KR20160008883A - Adipic acid production from 6-aminocaproic acid by two step enzyme conversion - Google Patents
Adipic acid production from 6-aminocaproic acid by two step enzyme conversion Download PDFInfo
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Abstract
Description
본 발명은 아미노카프로익산(6-aminocaproic acid)에서 6-옥소헥사노에이트(6-oxohexanoate;adipate semialdehyde)를 거쳐 아디프산(adipic acid;adipate)을 만드는 두 단계 효소반응에 관한 것이다. 보다 구체적으로 아미노카프로익산에서 6-옥소헥사노에이트는 4-aminobutyrate transaminase(2.6.1.19)를, 6-옥소헥사노에이트부터 아디프산까지는 6-oxohexanoate dehydrogenase(1.2.1.63)를 이용하여 전환하는 것을 특징으로 한다. The present invention relates to a two-step enzyme reaction in which 6-oxohexanoate (adipate semialdehyde) is used to make adipic acid (adipate) in 6-aminocaproic acid. More specifically, 6-oxohexanoate in aminocaproic acid converts 4-aminobutyrate transaminase (2.6.1.19) from 6-oxohexanoate to adipic acid using 6-oxohexanoate dehydrogenase (1.2.1.63) .
바이오화학산업은 화석원료를 바이오매스로 대체하여 지속가능한 탄소경제로 전환하고 기존 화학공정을 바이오공정으로 대체하여 화학제품을 생산하고 활용하는 산업으로서, 향후 전·후방 산업의 패러다임을 전환할 수 있는 새로운 산업군으로 온실가스 및 폐기물 발생과 에너지 소비를 저감하는 친환경적 기술이다. 바이오화학산업에는 환경친화형 바이오폴리머 생산 기술, 석유화학 대체 바이오리파이너리 기술, 기능성 바이오소재 생산기술 등이 포함되며 기존 주력산업의 경쟁력 제고 및 신산업창출을 위한 산업원천기술로 주목받고 있다. 그러므로 석유자원을 기반으로 하는 정밀화학 소재나 고분자 플라스틱 소재생산산업을 바이오화학 산업으로 응용범위의 확대, 기술융합의 심화, 산업화의 진전은 21세기 세계경제를 이끌어 갈 새로운 성장 동력으로 확대 발전시켜나갈 필요성이 점차 부각되고 있다. 특히 유럽의 신(新)화학물질관리제도(REACH)와 교토의정서 등 세계적인 환경 규제 강화로 바이오 화학 산업에 대한 정부 및 기업들의 관심과 투자의 필요성이 꾸준히 증가하고 있다. 해마다 전 세계적으로 25억 킬로그램에 달하는 아디프산(adipic acid)이 생산되고, 이들 대다수가 나일론섬유의 전구체인 나일론6-6을 생산하는데 사용된다. 하지만 현재까지 대부분의 아디프산은 석유화학물질에서 생산되고, 석유화학물질들의 상당한 부분은 아산화질소의 형태로 엄청난 양의 온실가스를 방출하고 있다. 그리하여 아디프산의 환경친화적 생산이 대두되고 있고, 아직 그 영향은 미미하다. 아디프산을 생산하는 pathway 등이 제시되어 왔지만 실제 효소 공정을 통한 예는 아직 보고된 바 없다. The bio-chemical industry is an industry that converts fossil raw materials to biomass to convert to a sustainable carbon economy and replaces existing chemical processes with bio-processes to produce and utilize chemical products. It is an eco-friendly technology that reduces greenhouse gas and waste generation and energy consumption. The bio-chemical industry includes eco-friendly biopolymer production technology, alternative biorefinery technology for petrochemicals, and functional biomaterial production technology. It is attracting attention as an industry-leading technology for enhancing competitiveness of existing main industries and creating new industries. Therefore, the expansion of the application range of fine chemical materials and polymer plastic materials based on petroleum resources to the biochemical industry, the deepening of technology convergence and the advancement of industrialization will be expanded to become a new growth engine that will lead the 21st century world economy. The need is increasing. In particular, with the strengthening of global environmental regulations such as Europe's new chemical management system (REACH) and the Kyoto Protocol, the interest of the government and companies in the bio chemical industry and the need for investment are steadily increasing. Each year, around 2.5 billion kilograms of adipic acid are produced worldwide, most of which are used to produce nylon 6-6, the precursor to nylon fibers. To date, however, most adipic acid has been produced in petrochemicals, and a significant portion of its petrochemicals emit enormous amounts of greenhouse gases in the form of nitrous oxide. Thus, the environmentally friendly production of adipic acid is emerging, yet its effect is minimal. And adipic acid-producing pathways have been proposed. However, no examples have been reported through actual enzyme processes.
본 발명은 상기와 같은 문제점을 해결하기 위하여 안출된 것으로서, 아미노카프로익산에서 아디프산까지의 두 단계 효소반응의 효소를 명확히 제시하고 실제 아디프산 생성을 실시예로 보여줌을 특징으로 한다.Disclosure of Invention Technical Problem [8] The present invention is conceived to solve the above-mentioned problems, and it is an object of the present invention to provide an enzyme for two-step enzyme reaction from aminocaproic acid to adipic acid and to show actual adipic acid production.
또한, 본 발명은 상기 효소 중 아미노카프로익산으로부터 6-옥소헥사노에이트로의 반응 조건을 제공하는 것을 또 다른 목적으로 한다.It is another object of the present invention to provide conditions for the reaction of aminocaproic acid to 6-oxohexanoate in the enzyme.
또한, 본 발명은 상기 효소 중 6-옥소헥사노에이트로부터 아디프산으로의 반응 조건을 제공하는 것을 또 다른 목적으로 한다.It is another object of the present invention to provide conditions for the reaction of 6-oxohexanoate to adipic acid among the above enzymes.
본 발명은 상술한 바와 같은 목적을 달성하기 위하여, 두 가지 효소 4-aminobutyrate transaminase(2.6.1.19)와 6-oxohexanoate dehydrogenase(1.2.1.63)를 포함하는 것을 특징으로 한다.The present invention is characterized by including two enzymes 4-aminobutyrate transaminase (2.6.1.19) and 6-oxohexanoate dehydrogenase (1.2.1.63) in order to achieve the above-mentioned object.
또한, 4-aminobutyrate transaminase(2.6.1.19)는 아미노카프로익산을 6-옥소헥사노에이트로 전환할 수 있다.In addition, 4-aminobutyrate transaminase (2.6.1.19) can convert aminocaproic acid to 6-oxohexanoate.
또한, 6-oxohexanoate dehydrogenase(1.2.1.63)는 6-옥소헥사노에이트를 아디프산으로 전환할 수 있다.In addition, 6-oxohexanoate dehydrogenase (1.2.1.63) can convert 6-oxohexanoate into adipic acid.
또한, 본 발명은 각각의 단계의 개별 반응과 두 단계의 연속 반응 모두 포함하는 것을 특징으로 한다. In addition, the present invention is characterized in that it includes both an individual reaction at each step and a continuous reaction in two steps.
본 발명은 아미노카프로익산으로부터 아디프산 생산을 효소를 이용한 두 단계 반응으로 전환함으로서 기존의 화학적인 방법을 대체하고 친환경적인 효과가 있다. 그리고 전구체인 나일론6-6을 생산하게 됨으로서 나일론 생산에 새로운 pathway를 제시할 수 있다. 덧붙여, 아디프산을 전구체로 하는 모든 생촉매적 공정에 교두보가 될 수 있는 반응이다. The present invention replaces the existing chemical method by converting adipic acid production from aminocaproic acid into a two-step reaction using an enzyme, and has an eco-friendly effect. And by producing the precursor, nylon 6-6, a new pathway for nylon production can be presented. In addition, it is a reaction that can be a bridgehead for all biocatalytic processes that use adipic acid as a precursor.
도 1은 아미노카프로익산이 6-옥소헥사노에이트를 거쳐 아디프산을 만드는 두 단계 효소반응에 관한 모식도이다.1 is a schematic diagram of a two-step enzyme reaction in which an aminocaproic acid is reacted with 6-oxohexanoate to form adipic acid.
이하, 본 발명의 바람직한 실시예에 대하여 상세히 설명한다. 또한, 하기의 설명에서는 구체적인 구성요소 등과 같은 많은 특정사항들이 설명되어 있는데, 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐 이러한 특정 사항들 없이도 본 발명이 실시될 수 있음은 이 기술분야에서 통상의 지식을 가진 자에게는 자명하다 할 것이다. 그리고, 본 발명을 설명함에 있어서, 관련된 공지 기능 혹은 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, numerous specific details, such as specific elements, are set forth in order to provide a thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without these specific details, It will be obvious to those who have knowledge of. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.
본 발명은 두 단계의 효소반응을 이용하여 아미노카프로익산을 전환하여 아디프산을 생산하는 것을 목표로 한다. 대부분의 아디프산 생산 방식은 고온 고압의 조건에서 화학촉매의 사용을 통해 이루어지기 때문에 에너지 소비량이 많고 운전 시 위험성이 높으며, 설비 투자비가 높고 환경친화도가 낮은 단점들이 있다. 효소를 이용하는 경우에는 저온 저압의 안전하고 에너지 소모량이 낮은 조건에서 환경 친화적으로 반응을 진행할 수 있기 때문에 본 발명에서는 효소를 이용한 생물학적 반응을 이용한 아디프산을 생산하고자 하였다.The present invention aims at producing adipic acid by converting aminocaproic acid using two-step enzyme reaction. Most adipic acid production methods are performed through the use of chemical catalysts under high temperature and high pressure conditions, so there are disadvantages of high energy consumption, high risk of operation, high facility investment cost, and low environmental friendliness. In the case of using the enzyme, since the reaction can be carried out environmentally friendly under low temperature and low pressure safety and low energy consumption conditions, the present invention aims to produce adipic acid using the biological reaction using the enzyme.
아미노카프로익산에서 6-옥소헥사노에이트를 만드는 반응에 이용되는 효소인 4-aminobutyrate transaminase(2.6.1.19)는 카본이 4개인 4-아미노부티레이트를 기질로 반응 한다. 아민기를 떼어내는 반응은 동일하지만 실제로 카본이 6개인 아미노카프로익산에서도 동일한 작용을 하는지는 확인되지 않았다. 선행 연구 중에 아미노카프로익산에 대한 활성이 존재하는 보고는 있지만, 실제로 생성물인 6-옥소헥사노에이트를 검출한 바가 없다. 4-aminobutyrate transaminase (2.6.1.19), an enzyme used in the reaction to make 6-oxohexanoate in aminocaproic acid, reacts with 4-aminobutyrate as a substrate with 4 carbon atoms. The reaction to remove the amine group is the same, but it is not confirmed whether the action is the same even in the case of aminocaproic acid having 6 carbon atoms. Although there have been reports of activity against aminocaproic acid in previous studies, the actual product, 6-oxohexanoate, has not been detected.
6-옥소헥사노에이트에서부터 아디프산까지는 6-oxohexanoate dehydrogenase(1.2.1.63)를 이용하여 전환을 시도하였다. 이 효소의 6-옥소헥사노에이트의 활성은 cofactor의 변화량으로 검출하여 보고 되었다. 하지만 첫 번째 반응과 마찬가지로 실제로 검출은 확인되지 않았다. Conversion from 6-oxohexanoate to adipic acid was attempted using 6-oxohexanoate dehydrogenase (1.2.1.63). The activity of this enzyme, 6-oxohexanoate, was detected and reported as a change in cofactor. However, as with the first reaction, no detection was actually found.
이 두 가지의 효소를 이용하여 반응을 설계하고 반응물을 검출하는 것으로 과제를 해결하고자 하였다. 첫 번째 반응은 24시간 반응으로 6-옥소헥사노에이트의 검출과 25%의 전환율을 확인하였다. 두 번째 반응은 16시간의 반응으로 아디프산의 검출과 13%의 전환율을 확인하였다.
We designed the reaction using these two enzymes and tried to solve the problem by detecting the reactants. The first reaction was 24 hour reaction with detection of 6-oxohexanoate and 25% conversion. In the second reaction, adipic acid was detected by the reaction of 16 hours and 13% conversion was confirmed.
이하, 본 발명의 실시예에 대하여 설명한다.
Hereinafter, embodiments of the present invention will be described.
실시예Example
1. 시약1. Reagents
효소인 4-aminobutyrate transaminase(2.6.1.19)와 6-oxohexanoate dehydrogenase(1.2.1.63)의 유전자는 Bioneer (Daejeon, Korea)에서 상업적으로 합성하였다. 제한효소 (NdeI, XhoI) 및 DNA 리가아제는 Enzynomics (Daejeon, Korea)에서 구입하였다. Competent Escherichia coli Top 10과 E. coli BL21 (DE3)는 Invitrogen (Carlsbad, CA, USA)과 Novagen (Madison, WI, USA)에서 각각 구매하였다. 아디프산과 아미노카프로익산, glutamate, NADH, NAD, a-keto glutarate는 Sigma (St. Louis, MO, USA)에서 구매하였다. 6-옥소헥사노에이트는 기존 문헌을 참고하여 합성하였다(Angew Chem Int Ed Engl, 2012;51(31):7684-7687). The genes for the enzymes 4-aminobutyrate transaminase (2.6.1.19) and 6-oxohexanoate dehydrogenase (1.2.1.63) were commercially synthesized in Bioneer (Daejeon, Korea). Restriction enzymes (NdeI, XhoI) and DNA ligase were purchased from Enzynomics (Daejeon, Korea). Competent Escherichia coli Top 10 and E. coli BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA) and Novagen (Madison, Wis., USA). Adipic acid, aminocaproic acid, glutamate, NADH, NAD and a-keto glutarate were purchased from Sigma (St. Louis, MO, USA). 6-oxohexanoate was synthesized with reference to the existing literature (Angew Chem Int Ed Engl, 2012; 51 (31): 7684-7687).
2. 효소 발현 및 정제2. Enzyme expression and purification
4-aminobutyrate transaminase와 6-oxohexanoate dehydrogenase는 각각 pET-22b(+) vector와 pET-23a vector에 클로닝하였다. 클로닝된 플라스미드는 단백질 발현을 위해 competent E. coli BL21 (DE3)에 형질전환하였다. 형질전환된 competent E. coli BL21 (DE3)에 1 mM IPTG (isopropyl b-D-1-thiogalactopyranoside)를 추가로 넣고, 효소를 발현시키기 위해 총 24시간 동안 섭씨 20도에서 배양하였다. 효소는 Ni-NTA agarose column을 이용하여 정제하였고, 순도는 12% SDS-PAGE로 측정하였다. 효소 농도는 Bradford assay (Bradford, 1976)로 측정하였다. 4-aminobutyrate transaminase and 6-oxohexanoate dehydrogenase were cloned into pET-22b (+) vector and pET-23a vector, respectively. The cloned plasmid was transformed into competent E. coli BL21 (DE3) for protein expression. 1 mM IPTG (isopropyl b-D-1-thiogalactopyranoside) was added to the transformed competent E. coli BL21 (DE3) and cultured at 20 ° C. for 24 hours for expression of the enzyme. The enzyme was purified using Ni-NTA agarose column and purity was measured by 12% SDS-PAGE. Enzyme concentrations were measured by Bradford assay (Bradford, 1976).
3. 효소 반응 및 HPLC 분석3. Enzyme Reaction and HPLC Analysis
3.1 아미노카프로익산에서 6-옥소헥사노에이트로의 반응 (4-aminobutyrate transaminase)3.1 Reaction of 4-aminobutyrate transaminase with 6-oxohexanoate in aminocaproic acid
pH 8의 sodium phosphate buffer에 10mM 6-aminocaproic acid, 10mM 2-keto glutarate, 100uM cofactor PLP를 넣고, 효소는 5.0uM을 반응 조건으로 하여 총 24시간 동안 섭씨 30도에서 반응을 진행하였다. 반응이 끝난 후 섭씨 99도에서 5분간 효소의 비활성화를 진행하였다. 10 mM 6-aminocaproic acid, 10 mM 2-keto glutarate, and 100 uM cofactor PLP were added to sodium phosphate buffer at pH 8 and the reaction was conducted at 30 ° C. for 24 hours under the reaction conditions of 5.0 uM of enzyme. After the reaction was completed, the enzyme was inactivated at 99 ° C for 5 minutes.
3.2 6-옥소헥사노에이트에서 아디프산으로의 반응 (6-oxohexanoate dehydrogenase)3.2 Reaction of 6-oxohexanoate to adipic acid (6-oxohexanoate dehydrogenase)
pH 8.5 의 Tris-HCl buffer에 10mM adipate semialdehyde, 10mM NAD를 넣고, 효소는 100ul를 반응 조건으로 하여 총 16시간 동안 섭씨 30도에서 반응을 진행하였다. 반응이 끝난 후 섭씨 99도에서 5분간 효소의 비활성화를 진행하였다. 10 mM adipate semialdehyde and 10 mM NAD were added to Tris-HCl buffer (pH 8.5), and the reaction was performed at 30 ° C. for 16 hours under the reaction conditions of 100 μl of the enzyme. After the reaction was completed, the enzyme was inactivated at 99 ° C for 5 minutes.
3.3 HPLC 분석3.3 HPLC analysis
아디프산과 6-옥소헥사노에이트는 고속액체크로마토그래피(HPLC) 방법으로 분석하였다. 기계는 Thermo system을 사용하였고 검출기는 photodiode array detector, 파장은 210nm에서 진행하였다. 유기산 컬럼은 Aminex HPX-87H ion exclusion column (300 by 7.8 mm)를 사용하였고 이때 컬럼의 온도는 섭씨 60도, buffer는 0.4mM sulphamic acid, 유속은 0.6ml/min이다. Adipic acid and 6-oxohexanoate were analyzed by a high performance liquid chromatography (HPLC) method. The instrument was a Thermo system and the detector was a photodiode array detector and the wavelength was 210 nm. For the organic acid column, Aminex HPX-87H ion exclusion column (300 by 7.8 mm) was used. The temperature of the column was 60 ° C, the buffer was 0.4 mM sulphamic acid, and the flow rate was 0.6 ml / min.
4. 효소 전환 결과4. Enzyme Conversion Results
HPLC 분석결과 기준시약 기준으로 아디프산은 14분에, 6-옥소헥사노에이트는 약 18분에 검출되었다. 4-aminobutyrate transaminase로 24시간 반응 후 아미노카프로익산이 6-옥소헥사노에이트로 24.51% 전환됨을 확인하였다. 6-oxohexanoate dehydrogenase로 16시간 반응 후 6-옥소헥사노에이트가 아디프산으로 13.35% 전환됨을 확인 하였다.As a result of HPLC analysis, adipic acid was detected at 14 minutes and 6-oxohexanoate was detected at about 18 minutes based on the standard reagent. 4-aminobutyrate transaminase, 24.51% conversion of aminocaproic acid to 6-oxohexanoate was observed after 24 hours of reaction. After 16 hours of reaction with 6-oxohexanoate dehydrogenase, it was confirmed that 6-oxohexanoate was converted to adipic acid by 13.35%.
(분)Detection time
(minute)
(mAU*분)area
(mAU * min)
(분)Detection time
(minute)
(mAU*분)area
(mAU * min)
상기 표 1은 아미노카프로익산으로부터 6-옥소헥사노에이트로으로 4-aminobutyrate transaminase 반응의 결과이다. 약 18분에 6-옥소헥사노에이트가 검출된 것을 확인 하였다. Table 1 above shows the results of 4-aminobutyrate transaminase reaction from aminocaproic acid to 6-oxohexanoate. It was confirmed that 6-oxohexanoate was detected in about 18 minutes.
(분)Detection time
(minute)
(mAU*분)area
(mAU * min)
(분)Detection time
(minute)
(mAU*분)area
(mAU * min)
상기 표 2는 6-옥소헥사노에이트로부터 아디프산으로 6-oxohexanoate dehydrogenase 반응의 결과이다. 14분에 아디프산이 검출된 것을 확인 하였다.Table 2 above shows the results of 6-oxohexanoate dehydrogenase reaction from 6-oxohexanoate to adipic acid. It was confirmed that adipic acid was detected at 14 minutes.
이상에서는 본 발명의 바람직한 실시예에 대해서 설명하였으나, 본 발명은 상술한 특정의 실시예에 한정되지 아니하며, 당해 기술분야에서 통상의 지식을 가진 자라면 본원 발명의 요지를 벗어남이 없이 다양한 변형 실시가 가능함은 물론이다. 따라서, 본 발명의 범위는 위의 실시예에 국한해서 해석되어서는 안되며, 후술하는 특허청구범위 뿐만 아니라 이 특허청구범위와 균등한 것들에 의해 정해져야 할 것이다.While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course it is possible. Accordingly, the scope of the present invention should not be construed as being limited to the above-described embodiments, but should be determined by equivalents to the appended claims, as well as the following claims.
Claims (3)
상기 효소를 동시에 이용하여 6-옥사헥사노에이트나 아디프산을 생산하는 방법.
3. The method according to claim 1 or 2,
Wherein said enzyme is used simultaneously to produce 6-oxahexanoate or adipic acid.
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| Title |
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| 비특허문헌 1: Journal of Experimental Botany, 2009;60(6):1743-1757,"Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis g-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate", Shawn M. Clark, Rosa Di Leo, Preetinder K. Dhanoa, Owen R. Van Cauwenberghe, Robert T. Mullen, and Barry J. Shelp. |
| 비특허문헌 2: Applied and Environmental Microbiology, 1999;65(11):5158-5162, "Identification of a transcriptional activator (ChnR) and a 6-oxohexanoate dehydrogenase (ChnE) in the cyclohexanol catabolic pathway in Acinetobacter sp. Strain NCIMB 9871 and localization of the genes that encode them", Hiroaki Iwaki, Yoshie Hasegawa, Masahiro Teraoka, Tai Tokuyama, Tokuyama, Helene Bergeron, and Peter C. K. Lau. |
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