avellanae
  • Lip PS pseudomonas stutzeri
  • Lipase commercial lipase derived from candida rugose
  • FIG. 8 is a graph analyzing the production rate of beta-amino acids through the chain reaction of each expressed novel ester hydrolase (lipase/esterase) and transaminase OD 100.
  • FIG. Reaction conditions ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (100 mM), omega-transaminase (100 OD), lipase/esterase (10-90 OD), DMSO (15%), ( S )- ⁇ -MBA (200 mM), PLP (Pyridoxal 5′-phosphate) (0.5 mM), buffer (200 mM Tris-HCl pH 7.0), temperature (37 °C), reaction hours (48 hours).
  • FIG. 9 is a graph analyzing the production rate of beta-amino acids through the chain reaction of the expressed novel lipase/esterase and transaminase OD 120, respectively.
  • Reaction conditions ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (100 mM), omega-transaminase (120 OD), lipase/esterase (10-90 OD), DMSO (15%), (S)- ⁇ -MBA (200 mM), PLP (0.5 mM), buffer (200 mM Tris-HCl pH 7.0), temperature (37° C.), reaction time (48 hours).
  • FIG. 10 is a graph analyzing the production rate of beta-amino acids produced using a system in which pET24ma omega-trans aminase (TAIC) and pQE-80L Lip PA/Lip PS were co-expressed in a single cell (E. coli BL21). .
  • TAIC pET24ma omega-trans aminase
  • pQE-80L Lip PA/Lip PS were co-expressed in a single cell (E. coli BL21).
  • FIG. 11 is a graph analyzing the production rate of beta-amino acids produced using a system in which pCDF-Duet omega-transaminase (TAIC) and pQE-80L Lip PA/Lip PS were co-expressed in a single cell (E. coli). to be.
  • TAIC pCDF-Duet omega-transaminase
  • pQE-80L Lip PA/Lip PS were co-expressed in a single cell (E. coli).
  • the lipase used in the prior patent (application number 10-2014-0098351) of the present invention is commercially available, and has low activity, so there is a restriction that a large amount of enzyme is required for the synthesis of beta amino acids.
  • the commercial lipase is a fungal-derived enzyme and has a problem in that it is not easy to express in E. coli.
  • a novel lipase with higher activity than the existing lipase was screened, and using this, the omega-trans amenase and lipase in E. coli were expressed by replacing the existing lipase, thereby producing a sitagliptin precursor and various beta-amino acids. was improved.
  • Scheme 1 A reaction scheme for producing beta amino acids using lipase and transaminase from beta-keto ester as a starting material.
  • the industrial Candida rugosa lipase (Sigma-Aldrich catalog number L1754) used in the previous study hydrolyzed beta-ketoester to beta-keto acid followed by amination using omega-transaminase to produce 3-ATfBA (3-amino-4 -(2,4,5-trifluorophenyl)butanoic acid) was successfully used to form various beta-amino acids.
  • omega-transaminase chain reaction and a study for identification of an ester hydrolase (lipase / esterase) more suitable than the existing ester hydrolase (lipase / esterase) was conducted. It was confirmed that the lipase protein BioH possessed by E. coli can hydrolyze beta-ketoester, and among the 42 lipases possessed by E.
  • FIG. 1 the amino acid sequence of Candida rugosa lipase was searched using BLAST (Basic Local Alignment Search Tool) based on this standard. was performed (FIG. 1). Among them, six types of E. coli-derived ester hydrolases (lipases/esterases) predicted to show activity based on phylogenetic analysis were selected ( FIG. 1 ).
  • Pseudomonas- derived ester hydrolase lipase / esterase
  • E. coli- derived six genes primer (Sequences (5' ⁇ 3'), ent ry 1: F1 primer - attaggatccatgaccaatataccg (SEQ ID NO: 3) R1p rimer - gccggagctcttatttctgcttcgt (SEQ ID NO: 4), entry 2: F2 primer - atta ggatccatgaaacatgaccat (SEQ ID NO: 5) R2 primer - gccggagctc ttacatcatttcaat (SEQ ID NO: 6), entry 3: F3 primer - gccgggatcc atgatgaataataaa (SEQ ID NO: 7) R3 primer - cggcgagctttacagagttgaatctcatcacagagtttcctc (SEQ ID NO: 8), F3 primer - cggcgagcttt
  • Beta-keto ester using a reactive substrate [ethyl ester (ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate)] and DMSO at a pH of 7.0 using a purified enzyme was tested for activity ( FIG. 3 ).
  • Reaction conditions were as follows: ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (10 mM), lipase/esterase (0.1 mg/mL), DMSO (15%), buffer solution (200 mM Tris-HCl pH 7.0), temperature (37° C.), reaction time (30 min).
  • Lip PA and Lip PS derived from Pseudomonas showed high activity of 6.2U/mg and 10.3U/mg, respectively.
  • 6 types of lipases derived from E. coli it was confirmed that they were formed with a large difference and low with an activity of less than 1U/mg. Accordingly, additional experiments were carried out by selecting Lip PA and Lip PS, which are highly reactive to beta-keto esters used as substrates.
  • Lip PA and Lip PS showed activity in the range of pH 6 - 9, and the reaction was carried out in 50 mM Phosphate buffer (pH 6.0-7.0) containing ⁇ -nitrophenyl acetate, 50 mM Tris-HCl buffer (pH 7.0) -9.0). In this reaction, ⁇ -nitrophenyl acetate was converted to ⁇ -nitrophenol using Lip PA and Lip PS, and a yellow substance was confirmed as a result of observation at a wavelength of 405 nm using a UV-spectrophotometer (Scheme 2).
  • Lip PA SEQ ID NO: 1 from Pseudomonas avellanae exhibited the maximum activity at 14.5 U/mg at pH 8.5, and Lip PS from Pseudomonas stutzeri at pH 8.0 at 55 U/mg. mg showed optimal activity (FIG. 4). Even at pH 7.0, which is the reaction condition of omega-trans aminase, it was confirmed that the reactivity was high at about 2U/mg for Lip PA and 20U/mg for Lip PS. 5 shows the results of analyzing the amount of decrease in activity according to the optimum temperature and time of Lip PA and Lip PS. The reaction of FIG. 5 was performed in a similar manner to the reaction at pH 7.0, incubated and reacted at 30-60° C.
  • Lip PA and Lip PS were measured under the same reaction conditions as above and different concentrations of DMSO (5%, 10%, 15%, 20%, 25%, 30%). It was observed that the enzymatic activity of Lip PA and PS decreased when the concentration of DMSO was increased from 5% to 30%.
  • lipase was confirmed by the following method: Lip PA, PS and commercial lipase Candida rugosa Confirmation of the specific activity of ( candida rugosa ) was carried out in a 0.5 ml reaction volume of (ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate, a substrate, and the commercial lipase Candida rugosa ( candida rugosa ) 1mg/ml, the novel lipase Lip PA and Lip PS were reacted in 200mM Tris-HCl buffer (pH 7.0) using 0.5 OD at 37°C and 180rpm for 20 minutes, and after reaction 1:1 (v/v) ) was added to stop the reaction by adding 10% perchloric acid.
  • Tris-HCl buffer pH 7.0
  • E. After cloning into the pET24ma vector, E. It was introduced into coli BL21. Transformants of E. coli BL21 were grown in 1 L of LB medium containing 50 mg/L kanamycin at 37° C. When the OD600 of the transformants reached 0.4 to 0.6, 0.5 The expression of w-TA was induced by the addition of mM IPTG (Isopropyl- ⁇ -D-thiogalactopyranoside) (Carbosynth) After 12 hours, the cells were harvested and once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction.
  • IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
  • Beta-amino acid synthesis was performed in a single cell system by co-expressing both vectors in a single cell after optimizing the ratio using the two cell system.
  • Omega-trans aminase (TAIC) was cloned in pET24ma and Lip PA and Lip PS were cloned in pQE-80L, and they were co-expressed in single cells.
  • omega-trans aminase (TAIC) was cloned into the pET24ma vector and then introduced into E. coli BL21. Transformants of E. coli BL21 were grown in 1 L of LB medium containing 50 mg/L of kanamycin at 37°C.
  • omega-trans aminase was cloned from the pCDF-Duet vector, whereas Lip PA and Lip PS were cloned from the pQE-80L vector, and they were co-expressed in a single cell to perform the above reaction.
  • the omega-trans aminase was cloned into the pCDF-Duet vector and the clone ester hydrolases Lip PA and Lip PS were cloned into the pQE80L vector. These two vectors were simultaneously introduced into E. coli BL21. Transformants of E.
  • coli BL21 were grown in 1 L of LB medium containing 25 mg/L of chloramphenical and 100 mg/L of ampicillin at 37°C.
  • OD600 of the transformant reached 0.4 to 0.6
  • 0.5 mM Isopropyl- ⁇ -D-thiogalactopyranoside was added to induce co-expression of w-TA and ester gas lyase.
  • the cells were harvested and washed once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction.
  • ester hydrolases Lip PA and Lip PS were cloned into the pQE80L vector, and then introduced into E. coli BL21.
  • Transformants of E. coli BL21 were grown in 1 L of LB medium containing 100 mg/L of ampicillin at 37°C. When the OD600 of the transformant reached 0.4 to 0.6, 0.5 mM Isopropyl- ⁇ -D-thiogalactopyranoside (Carbosynth) was added to induce expression of the ester hydrolase. After 12 hours, the cells were harvested and washed once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction.
  • phosphate buffer pH 8.0

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Abstract

The present invention relates to a method for enzymatically producing beta amino acids by using a novel ester hydrolase, and more specifically, to a method whereby the productivity of a beta-amino acid can be increased by using a novel ester hydrolase (lipase/esterase) when synthesizing a beta-keto acid from a beta-keto ester during the process of synthesizing the beta-amino acid.

Description

신규 에스터 가수분해효소를 이용한 베타 아미노산의 효소적 생산방법Enzymatic production method of beta amino acids using novel ester hydrolase

본 출원은 2019년 12월 10일 출원된 대한민국 특허출원 제10-2019-0164157호를 우선권으로 주장하고, 상기 명세서 전체는 본 출원의 참고문헌이다. This application claims priority to Republic of Korea Patent Application No. 10-2019-0164157 filed on December 10, 2019, and the entire specification is a reference to the present application.

본 발명은 신규 에스터 가수분해효소를 이용한 베타 아미노산의 효소적 생산방법에 관한 것으로, 더욱 구체적으로 베타 아미노산의 합성 과정 중 베타-케토 에스테르로부터 베타-케토산을 합성하는데 있어서 신규 에스터 가수분해효소 (리파아제/에스테라아제)를 적용함으로써 베타 아미노산의 생산성을 증대시킬 수 있는 방법에 관한 것이다. The present invention relates to an enzymatic production method of beta amino acids using a novel ester hydrolase, and more particularly, to a novel ester hydrolase (lipase) in synthesizing beta-keto acid from beta-keto ester during the synthesis of beta amino acid. / esterase) relates to a method that can increase the productivity of beta amino acids by applying.

소분자 제약, 농약 및 기타 산업에 있어서 중요한 화합물의 제조를 위해 필수 구성요소인 베타 아미노산의 중요성이 부각되고 있다. 특히 베타 아미노산은 독특한 약리 활성으로 인하여 약리화합물로 중요하게 인식되어 있으며, 생리조건에서 단백질 분해 효소 대하여 저항성이 있어 각종 펩티드 신약 연구에도 중요하게 사용되어져 왔다.The importance of beta amino acids as essential components for the preparation of important compounds in small molecule pharmaceuticals, pesticides and other industries is emerging. In particular, beta amino acids are recognized as important pharmacological compounds due to their unique pharmacological activity, and have been used importantly in the research of various peptide drugs because of their resistance to proteolytic enzymes under physiological conditions.

한편, 리파아제와 같은 에스터 가수분해효소는 베타 아미노산을 효소 공학적으로 합성하는 과정에 필수적인 효소이다. 기존 보고된 리파아제(특허출원 제10-2014-0098351호 참조)는 상업적으로 이용 가능한 반면 곰팡이 유래의 리파아제이기 때문에 대장균에서의 발현이 용이하지 않다는 문제점과 비용적인 문제가 있었고, 또한 낮은 활성도로 인해 베타 아미노산의 합성 공정상 문제가 있었다.On the other hand, ester hydrolases such as lipase are essential enzymes for enzymatically synthesizing beta amino acids. While the previously reported lipase (see Patent Application No. 10-2014-0098351) is commercially available, it has a problem in that expression in E. coli is not easy and a cost problem because it is a lipase derived from a fungus, and also has a low activity of beta There was a problem in the synthesis process of amino acids.

본 발명에서는 상기의 문제점을 해결하기 위하여, 종래의 상업적 리파아제(Candida rugose)보다 활성이 뛰어난 신규 에스터 가수분해효소 (리파아제/에스테라아제)를 적용함으로써 베타 아미노산의 생산성을 증대시킬 수 있는 방법을 제공하고자 한다.In the present invention, in order to solve the above problems, a method capable of increasing the productivity of beta amino acids by applying a novel ester hydrolase (lipase / esterase) that is more active than the conventional commercial lipase (Candida rugose) It is intended to provide a method .

선행기술문헌Prior art literature

대한민국 등록특허 제10-1565439호Republic of Korea Patent No. 10-1565439

상기와 같은 문제점을 해결하기 위하여, 본 발명은 신규의 에스터 가수분해효소 (리파아제/에스테라아제)를 이용한 베타 아미노산의 생산 방법을 제공한다.In order to solve the above problems, the present invention provides a method for producing beta amino acids using a novel ester hydrolase (lipase / esterase).

또한 본 발명은 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소, 트랜스 아미나제 및 아미노기 공여체를 포함하는 베타 케토 에스터로부터 베타 아미노산의 생산을 위한 조성물을 제공한다.The present invention also provides a composition for the production of beta amino acids from beta-keto esters comprising an ester hydrolase, a transaminase and an amino group donor derived from Pseudomonas sp. strain.

또한 본 발명은 에틸 4-(2,4,5-트리플루오로페닐)-3-옥소부타노에이트[ethyl 4-(2,4,5-trifluorophenyl)-3-oxobutanoate]를 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소와 반응시켜, 4-(2,4,5-트리플루오로페닐)-3-옥소부탄산[4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid]을 합성한 후, 상기 4-(2,4,5-트리플루오로페닐)-3-옥소부탄산[4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] 을 트랜스 아미나아제 및 아미노기 공여체와 반응시켜, (R)-3-아미노-4-(2,4,5-트리플루오로페닐)부탄산을 생산하는 방법을 제공한다.In addition, the present invention is ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate [ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate] Pseudomonas genus By reacting with an ester hydrolase derived from the strain, 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] After synthesis, the 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] was used as a transaminase and an amino group donor A method for producing (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid is provided.

또한 본 발명은 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소, 트랜스 아미나아제 및 아미노기 공여체를 포함하는, 에틸 4-(2,4,5-트리플루오로페닐)-3-옥소부타노에이트로부터 (R)-3-아미노-4-(2,4,5-트리플루오로페닐)부탄산의 생산을 위한 조성물을 제공한다.In addition, the present invention is from ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate, including an ester hydrolase, a transaminase and an amino group donor derived from Pseudomonas sp. strain Provided is a composition for the production of (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid.

그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

본 발명은 하기 화학식 1로 표시되는 베타 케토 에스터를 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소와 반응시켜 하기 화학식 2로 표시되는 베타 케토산을 합성한 후, 상기 베타 케토산을 트랜스 아미나제 및 아미노기 공여체와 반응시켜, 하기 화학식 3으로 표시되는 베타 아미노산을 생산하는 방법을 제공한다.The present invention reacts a beta-keto ester represented by the following formula (1 ) with an ester hydrolase derived from a Pseudomonas sp . strain to synthesize a beta-keto acid represented by the following formula (2), and then converts the beta-keto acid to a transaminase And by reacting with an amino group donor, it provides a method for producing a beta amino acid represented by the following formula (3).

[화학식 1][Formula 1]

Figure PCTKR2020017233-appb-I000001
Figure PCTKR2020017233-appb-I000001

[화학식 2][Formula 2]

Figure PCTKR2020017233-appb-I000002
Figure PCTKR2020017233-appb-I000002

[화학식 3][Formula 3]

Figure PCTKR2020017233-appb-I000003
Figure PCTKR2020017233-appb-I000003

상기 화학식 1, 화학식 2, 화학식 3에서 R은

Figure PCTKR2020017233-appb-I000004
,
Figure PCTKR2020017233-appb-I000005
,
Figure PCTKR2020017233-appb-I000006
,
Figure PCTKR2020017233-appb-I000007
,
Figure PCTKR2020017233-appb-I000008
,
Figure PCTKR2020017233-appb-I000009
,
Figure PCTKR2020017233-appb-I000010
및
Figure PCTKR2020017233-appb-I000011
로 이루어진 군에서 선택됨.In Formula 1, Formula 2, and Formula 3, R is
Figure PCTKR2020017233-appb-I000004
,
Figure PCTKR2020017233-appb-I000005
,
Figure PCTKR2020017233-appb-I000006
,
Figure PCTKR2020017233-appb-I000007
,
Figure PCTKR2020017233-appb-I000008
,
Figure PCTKR2020017233-appb-I000009
,
Figure PCTKR2020017233-appb-I000010
and
Figure PCTKR2020017233-appb-I000011
selected from the group consisting of

상기 슈도모나스 속 균주는 슈도모나스 아벨라나에(Pseudomonas avellanae) 또는 슈도모나스 스투트제리(Pseudomonas stutzeri) 둘 중 하나 이상을 포함할 수 있다. The Pseudomonas sp. strain may include one or more of Pseudomonas avellanae (Pseudomonas avellanae) or Pseudomonas stutzeri (Pseudomonas stutzeri).

상기 가수분해효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성될 수 있다. The hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

상기 트랜스 아미나제는 오메가 트랜스 아미나제일 수 있다. The transaminase may be an omega transaminase.

상기 베타 케토 에스터와 에스터 가수분해효소의 반응은 20 ~ 45 ℃에서 20 ~ 180 분 동안 수행될 수 있다.The reaction of the beta-keto ester with the ester hydrolase may be performed at 20 to 45° C. for 20 to 180 minutes.

상기 베타 케토산과, 트랜스 아미나제 및 아미노기 공여체와의 반응은 20 ~ 45 ℃에서 12 ~ 60시간 동안 수행될 수 있다. The reaction of the beta-keto acid with the transaminase and the amino group donor may be performed at 20 to 45° C. for 12 to 60 hours.

상기 베타 케토 에스터와 에스터 가수분해효소의 반응 및/또는 상기 베타 케토산과, 트랜스 아미나제 및 아미노기 공여체와의 반응에서 디메틸설폭시드(dimethyl sulfoxide, DMSO), 테트라하이드로퓨란(tetrahydrofuran, THF), 에탄올, 메탄올 및 아세톤로 구성된 군으로부터 선택된 어느 하나의 유기용매를 더 포함할 수 있다.dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), ethanol in the reaction of the beta keto ester with an ester hydrolase and/or the beta keto acid with a transaminase and an amino group donor , may further include any one organic solvent selected from the group consisting of methanol and acetone.

상기 아미노기 공여체는 L-알라닌, 베타-알라닌, 3-아미노프로피오닉산(3-aminopropionic acid;3-APA), 아이소프로필아민(isopropyl amine; iPrNH2), 벤질아민(benzylamine; BnNH2) 및 (S)-알파메틸벤질아민(α-methylbenzylamine; MBA)으로 구성된 군으로부터 선택되는 하나 이상을 포함할 수 있다. The amino group donor is L-alanine, beta-alanine, 3-aminopropionic acid (3-aminopropionic acid; 3-APA), isopropyl amine (iPrNH2), benzylamine (benzylamine; BnNH2) and (S) -Alpha methylbenzylamine (α-methylbenzylamine; MBA) may include one or more selected from the group consisting of.

상기 에스터 가수분해효소와 트랜스 아미나제의 농도비는 1: 1 ~ 12일 수 있다.The concentration ratio of the ester hydrolase and the transaminase may be 1: 1 to 12.

상기 에스터 가수분해효소와 트랜스 아미나제는 대장균에서 발현된 것일 수 있다. 더욱 바람직하게는 동일한 대장균에서 공동 발현된 것일 수 있다.The ester hydrolase and transaminase may be expressed in E. coli. More preferably, it may be co-expressed in the same E. coli.

또한 본 발명은 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소, 트랜스 아미나제 및 아미노기 공여체를 포함하는, 상기 화학식 1로 표시되는 베타 케토 에스터로부터 상기 화학식 3으로 표시되는 베타 아미노산의 생산을 위한 조성물을 제공할 수 있다. 상기 가수분해효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성될 수 있다. The present invention also provides a composition for the production of beta amino acids represented by Formula 3 from the beta-keto ester represented by Formula 1, comprising an ester hydrolase, a transaminase, and an amino group donor derived from Pseudomonas sp. strain can provide The hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

또한 본 발명은, 에틸 4-(2,4,5-트리플루오로페닐)-3-옥소부타노에이 트[ethyl 4-(2,4,5-trifluorophenyl)-3-oxobutanoate]를 슈도모나스(Pseudomonas) 속 균주 유래의 에스터 가수분해효소와 반응시켜, 4-(2,4,5-트리플루오로페닐)-3- 옥소부탄산[4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid]을 합성한 후, 상기 4-(2,4,5-트리플루오로페닐)-3-옥소부탄산[4-(2,4,5-trifluorophenyl)-3- oxobutanoic acid]을 트랜스 아미나제 및 아미노기 공여체와 반응시켜, (R)-3-아미노-4-(2,4,5-트리플루오로페닐)부탄산을 생산하는 방법을 제공한다. 상기 가수분해 효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성될 수 있다. 상기 (R)-3-아미노-4-(2,4,5-트리플루오로페닐)부탄산은 시타글립틴의 전구체일 수 있다. In addition, the present invention, ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate [ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate] Pseudomonas ) by reaction with an ester hydrolase derived from the genus strain, 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid ], and then 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] was synthesized by transaminase and A method for producing (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid by reaction with an amino group donor is provided. The hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid may be a precursor of sitagliptin.

또한 본 발명은, 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소, 트랜스 아미나제 및 아미노기 공여체를 포함하는, 에틸 4-(2,4,5-트리플루오로페닐)-3-옥소부타노에이트로부터 (R)-3-아미노-4-(2,4,5-트리플루오로페닐)부탄산의 생산을 위한 조성물을 제공한다. 상기 가수분해효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성될 수 있다. In addition, the present invention, ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate containing an ester hydrolase, a transaminase and an amino group donor derived from Pseudomonas sp. strain Provided is a composition for the production of (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid from The hydrolase may be composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

본 발명에 따르면 베타-아미노산의 효소적 생산 과정에 있어서, 종래의 상업적 리파아제(Candida rugose)보다 활성이 뛰어난 신규의 에스터 가수분해효소 (리파아제/에스테라아제)를 적용함으로써, 높은 수율로 베타 아미노산을 생산할 수 있다.According to the present invention, in the enzymatic production process of beta-amino acids, by applying a novel ester hydrolase (lipase / esterase) that is more active than the conventional commercial lipase (Candida rugose), beta amino acids can be produced in high yield. have.

도 1은 BLAST를 통해 선별된 후보 중 상대적으로 서열의 길이를 비교하여 선별된 42개의 에스터 가수분해효소 (리파아제/에스테라아제)의 후보군을 나타내는 것이다.1 shows a candidate group of 42 ester hydrolases (lipase/esterase) selected by relatively comparing the lengths of sequences among candidates selected through BLAST.

도 2는 선별된 E.Coli 유래의 에스터 가수분해효소 (리파아제/에스테라아제) 및 슈도모나스 (Pseudomonas) 유래의 에스터 가수분해효소 (리파아제/에스테라아제)의 SDS-PAGE 사진이다.Figure 2 shows selected E. coli- derived ester hydrolase (lipase / esterase) and Pseudomonas ( Pseudomonas ) It is an SDS-PAGE picture of the derived ester hydrolase (lipase/esterase).

도 3은 선별된 8개의 리파아제/에스테라아제의 활성측정 결과를 나타내는 그래프이다.3 is a graph showing the results of measuring the activity of the selected 8 lipase / esterase.

도 4는 Lip PA (서열번호 1) 와 Lip PS (서열번호 2)의 최적 pH를 나타내는 그래프이다. 4 is a graph showing the optimum pH of Lip PA (SEQ ID NO: 1) and Lip PS (SEQ ID NO: 2).

도 5는 Lip PA 와 Lip PS의 최적온도와 시간에 따른 활성감소량을 분석한 그래프이다.5 is a graph analyzing the amount of decrease in activity according to the optimum temperature and time of Lip PA and Lip PS.

도 6은 DMSO의 농도에 따른 Lip PA 와 Lip PS의 활성 차이를 분석한 그래프이다.6 is a graph analyzing the difference in the activity of Lip PA and Lip PS according to the concentration of DMSO.

도 7은 Pseudomonas avellanae 유래의 리파아제(Lip PA)와 pseudomonas stutzeri 유래의 리파아제(Lip PS), candida rugose 유래의 상업적 리파아제(Lipase)의 특이적 활성을 비교한 결과를 나타내는 그래프이다.7 is a graph showing the results of comparing the specific activity of a lipase derived from Pseudomonas avellanae (Lip PA), a lipase derived from pseudomonas stutzeri (Lip PS), and a commercial lipase derived from candida rugose (Lipase).

도 8은 각각 발현된 신규 에스터 가수 분해효소 (리파아제/에스테라아제) 및 트랜스 아미나제 OD 100의 연쇄반응을 통한 베타-아미노산의 생성률을 분석한 그래프이다. 반응조건: 에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트(100 mM), 오메가-트랜스 아미나제(100 OD), 리파아제/에스테라아제(10-90 OD), DMSO(15%), (S)-α-MBA (200 mM), PLP (Pyridoxal 5′-phosphate) (0.5 mM), 완충용액 (200 mM Tris-HCl pH 7.0), 온도 (37 ℃), 반응시간 (48 시간).8 is a graph analyzing the production rate of beta-amino acids through the chain reaction of each expressed novel ester hydrolase (lipase/esterase) and transaminase OD 100. FIG. Reaction conditions: ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (100 mM), omega-transaminase (100 OD), lipase/esterase (10-90 OD), DMSO (15%), ( S )-α-MBA (200 mM), PLP (Pyridoxal 5′-phosphate) (0.5 mM), buffer (200 mM Tris-HCl pH 7.0), temperature (37 °C), reaction hours (48 hours).

도 9는 각각 발현된 신규 리파아제/에스테라아제 및 트랜스 아미나제 OD 120의 연쇄반응을 통한 베타-아미노산의 생성률을 분석한 그래프이다. 반응조건: 에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트(100 mM), 오메가-트랜스 아미나제(120 OD), 리파아제/에스테라아제(10-90 OD), DMSO(15%), (S)-α-MBA(200 mM), PLP(0.5 mM), 완충용액(200 mM Tris-HCl pH 7.0), 온도 (37 ℃), 반응시간 (48 시간).9 is a graph analyzing the production rate of beta-amino acids through the chain reaction of the expressed novel lipase/esterase and transaminase OD 120, respectively. Reaction conditions: ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (100 mM), omega-transaminase (120 OD), lipase/esterase (10-90 OD), DMSO (15%), (S)-α-MBA (200 mM), PLP (0.5 mM), buffer (200 mM Tris-HCl pH 7.0), temperature (37° C.), reaction time (48 hours).

도 10은 pET24ma의 오메가-트랜스 아미나제(TAIC)와 pQE-80L의 Lip PA/Lip PS를 단일 세포(대장균 BL21)에서 공동 발현시킨 시스템을 이용하여 생산한 베타-아미노산의 생성률을 분석한 그래프이다. 반응조건: 에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트(100 mM), 공동 발현된 오메가-트렌스 아미나제/리파아제(30-150 OD) DMSO(15%), (S)-α-MBA(200 mM), PLP(0.5 mM), 완충용액(200 mM Tris-HCl pH 7.0), 온도 (37 ℃), 반응시간 (48 시간).10 is a graph analyzing the production rate of beta-amino acids produced using a system in which pET24ma omega-trans aminase (TAIC) and pQE-80L Lip PA/Lip PS were co-expressed in a single cell (E. coli BL21). . Reaction conditions: ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (100 mM), co-expressed omega-transaminase/lipase (30-150 OD) DMSO (15%) ), (S)-α-MBA (200 mM), PLP (0.5 mM), buffer (200 mM Tris-HCl pH 7.0), temperature (37 °C), reaction time (48 hours).

도 11는 pCDF-Duet의 오메가-트랜스 아미나제(TAIC)와 pQE-80L의 Lip PA/Lip PS를 단일 세포(대장균)에 공동 발현시킨 시스템을 이용하여 생산한 베타-아미노산의 생성률을 분석한 그래프이다. 반응조건: 에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트(100 mM), 공동 발현된 오메가-트랜스 아미나제/리파아제(30-150 OD) DMSO(15%), (S)-α-MBA(200 mM), PLP(0.5 mM), 완충용액(200 mM Tris-HCl pH 7.0), 온도 (37 ℃), 반응시간 (48 시간).11 is a graph analyzing the production rate of beta-amino acids produced using a system in which pCDF-Duet omega-transaminase (TAIC) and pQE-80L Lip PA/Lip PS were co-expressed in a single cell (E. coli). to be. Reaction conditions: ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (100 mM), co-expressed omega-transaminase/lipase (30-150 OD) DMSO (15%) ), (S)-α-MBA (200 mM), PLP (0.5 mM), buffer (200 mM Tris-HCl pH 7.0), temperature (37 °C), reaction time (48 hours).

이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 본 발명의 목적, 특징, 장점은 이하의 실시예를 통하여 쉽게 이해될 것이다. 본 발명은 여기서 설명하는 실시예에 한정되지 않고, 다른 형태로 구체화될 수도 있다. 여기서 소개되는 실시예는 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 본 발명의 사상이 충분히 전달될 수 있도록 하기 위해 제공되는 것이다. 따라서 이하의 실시 예에 의해 본 발명이 제한되어서는 안 된다.Hereinafter, the present invention will be described in more detail through examples. Objects, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the embodiments described herein, and may be embodied in other forms. The embodiments introduced herein are provided so that the spirit of the present invention can be sufficiently conveyed to those of ordinary skill in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

본 발명의 선행특허 (출원번호 10-2014-0098351)에서 사용되었던 리파아제는 상업적으로 구입 가능한 것으로, 활성이 낮아서 베타 아미노산의 합성 시 많은 양의 효소가 필요한 제약이 있었다. 무엇보다도 상기 상업적 리파아제는 곰팡이 유래의 효소로 대장균에서 발현이 용이하지 않은 문제점이 있었다. 본 발명에서는 기존의 리파아제 보다 활성이 높은 신규 리파아제를 스크리닝하였고, 이를 이용하여 기존에 리파아제를 대체하여 대장균에서의 오메가-트렌스 아메나제와 리파아제를 발현시켜 시타글립틴 전구체 및 각종 베타-아미노산의 공정법을 개선하였다. The lipase used in the prior patent (application number 10-2014-0098351) of the present invention is commercially available, and has low activity, so there is a restriction that a large amount of enzyme is required for the synthesis of beta amino acids. Above all, the commercial lipase is a fungal-derived enzyme and has a problem in that it is not easy to express in E. coli. In the present invention, a novel lipase with higher activity than the existing lipase was screened, and using this, the omega-trans amenase and lipase in E. coli were expressed by replacing the existing lipase, thereby producing a sitagliptin precursor and various beta-amino acids. was improved.

본 발명의 일 실시예에 따르면, 하기와 같은 반응식에 따라 시타글립틴 전구체인 베타 아미노산을 효율적으로 합성할 수 있다. According to an embodiment of the present invention, it is possible to efficiently synthesize a beta amino acid, a sitagliptin precursor, according to the following reaction scheme.

[반응식 1] 출발물질인 베타-케토 에스테르로부터 리파아제 및 트랜스 아미나제를 이용하여 베타 아미노산을 생산하는 반응 스킴.[Scheme 1] A reaction scheme for producing beta amino acids using lipase and transaminase from beta-keto ester as a starting material.

Figure PCTKR2020017233-appb-I000012
Figure PCTKR2020017233-appb-I000012

베타 아미노산의 효소반응공정 과정에서 베타-케토 에스테르로부터 베타-케토산을 합성할 때, 기존에 알려진 상업적 리파아제 대신 본 발명에서 새롭게 스크리닝한 리파아제를 이용하는 경우 빠른 베타-케토산의 전환으로 높은 수준의 수득률을 얻을 수 있다. When synthesizing beta-keto acid from beta-keto ester in the enzymatic reaction process of beta amino acids, when using the newly screened lipase in the present invention instead of the previously known commercial lipase, a high level of yield due to rapid beta-keto acid conversion can get

실시예 : 에스터 가수분해효소 (리파아제/에스테라아제) 스크리닝 및 베타-아미노산의 효소적 생산Example: ester hydrolase (lipase/esterase) screening and enzymatic production of beta-amino acids

1. 에스터 가수분해효소 (리파아제/에스테라아제) 스크리닝1. Esterase (lipase/esterase) screening

이전 연구에서 사용된 산업적 Candida rugosa 리파아제(시그마 알드리치 카탈로그 번호 L1754)는 베타-케토에스테르를 베타-케토산으로 가수분해 한 다음 오메가-트랜스 아미나제를 이용해 아민화하여 3-ATfBA (3-amino-4-(2,4,5-trifluorophenyl)butanoic acid)를 포함한 다양한 베타-아미노산을 형성하는데 성공적으로 활용되었다. 본 실시예에서는 베타-케토에스테르에 해당하는 [에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트로부터 베타-케토산인 3-ATfBA 합성에 필요한 에스터 가수분해효소(리파아제/에스테라아제)를 확보하기 위하여, 오메가-트랜스 아미나제의 연쇄반응 및 기존 에스터 가수분해효소 (리파아제/에스테라아제) 보다 적합한 에스터 가수분해효소 (리파아제/에스테라아제)의 동정을 위한 연구를 수행하였다. 대장균이 가지고 있는 BioH라는 리파아제 단백질이 베타-케토에스테르를 가수분해할 수 있음을 확인하고, 대장균이 보유한 42개의 리파아제 중에서, Candida rugosa 리파아제의 아미노산 서열을 이 기준으로 BLAST (Basic Local Alignment Search Tool) 검색을 수행하였다 (도 1). 그 중 계통 발생학적 분석에 기초하여 활성을 보일 것으로 예측되는 6종류의 대장균 유래의 에스터 가수분해효소 (리파아제/에스테라아제)를 선정하였다 (도 1). The industrial Candida rugosa lipase (Sigma-Aldrich catalog number L1754) used in the previous study hydrolyzed beta-ketoester to beta-keto acid followed by amination using omega-transaminase to produce 3-ATfBA (3-amino-4 -(2,4,5-trifluorophenyl)butanoic acid) was successfully used to form various beta-amino acids. In this example, beta-keto ester from [ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate corresponding to beta- ester hydrolase required for the synthesis of keto acid 3-ATfBA To secure (lipase / esterase), omega-transaminase chain reaction and a study for identification of an ester hydrolase (lipase / esterase) more suitable than the existing ester hydrolase (lipase / esterase) was conducted. It was confirmed that the lipase protein BioH possessed by E. coli can hydrolyze beta-ketoester, and among the 42 lipases possessed by E. coli, the amino acid sequence of Candida rugosa lipase was searched using BLAST (Basic Local Alignment Search Tool) based on this standard. was performed (FIG. 1). Among them, six types of E. coli-derived ester hydrolases (lipases/esterases) predicted to show activity based on phylogenetic analysis were selected ( FIG. 1 ).

대장균 유래 리파아제 단백질 외 추가적으로 슈도모나스 (Pseudomonas) 유래의 에스터 가수분해효소 (리파아제/에스테라아제)에 대하여 BLAST를 통하여 비교를 진행하였다. 슈도모나스 (Pseudomonas) 유래의 일부 리파아제/에스테라아제의 경우 사용하고자 하는 기질인 베타-케토 에스테르와 크기 및 구조가 비슷한 케토 프로펜 에틸 에스테르의 에스테르 구조를 잘라낸다고 보고되었다. 이를 통하여 베타-케토 에스테르를 분해할 수 있을 것으로 예상하고 보고된 에스터 가수분해효소 (리파아제/에스테라아제)를 이용하여 BLAST를 진행하였고, 이를 통하여 Pseudomonas avellanae 유래의 Lip PA (서열번호 1) 및 Pseudomonas stutzeri 유래의 Lip PS (서열번호 2) 2종류의 신규 에스터 가수분해효소 (리파아제/에스테라아제)를 선정 하였다. 선택된 총 8개의 에스터 가수분해효소 (리파아제/에스테라아제)에 대한 유전자은행 고유 식별 번호를 하기 표 1에 나타내었다.In addition to E. coli-derived lipase protein, Pseudomonas- derived ester hydrolase (lipase / esterase) was compared through BLAST. It has been reported that some lipases/esterases derived from Pseudomonas cut the ester structure of ketoprofen ethyl ester, which is similar in size and structure to beta-keto ester, which is a substrate to be used. Through this, it was expected that beta-keto ester could be degraded and BLAST was performed using the reported ester hydrolase (lipase/esterase), and through this, Lip PA (SEQ ID NO: 1) derived from Pseudomonas avellanae and Pseudomonas stutzeri derived of Lip PS (SEQ ID NO: 2), two novel ester hydrolases (lipase/esterase) were selected. GenBank unique identification numbers for a total of 8 selected ester hydrolases (lipase/esterase) are shown in Table 1 below.

[표 1][Table 1]

선별된 42개의 후보군 중 E.coli 유래의 6개의 에스터 가수분해효소 (리파아제/에스테라아제) 및 슈도모나스 (Pseudomonas) 유래의 2개 에스터 가수분해효소 (리파아제/에스테라아제)Of the 42 candidates selected , 6 ester hydrolases from E. coli (lipase/esterase) and 2 ester hydrolases from Pseudomonas (lipase/esterase)

Figure PCTKR2020017233-appb-T000001
Figure PCTKR2020017233-appb-T000001

E.coli 유래의 6개의 유전자를 프라이머 (Sequences (5'→3'), ent ry 1: F1 primer - attaggatccatgaccaatataccg (서열번호 3) R1p rimer - gccggagctcttatttctgcttcgt (서열번호 4), entry 2: F2 primer - atta ggatccatgaaacatgaccat (서열번호 5) R2 primer - gccggagctc ttacatcatttcaat (서열번호 6), entry 3: F3 primer - gccgggatcc atgatgaataataaa (서열번호 7) R3 primer - cggcgagctcttacagagtttcctc (서열번호 8), entry 4: F4 primer - attaggatccatgattgaaatagaatcacgcga (서열번호 9) R4 primer - gccggagctcttaaagatgctggcggaaaaatgtcac (서열번호 10), entry 5: F5 primer - attaggatccatggaaaattcacgcatccctggg (서열번호 11) R5 primer - agatggatcctcagttatcacgacgcaccgccatcg (서열번호 12), entry 8: F8 primer - attaggatccatgaacttcaaca (서열번호 13) R8 primer - gccggagctcttatgagtcatga (서열번호 14))를 이용하여 pQE-80L 벡터에 클로닝하였고, 슈도모나스 (Pseudomonas) 유래의 2개 유전자는 코돈 최적화 후 합성(bionics) 및 pQE-80L 벡터 내에 클로닝하고 대장균 BL21(DE3) 내로 도입하였다. 총 8개의 리파아제/에스테라아제는 하기와 같이 Ni-NTA 친화성 컬럼을 사용하여 발현 및 정제하였다 (도 2). E. coli BL21의 형질 전환체는 37℃에서 50 mg/L의 카나마이신을 포함하는 1L의 LB 배지에서 성장시켰다. 형질전환체의 OD600이 0.4 ~ 0.6에 도달하였을 때, 0.5 mM의 IPTG(Isopropyl-β-D-thiogalactopyranoside)(Carbosynth)를 첨가하여 리파아제/에스테라아제의 발현을 유도하였다. 12시간 후, 상기 세포를 채취한 후 50 mM 인산완충용액(pH 8.0)으로 두 번 세척하였다. 원심분리 후, 세포 침전물을 20 uM 피리독살 50 인산염(pyridoxal 5-phosphate, PLP), 2 mM EDTA, 1 mM PMSF, 포함하는 20 mL 의 50 mM 인산완충용액(pH 8.0)으로 재현탁하였다. 초음파 분해를 이용한 세포 파괴를 통해 세포추출물을 획득 하였다. His-태그된(tagged) 리파아제/에스테라아제를 포함하는 재조합 E. coli 세포의 조추출물을 Ni-NTA 아가로즈컬럼 (Qiagen, Hilden, Germany)에 적용시켜 효소를 정제하였다(도 2). E. coli- derived six genes primer (Sequences (5'→3'), ent ry 1: F1 primer - attaggatccatgaccaatataccg (SEQ ID NO: 3) R1p rimer - gccggagctcttatttctgcttcgt (SEQ ID NO: 4), entry 2: F2 primer - atta ggatccatgaaacatgaccat (SEQ ID NO: 5) R2 primer - gccggagctc ttacatcatttcaat (SEQ ID NO: 6), entry 3: F3 primer - gccgggatcc atgatgaataataaa (SEQ ID NO: 7) R3 primer - cggcgagctttacagagttgaatctcatcacagagtttcctc (SEQ ID NO: 8), F3 primer - cggcgagctttacagagttgaat 9) R4 primer - gccggagctcttaaagatgctggcggaaaaatgtcac (SEQ ID NO: 10), entry 5: F5 primer - attaggatccatggaaaattcacgcatccctggg (SEQ ID NO: 11) R5 primer - agatggatcctctcagctggcggaaaaaatgtcac (SEQ ID NO. (SEQ ID NO: 14)) was cloned into the pQE-80L vector, and two genes from Pseudomonas were synthesized after codon optimization (bionics) and cloned into the pQE-80L vector and introduced into E. coli BL21 (DE3). . A total of eight lipases/esterases were expressed and purified using a Ni-NTA affinity column as follows ( FIG. 2 ). Transformants of E. coli BL21 were grown in 1 L of LB medium containing 50 mg/L of kanamycin at 37°C. When the OD600 of the transformant reached 0.4 to 0.6, 0.5 mM Isopropyl-β-D-thiogalactopyranoside (Carbosynth) was added to induce expression of lipase/esterase. After 12 hours, the cells were harvested and washed twice with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cell precipitate was resuspended in 20 mL of 50 mM phosphate buffer (pH 8.0) containing 20 uM pyridoxal 5-phosphate (PLP), 2 mM EDTA, and 1 mM PMSF. Cell extracts were obtained through cell disruption using sonication. A crude extract of recombinant E. coli cells containing His-tagged lipase/esterase was applied to a Ni-NTA agarose column (Qiagen, Hilden, Germany) to purify the enzyme (FIG. 2).

정제된 효소를 사용하여 pH 7.0의 조건에서 반응기질[에틸에스테르(에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트)] 및 DMSO를 사용하여 베타-케토 에스테르에 대한 활성을 시험하였다 (도 3). 반응조건은 다음과 같다: 에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트 (10mM), 리파아제/에스테라아제 (0.1m g/mL), DMSO (15%), 완충용액 (200 mM Tris-HCl pH 7.0), 온도 (37℃), 반응 시간 (30분). 8개의 리파아제/에스테라아제 중에서 슈도모나스 (Pseudomonas)에서 유래한 Lip PA와 Lip PS가 각각 6.2U/mg, 10.3U/mg의 높은 활성을 보여주었다. 반면에, E.coli에서 유래한 6종류의 리파아제의 경우 1U/mg미만의 활성으로 큰 차이로 낮게 형성되는 것을 확인할 수 있었다. 이에 따라, 기질로 사용되는 베타-케토 에스테르 에 반응성이 높게 형성되는 Lip PA와 Lip PS를 선택하여 추가적인 실험을 진행하였다. Beta-keto ester using a reactive substrate [ethyl ester (ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate)] and DMSO at a pH of 7.0 using a purified enzyme was tested for activity ( FIG. 3 ). Reaction conditions were as follows: ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate (10 mM), lipase/esterase (0.1 mg/mL), DMSO (15%), buffer solution (200 mM Tris-HCl pH 7.0), temperature (37° C.), reaction time (30 min). Among the eight lipases/esterases, Lip PA and Lip PS derived from Pseudomonas showed high activity of 6.2U/mg and 10.3U/mg, respectively. On the other hand, in the case of 6 types of lipases derived from E. coli , it was confirmed that they were formed with a large difference and low with an activity of less than 1U/mg. Accordingly, additional experiments were carried out by selecting Lip PA and Lip PS, which are highly reactive to beta-keto esters used as substrates.

2. Lip PA 와 Lip PS 세부 활성도 측정2. Lip PA and Lip PS detailed activity measurement

2-1) Lip PA 와 Lip PS의 최적 pH와 온도 확인2-1) Check the optimum pH and temperature of Lip PA and Lip PS

Lip PA와 Lip PS는 pH 6 - 9의 범위에서 활성을 나타내었고, 반응은 ρ-니트로페닐 아세테이트를 함유하는 50 mM 포스페이트(Phosphate) 완충액(pH 6.0-7.0), 50mM Tris-HCl 완충액 (pH 7.0-9.0)에서 수행되었다. 이 반응에서, ρ-니트로 페닐 아세테이트를 Lip PA 및 Lip PS를 사용하여 ρ-니트로페놀로 전환시키고, UV-분광 광도계를 사용하여 405 nm 파장에서 관찰한 결과 황색 물질이 확인되었다 (반응식 2). Lip PA and Lip PS showed activity in the range of pH 6 - 9, and the reaction was carried out in 50 mM Phosphate buffer (pH 6.0-7.0) containing ρ-nitrophenyl acetate, 50 mM Tris-HCl buffer (pH 7.0) -9.0). In this reaction, ρ-nitrophenyl acetate was converted to ρ-nitrophenol using Lip PA and Lip PS, and a yellow substance was confirmed as a result of observation at a wavelength of 405 nm using a UV-spectrophotometer (Scheme 2).

[반응식 2] Lip PA 및 Lip PS의 활성 측정을 위한 스킴.[Scheme 2] Scheme for measuring the activity of Lip PA and Lip PS.

Figure PCTKR2020017233-appb-I000013
Figure PCTKR2020017233-appb-I000013

슈도모나스 아벨라나에(Pseudomonas avellanae) 유래의 Lip PA (서열번호 1)는 p H 8.5에서 14.5U/mg으로 최대 활성을 나타내었고 슈도모나스 스투트제리(Pseudomonas stutzeri)유래의 Lip PS는 pH 8.0에서 55U/mg으로 최적 활성을 보였다(도 4). 오메가-트랜스 아미나제의 반응조건인 pH 7.0에서도 Lip PA의 경우 약 2U/mg, Lip PS의 경우 20U/mg으로 반응성이 높게 나타나는 것으로 확인되었다. 도 5는 Lip PA 와 Lip P S의 최적온도와 시간에 따른 활성감소량을 분석한 결과를 나타내는 것이다. 도 5의 반응은 상기 pH 7.0에서의 반응과 유사한 방식으로 수행되었고, 정제된 효소를 사용하여 30-60℃에서 0-15시간 동안 배양 및 반응하고 샘플을 특정시간 간격 동안 추출하고 405 nm에서 UV-분광 광도계를 사용하여 분석 하였고 분석 결과, Lip PA 및 Lip PS의 최적 온도는 30 ℃로 확인하였다 (도 5). Lip PA (SEQ ID NO: 1) from Pseudomonas avellanae exhibited the maximum activity at 14.5 U/mg at pH 8.5, and Lip PS from Pseudomonas stutzeri at pH 8.0 at 55 U/mg. mg showed optimal activity (FIG. 4). Even at pH 7.0, which is the reaction condition of omega-trans aminase, it was confirmed that the reactivity was high at about 2U/mg for Lip PA and 20U/mg for Lip PS. 5 shows the results of analyzing the amount of decrease in activity according to the optimum temperature and time of Lip PA and Lip PS. The reaction of FIG. 5 was performed in a similar manner to the reaction at pH 7.0, incubated and reacted at 30-60° C. for 0-15 hours using the purified enzyme, and samples were extracted for a specific time interval and UV at 405 nm. - It was analyzed using a spectrophotometer, and as a result of the analysis, it was confirmed that the optimum temperature of Lip PA and Lip PS was 30 °C (Fig. 5).

2-2) DMSO의 농도 에 따른 Lip PA 와 Lip PS의 활성2-2) Activity of Lip PA and Lip PS according to the concentration of DMSO

위 실험의 반응조건을 동일하고 서로 다른 농도의 DMSO (5 %, 10 %, 15 %, 20 %, 25 %, 30 %)의 조건 하에 Lip PA 및 Lip PS의 효소 활성을 측정하였다. DMSO의 농도를 5%에서 30%로 증가시키면 Lip PA 및 PS의 효소 활성이 감소하는 것으로 관찰되었다.The enzyme activities of Lip PA and Lip PS were measured under the same reaction conditions as above and different concentrations of DMSO (5%, 10%, 15%, 20%, 25%, 30%). It was observed that the enzymatic activity of Lip PA and PS decreased when the concentration of DMSO was increased from 5% to 30%.

2-3) Lip PA 및 Lip PS와 오메가-트랜스 아미나제 연쇄반응을 통한 리파아제의 농도 최적화2-3) Optimization of Lipase Concentration through Lip PA and Lip PS and Omega-Trans Aminase Chain Reaction

이전 연구에서 본 발명자들은 두 세포 시스템인 상업용 리파아제와 오메가-트랜스 아미나제를 사용하여 베타-아미노산을 합성하였다. 본 연구에서는 기존 리파아제 보다 활성이 좋은 신규 에스터 가수분해효소 (리파아제/에스테라아제)를 사용하여 베타 아미노산을 합성하려고 시도하였다. 이를 위해, 본 연구는 신규 에스터 가수분해효소 (리파아제/에스테라아제)의 활성이 있는지에 대한 여부를 확인하였다. 이전 연구에서 사용된 오메가-트랜스 아미나제 (TAIC:(Ilumatobacter coccineus) 유래 오메가트랜스 아미나제 (protein_id =WP_015443725.1")와 신규 리파아제인 Lip PA, Lip PS가 각각 거의 동일한 분자량인 45, 43, 43 kDa으로 SDS-PAGE로는 공동 발현된 세포 시스템의 경우에 효소의 발현량을 밴드로 구별할 수 없다. 따라서 다음의 방법으로 리파아제의 특이적 활성을 확인하였다. Lip PA, PS 및 상업용 리파아제 칸디다 루고사 (candida rugosa)의 특이적 활성의 확인은, 기질인 (에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트 0.5 ml 반응 부피에서 수행 되었고 상업용 리파아제 칸디다 루고사 (candida rugosa) 1mg/ml, 신규 리파아제 Lip PA, Lip PS는 각각 0.5 OD를 사용하여 200mM Tris-HCl 완충액 (pH 7.0)에서 37 ℃ 180 rpm으로 20분 동안 반응을 진행하고, 반응 후 1 : 1 (v/v)의 비율로 10% 과염소산을 첨가하여 반응을 정지시켰다. 반응 정지 후 혼합물을 17000 g에서 30분 동안 원심 분리하고, 투명한 상등액을 분석하였다. 기질인 에틸3-옥소-4-(2,4,5-트리플루오로페닐)부타노에이트의 감소량으로 C18 대칭 컬럼을 사용하여 HPLC로 분석 하였다. 그 결과 Lip PS (12U/mg)에 이어 Lip PA (9.8U/mg)가 특이적 활성을 나타내었으며 상업적인 리파아제 칸디다 루고사 (candida rugosa) (0.324U/mg) 보다 훨 씬 좋은 활성을 나타내는 것으로 확인되었다 (도 7).In a previous study, we synthesized beta-amino acids using two cellular systems, commercial lipase and omega-trans aminase. In this study, an attempt was made to synthesize beta amino acids using a novel ester hydrolase (lipase/esterase), which is more active than existing lipases. To this end, this study confirmed whether there is activity of a novel ester hydrolase (lipase/esterase). The omega-trans aminase (TAIC:(Ilumatobacter coccineus)-derived omega-trans aminase (protein_id = WP_015443725.1")) and the novel lipases Lip PA and Lip PS used in the previous study had almost the same molecular weight, 45, 43, 43, respectively. In case of co-expressed cell system with kDa SDS-PAGE, the expression level of the enzyme could not be distinguished by band Therefore, the specific activity of lipase was confirmed by the following method: Lip PA, PS and commercial lipase Candida rugosa Confirmation of the specific activity of ( candida rugosa ) was carried out in a 0.5 ml reaction volume of (ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate, a substrate, and the commercial lipase Candida rugosa ( candida rugosa ) 1mg/ml, the novel lipase Lip PA and Lip PS were reacted in 200mM Tris-HCl buffer (pH 7.0) using 0.5 OD at 37°C and 180rpm for 20 minutes, and after reaction 1:1 (v/v) ) was added to stop the reaction by adding 10% perchloric acid. After the reaction was stopped, the mixture was centrifuged at 17000 g for 30 minutes, and the clear supernatant was analyzed. The reduced amount of ethyl 3-oxo-4- (2,4,5-trifluorophenyl) butanoate as a substrate was analyzed by HPLC using a C18 symmetric column. As a result, it was confirmed that Lip PS (12U/mg) followed by Lip PA (9.8U/mg) showed specific activity and showed much better activity than the commercial lipase Candida rugosa (0.324U/mg). became (Fig. 7).

상기와 같은 결과에 기초하여 우리는 각각 대장균에 발현된 신규 리파아제/에스테라아제를 오메가-트랜스 아미나제와 연쇄반응을 통해 베타-아미노산을 생합성 하였다. 트랜스 아미나제는 다음과 같이 최적화된 Ilumatobacter coccineus 유래의 오메가-트랜스 아미나제를 pET-24ma 백터를 통해 대장균에 과발현 후 정제하여 사용하였다. 구체적으로 TAIC:(Ilumatobacter coccineus) 유래 오메가트랜스 아미나제 (protein_id =WP_015443725.1")를 바이오닉스 회사에 의뢰하여 유전자 합성 후, IPTG 유도 발현벡터 pET24ma 벡터에 클로닝시켰다. pET24ma 벡터 내로 클론 시킨 다음, E. coli BL21 내로 도입하였다. E. coli BL21의 형질전환체는 37℃에서 50 mg/L의 카나마이신을 포함하는 1L의 LB 배지에서 성장시켰다. 형질전환체의 OD600이 0.4 ~ 0.6에 도달하였을 때, 0.5 mM의 IPTG(Isopropyl-β-D-thiogalactopyranoside)(Carbosynth)를 첨가하여 w-TA의 발현을 유도하였다. 12시 간 후, 상기 세포를 채취한 후 50 mM 인산완충용액(pH 8.0)으로 한 번 세척하였다. 원심분리 후, 세포를 반응에 사용하였다.Based on the above results, we biosynthesized beta-amino acids through a chain reaction with omega-transaminase of novel lipase/esterase expressed in E. coli, respectively. Transaminase was used after overexpression of Ilumatobacter coccineus- derived omega-transaminase optimized as follows in E. coli through pET-24ma vector and then purified. Specifically, TAIC:(Ilumatobacter coccineus)-derived omegatrans aminase (protein_id = WP_015443725.1") was requested to Bionics company, and after gene synthesis, it was cloned into the IPTG-induced expression vector pET24ma vector. After cloning into the pET24ma vector, E. It was introduced into coli BL21. Transformants of E. coli BL21 were grown in 1 L of LB medium containing 50 mg/L kanamycin at 37° C. When the OD600 of the transformants reached 0.4 to 0.6, 0.5 The expression of w-TA was induced by the addition of mM IPTG (Isopropyl-β-D-thiogalactopyranoside) (Carbosynth) After 12 hours, the cells were harvested and once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction.

100 OD, 120 OD의 오메가-트랜스 아미나제에서의 신규 에스터 가수분해효소 (리파아제/에스테라아제) 농도에 따른 베타아미노산의 생성률을 확인하고 그 결과를 도 8에 나타내었다. 오메가-트랜스 아미나제의 100 OD의 조건 하에서 Lip PA OD50, Lip PS OD10일 때 각각 49 mM, 61 mM의 베타-아미노산 생성률을 나타내는 것으로 확인되었다 (도 8). 오메가-트랜스 아미나제의 120OD 조건하에서는 Lip PA OD50, Lip PS OD10일 때 각각 39mM, 56.6mM로 베타-아미노산 생성률이 감소하는 결과를 나타내었다 (도 9). 이러한 결과를 바탕으로 오메가-트랜스 아미나제와 신규 리파아제/에스테라아제의 비율이 Lip PA는 2:1, Lip PS는 10:1이 최적임을 확인할 수 있었다.100 OD, 120 OD of omega- The production rate of beta amino acids according to the new ester hydrolase (lipase / esterase) concentration in the transaminase was confirmed, and the results are shown in FIG. 8 . It was confirmed that beta-amino acid production rates of 49 mM and 61 mM, respectively, were exhibited at Lip PA OD50 and Lip PS OD10 under a condition of 100 OD of omega-trans aminase ( FIG. 8 ). Omega-transaminase under 120OD condition, when Lip PA OD50 and Lip PS OD10, respectively, 39mM and 56.6mM, the beta-amino acid production rate was decreased (FIG. 9). Based on these results, it was confirmed that the ratio of omega-trans aminase and novel lipase/esterase was 2:1 for Lip PA and 10:1 for Lip PS.

2-4) 벡터시스템 확립 및 동시발현을 통한 최적화2-4) Optimization through establishment of vector system and co-expression

두 세포 시스템을 사용하여 비율을 최적화 한 후 단일 세포에서 두 벡터를 공동 발현함으로써 단일 세포 시스템으로 베타-아미노산 합성을 수행하였다. 오메가-트랜스 아미나제(TAIC)는 pET24ma에서 클로닝되었고 Lip PA 및 Lip PS는 pQE-80L에서 클로닝되었으며, 이들을 단일 세포에서 공동 발현시켰다. 구체적으로 오메가-트랜스 아미나제(TAIC)는 pET24ma 벡터 내로 클론시킨 다음, E. coli BL21 내 로 도입하였다. E. coli BL21의 형질전환체는 37℃에서 50 mg/L의 카나마이신을 포함하는 1L의 LB 배지에서 성장시켰다. 형질전환체의 OD600이 0.4 ~ 0.6에 도달하였을 때, 0.5 mM의 IPTG(Isopropyl-β-D-thiogalactopyranoside)(Carbosynth)를 첨가하여 w-TA의 발현을 유도하였다. 12시간 후, 상기 세포를 채취한 후 50 mM 인산 완충용액(pH 8.0)으로 한 번 세척하였다. 원심분리 후, 세포를 반응에 사용하였다. 그리고 에스터 가수분해효소 Lip PA 및 Lip PS는 pQE80L 벡터 내로 클론 시킨 다음, E. coli BL21 내로 도입하였다. E. coli BL21의 형질전환체는 37℃에서 100 mg/L의 엠피실린을 포함하는 1L의 LB 배지에서 성장시켰다. 형질전환체의 OD600이 0.4 ~ 0.6에 도달하였을 때, 0.5 mM의 IPTG(Isopropyl-β-D-thiogalactopyranoside)(Carbosynth)를 첨가하여 에스터 가수분해 효소의 발현을 유도하였다. 12시간 후, 상기 세포를 채취한 후 50 mM 인산완충용액(pH 8.0)으로 한 번 세척하였다. 원심분리 후, 세포를 반응에 사용하였다. Beta-amino acid synthesis was performed in a single cell system by co-expressing both vectors in a single cell after optimizing the ratio using the two cell system. Omega-trans aminase (TAIC) was cloned in pET24ma and Lip PA and Lip PS were cloned in pQE-80L, and they were co-expressed in single cells. Specifically, omega-trans aminase (TAIC) was cloned into the pET24ma vector and then introduced into E. coli BL21. Transformants of E. coli BL21 were grown in 1 L of LB medium containing 50 mg/L of kanamycin at 37°C. When the OD600 of the transformant reached 0.4 to 0.6, 0.5 mM Isopropyl-β-D-thiogalactopyranoside (Carbosynth) was added to induce w-TA expression. After 12 hours, the cells were harvested and washed once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction. And the ester hydrolases Lip PA and Lip PS were cloned into the pQE80L vector, and then introduced into E. coli BL21. Transformants of E. coli BL21 were grown in 1 L of LB medium containing 100 mg/L of ampicillin at 37°C. When the OD600 of the transformant reached 0.4 to 0.6, 0.5 mM Isopropyl-β-D-thiogalactopyranoside (Carbosynth) was added to induce expression of the ester hydrolase. After 12 hours, the cells were harvested and washed once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction.

그 후 단일 세포에서 공동 발현된 대장균을 30-150OD의 농도로 반응을 수행하였다 (도 11). 오메가-트랜스 아미나제(TAIC) 및 Lip PA 공동 발현 시스템에서 최대 전환율은 28. 30 mM (120 OD)인 반면, 오메가-트랜스 아미나제(TAIC) 및 Lip PS 공동 발현된 셀 시스템의 경우 약 48.22 mM (150 OD) 전환에 이르는 것으로 확인되었다. Thereafter, the reaction was performed at a concentration of 30-150OD for co-expressed E. coli in single cells (FIG. 11). The maximum conversion in the omega-trans aminase (TAIC) and Lip PA co-expression system is 28. 30 mM (120 OD), while about 48.22 mM in the omega-trans aminase (TAIC) and Lip PS co-expressed cell system. (150 OD) conversion was found.

또 다른 공동 발현된 세포 시스템은 다음과 같다. 오메가-트랜스 아미나제(TAIC)는 pCDF-Duet 벡터에서 클로닝되는 반면, Lip PA 및 Lip PS는 pQE-80L 벡터에서 클로닝되었으며, 이들을 단일 세포에서 공동 발현시켜 상기와 같은 반응을 수행하였다. 구체적으로 오메가-트랜스 아미나제(TAIC)는 pCDF-Duet 벡터 내로 클론 에스터 가수분해효소 Lip PA 및 Lip PS는 pQE80L 벡터 내로 클론 시킨 이 두 개의 벡터를 동시에, E. coli BL21 내로 도입하였다. E. coli BL21의 형질 전환체는 37℃에서 25 mg/L의 클로람페니칼과 100mg/L 엠피실린을 포함하는 1L의 LB 배지에서 성장시켰다. 형질전환체의 OD600이 0.4 ~ 0.6에 도달하였을 때, 0.5 mM의 IPTG(Isopropyl-β-D-thiogalactopyranoside)(Carbosynth)를 첨가하여 w-TA와 에스터 가스분해효소의 공동발현을 유도하였다. 12시간 후, 상기 세포를 채취한 후 50 mM 인산완충용액(pH 8.0)으로 한 번 세척하였다. 원심분리 후, 세포를 반응에 사용 하였다. 그리고 에스터 가수분해효소 Lip PA 및 Lip PS는 pQE80L 벡터 내로 클론 시킨 다음, E. coli BL21 내로 도입하였다. E. coli BL21의 형질전환체는 37℃에서 100 mg/L의 엠피실린을 포함하는 1L의 LB 배지에서 성장시켰다. 형질전환체의 OD600이 0.4 ~ 0.6에 도달하였을 때, 0.5 mM의 IPTG(Isopropyl-β-D-thiogalactopyranoside)(Carbosynth)를 첨가하여 에스터 가수분해 효소의 발현을 유도하였다. 12시간 후, 상기 세포를 채취한 후 50 mM 인산완충용액(pH 8.0)으로 한 번 세척하였다. 원심분리 후, 세포를 이용하여 반응에 사용되었다.Another co-expressed cell system is as follows. Omega-trans aminase (TAIC) was cloned from the pCDF-Duet vector, whereas Lip PA and Lip PS were cloned from the pQE-80L vector, and they were co-expressed in a single cell to perform the above reaction. Specifically, the omega-trans aminase (TAIC) was cloned into the pCDF-Duet vector and the clone ester hydrolases Lip PA and Lip PS were cloned into the pQE80L vector. These two vectors were simultaneously introduced into E. coli BL21. Transformants of E. coli BL21 were grown in 1 L of LB medium containing 25 mg/L of chloramphenical and 100 mg/L of ampicillin at 37°C. When the OD600 of the transformant reached 0.4 to 0.6, 0.5 mM Isopropyl-β-D-thiogalactopyranoside (Carbosynth) was added to induce co-expression of w-TA and ester gas lyase. After 12 hours, the cells were harvested and washed once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction. And the ester hydrolases Lip PA and Lip PS were cloned into the pQE80L vector, and then introduced into E. coli BL21. Transformants of E. coli BL21 were grown in 1 L of LB medium containing 100 mg/L of ampicillin at 37°C. When the OD600 of the transformant reached 0.4 to 0.6, 0.5 mM Isopropyl-β-D-thiogalactopyranoside (Carbosynth) was added to induce expression of the ester hydrolase. After 12 hours, the cells were harvested and washed once with 50 mM phosphate buffer (pH 8.0). After centrifugation, the cells were used for the reaction.

오메가-트렌스 아미나제(TAIC) 및 Lip PA 공동 발현 세포 시스템을 이용한 경우, 최대 전환율(베타-아미노산 생성률)은 35.58 mM (150 OD) 인 것으로 나타났으며, 오메가 -트랜스 아미나제(TAIC) 및 Lip PS 공동발현된 세포 시스템을 이용한 경우 약 42.34 mM (150 OD) 전환율 (베타-아미노산 생성률)이 관찰되었다 (도 11).When using the omega-transaminase (TAIC) and Lip PA co-expression cell system, the maximum conversion (beta-amino acid production) was found to be 35.58 mM (150 OD), and the omega-transaminase (TAIC) and Lip PA About 42.34 mM (150 OD) conversion (beta-amino acid production) was observed when using the PS co-expressed cell system ( FIG. 11 ).

서열정보sequence information

서열번호 1 (Lip PA : 진뱅크 접근번호 WP_005619979.1)SEQ ID NO: 1 (Lip PA: GenBank Accession Number WP_005619979.1)

mqiqghyelqfeavreafaalfddpqergaalciqvggqtvvd lwagtadkdgaeawhtdtianlfsctktftsvavlqlveegklkldepvarlwpefavagkas itlrqllchqaglpalreplpaealyqwdtmtaalaaeepwwtpgqghgyaaitygwlvgeml rradgrgpgesiaarisrplgldfhvgladeqfyrvahiargkgnagdaaaqrvlqatmrepa sitakaftnppsimtstnkpewrrmqqpaanghgnarslagfynglldgslleadmlneltrehslgdkt lltstrlglgcmldqpaepnatfglgpkafghpgaggsvgfadpdydvafgfvtntlgpyilmdpraqklvgvlreclqmqiqghyelqfeavreafaalfddpqergaalciqvggqtvvd lwagtadkdgaeawhtdtianlfsctktftsvavlqlveegklkldepvarlwpefavagkas itlrqllchqaglpalreplpaealyqwdtmtaalaaeepwwtpgqghgyaaitygwlvgeml rradgrgpgesiaarisrplgldfhvgladeqfyrvahiargkgnagdaaaqrvlqatmrepa sitakaftnppsimtstnkpewrrmqqpaanghgnarslagfynglldgslleadmlneltrehslgdkt lltstrlglgcmldqpaepnatfglgpkafghpgaggsvgfadpdydvafgfvtntlgpyilmdpraqklvgvlreclq

서열번호 2 (Lip PS : 진뱅크 접근번호 WP_020308675.1)SEQ ID NO: 2 (Lip PS: GenBank Accession Number WP_020308675.1)

mqvqgyfdlrfeavrdafaalfdgtqqrgaalcvqiggetvidlwagvadnqgeqawhsdtlvnlfsctktftavaalqlveegkleldapvarvwpefaangkesitlrqllchraglpairrplapealydwtcmtdalaaeqpwwapgtdqgyaamtygwlvgellrrvdgcgagesivrrtaaplgldfhvglddsqadrvayltrtkndfgdacaqrllkalmsdpasisacafnnppsimssgnkpewrrmaqpaanghgnarslaglytgllqgrlldeavlremthehsagedrtllastrfglgcwldqpdvanatfamgprafghpgaggcigfadperelgfgfvtntlgpyvlmdpraqslarcvaaclhmqvqgyfdlrfeavrdafaalfdgtqqrgaalcvqiggetvidlwagvadnqgeqawhsdtlvnlfsctktftavaalqlveegkleldapvarvwpefaangkesitlrqllchraglpairrplapealydwtcmtdalaaeqpwwapgtdqgyaamtygwlvgellrrvdgcgagesivrrtaaplgldfhvglddsqadrvayltrtkndfgdacaqrllkalmsdpasisacafnnppsimssgnkpewrrmaqpaanghgnarslaglytgllqgrlldeavlremthehsagedrtllastrfglgcwldqpdvanatfamgprafghpgaggcigfadperelgfgfvtntlgpyvlmdpraqslarcvaaclh

Claims (14)

하기 화학식 1로 표시되는 베타 케토 에스터를 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소와 반응시켜, 하기 화학식 2로 표시되는 베타 케토산을 합성한 후, 상기 베타 케토산을 트랜스 아미나제 및 아미노기 공여체와 반응시켜, 하기 화학식 3으로 표시되는 베타 아미노산을 생산하는 방법.Beta-keto ester represented by the following formula (1 ) is reacted with an ester hydrolase derived from a Pseudomonas sp . strain to synthesize beta-keto acid represented by the following formula (2), and then the beta-keto acid is converted to a transaminase and an amino group A method for producing a beta amino acid represented by the following formula (3) by reacting with a donor. [화학식 1][Formula 1]
Figure PCTKR2020017233-appb-I000014
Figure PCTKR2020017233-appb-I000014
 [화학식 2][Formula 2]
Figure PCTKR2020017233-appb-I000015
Figure PCTKR2020017233-appb-I000015
 [화학식 3][Formula 3]
Figure PCTKR2020017233-appb-I000016
Figure PCTKR2020017233-appb-I000016
 상기 화학식 1, 화학식 2, 화학식 3에서 R은
Figure PCTKR2020017233-appb-I000017
,
Figure PCTKR2020017233-appb-I000018
,
Figure PCTKR2020017233-appb-I000019
,
Figure PCTKR2020017233-appb-I000020
,
Figure PCTKR2020017233-appb-I000021
,
Figure PCTKR2020017233-appb-I000022
,
Figure PCTKR2020017233-appb-I000023
및
Figure PCTKR2020017233-appb-I000024
로 이루어진 군에서 선택됨.In Formula 1, Formula 2, and Formula 3, R is
Figure PCTKR2020017233-appb-I000017
,
Figure PCTKR2020017233-appb-I000018
,
Figure PCTKR2020017233-appb-I000019
,
Figure PCTKR2020017233-appb-I000020
,
Figure PCTKR2020017233-appb-I000021
,
Figure PCTKR2020017233-appb-I000022
,
Figure PCTKR2020017233-appb-I000023
and
Figure PCTKR2020017233-appb-I000024
selected from the group consisting of 청구항 1에 있어서,The method according to claim 1, 상기 슈도모나스 속 균주는 슈도모나스 아벨라나에(Pseudomonas avellanae) 또는 슈도모나스 스투트제리(Pseudomonas stutzeri) 둘 중 하나 이상을 포함하는 것을 특징으로 하는 베타 아미노산을 생산하는 방법.The Pseudomonas sp. strain is Pseudomonas avellanae ( Pseudomonas avellanae ) or Pseudomonas stutzeri ( Pseudomonas stutzeri ) Method for producing beta amino acids, characterized in that it comprises one or more of the two. 청구항 1에 있어서,The method according to claim 1, 상기 가수분해효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성되는 것을 특징으로 하는 베타 아미노산을 생산하는 방법.The hydrolase is a method for producing beta amino acids, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 청구항 1에 있어서,The method according to claim 1, 상기 트랜스 아미나제는 오메가 트랜스 아미나제인 것을 특징을 하는 베타 아미노산을 생산하는 방법.The method for producing beta amino acids, characterized in that the trans-aminase is an omega trans-aminase. 청구항 1에 있어서,The method according to claim 1, 상기 아미노기 공여체는 L-알라닌, 베타-알라닌, 3-아미노프로피오닉산(3-aminopropionic acid;3-APA), 아이소프로필아민(isopropyl amine; iPrNH2), 벤질아민(benzylamine; BnNH2) 및 (S)-알파메틸벤질아민(α-methylbenzylamine; MBA)으로 구성된 군으로부터 선택되는 하나 이상을 포함하는 것을 특징으로 하는 베타 아미노산을 생산하는 방법.The amino group donor is L-alanine, beta-alanine, 3-aminopropionic acid (3-aminopropionic acid; 3-APA), isopropyl amine (iPrNH2), benzylamine (benzylamine; BnNH2) and (S) - A method for producing a beta amino acid, characterized in that it comprises one or more selected from the group consisting of alpha-methylbenzylamine (MBA). 청구항 1에 있어서,The method according to claim 1, 상기 에스터 가수분해효소와 트랜스 아미나제의 농도비는 1: 1 ~ 12인 것을 특징으로 하는 베타 아미노산을 생산하는 방법.The method for producing beta amino acids, characterized in that the concentration ratio of the ester hydrolase and transaminase is 1: 1 to 12. 청구항 1에 있어서,The method according to claim 1, 상기 에스터 가수분해효소와 트랜스 아미나제는 대장균에서 발현된 것을 특징으로 하는 베타 아미노산을 생산하는 방법.The method for producing beta amino acids, characterized in that the ester hydrolase and transaminase expressed in E. coli. 청구항 1에 있어서,The method according to claim 1, 상기 에스터 가수분해효소와 트랜스 아미나제는 동일한 대장균에서 공동 발현된 것을 특징으로 하는 베타 아미노산을 생산하는 방법.The method for producing a beta amino acid, characterized in that the ester hydrolase and transaminase co-expressed in the same E. coli. 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소, 트랜스 아미나제 및 아미노기 공여체를 포함하는, 하기 화학식 1로 표시되는 베타 케토 에스터로부터 하기 화학식 3으로 표시되는 베타 아미노산의 생산을 위한 조성물.Pseudomonas ( Pseudomonas ) A composition for the production of beta amino acids represented by the following formula (3) from a beta keto ester represented by the following formula (1), comprising an ester hydrolase, a transaminase and an amino group donor derived from a sp. strain. [화학식 1][Formula 1]
Figure PCTKR2020017233-appb-I000025
Figure PCTKR2020017233-appb-I000025
 [화학식 3][Formula 3]
Figure PCTKR2020017233-appb-I000026
Figure PCTKR2020017233-appb-I000026
 상기 화학식 1, 화학식 2, 화학식 3에서 R은
Figure PCTKR2020017233-appb-I000027
,
Figure PCTKR2020017233-appb-I000028
,
Figure PCTKR2020017233-appb-I000029
,
Figure PCTKR2020017233-appb-I000030
,
Figure PCTKR2020017233-appb-I000031
,
Figure PCTKR2020017233-appb-I000032
,
Figure PCTKR2020017233-appb-I000033
및
Figure PCTKR2020017233-appb-I000034
로 이루어진 군에서 선택됨.In Formula 1, Formula 2, and Formula 3, R is
Figure PCTKR2020017233-appb-I000027
,
Figure PCTKR2020017233-appb-I000028
,
Figure PCTKR2020017233-appb-I000029
,
Figure PCTKR2020017233-appb-I000030
,
Figure PCTKR2020017233-appb-I000031
,
Figure PCTKR2020017233-appb-I000032
,
Figure PCTKR2020017233-appb-I000033
and
Figure PCTKR2020017233-appb-I000034
selected from the group consisting of 청구항 9에 있어서,10. The method of claim 9, 상기 가수분해효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성되는 것을 특징으로 하는 조성물.The hydrolase composition, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 에틸 4-(2,4,5-트리플루오로페닐)-3-옥소부타노에이트[ethyl 4-(2,4,5-trifluorophenyl)-3-oxobutanoate]를 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소와 반응시켜, 4-(2,4,5-트리플루오로페닐)-3-옥소부탄산[4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid]을 합성한 후, 상기 4-(2,4,5-트리플루오로페닐)-3-옥소부탄산[4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] 을 트랜스 아미나제 및 아미노기 공여체와 반응시켜, (R)-3-아미노-4-(2,4,5-트리플루오로페닐) 부탄산을 생산하는 방법.Ester of ethyl 4-(2,4,5-trifluorophenyl)-3-oxobutanoate [ethyl 4-(2,4,5-trifluorophenyl)-3-oxobutanoate] to Pseudomonas sp. strain After reacting with a hydrolase to synthesize 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid], The 4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid [4-(2,4,5-trifluorophenyl)-3-oxobutanoic acid] is reacted with a transaminase and an amino group donor, A process for producing (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid. 청구항 11에 있어서,12. The method of claim 11, 상기 가수분해효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성되는 것을 특징으로 하는 방법.The hydrolase method, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 슈도모나스(Pseudomonas)속 균주 유래의 에스터 가수분해효소, 트랜스 아미나제 및 아미노기 공여체를 포함하는, 에틸 4-(2,4,5-트리플루오로페닐)-3-옥소부 타노에이트로부터 (R)-3-아미노-4-(2,4,5-트리플루오로페닐)부탄산의 생산을 위한 조성물.From ethyl 4- (2,4,5-trifluorophenyl) -3-oxobutanoate containing an ester hydrolase, a transaminase and an amino group donor from Pseudomonas sp. strain (R)- A composition for the production of 3-amino-4-(2,4,5-trifluorophenyl)butanoic acid. 청구항 13에 있어서, 14. The method of claim 13, 상기 가수분해효소는 서열번호 1 또는 서열번호 2의 아미노산 서열로 구성되는 것을 특징으로 하는 조성물.The hydrolase composition, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
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