KR102675089B1 - Protein having synthetic activity for dipeptide and production method of dipeptide using thereof - Google Patents
Protein having synthetic activity for dipeptide and production method of dipeptide using thereof Download PDFInfo
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- KR102675089B1 KR102675089B1 KR1020240018462A KR20240018462A KR102675089B1 KR 102675089 B1 KR102675089 B1 KR 102675089B1 KR 1020240018462 A KR1020240018462 A KR 1020240018462A KR 20240018462 A KR20240018462 A KR 20240018462A KR 102675089 B1 KR102675089 B1 KR 102675089B1
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- carnosine
- protein
- present
- polypeptide
- producing
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Abstract
본 발명은 L-카르노신(L-carnosine) 합성 활성을 갖는 단백질 및 이를 이용한 L-카르노신의 생산 방법에 관한 것으로, 보다 상세하게는 서열번호 1로 표시되는 아미노산 서열로 이루어진 단리된 폴리펩티드, 및 (a) 서열번호 1로 표시되는 아미노산 서열로 이루어진 단백질을 발현하는 재조합 균주를 상기 단백질을 생산하는데 적합한 시간 및 조건 하에 배지에서 배양하는 단계; (b) 상기 배양물에 i) 글루코스(glucose) 및 베타 알라닌(β-alanine) 중 어느 하나; 및 ii) L-히스티딘(L-histidine)을 혼합하는 단계; 및 (c) L-카르노신(L-carnosine)을 수득하는 단계;를 포함하는 L-카르노신(L-carnosine)의 생산 방법에 관한 것이다. 본 발명에 따른 L-카르노신 합성 활성이 있는 단백질 및 이를 이용한 L-카르노신의 생산 방법은 본 발명자가 새롭게 구축한 것으로서 L-카르노신의 생산 효율이 뛰어나므로 이의 생산에 유용하게 이용될 수 있다.The present invention relates to a protein having L-carnosine synthesis activity and a method for producing L-carnosine using the same, and more specifically, to an isolated polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and ( a) cultivating a recombinant strain expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in a medium under time and conditions suitable for producing the protein; (b) i) any one of glucose and beta-alanine; and ii) mixing L-histidine; and (c) obtaining L-carnosine. The protein with L-carnosine synthesis activity according to the present invention and the production method of L-carnosine using the same were newly constructed by the present inventor and have excellent production efficiency of L-carnosine, so they can be usefully used for its production.
Description
본 발명은 L-카르노신(L-carnosine) 합성 활성을 갖는 단백질 및 이를 이용한 L-카르노신의 생산 방법에 관한 것으로, 보다 상세하게는 서열번호 1로 표시되는 아미노산 서열로 이루어진 단리된 폴리펩티드, 및 (a) 서열번호 1로 표시되는 아미노산 서열로 이루어진 단백질을 발현하는 재조합 균주를 상기 단백질을 생산하는데 적합한 시간 및 조건 하에 배지에서 배양하는 단계; (b) 상기 배양물에 i) 글루코스(glucose) 및 베타 알라닌(β-alanine) 중 어느 하나 이상; 및 ii) L-히스티딘(L-histidine)을 혼합하는 단계; 및 (c) L-카르노신(L-carnosine)을 수득하는 단계;를 포함하는 L-카르노신(L-carnosine)의 생산 방법에 관한 것이다.The present invention relates to a protein having L-carnosine synthesis activity and a method for producing L-carnosine using the same, and more specifically, to an isolated polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and ( a) cultivating a recombinant strain expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in a medium under time and conditions suitable for producing the protein; (b) the culture containing i) at least one of glucose and beta-alanine; and ii) mixing L-histidine; and (c) obtaining L-carnosine.
L-카르노신(L-carnosine, β-alanyl-L-histidine, Cas No. 305-84-0)은 아미노산인 β-알라닌과 히스티딘으로 구성된 디펩티드로, C9H14N4O3의 화학식을 갖는 화합물이며, 체내의 간에서 자연적으로 합성되어 뇌, 근육 및 눈의 수정체에 주로 분포한다. L-카르노신은 활성 산소종과 전이 금속 이온을 제거하는 강력한 항산화제로 알려져 있고, 아세트알데히드, 포름알데히드 및 지질 과산화의 주요 산물인 말론알데히드와 같은 독성 알데히드와 차아염소산 음이온에 의해 유도되는 단백질-단백질 및 단백질-DNA cross-link를 차단하며, 또한, 알도스 및 케토오스 환원당에 의해 유도되는 비효소성 단백질 당화를 억제하고 독성 최종 당화 산물(advanced glycation end products, AGEs)의 형성을 억제한다(Hipkiss AR et al,. "Carnosine: can understanding its actions on energy metabolism and protein homeostasis inform its therapeutic potential?" Chem Cent J. 2013 Feb 25;7(1):38.)L-carnosine (β-alanyl-L-histidine, Cas No. 305-84-0) is a dipeptide composed of the amino acids β-alanine and histidine, with the chemical formula C 9 H 14 N 4 O 3 It is a compound that is naturally synthesized in the liver of the body and is mainly distributed in the brain, muscles, and lens of the eye. L-carnosine is known as a powerful antioxidant that scavenges reactive oxygen species and transition metal ions, and is a protein-protein and It blocks protein-DNA cross-links, and also inhibits non-enzymatic protein glycation induced by aldose and ketose reducing sugars and inhibits the formation of toxic advanced glycation end products (AGEs) (Hipkiss AR et al,. "Carnosine: can understanding its actions on energy metabolism and protein homeostasis inform its therapeutic potential?"
이에 따라 운동 능력을 향상시키거나 노화 방지 등 다양한 목적의 식이 보충제부터, 피부 질환, 암, 알츠하이머병, 파킨슨 병 및 당뇨병을 비롯한 여러 가지 질병의 잠재적인 치료제 및 화장품 제제로서 널리 이용되고 있어, 식품, 의약, 사료 산업 등 다양한 분야에서 그 필요성과 수요가 점점 증가하는 추세이다.Accordingly, it is widely used as a dietary supplement for various purposes such as improving exercise ability or preventing aging, as a potential treatment for various diseases including skin disease, cancer, Alzheimer's disease, Parkinson's disease, and diabetes, and as a cosmetic agent. The need and demand are increasing in various fields such as medicine and feed industry.
이러한 L-카르노신의 산업적인 생산을 위하여, 종래에는 산무수물화 된 β-알라닌 및 유기 용매에 용해된 L-히스티딘에 산성화제를 첨가하거나(등록특허 제10-0739448호), L-히스티딘 또는 그 유도체와 시아노아세트산에스테르를 반응시키는 방법(WO 2001/064638 A1)을 비롯한 다양한 화학적인 방법이 이용되었으며, 그 밖에 베타-아미노펩티다아제(β-aminopeptidase)를 이용한 효소적인 방법(She, Jiajia et al. "Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome" Microbial cell factories vol. 21,1 129. 27 Jun. 2022)이 알려져 있다. 그러나, 상기 화학적 방법의 경우 화학 폐기물 및 유기 용매로 인한 환경 오염의 문제가 발생하고, 유기 용매 제거를 위한 후처리 공정에 의해 제조 비용이 높으며, 수율이 낮다는 단점이 있어, 종래의 L-카르노신 합성 방법을 대체할 수 있고 친환경적이며 높은 수율을 갖는 새로운 합성 기술의 개발이 필요하다.For the industrial production of L-carnosine, an acidifying agent was conventionally added to acid anhydride β-alanine and L-histidine dissolved in an organic solvent (Registration Patent No. 10-0739448), or L-histidine or its Various chemical methods were used, including a method of reacting a derivative with a cyanoacetic acid ester (WO 2001/064638 A1), and an enzymatic method using beta-aminopeptidase (She, Jiajia et al. “Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome” Microbial cell factories vol. 21,1 129. 27 Jun. However, in the case of the above chemical method, problems of environmental pollution due to chemical waste and organic solvents occur, manufacturing costs are high due to a post-treatment process to remove organic solvents, and the yield is low, so the conventional L-carnodine method has the disadvantages of There is a need to develop new synthesis technologies that can replace new synthesis methods, are environmentally friendly, and have high yields.
이에 본 발명자들은 L-카르노신 생산을 위한 신규한 합성 효소를 탐색하던 중, Bacillus altitudinis에서 유래한 ATP-grasp domain-containing protein 효소에 L-카르노신 생산능이 있음을 확인하였고, 여기에 3차원 구조분석을 통해 일부 아미노산에 돌연변이를 도입한 결과, 이의 L-카르노신의 생산 능력이 크게 향상되었음을 확인하고 본 발명을 완성하였다. Accordingly, while searching for a new synthetic enzyme for the production of L-carnosine, the present inventors confirmed that the ATP-grasp domain-containing protein enzyme derived from Bacillus altitudinis has the ability to produce L-carnosine, and presented a three-dimensional structure here. Through analysis, it was confirmed that the L-carnosine production ability was significantly improved by introducing mutations into some amino acids, and the present invention was completed.
따라서, 본 발명의 목적은 서열번호 1로 표시되는 아미노산 서열로 이루어진 단리된 폴리펩티드를 제공하는 것이다. Therefore, the object of the present invention is to provide an isolated polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
본 발명의 다른 목적은 상기 폴리펩티드를 암호화하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현 벡터, 및 상기 발현 벡터를 포함하는 숙주 세포를 제공하는 것이다. Another object of the present invention is to provide a polynucleotide encoding the polypeptide, an expression vector containing the polynucleotide, and a host cell containing the expression vector.
본 발명의 또 다른 목적은 상기 폴리펩티드 및 상기 숙주 세포 중 어느 하나 이상을 포함하는 L-카르노신 생산용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for producing L-carnosine containing at least one of the polypeptide and the host cell.
본 발명의 또 다른 목적은 (a) 서열번호 1로 표시되는 아미노산 서열로 이루어진 단백질을 발현하는 재조합 균주를 상기 단백질을 생산하는데 적합한 시간 및 조건 하에 배지에서 배양하는 단계; (b) 상기 배양물에 i) 글루코스(glucose) 및 베타 알라닌(β-alanine) 중 어느 하나 이상, 및 ii) L-히스티딘(L-histidine)을 혼합하는 단계; 및 (c) L-카르노신(L-carnosine)을 수득하는 단계; 를 포함하는 L-카르노신(L-carnosine)의 생산 방법을 제공하는 것이다.Another object of the present invention is (a) culturing a recombinant strain expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in a medium under time and conditions suitable for producing the protein; (b) mixing i) one or more of glucose and beta-alanine, and ii) L-histidine into the culture; and (c) obtaining L-carnosine; To provide a method for producing L-carnosine containing.
상기와 같은 목적을 달성하기 위하여, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 단리된 폴리펩티드를 제공한다. In order to achieve the above object, the present invention provides an isolated polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 폴리펩티드를 암호화하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현 벡터, 및 상기 발현 벡터를 포함하는 숙주 세포를 제공한다. In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the polypeptide, an expression vector containing the polynucleotide, and a host cell containing the expression vector.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 폴리펩티드 및 상기 숙주 세포 중 어느 하나 이상을 포함하는 L-카르노신 생산용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a composition for producing L-carnosine containing at least one of the polypeptide and the host cell.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 (a) 서열번호 1로 표시되는 아미노산 서열로 이루어진 단백질을 발현하는 재조합 균주를 상기 단백질을 생산하는데 적합한 시간 및 조건 하에 배지에서 배양하는 단계; (b) 상기 배양물에 i) 글루코스(glucose) 및 베타 알라닌(β-alanine) 중 어느 하나 이상, 및 ii) L-히스티딘(L-histidine)을 혼합하는 단계; 및 (c) L-카르노신(L-carnosine)을 수득하는 단계; 를 포함하는 L-카르노신(L-carnosine)의 생산 방법을 제공한다.In order to achieve another object of the present invention, the present invention includes the steps of (a) culturing a recombinant strain expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in a medium under time and conditions suitable for producing the protein; (b) mixing i) one or more of glucose and beta-alanine, and ii) L-histidine into the culture; and (c) obtaining L-carnosine; It provides a method for producing L-carnosine including.
이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 β-알라닌(β-alanine)과 L-히스티딘(L-histidine)이 결합된, L-카르노신(L-carnosine) 생합성 효소에 관한 것으로서, 상기 효소는 이하의 아미노산 서열로 이루어진 단백질을 의미한다.The present invention relates to an L-carnosine biosynthetic enzyme combining β-alanine and L-histidine, which enzyme produces a protein consisting of the following amino acid sequence: it means.
서열번호 1 :SEQ ID NO: 1:
MSKKTVLVIADLGGCPPHMFYESVAASYHIVSYIPRPFAITKGHAELIEKYSIAVIKDRDYFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTN E ELFIAPMAKAAERLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPTYLASSIGVTLFHDKAGSDDLFLQVQSYLETIPVPDAVTYEAPFVAETYLEGAYEDWYEDEGYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEGLGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKAVLPKQLLSGHTFYVADC K LYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVSQSFPTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKGMSKKTVLVIADLGGCPPHMFYESVAASYHIVSYIPRPFAITKGHAELIEKYSIAVIKDRDYFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTN EELFIAPMAKAAERLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPTYLASSIGVTLFHDKAGSDDLFLQVQSYLETIPV PDAVTYEAPFVAETYLEGAYEDWYEDEGYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEGLGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKAVLPKQLLSGHTFYVADC K LYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVS QSFPTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKG
상기 단백질은 Bacillus altitudinis에서 유래한 야생형의 단백질로서, 서열번호 2의 아미노산 서열을 갖는 단백질인 ATP-grasp domain-containing protein (WP_045033870)에, 상기 밑줄로 표시된 2개의 아미노산에 변이(108번째 아미노산인 아스파라긴(N)을 글루탐산(E)으로, 378번째 아미노산인 히스티딘(H)을 라이신(K)으로 변이)를 도입한 것으로서, 그 결과 상기 단백질의 L-카르노신 생성능이 돌연변이가 도입되기 전과 비교하여 141배로 크게 향상되었음을 확인하였다.The protein is a wild-type protein derived from Bacillus altitudinis . ATP-grasp domain-containing protein (WP_045033870), which is a protein with the amino acid sequence of SEQ ID NO: 2, has mutations in the two underlined amino acids (asparagine, the 108th amino acid). (N) to glutamic acid (E) and histidine (H), the 378th amino acid, to lysine (K)) were introduced, and as a result, the L-carnosine production ability of the protein increased by 141 compared to before the mutation was introduced. It was confirmed that it was greatly improved twofold.
서열번호 2 :SEQ ID NO: 2:
MSKKTVLVIADLGGCPPHMFYESVAASYHIVSYIPRPFAITKGHAELIEKYSIAVIKDRDYFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTNNELFIAPMAKAAERLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPTYLASSIGVTLFHDKAGSDDLFLQVQSYLETIPVPDAVTYEAPFVAETYLEGAYEDWYEDEGYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEGLGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKAVLPKQLLSGHTFYVADCHLYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVSQSFPTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKGMSKKTVLVIADLGGCPPHMFYESVAASYHIVSYIPRPFAITKGHAELIEKYSIAVIKDRDYFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTN ELFIAPMAKAAERLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPTYLASSIGVTLFHDKAGSDDLFLQVQSYLETIPV PDAVTYEAPFVAETYLEGAYEDWYEDEGYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEGLGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKAVLPKQLLSGHTFYVADC H LYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVS QSFPTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKG
따라서, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 단리된 폴리펩티드를 제공한다. Accordingly, the present invention provides an isolated polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
본 발명에서 상기 β-알라닌(beta-alanine, β-alanine)은 β탄소에 아미노기가 결합하고 있는 자연적으로 생성되는 β-아미노산으로, IUPAC 명명법으로는 3-아미노프로판산(3-aminopropanoic acid)이라고 한다. β-알라닌은 주요 단백질이나 효소에서 발견되지 않으며, 자연적으로 생성되는 펩티드인 카르노신과 안세린(anserine)의 구성 성분이다. 또한, 조효소 A의 구성 요소인 판토텐산(비타민 B5)의 구성 성분으로 알려져 있고, 정상 상태에서는 아세트산으로 대사된다. In the present invention, β-alanine (beta-alanine) is a naturally occurring β-amino acid with an amino group bonded to the β carbon, and is called 3-aminopropanoic acid in IUPAC nomenclature. do. β-Alanine is not found in major proteins or enzymes, but is a component of the naturally occurring peptides carnosine and anserine. In addition, it is known to be a component of pantothenic acid (vitamin B5), a component of coenzyme A, and is metabolized into acetic acid under normal conditions.
본 발명에서 상기 히스티딘(L-histidine)은 20개의 아미노산 중 하나로서, 필수 아미노산이므로 인체에서 동화작용으로 합성되지 않아 인간과 같은 동물들은 반드시 이를 포함하는 단백질을 섭취해야 한다. 히스티딘은 생물학적 활성을 가지는 다른 아민으로 변환되기도 하는데, 구체적으로 일종의 면역 자극제로써 염증 반응에서 중요한 역할을 하는 히스타민(histamine)의 전구물질이다. 또한, 히스티딘은 3-메틸 히스티딘으로 전환될 수 있는데, 이는 골격근 손상을 나타내는 생물 지표가 되며 특정 메틸기 전달 효소가 작용한다. In the present invention, histidine (L-histidine) is one of the 20 amino acids. Since it is an essential amino acid, it is not synthesized through anabolism in the human body, so animals such as humans must consume proteins containing it. Histidine can also be converted into other amines with biological activity, and is specifically a precursor to histamine, which plays an important role in inflammatory reactions as a type of immune stimulant. Additionally, histidine can be converted to 3-methyl histidine, which is a biomarker for skeletal muscle damage, upon the action of certain methyl transfer enzymes.
상기 β-알라닌과 히스티딘은 L-아미노산 리가아제(L-amino acid ligase, Lal)인 본 발명에 따른 폴리펩티드의 촉매 작용(도 1 참조)에 의하여 하기의 화학 구조를 갖는 화합물인 L-카르노신(L-carnosine)으로 합성되며, 본 발명자가 확인한 결과 그 합성 효율이 매우 우수하였다.The β-alanine and histidine are converted to L-carnosine (L-carnosine, a compound having the following chemical structure) by the catalytic action of the polypeptide according to the present invention, which is L-amino acid ligase (Lal) (see Figure 1). L-carnosine), and the present inventor confirmed that its synthesis efficiency was very excellent.
본 명세서 사용된 용어 '폴리펩티드(polypeptide)' 및 '단백질'은 통상의 의미에 따라 사용되는 것으로, 즉 아미노산 잔기의 중합체를 의미한다. 폴리펩티드는 특정의 길이로 한정되지 않지만, 본 발명의 문맥에서는 일반적으로 전장(full length) 단백질의 단편을 지칭하는 것일 수 있다. 상기 폴리펩티드 또는 단백질은 번역 후의 수식, 예를 들면 글리코실화, 아세틸화, 인산화 등 및 해당 분야에 공지된 다른 수식(자연적으로 발생하는 수식 및 비자연적 발생의 수식)을 포함할 수 있다. 본 발명의 폴리펩티드 및 단백질은 임의의 다양한 공지의 재조합 및/또는 합성의 기술을 이용하여 제조될 수 있다.As used herein, the terms 'polypeptide' and 'protein' are used according to their usual meaning, that is, they mean a polymer of amino acid residues. Polypeptide is not limited to a particular length, but in the context of the present invention may generally refer to a fragment of a full length protein. The polypeptide or protein may include post-translational modifications, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art (both naturally occurring and non-naturally occurring). Polypeptides and proteins of the present invention can be produced using any of a variety of known recombinant and/or synthetic techniques.
본 명세서에 사용된 아미노산의 일문자(삼문자)는 생화학 분야에서의 표준 약어 규정에 따라 다음의 아미노산을 의미한다: A(Ala): 알라닌; C(Cys): 시스테인; D(Asp):아스파르트산; E(Glu): 글루탐산; F(Phe): 페닐알라닌; G(Gly): 글라이신; H(His): 히스티딘; I(IIe): 이소류신; K(Lys): 라이신; L(Leu): 류신; M(Met): 메티오닌; N(Asn): 아스파라긴; O(Ply)피롤라이신; P(Pro): 프롤린; Q(Gln): 글루타민; R(Arg): 아르기닌; S(Ser): 세린; T(Thr): 트레오닌; U(Sec):셀레노시스테인, V(Val): 발린; W(Trp): 트립토판; Y(Tyr): 티로신.As used herein, the single letter (triple letter) of amino acids refers to the following amino acids according to standard abbreviation conventions in the field of biochemistry: A (Ala): alanine; C(Cys): Cysteine; D(Asp): Aspartic acid; E(Glu): glutamic acid; F(Phe): Phenylalanine; G(Gly): glycine; H(His): histidine; I(IIe): Isoleucine; K(Lys): Lysine; L(Leu): leucine; M(Met): methionine; N(Asn): Asparagine; O(Ply)pyrrolysine; P(Pro): Proline; Q(Gln): Glutamine; R(Arg): arginine; S(Ser): Serine; T(Thr): Threonine; U(Sec): selenocysteine, V(Val): valine; W(Trp): tryptophan; Y(Tyr): Tyrosine.
한편, 본 발명에 따른 상기 폴리펩티드는 해당 분야의 숙련자에게 공지된 임의의 적합한 절차, 즉 유전공학적 방법, 예컨대 재조합 기법에 의해 제조(제작)될 수 있다. 예를 들면, 통상적인 방법에 따라 상기 폴리펩티드 또는 이의 기능적 동등물을 암호화하는 핵산을 제작한다. 상기 핵산은 적절한 프라이머를 사용하여 PCR 증폭함으로써 제작할 수 있다. 다른 방법으로 당업계에 공지된 표준 방법, 예컨대, 자동 DNA 합성기(Biosearch 또는 Applied Biosystems 사에서 판매하는 것)을 사용하여 DNA 서열을 합성할 수도 있다. 제작된 핵산은 이에 작동 가능하게 연결되어 (operatively linked) 핵산의 발현을 조절하는 하나 이상의 발현 조절 서열(expression control sequence)(예: 프로모터, 인핸서 등)을 포함하는 벡터에 삽입시키고, 이로부터 형성된 재조합 발현 벡터로 숙주세포를 형질전환 시킨다. 생성된 형질전환체를 상기 핵산이 발현되기에 적절한 배지 및 조건 하에서 배양하여, 배양물로부터 상기 핵산에 의해 발현된, 실질적으로 순수한 폴리펩티드를 회수한다. 상기 회수는 당업계에 공지된 방법(예컨대, 크로마토그래피)을 이용하여 수행할 수 있다. 상기에서 '실질적으로 순수한 폴리펩티드(substantially pure polypeptide)'라 함은 본 발명에 따른 폴리펩티드가 숙주세포로부터 유래된 어떠한 다른 단백질도 실질적으로 포함하지 않는 것을 의미한다.Meanwhile, the polypeptide according to the present invention can be produced (manufactured) by any suitable procedure known to those skilled in the art, that is, genetic engineering methods, such as recombinant techniques. For example, a nucleic acid encoding the polypeptide or its functional equivalent is prepared according to a conventional method. The nucleic acid can be produced by PCR amplification using appropriate primers. Alternatively, DNA sequences can be synthesized using standard methods known in the art, such as automated DNA synthesizers (available from Biosearch or Applied Biosystems). The prepared nucleic acid is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) that are operably linked to control the expression of the nucleic acid, and the recombination formed therefrom Transform host cells with expression vectors. The resulting transformant is cultured under media and conditions appropriate for expression of the nucleic acid, and substantially pure polypeptide expressed by the nucleic acid is recovered from the culture. The recovery can be performed using methods known in the art (eg, chromatography). In the above, 'substantially pure polypeptide' means that the polypeptide according to the present invention substantially does not contain any other proteins derived from host cells.
또한, 본 발명의 폴리펩티드는 재조합 제조 방법 뿐만 아니라 당업계에 공지된 화학적 합성 방법에 의해 제조될 수 있다. 대표적인 방법으로서 이들로 한정되는 것은 아니지만 액체 또는 고체상 합성, 단편 응축, F-MOC 또는 T-BOC 화학법이 포함된다.Additionally, the polypeptide of the present invention can be produced by recombinant production methods as well as chemical synthesis methods known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.
일례로 본 발명의 폴리펩티드는 고체상 기법을 이용한 직접적 펩티드 합성에 의해 제조될 수 있다(Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). 고체상 펩티드 합성(SPPS) 방법은 작은 다공성의 비드(beads)에 링커(linkers)라 불리는 기능성 유닛(functional units)을 부착하여 펩티드 사슬을 이어 나갈 수 있도록 유도함으로써 합성을 개시할 수 있다. 액체상 방법과 달리 펩티드는 비드와 공유 결합하여 TFA (trifluoroacetic acid)와 같은 특정 반응물에 의해 절단되기 전까지 여과(filtration) 과정에 의해 떨어져 나가는 것을 방지한다. 고체상에 부착된 펩티드의 N-말단 아민과 N-보호 아미노산 유닛(N-protected amino acid unit)이 결합하는 보호(protection) 과정, 탈보호(deprotection) 과정, 다시 드러난 아민 그룹(amine group)과 새로운 아미노산이 결합하는 커플링(coupling) 과정의 사이클(cycle, deprotection-wash-coupling-wash)이 반복되면서 합성이 이루어지게 된다. 상기 SPPS 방법은 마이크로파(microwave) 기술을 함께 이용하여 수행할 수 있으며, 마이크로파 기술은 펩티드 합성 과정에서 열을 가해줌으로써 각 사이클의 커플링과 탈보호에 필요 시간을 단축시킬 수 있다. 상기 열 에너지는 확장되는 펩티드 사슬이 접히거나(folding) 집합체를 형성하는 것(aggregation)을 방지하고 화학적 결합을 촉진시킬 수 있다.For example, the polypeptide of the present invention can be prepared by direct peptide synthesis using solid phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). The solid-phase peptide synthesis (SPPS) method can initiate synthesis by attaching functional units called linkers to small porous beads to connect the peptide chain. Unlike liquid phase methods, the peptide is covalently bonded to the beads and is prevented from falling off during the filtration process until it is cleaved by a specific reactant such as TFA (trifluoroacetic acid). The protection process, deprotection process, which combines the N-terminal amine of the peptide attached to the solid phase with the N-protected amino acid unit, the re-revealed amine group and the new Synthesis occurs by repeating the cycle of coupling process (deprotection-wash-coupling-wash) in which amino acids combine. The SPPS method can be performed using microwave technology, which can shorten the time required for coupling and deprotection of each cycle by applying heat during the peptide synthesis process. The heat energy can prevent folding or aggregation of the extended peptide chain and promote chemical bonding.
또한, 액체상 펩티드 합성법에 의해 본 발명의 펩티드를 제작할 수 있으며, 이의 구체적 방법은 하기의 문헌들을 참조로 한다: US 등록특허 제 5,516,891호. 또한 본 발명의 펩티드는 상기 고체상 합성법과 액체상 합성법을 혼합하는 방법 등의 다양한 방법으로 합성 가능하며, 본 명세서에 기술된 수단에 그 제조 방법이 제한되지 않는다. In addition, the peptide of the present invention can be produced by liquid phase peptide synthesis, and for specific methods, refer to the following documents: US Patent No. 5,516,891. In addition, the peptide of the present invention can be synthesized by various methods, such as a method of mixing the solid phase synthesis method and the liquid phase synthesis method, and the production method is not limited to the means described herein.
단백질 합성은 수동 기법을 이용해서 또는 자동화에 의해 수행될 수 있다. 자동화된 합성은, 예를 들어 Applied Biosystems 431A 펩티드 합성기(Perkin Elmer)를 이용해서 달성될 수 있다. 대안적으로, 다양한 단편이 별도로 화학적으로 합성되고 화학적 방법을 이용하여 조합되어 목적 분자를 제조할 수 있다.Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished using, for example, the Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and combined using chemical methods to produce the target molecule.
본 발명에 따른 폴리펩티드의 범위에는 전술한 본 발명 폴리펩티드의 기능적 동등물 및 그들의 염을 포함한다. 상기 '기능적 동등물'이란 전술한 본 발명의 폴리펩티드와 적어도 80% 이상의, 바람직하게는 90%, 더욱 바람직하게는 95% 이상의 아미노산기 서열 상동성(즉, 동일성)을 갖는 것으로 예를 들면, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%의 서열 상동성을 갖는 것을 포함하며, 본 발명의 폴리펩티드와 실질적으로 동질의 생리활성을 나타내는 펩티드를 말한다. 상기에서 '실질적으로 동질의 생리활성'이란, 이에 제한되지 않으나, 일례로 β-알라닌 및 L-히스티딘으로부터 L-카르노신을 합성하는 능력을 의미하는 것일 수 있다.The scope of polypeptides according to the present invention includes functional equivalents of the above-described polypeptides of the present invention and their salts. The term 'functional equivalent' refers to an amino acid sequence homology (i.e., identity) of at least 80% or more, preferably 90%, more preferably 95% or more, with the polypeptide of the present invention described above, for example, 80% or more. %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, It refers to a peptide that has 97%, 98%, 99%, and 100% sequence homology, and exhibits substantially the same physiological activity as the polypeptide of the present invention. In the above, 'substantially homogeneous physiological activity' is not limited thereto, but may mean, for example, the ability to synthesize L-carnosine from β-alanine and L-histidine.
하나의 실시 양태에서, 본 발명에서 기능적 동등물은 전술한 본 발명 폴리펩티드의 아미노산 서열 중 일부가 부가, 삽입, 치환(비보전적 또는 보전적 치환), 결실 또는 이들의 조합에 의해 생성된 것일 수 있다. 상기에서 아미노산의 치환은 바람직하게는 보존적 치환일 수 있다. 천연에 존재하는 아미노산의 보존적 치환의 예는 다음과 같다; 지방족 아미노산(Gly, Ala, Pro), 소수성 아미노산(Ile, Leu, Val), 방향족 아미노산(Phe, Tyr, Trp), 산성 아미노산(Asp, Glu), 염기성 아미노산(His, Lys, Arg, Gln, Asn) 및 황함유 아미노산(Cys, Met). 분자의 활성을 전체적으로 변경시키지 않는 아미노산 교환은 당해 분야에 공지되어 있다(H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 또한 상기 기능적 동등물에는, 본 발명 폴리펩티드의 아미노산 서열상에서 아미노산의 일부가 결실된 변형체도 포함된다. 상기 아미노산의 결실 또는 치환은 바람직하게는 본 발명에서 제공하는 폴리펩티드의 생리활성에 직접적으로 관련되지 않은 영역에 위치해 있다. 아울러 상기 본 발명 폴리펩티드의 아미노산 서열의 양 말단 또는 서열 내에 몇몇의 아미노산이 부가된 변형체도 포함된다. 상기에서 부가된 아미노산이란, 예를 들어 단백질 분리/정제를 위한 히스티딘 태그(Histidine-tag)로서 '-GSHHHHHH' 서열일 수 있다.In one embodiment, the functional equivalent in the present invention may be one produced by addition, insertion, substitution (non-conservative or conservative substitution), deletion, or a combination thereof of some of the amino acid sequences of the above-described polypeptide of the present invention. . In the above, amino acid substitution may preferably be a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) ) and sulfur-containing amino acids (Cys, Met). Amino acid exchanges that do not overall alter the activity of the molecule are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The functional equivalents also include variants in which some amino acids are deleted in the amino acid sequence of the polypeptide of the present invention. The deletion or substitution of the amino acid is preferably located in a region not directly related to the physiological activity of the polypeptide provided by the present invention. In addition, variants in which several amino acids are added to both ends of the amino acid sequence of the polypeptide of the present invention or within the sequence are also included. The amino acid added above may be, for example, a '-GSHHHHHH' sequence as a histidine tag for protein separation/purification.
또한, 상기 기능적 동등물의 범위에는 폴리펩티드의 기본 골격 및 이의 생리 활성을 유지하면서 폴리펩티드의 일부 화학 구조가 변형된 폴리펩티드 유도체도 포함된다. 예를 들어, 본 발명의 폴리펩티드의 안정성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한 구조변경 및 생리활성을 유지하면서 다른 단백질과 융합으로 만들어진 융합단백질 등이 이에 포함된다.In addition, the scope of the functional equivalents also includes polypeptide derivatives in which part of the chemical structure of the polypeptide is modified while maintaining the basic skeleton of the polypeptide and its physiological activity. For example, these include structural changes to change the stability, storage, volatility, or solubility of the polypeptide of the present invention, and fusion proteins made by fusing with other proteins while maintaining physiological activity.
하나의 실시 양태에서, 본 발명의 폴리펩티드는 경우에 따라 인산화(phosphorylation), 황화(sulfation), 아크릴화(acrylation), 당화(glycosylation), 메틸화(methylation), 파네실화(farnesylation) 등으로 수식(modification)될 수도 있다.In one embodiment, the polypeptide of the present invention is optionally modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. It could be.
또한, 본 발명은 상기 본 발명의 폴리펩티드를 암호화(코딩)하는 폴리뉴클레오티드를 제공한다.Additionally, the present invention provides a polynucleotide encoding the polypeptide of the present invention.
상기 폴리뉴클레오티드는 본 발명의 폴리펩티드를 암호화할 수 있는 한 폴리뉴클레오티드의 염기 조합이 특별히 제한되지 않는다. 상기 폴리뉴클레오티드는 DNA, cDNA 및 RNA 서열을 모두 포함하여 단쇄 또는 이중쇄의 형태의 핵산 분자로서 제공될 수 있다.The base combination of the polynucleotide is not particularly limited as long as it can encode the polypeptide of the present invention. The polynucleotide may be provided as a nucleic acid molecule in the form of a single strand or a double strand, including all DNA, cDNA, and RNA sequences.
본 명세서에서 사용된 용어 '폴리뉴클레오티드', '핵산'은 단일-가닥 또는 이중-가닥의 형태로 된 데옥시리보뉴클레오티드(DNA) 또는 리보뉴클레오티드(RNA)를 말한다. 다른 제한이 없는한, 자연적으로 생성되는 뉴클레오티드와 비슷한 방법으로 핵산에 혼성화되는 자연적 뉴클레오티드의 공지된 아날로그도 포함된다.As used herein, the terms 'polynucleotide' and 'nucleic acid' refer to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single-stranded or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.
본 발명의 폴리펩티드를 코딩하는 핵산 서열은 이를 발현할 수 있는 벡터에 작동적으로 연결시켜 상기 본 발명의 폴리펩티드를 제공할 수 있다. 따라서, 본 발명은 상기 폴리뉴클레오티드를 포함하는 발현 벡터 또는 재조합 벡터를 제공한다.A nucleic acid sequence encoding a polypeptide of the invention can be operably linked to a vector capable of expressing it to provide the polypeptide of the invention. Accordingly, the present invention provides an expression vector or recombinant vector containing the above polynucleotide.
본 발명에서 용어 '발현 벡터' 또는 '재조합 벡터'란 적당한 숙주 세포에서 목적 단백질 또는 목적 RNA을 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동 가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 말한다.In the present invention, the term 'expression vector' or 'recombinant vector' refers to a vector capable of expressing a target protein or target RNA in a suitable host cell, and refers to a gene construct containing essential regulatory elements operably linked to express the gene insert. .
상기 용어 '작동 가능하게 연결된(operably linked)'는 일반적 기능을 수행하도록 핵산 발현 조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 것을 말한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동 가능하게 연결되어 코딩하는 핵산 서열의 발현에 영향을 미칠 수 있다. 재조합 벡터와의 작동적 연결은 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다.The term 'operably linked' refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect expression of the encoding nucleic acid sequence. Operational linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be done using enzymes generally known in the art.
본 발명의 일 실시예에서는 pET28a 벡터를 이용하였으나, 이에 제한되는 것은 아니며, 그 밖에 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터 및 바이러스 벡터 등을 이용할 수 있다. 적합한 발현 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 등과 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 또한 발현 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함하고, 복제 가능한 발현 벡터인 경우 복제 기원을 포함할 수 있다.In one embodiment of the present invention, the pET28a vector was used, but it is not limited thereto, and other plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. can be used. A suitable expression vector includes expression control elements such as a promoter, operator, start codon, stop codon, polyadenylation signal, and enhancer, as well as a signal sequence or leader sequence for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The expression vector may also include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, may include an origin of replication.
시그널 서열에는 숙주가 에스케리치아 속(Escherichia sp.) 균인 경우에는 PhoA 시그널 서열, OmpA 시그널 서열 등이, 숙주가 바실러스속 균인 경우에는 α-아밀라아제 시그널 서열, 서브틸리신 시그널 서열 등이, 숙주가 효모인 경우에는 MFα 시그널 서열, SUC2 시그널 서열 등이, 숙주가 동물세포인 경우에는 인슐린 시그널서열, α-인터페론 시그널 서열, 항체 분자 시그널 서열 등을 이용할 수 있으나, 이에 제한되지 않는다.The signal sequence includes the PhoA signal sequence and OmpA signal sequence when the host is a genus Escherichia sp., and the α-amylase signal sequence and subtilisin signal sequence when the host is a bacterium of the genus Bacillus. In the case of yeast, the MFα signal sequence, SUC2 signal sequence, etc. can be used, and if the host is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, but are not limited thereto.
또한, 본 발명은 상기 발현 벡터를 포함하는 숙주 세포를 제공한다. 즉, 본 발명은 상기 발현 벡터(재조합 벡터)로 형질전환된 형질전환체(숙주 세포)를 제공한다.Additionally, the present invention provides a host cell containing the expression vector. That is, the present invention provides a transformant (host cell) transformed with the expression vector (recombinant vector).
상기 형질전환은 핵산을 유기체, 세포, 조직 또는 기관에 도입할 수 있는 것으로 공지된 것이라면 어떤 방법이라도 사용 가능하며, 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 그 방법에는 미세사출법(microprojectile bombardment), 전기충격유전자전달법(electroporation), 원형질 융합, 인산 칼슘(CaPO4) 침전, 염화 칼슘(CaCl2) 침전, 실리콘 카바이드 섬유 이용한 교반, 아그로 박테리아 매개된 형질전환, PEG-매개 융합법(PEG-mediated fusion), 미세주입법(microinjection), 리포좀 매개법(liposome-mediated method), 덱스트란 설페이트, 리포펙타민, 열충격법 등이 포함되나, 이로 제한되지 않는다.The transformation can be performed by any method known to be capable of introducing nucleic acids into an organism, cell, tissue or organ, and can be performed by selecting an appropriate standard technique depending on the host cell, as known in the art. . The methods include microprojectile bombardment, electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, stirring using silicon carbide fibers, and agrobacteria-mediated transformation. Conversion, PEG-mediated fusion, microinjection, liposome-mediated method, dextran sulfate, lipofectamine, heat shock method, etc. are included, but are not limited to these.
상기 용어 '형질전환체'는 '숙주 세포', '재조합 세포' 및 '재조합 균주' 등과 호환성 있게 사용될 수 있으며, 임의의 수단(예: 전기충격법, 칼슘 포스파타제 침전법, 미세주입법, 형질전환법, 바이러스 감염 등)에 의해 세포 내로 도입된 이종성 DNA를 포함하는 원핵 또는 진핵 세포를 의미한다.The term 'transformant' can be used interchangeably with 'host cell', 'recombinant cell', and 'recombinant strain', and can be used by any means (e.g., electroshock method, calcium phosphatase precipitation method, microinjection method, transformation method). , virus infection, etc.) refers to a prokaryotic or eukaryotic cell containing heterologous DNA introduced into the cell.
본 발명에서 상기 형질전환체는 클로닝 분야에서 통상적으로 사용되는 모든 종류의 단세포 유기체, 예컨대 각종 박테리아(예컨대, Clostridia 속, 대장균, 등) 등의 원핵세포 미생물, 효모 등의 하등 진핵세포 미생물과 곤충 세포, 식물 세포, 포유동물 등을 포함하는 고등 진핵생물 유래의 세포를 숙주세포로 사용할 수 있으며, 이에 제한되지 않는다. 숙주세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로 당업자가 목적하는 바에 가장 적합한 숙주세포를 선택하여 사용할 수 있다. 본 발명의 상기 형질전환체는 바람직하게 형질전환 미생물을 의미하는 것일 수 있다. 구체적으로, 이에 제한되지 않으나, 예를 들어 숙주세포로는 에스케리치아 콜라이(대장균, Escherichia coli), 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)와 같은 원핵 숙주 세포일 수 있다. 또한, 진균(예를 들어, 아스페르길러스(Aspergillus)), 효모(예를 들어, 피치아 파스토리스(Pichia pastoris), 사카로마이세스 세르비지애(Saccharomyces cerevisiae), 쉬조사카로미세스(Schizosaccharomyces), 뉴로스포라크라사(Neurospora crassa))등과 같은 하등 진핵 세포, 곤충 세포, 식물 세포, 포유동물 등을 포함하는 고등 진핵생물 유래의 세포를 숙주세포로 사용할 수 있으며, 이에 제한되지 않는다. In the present invention, the transformant includes all types of single-celled organisms commonly used in the cloning field, such as prokaryotic microorganisms such as various bacteria (e.g., Clostridia genus, Escherichia coli, etc.), lower eukaryotic microorganisms such as yeast, and insect cells. , cells derived from higher eukaryotes, including plant cells, mammals, etc., can be used as host cells, but are not limited thereto. Since the expression level and modification of proteins varies depending on the host cell, a person skilled in the art can select and use the host cell most suitable for the purpose. The transformant of the present invention may preferably refer to a transformed microorganism. Specifically, but not limited thereto, host cells include, for example , Escherichia coli, Bacillus subtilis, Streptomyces , Pseudomonas , and Proteus mirabili. It may be a prokaryotic host cell such as Proteus mirabilis or Staphylococcus . Additionally, fungi (e.g., Aspergillus ), yeast (e.g., Pichia pastoris , Saccharomyces cerevisiae ), Schizosaccharomyces ), Neurospora crassa ( Neurospora crassa ), etc. Cells derived from higher eukaryotes including lower eukaryotes, insect cells, plant cells, mammals, etc. can be used as host cells, but are not limited thereto.
상기 형질전환체(또는 형질전환 미생물, 숙주 세포, 재조합 균주)로서 Rosetta2 (DE3), C41 (DE3), SoluBL21 등이 사용될 수 있으며, 본 발명의 일 실시예에서는 E. coli BL21 (DE3)이 사용되었다. 특히 본 발명의 일 실시예에서 사용된 상기 BL21 (DE3) 균주는 글루코스(glucose)로부터 β-알라닌를 생합성 할 수 있도록 대사 부산물 생합성 효소(PoxB, PflB, LdhA, AdhE) 및 펩티다아제(peptidase)(PepB, PepN)를 암호화하는 유전자들의 위치에 α-알라닌 생합성 효소를 암호화하는 유전자를 대체 삽입한 것이다. 구체적으로 본 발명의 일 실시예에서 사용된 상기 BL21 (DE3) 균주는 poxB, pflB, ldhA, adhE, pepB, 및 pepN이 결손되고, Cg_panD, Ec_ppc, Cg_aspB, Ec_aspA, Cg_pyc, 및 Ec_gdh가 각각 4, 3, 2, 1, 1, 및 1 카피 씩 삽입된 β-알라닌 생산 균주를 의미한다. 다만, 본 발명에 있어서 상기 각각의 유전자가 삽입된 카피 수는 이에 제한되는 것은 아니고, 상기 기재된 유전자가 적절하게 기능할 수 있는 정도로 발현되어 그 기능을 다 할 수 있는 정도라면 그 어떤 카피 수라도 적용될 수 있음은 통상의 기술자에게 자명하다.Rosetta2 (DE3), C41 (DE3), SoluBL21, etc. can be used as the transformant (or transformed microorganism, host cell, recombinant strain), and in one embodiment of the present invention , E. coli BL21 (DE3) is used. It has been done. In particular, the BL21 (DE3) strain used in one embodiment of the present invention has metabolic by-product biosynthetic enzymes (PoxB, PflB, LdhA, AdhE) and peptidase (PepB, A gene encoding an α-alanine biosynthetic enzyme was substituted and inserted into the position of the genes encoding PepN). Specifically, the BL21 (DE3) strain used in one embodiment of the present invention is deficient in poxB , pflB , ldhA , adhE , pepB , and pepN , and has 4 Cg_panD , Ec_ppc , Cg_aspB , Ec_aspA , Cg_pyc , and Ec_gdh , respectively. Refers to a β-alanine producing strain with 3, 2, 1, 1, and 1 copies inserted. However, in the present invention, the number of copies into which each of the genes is inserted is not limited to this, and any copy number can be applied as long as the genes described above are expressed to the extent that they can function properly and perform their functions. This is obvious to those skilled in the art.
여기서, 상기 poxB는 Pyruvate dehydrogenase 단백질을 암호화하는 유전자로서, 상기 단백질은 피루브산의 산화적 탈카르복실화를 촉매하여 아세테이트와 CO2를 형성하는 말초 세포막 효소이다.Here, poxB is a gene encoding Pyruvate dehydrogenase protein, which is a peripheral cell membrane enzyme that catalyzes oxidative decarboxylation of pyruvate to form acetate and CO 2 .
상기 pflB는 Formate acetyltransferase 1 단백질을 암호화하는 유전자로서, 상기 단백질은 피루브산염이 포른산염과 아세틸-CoA로 전환되는 것을 촉매하는 효소이다.The pflB is a gene encoding the Formate acetyltransferase 1 protein, which is an enzyme that catalyzes the conversion of pyruvate into formate and acetyl-CoA.
상기 ldhA는 D-lactate dehydrogenase 단백질을 의미하며, 상기 단백질은 발효성 젖산 탈수소효소이다.The ldhA refers to D-lactate dehydrogenase protein, and the protein is a fermentative lactate dehydrogenase.
상기 adhE는 Bifunctional aldehyde-alcohol dehydrogenase AdhE 단백질을 암호화하는 유전자로서, 상기 단백질은 발효 조건에서 아세틸-CoA가 아세트알데히드로, 그 다음 에탄올로 순차적으로 NADH 의존적으로 환원되는 것을 촉매하는 한편, 호기성 조건에서는 AdhE는 과산화수소 제거제 역할을 할 수 있으며 산화 스트레스에 대한 보호 역할을 한다.The adhE is a gene encoding the bifunctional aldehyde-alcohol dehydrogenase AdhE protein, which catalyzes the sequential NADH-dependent reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentation conditions, while AdhE under aerobic conditions Can act as a hydrogen peroxide scavenger and protect against oxidative stress.
상기 pepB는 Peptidase B 단백질을 암호화하는 유전자로서, 상기 단백질은 세포 내 펩티드 분해에 중요한 역할을 한다. The pepB is a gene encoding the Peptidase B protein, and the protein plays an important role in decomposing peptides in cells.
상기 pepN은 Aminopeptidase N 단백질을 암호화하는 유전자로서, 상기 단백질은 영양 결핍에 대한 반응 뿐만 아니라 정상적인 성장 동안 단백질 분해에 의해 생성된 세포내 펩타이드의 분해에 관여한다. The pepN is a gene encoding the Aminopeptidase N protein, which is involved in the degradation of intracellular peptides produced by protein degradation during normal growth as well as in response to nutrient deficiency.
상기 panD는 Aspartate 1-decarboxylase 단백질을 암호화하는 유전자로서, 상기 단백질은 β-알라닌을 생성하기 위해 아스파테이트의 피루보일 의존성 탈카르복실화를 촉매한다. The panD is a gene encoding the Aspartate 1-decarboxylase protein, which catalyzes the pyruvyl-dependent decarboxylation of aspartate to produce β-alanine.
상기 ppc는 Phosphoenolpyruvate carboxylase 단백질을 암호화하는 유전자로서, 상기 단백질은 트리카르복실산 회로의 4탄소 디카르복실산 공급원인 옥살로아세트산을 형성한다. The ppc is a gene encoding the Phosphoenolpyruvate carboxylase protein, which forms oxaloacetic acid, a 4-carbon dicarboxylic acid source of the tricarboxylic acid cycle.
상기 aspB는 Aspartate aminotransferase 단백질을 암호화하는 유전자로서, 상기 단백질은 아스파르트산과 글루탐산 사이에서 α-아미노기의 가역적인 전이를 촉매하며, 아미노산 대사에서 중요한 효소이다.The aspB is a gene encoding the Aspartate aminotransferase protein, which catalyzes the reversible transfer of an α-amino group between aspartic acid and glutamic acid and is an important enzyme in amino acid metabolism.
상기 aspA는 Aspartate ammonia-lyase 단백질을 암호화하는 유전자로서, 상기 단백질은 아스파르트산염을 푸마르산염과 암모니아로 분해하는 과정을 촉매한다.The aspA is a gene encoding the Aspartate ammonia-lyase protein, which catalyzes the process of decomposing aspartate into fumarate and ammonia.
상기 pyc는 Pyruvate carboxylase 단백질을 암호화하는 유전자로서, 상기 단백질은 공유 결합된 비오틴을 ATP 의존적 카르복실화 하는 첫 번째 단계 및 카르복실기가 피루브산으로 전달되는 두 번째 단계의 2 단계 반응을 촉매한다.The pyc is a gene encoding the Pyruvate carboxylase protein, which catalyzes a two-step reaction of the first step of ATP-dependent carboxylation of covalently bound biotin and the second step of transferring the carboxyl group to pyruvate.
상기 gdh는 Glutamate dehydrogenase 단백질을 암호화하는 유전자로서, 상기 단백질은 글루타메이트의 α-케토글루타레이트로의 가역적인 산화 탈아민화를 촉매한다.The gdh is a gene encoding a glutamate dehydrogenase protein, which catalyzes the reversible oxidative deamination of glutamate into α-ketoglutarate.
한편, 상기 유전자 표기에 있어서 접두어로 사용된 "Cg_"는 Corynebacterium glutamicum을 의미하며, 또한 "Ec_"는 Escherichia coli를 의미하는 것으로서, 해당 균주에서 유래한 유전자임을 나타낸다.Meanwhile, " Cg_ " used as a prefix in the gene notation refers to Corynebacterium glutamicum , and " Ec_ " refers to Escherichia coli , indicating that the gene is derived from the corresponding strain.
한편, 상술한 바와 같이, 상기 본 발명에 따른 폴리펩티드 및 상기 폴리펩티드를 암호화하는 폴리뉴클레오티드가 포함된 발현 벡터를 포함하는 숙주 세포 중 어느 하나 이상을 포함하는 조성물은 L-카르노신의 생산을 위한 조성물로 이용될 수 있다. Meanwhile, as described above, a composition containing at least one of the polypeptide according to the present invention and a host cell containing an expression vector containing a polynucleotide encoding the polypeptide is used as a composition for the production of L-carnosine. It can be.
따라서, 본 발명은 본 발명의 폴리펩티드; 또는 상기 폴리펩티드를 암호화하는 폴리뉴클레오티드가 포함된 발현 벡터를 포함하는 숙주 세포 중 어느 하나 이상을 포함하는 L-카르노신 생산용 조성물을 제공한다.Accordingly, the present invention relates to polypeptides of the present invention; Or, it provides a composition for producing L-carnosine containing at least one of host cells containing an expression vector containing a polynucleotide encoding the polypeptide.
또한, 본 발명은 (a) 서열번호 1로 표시되는 아미노산 서열로 이루어진 단백질을 발현하는 재조합 균주를 상기 단백질을 생산하는데 적합한 시간 및 조건 하에 배지에서 배양하는 단계; (b) 상기 배양물에 i) 글루코스(glucose) 및 베타 알라닌(β-alanine) 중 어느 하나 이상, 및 ii) L-히스티딘(L-histidine)을 혼합하는 단계; 및 (c) L-카르노신(L-carnosine)을 수득하는 단계; 를 포함하는 L-카르노신(L-carnosine)의 생산 방법을 제공한다. In addition, the present invention includes the steps of (a) culturing a recombinant strain expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in a medium under time and conditions suitable for producing the protein; (b) mixing i) one or more of glucose and beta-alanine, and ii) L-histidine into the culture; and (c) obtaining L-carnosine; It provides a method for producing L-carnosine including.
본 발명에 따른 방법의 상기 (a) 단계에 있어서, 상기 '재조합 균주'는 상술한 숙주 세포, 형질전환체, 형질전환 미생물과 동일한 의미로 사용되었으며, 그 의미는 상술한 바와 같다. In step (a) of the method according to the present invention, the 'recombinant strain' is used in the same sense as the above-mentioned host cell, transformant, and transformed microorganism, and its meaning is as described above.
상기 (a) 단계에 있어서, 상기 단백질을 생산하는데 적합한 시간, 조건 및 배지는 상기 재조합 균주로 사용된 숙주균의 종류에 따라 달라질 수 있다.In step (a), the time, conditions, and medium suitable for producing the protein may vary depending on the type of host bacteria used as the recombinant strain.
상기 '배양'은 상기 재조합 균주를 적당히 조절된 환경 조건에서 생육시키는 것을 의미한다. 배양과정은 당업계에 알려진 적당한 배지와 배양조건에 따라 이루어질 수 있다. 이러한 배양 과정은 선택되는 미생물에 따라 통상의 기술자가 용이하게 조정하여 사용할 수 있다. 구체적으로 상기 배양은 회분식, 연속식 및/또는 유가식일 수 있으나, 이에 제한되는 것은 아니다.The 'culturing' means growing the recombinant strain under appropriately controlled environmental conditions. The culture process can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art depending on the selected microorganism. Specifically, the culture may be batch, continuous, and/or fed-batch, but is not limited thereto.
상기 '배지'는 상기 재조합 균주를 배양하기 위해 필요로 하는 영양물질을 주성분으로 혼합한 물질을 의미하며, 생존 및 발육에 불가결한 물을 비롯하여 영양물질 및 발육인자 등을 공급한다. 구체적으로, 상기 재조합 균주의 배양에 사용되는 배지 및 기타 배양 조건은 통상의 미생물의 배양에 사용되는 배지라면 특별한 제한 없이 어느 것이나 사용할 수 있으며, 상기 재조합 균주를 적당한 탄소원, 질소원, 인원, 무기화합물, 아미노산 및/또는 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.The 'medium' refers to a material that is mainly mixed with nutrients necessary for cultivating the recombinant strain, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the medium and other culture conditions used for cultivating the recombinant strain can be any medium used for cultivating ordinary microorganisms without particular limitation, and the recombinant strain can be grown with an appropriate carbon source, nitrogen source, personnel, inorganic compounds, It can be cultured under aerobic conditions in a normal medium containing amino acids and/or vitamins while controlling temperature, pH, etc.
상기 탄소원으로는 글루코오스, 사카로오스, 락토오스, 프룩토오스, 수크로오스, 말토오스 등과 같은 탄수화물; 만니톨, 소르비톨 등과 같은 당 알코올, 피루브산, 락트산, 시트르산 등과 같은 유기산; 글루탐산, 메티오닌, 리신 등과 같은 아미노산 등이 포함될 수 있다. 또한, 전분 가수분해물, 당밀, 블랙스트랩 당밀, 쌀겨울, 카사버, 사탕수수 찌꺼기 및 옥수수 침지액 같은 천연의 유기 영양원을 사용할 수 있으며, 구체적으로는 글루코오스 및 살균된 전처리 당밀(즉, 환원당으로 전환된 당밀) 등과 같은 탄수화물이 사용될 수 있으며, 그 외의 적정량의 탄소원을 제한없이 다양하게 이용할 수 있다. 이들 탄소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있으며, 이에 제한되는 것은 아니다.The carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; Sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; Amino acids such as glutamic acid, methionine, lysine, etc. may be included. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice bran, cassava, bagasse and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e. converted to reducing sugars). Carbohydrates such as molasses) can be used, and various other carbon sources in an appropriate amount can be used without limitation. These carbon sources may be used alone or in combination of two or more types, but are not limited thereto.
상기 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 질산암모늄 등과 같은 무기질소원; 글루탐산, 메티오닌, 글루타민 등과 같은 아미노산, 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해 생성물 등과 같은 유기 질소원이 사용될 수 있다. 이들 질소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있으며, 이에 제한되는 것은 아니다.The nitrogen source includes inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.
상기 인원으로는 인산 제1칼륨, 인산 제2칼륨, 또는 이에 대응되는 소디움-함유 염 등이 포함될 수 있다. 무기 화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간, 탄산칼슘 등이 사용될 수 있으며, 그 외에 아미노산, 비타민 및/또는 적절한 전구체 등이 포함될 수 있다. 이들 구성성분 또는 전구체는 배지에 회분식 또는 연속식으로 첨가될 수 있다. 그러나, 이에 제한되는 것은 아니다.The agent may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and/or appropriate precursors. These components or precursors can be added to the medium batchwise or continuously. However, it is not limited to this.
또한, 상기 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산, 황산 등과 같은 화합물을 배지에 적절한 방식으로 첨가하여, 배지의 pH를 조정할 수 있다. 또한, 배양 중에는 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 또한, 배지의 호기 상태를 유지하기 위하여, 배지 내로 산소 또는 산소 함유 기체를 주입하거나 혐기 및 미호기 상태를 유지하기 위해 기체의 주입 없이 혹은 질소, 수소 또는 이산화탄소 가스를 주입할 수 있으며, 이에 제한되는 것은 아니다.Additionally, during the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, and is limited thereto. That is not the case.
상기 배양에서 배양 온도는 20℃ 내지 45℃ 구체적으로는 25℃내지 40℃를 유지할 수 있고, 약 10 내지 160 시간 동안 배양할 수 있으나, 이에 제한되는 것은 아니다.In the above culture, the culture temperature can be maintained at 20°C to 45°C, specifically 25°C to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.
본 발명의 일 실시예에서는 E. coli BL21 (DE3)를 숙주균으로 하여, LB배지 (Luria-Bertani broth)에서 37℃에서 18시간 동안 200 rpm으로 진탕 배양하였으나, 이에 제한되는 것은 아니며, 통상의 기술자는 당업계에 잘 알려진 바에 따라 적절하게 배양 조건을 변경하여 적용할 수 있고, 본 발명의 서열번호 1로 이루어진 단백질이 발현될 수 있는 숙주균의 배양 조건이라면 상술한 내용 중 어느 것이라도 적용 가능하는 점은 통상의 기술자에게 자명하다.In one embodiment of the present invention , E. coli BL21 (DE3) was used as a host bacterium and cultured in LB medium (Luria-Bertani broth) at 37°C for 18 hours with shaking at 200 rpm, but the method is not limited to this and conventional Technicians can appropriately change and apply the culture conditions according to what is well known in the art, and any of the above-mentioned contents can be applied as long as the culture conditions of the host bacteria that can express the protein consisting of SEQ ID NO: 1 of the present invention This point is self-evident to those skilled in the art.
본 발명에 있어서, 상기 (b) 단계는 L-카르노신의 생합성을 위한 기질을 투입하여 실질적으로 L-카르노신을 합성하는 단계를 의미한다. In the present invention, step (b) refers to substantially synthesizing L-carnosine by adding a substrate for biosynthesis of L-carnosine.
상기 기질은 β-알라닌과 L-히스티딘을 의미하며, 한편 본 발명의 일 실시예에 따르면, 본 발명의 폴리펩티드를 발현하는 숙주 세포는 글루코스(glucose)를 β-알라닌으로 전환할 수 있도록 유전적으로 조작된 E. coli BL21 (DE3)인 점을 고려할 때, 기질로서 상기 β-알라닌을 대신하여, 또는 그와 함께 글루코스를 투입하는 것도 가능하다. The substrate refers to β-alanine and L-histidine, and according to one embodiment of the present invention, the host cell expressing the polypeptide of the present invention is genetically engineered to convert glucose to β-alanine. Considering that it is E. coli BL21 (DE3), it is also possible to add glucose instead of or together with β-alanine as a substrate.
상기 (b) 단계에 있어서, 상기 기질의 농도는 반응물의 비례반응관계에 따라 설정하는 것이 생산에 있어 유리하며, 반응 기질의 농도에 있어서 상한/하한의 제한이 없으나, 통상의 기술자는 본 발명에 있어서 필요한 기질이 농도를 적절하게 결정하여 투입할 수 있다. In step (b), it is advantageous for production to set the concentration of the substrate according to the proportional reaction relationship of the reactants. There is no upper/lower limit to the concentration of the reaction substrate, but those skilled in the art will Therefore, the required substrate can be added by appropriately determining its concentration.
한편, 상기 기질을 투여할 때 재조합 균주의 밀도는 O.D.600 = 0.1 내지 4.0 일 수 있으나 이에 제한되는 것은 아니다.Meanwhile, when administering the substrate, the density of the recombinant strain may be OD.600 = 0.1 to 4.0, but is not limited thereto.
본 발명에 있어서, 상기 (c) 단계는 반응을 종료하고 최종 생성된 L-카르노신을 회수하는 단계를 의미한다.In the present invention, step (c) refers to terminating the reaction and recovering the finally produced L-carnosine.
본 발명에 따른 L-카르노신 생산 방법에 있어서, 재조합 균주가 생산을 진행하여 필요한 정도로 반응 진행이 완료되면, 재조합 균주를 새로 합성된 L- 카르노신으로부터 물리적으로 분리, 예를 들어 원심분리를 이용하여 분리함으로써 반응을 종료시킬 수 있으며, 이렇게 분리된 재조합 균주는 공정균으로서 새로운 반응 시스템에 투입되어 반복적으로 이용될 수 있다.In the method for producing L-carnosine according to the present invention, when the recombinant strain is produced and the reaction is completed to the required extent, the recombinant strain is physically separated from the newly synthesized L-carnosine, for example, using centrifugation. The reaction can be terminated by separation, and the separated recombinant strain can be used repeatedly as a process bacteria by being introduced into a new reaction system.
상기 '회수'는 상술한 재조합 균주의 배양 방법, 즉, 회분식, 연속식 또는 유가식 등 배양 방법 등에 따라 당해 기술 분야에 공지된 적합한 방법을 이용하여 목적하는 L- 카르노신을 수집하는 것일 수 있다. 예를 들어, 원심 분리, 여과, 결정화 단백질 침전제에 의한 처리(염석법), 추출, 초음파 파쇄, 한외여과, 투석법, 분자체 크로마토그래피(겔여과), 흡착크로마토그래피, 이온교환 크로마토그래피, 친화도 크로마토그래피 등의 각종 크로마토그래피, HPLC 또는 이들의 방법을 조합하여 사용될 수 있으며, 당해 분야에 공지된 적합한 방법을 이용하여 배지 또는 재조합 균주로부터 목적하는 L- 카르노신을 회수할 수 있다The 'recovery' may be to collect the desired L-carnosine using a suitable method known in the art according to the culturing method of the recombinant strain described above, that is, a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity. Various chromatography such as chromatography, HPLC, or a combination of these methods can be used, and the desired L-carnosine can be recovered from the medium or recombinant strain using a suitable method known in the art.
본 발명에 따른 L-카르노신 합성 활성이 있는 단백질 및 이를 이용한 L-카르노신의 생산 방법은 본 발명자가 새롭게 구축한 것으로서 L-카르노신의 생산 효율이 뛰어나므로 L-카르노신의 생산에 유용하게 이용될 수 있다.The protein with L-carnosine synthesis activity according to the present invention and the production method of L-carnosine using the same were newly constructed by the present inventor and have excellent production efficiency of L-carnosine, so they can be usefully used for the production of L-carnosine. there is.
도 1은 본 발명에 따른 폴리펩티드를 포함하는 숙주 세포가 L-카르노신을 생산하는 과정을 모식도로 나타낸 것이다.
도 2는 돌연변이가 도입된 본 발명에 따른 폴리펩티드의 L-카르노신 생산량을 야생형의 BaBacD 효소와 비교하여 나타낸 것이다.Figure 1 schematically shows the process by which a host cell containing a polypeptide according to the present invention produces L-carnosine.
Figure 2 shows the L-carnosine production of the polypeptide according to the present invention into which the mutation is introduced compared to the wild-type BaBacD enzyme.
이하 본 발명을 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. β-알라닌 생산 균주 제작Example 1. Production of β-alanine producing strain
β-알라닌(β-alanine) 생산 균주는 야생형 BL21(DE3)의 대사 부산물 생합성 효소(PoxB, PflB, LdhA, AdhE) 및 펩티다제(peptidase) 효소(PepB, PepN)을 암호화 하는 유전자들의 위치에 β-알라닌 생합성 효소들을 암호화하는 유전자를 대체 삽입하여 구축하였다.The β-alanine producing strain is located at the location of genes encoding metabolic by-product biosynthetic enzymes (PoxB, PflB, LdhA, AdhE) and peptidase enzymes (PepB, PepN) of wild type BL21 (DE3). It was constructed by replacement insertion of genes encoding β-alanine biosynthetic enzymes.
이를 위하여, 먼저 표 1에 나열된 프라이머를 이용하여 상기 타겟 유전자들의 업스트림(upstream)과 다운스트림(downstream) 지역을 수득하고, EcoRV 제한효소로 절단된 염색체 형질전환용 벡터 pCBT1과 깁슨 어셈블리 방법을 이용하여 클로닝함으로써 재조합 플라스미드 pCBT1-ΔpoxB, pCBT1-ΔpflB, pCBT1-ΔldhA, pCBT1-ΔadhE, pCBT1-ΔpepB, pCBT1-ΔpepN 을 구축하였다.For this purpose, first obtain the upstream and downstream regions of the target genes using the primers listed in Table 1, and use the Gibson assembly method with the vector pCBT1 for chromosomal transformation cut with EcoRV restriction enzyme. By cloning, recombinant plasmids pCBT1-Δ poxB , pCBT1-Δ pflB , pCBT1-Δ ldhA , pCBT1-Δ adhE , pCBT1-Δ pepB , and pCBT1-Δ pepN were constructed.
번호order
number
업스트림 poxB
upstream
다운스트림 poxB
downstream
업스트림 pflB
upstream
다운스트림 pflB
downstream
업스트림 ldhA
upstream
다운스트림 ldhA
downstream
업스트림 adhE
upstream
다운스트림 adhE
downstream
업스트림 pepB
upstream
다운스트림 pepB
downstream
업스트림 pepN
upstream
다운스트림 pepN
downstream
다음으로, 표 2에 정리된 바와 같이, PCR 수행을 통해 수득한 대체/삽입 합성 오페론을 ScaI 제한효소로 절단된 상기 재조합 플라스미드와 깁슨 어셈블리 방법을 이용하여 클로닝함으로써, 다음의 재조합 플라스미드를 구축하였다.Next, as summarized in Table 2, the replacement/insertion synthetic operon obtained through PCR was cloned using the Gibson assembly method with the recombinant plasmid cut with ScaI restriction enzyme to construct the following recombinant plasmid.
pCBT1-ΔpoxB::PCP44_Cg_panD-PcysK_Ec_ppc, pCBT1-Δ poxB ::P CP44 _Cg_panD-P cysK _Ec_ppc;
pCBT1-ΔpflB::PCP44_Cg_panD-PcysK_Ec_gdh, pCBT1-Δ pflB ::P CP44 _Cg_panD-P cysK _Ec_gdh;
pCBT1-ΔldhA::PJ23100_Ec_ppc-PJ23100_Cg_pyc, pCBT1-Δ ldhA ::P J23100 _Ec_ppc-P J23100 _Cg_pyc,
pCBT1-ΔadhE::PJ23100_Cg_aspB-PJ23100_Ec_aspA, pCBT1-Δ adhE ::P J23100 _Cg_aspB-P J23100 _Ec_aspA,
pCBT1-ΔpepB::PCP44_Cg_panD-PcysK_Cg_aspB, 및pCBT1-ΔpepB::P CP44 _Cg_panD-P cysK _Cg_aspB, and
pCBT1-ΔpepN::PCP44_Cg_panD-PcysK_Ec_ppcpCBT1-ΔpepN::P CP44 _Cg_panD-P cysK _Ec_ppc
하기의 표 2는 타겟 유전자와 그에 상응하는 대체/삽입 합성 오페론을 요약 정리한 것이다.Table 2 below summarizes the target genes and the corresponding replacement/insertion synthetic operons.
유전자target
gene
합성 오페론Replace/Insert
synthetic operon
dehydrogenasePyruvate
dehydrogenase
PcysK_Ec_ppcP CP44 _Cg_panD
P cysK _Ec_ppc
Aspartate 1-decarboxylase Corynebacterium glutamicum
Aspartate 1-decarboxylase
Phosphoenolpyruvate carboxylase Escherichia coli
Phosphoenolpyruvate carboxylase
acetyltransferaseFormate
acetyltransferase
PcysK_Ec_gdhP CP44 _Cg_panD
P cysK _Ec_gdh
Aspartate 1-decarboxylase Corynebacterium glutamicum
Aspartate 1-decarboxylase
Glutamate dehydrogenase Escherichia coli
Glutamate dehydrogenase
dehydrogenaseLactate
dehydrogenase
PJ23100_Cg_pycP J23100 _Ec_ppc
P J23100 _Cg_pyc
Phosphoenolpyruvate carboxylase Escherichia coli
Phosphoenolpyruvate carboxylase
pyruvate carboxylase Corynebacterium glutamicum
pyruvate carboxylase
aldehyde-alcohol
dehydrogenaseBifunctional
aldehyde-alcohol
dehydrogenase
PJ23100_Ec_aspAP J23100 _Cg_aspB
P J23100_Ec_aspA
Aspartate aminotransferase Corynebacterium glutamicum
Aspartate aminotransferase
Aspartate ammonia-lyase Escherichia coli
Aspartate ammonia-lyase
PcysK_Cg_aspBP CP44 _Cg_panD
P cysK_Cg_aspB
Aspartate 1-decarboxylase Corynebacterium glutamicum
Aspartate 1-decarboxylase
Aspartate aminotransferase Corynebacterium glutamicum
Aspartate aminotransferase
PcysK_Ec_ppcP CP44 _Cg_panD
P cysK _Ec_ppc
Aspartate 1-decarboxylase Corynebacterium glutamicum
Aspartate 1-decarboxylase
Phosphoenolpyruvate carboxylase Escherichia coli
Phosphoenolpyruvate carboxylase
중합효소는 KOD OneTM 폴리머라제를 사용하였으며, PCR 증폭 조건은 98℃에서 1분간 변성 후, 98℃ 10초 변성, 55℃ 5초 어닐링, 68℃ 1kb 당 5초로 중합을 29회 반복한 후, 72℃에서 1분간 중합반응을 수행하였다. 깁슨 어셈블리를 이용한 클로닝은 깁슨 어셈블리 시약과 정제된 각 유전자 단편들을 혼합한 후 50℃에서 1시간 반응하여 수행하였다.The polymerase used was KOD One TM polymerase, and the PCR amplification conditions were denaturation at 98°C for 1 minute, denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and polymerization at 68°C for 5 seconds per 1 kb repeated 29 times. Polymerization was performed at 72°C for 1 minute. Cloning using Gibson assembly was performed by mixing Gibson assembly reagent and each purified gene fragment and reacting at 50°C for 1 hour.
상기 구축된 각각의 재조합 플라스미드 pCBT1-ΔpoxB::PCP44_Cg_panD-PcysK_Ec_ppc, pCBT1-ΔpflB::PCP44_Cg_panD-PcysK_Ec_gdh, pCBT1-ΔldhA::PJ23100_Ec_ppc-PJ23100_Cg_pyc, pCBT1-ΔadhE::PJ23100_Cg_aspB-PJ23100_Ec_aspA, pCBT1-ΔpepB::PCP44_Cg_panD-PcysK_Cg_aspB, 및 pCBT1-ΔpepN::PCP44_Cg_panD-PcysK_Ec_ppc를 전기천공법으로 E. coli BL21(DE3) 균주에 순차적으로 형질전환 후, 2차 교차 과정을 거쳐 poxB, pflB, ldhA, adhE, pepB, pepN이 결손되고, Cg_panD, Ec_ppc, Cg_aspB, Ec_aspA, Cg_pyc, 및 Ec_gdh가 각각 4, 3, 2, 1, 1, 및 1 카피 씩 삽입된 β-알라닌 생산 균주를 제작하였으며, CE01EC006으로 명명하였다. 해당 유전자적 조작은 아래의 표 3에 나열된 프라이머를 이용한 PCR 법을 통하여 확인하였다.Each of the recombinant plasmids constructed above pCBT1-Δ poxB ::P CP44 _Cg_panD-P cysK _Ec_ppc, pCBT1-Δ pflB ::P CP44 _Cg_panD-P cysK _Ec_gdh, pCBT1-Δ ldhA ::P J23100 _Ec_ppc-P J23100 _Cg_pyc, pCBT1 -Δ adhE ::P J23100 _Cg_aspB-P J23100 _Ec_aspA, pCBT1-ΔpepB::P CP44 _Cg_panD-P cysK _Cg_aspB, and pCBT1-ΔpepN::P CP44 _Cg_panD-P cysK _Ec_ppc were transferred to E. coli BL by electroporation. 21(DE3 ) After sequentially transforming the strains, poxB , pflB , ldhA , adhE , pepB , and pepN were deleted through a second crossing process, and Cg_panD , Ec_ppc , Cg_aspB , Ec_aspA , Cg_pyc , and Ec_gdh were 4, 3, and 2, respectively. A β-alanine producing strain with 1, 1, and 1 copies each was created and named CE01EC006. The genetic manipulation was confirmed through PCR using the primers listed in Table 3 below.
번호order
number
실시예 2. BaBacD를 이용한 L-카르노신 생산 균주 제작Example 2. Production of L-carnosine producing strain using BaBacD
도 1에서 확인할 수 있는 바와 같이, 생물학적으로 L-카르노신(L-carnosine)은 β-알라닌(β-alanine)과 L-히스티딘(L-histidine)을 이용한 L-아미노산 리가아제(L-amino acid ligase, Lal)의 촉매 작용으로 합성될 수 있다.As can be seen in Figure 1, biologically, L-carnosine is L-amino acid ligase using β-alanine and L-histidine. It can be synthesized through the catalytic action of ligase (Lal).
신규한 Lal를 탐색하기 위하여 Bacillus altitudinis 유래의 ATP-grasp domain-containing protein, WP_045033870을 암호화 하는 유전자를 합성하고, pET28a 벡터에 도입함으로써 재조합 플라스미드 1종을 획득하고, 이를 pBaBacD로 명명하였다.To discover new Lal, a gene encoding the ATP-grasp domain-containing protein, WP_045033870, from Bacillus altitudinis was synthesized and introduced into the pET28a vector to obtain a recombinant plasmid, which was named pBaBacD.
아래의 표 4는 BaBacD (WP_045033870)의 아미노산 서열이다.Table 4 below is the amino acid sequence of BaBacD (WP_045033870).
번호order
number
YFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTNNELFIAPMAKAAE
RLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPT
YLASSIGVTLFHDKAGSDDLFLQVQSYLETIPVPDAVTYEAPFVAETYLEGAYEDWYEDE
GYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEG
LGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKA
VLPKQLLSGHTFYVADCHLYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVSQSF
PTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKGMSKKTVLVIADLGGCPPHMFYESVAASYHIVSYIPRPFAITKGHAELIEKYSIAVIKDRD
YFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTNNELFIAPMAKAAE
RLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPT
YLASSIGVTLFHDKAGSDDLFLQVQSYLETIPVPDAVTYEAPFVAETYLEGAYEDWYEDE
GYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEG
LGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKA
VLPKQLLSGHTFYVADCHLYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVSQSF
PTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKG
BaBacD (WP_045033870)가 L-카르노신의 생합성 능이 있는지를 평가하기 위해서 상기 구축된 pBaBacD을 상기 실시예 1에서 제작한 β-알라닌 생산 균주 CE01EC006에 형질 전환하여 L-카르노신 생산 균주 1종을 획득하고, 이를 Ec_BaBacD로 명명하였다.In order to evaluate whether BaBacD (WP_045033870) has the ability to biosynthesize L-carnosine, the constructed pBaBacD was transformed into the β-alanine producing strain CE01EC006 prepared in Example 1 to obtain one L-carnosine producing strain, This was named Ec_BaBacD.
실시예 3. Ec_BaBacD의 L-카르노신 생산 능 평가Example 3. Evaluation of L-carnosine production ability of Ec_BaBacD
상기 실시예 2에서 제작한 Ec_BaBacD 균주의 L-카르노신 생산능을 확인하고자, 아래와 같은 방법으로 배양하여 평가하였다.To confirm the L-carnosine production ability of the Ec_BaBacD strain prepared in Example 2, it was cultured and evaluated in the following manner.
먼저, 50 ㎍/㎖ 농도의 카나마이신(kanamycin)이 첨가된 LB 배지(Luria-Bertani broth) 5 ml을 함유하는 15 ml 배양 튜브에 균주를 접종하고, 37℃에서 18시간 동안 200 rpm으로 진탕 배양하였다. 그 다음, 50 ㎍/㎖ 농도의 카나마이신이 첨가된 생산 배지 25 ml을 함유하는 250 ml 코너-바플 플라스크에 0.25 ml의 종 배양액을 접종하고 O.D.600 값이 0.4~0.6이 되었을 때 IPTG (Isopropyl-β-D-thio-galactoside) 0.1 mM를 첨가하여 재조합 단백질의 발현을 유도하였다. 37℃에서 18 시간 동안 200 rpm에서 진탕 배양하였으며, 배양 종료 후, HPLC를 이용하여 L-카르노신의 생산량을 측정하였다. 모든 실험은 세트 당 2회 반복되었다.First, the strain was inoculated into a 15 ml culture tube containing 5 ml of LB medium (Luria-Bertani broth) supplemented with kanamycin at a concentration of 50 μg/ml, and cultured with shaking at 200 rpm for 18 hours at 37°C. . Then, 0.25 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 25 ml of production medium supplemented with kanamycin at a concentration of 50 ㎍/㎖, and when the O.D.600 value reached 0.4~0.6, IPTG (Isopropyl-β -D-thio-galactoside) 0.1 mM was added to induce expression of the recombinant protein. The culture was performed with shaking at 200 rpm for 18 hours at 37°C, and after completion of the culture, the production amount of L-carnosine was measured using HPLC. All experiments were repeated twice per set.
<생산 배지 (pH 7.0)><Production medium (pH 7.0)>
포도당 50 g, KH2PO4 0.3 g, K2HPO4 0.6 g, (NH4)2SO4 15 g, NaCl 2.5 g, 효모추출물 2.5 g, MgSO4ㆍ7H20 1 g, FeSO4ㆍH2O 0.005 g, MnSO4ㆍH20 0.005 g, L-히스티딘 15.5 g, CaCO3 30 g (증류수 1리터 기준) Glucose 50 g, KH 2 PO 4 0.3 g, K 2 HPO 4 0.6 g, (NH 4 ) 2 SO 4 15 g, NaCl 2.5 g, yeast extract 2.5 g, MgSO 4 ㆍ7H 2 0 1 g, FeSO 4 ㆍH 2 O 0.005 g, MnSO 4 ㆍH 2 0 0.005 g, L-histidine 15.5 g, CaCO 3 30 g (based on 1 liter of distilled water)
상기 실험 결과, Ec_BaBacD는 72시간의 배양시간 동안 4.77±1.08 mg/L의 L-카르노신을 생산하였고, 이로써, BaBacD 효소의 L-카르노신 생산 능을 확인 할 수 있었다(표 5 참조).As a result of the above experiment, Ec_BaBacD produced 4.77 ± 1.08 mg/L of L-carnosine during 72 hours of culture, thereby confirming the L-carnosine production ability of the BaBacD enzyme (see Table 5).
실시예 4. CutisLal을 이용한 L-카르노신 생산 균주 제작Example 4. Production of L-carnosine producing strain using CutisLal
상기 실시예 3에서 확인된 BaBacD 효소의 L-카르노신 생산 능을 향상시키기 위하여, 3차원 구조 분석을 통해 아래의 표 6과 같이 타겟 아미노산을 선정하고, PCR 수행을 통해 수득한 BaBacD 변이형을 깁슨 어셈블리 방법을 이용하여 pET28a 벡터에 도입함으로써 재조합 플라스미드 1종을 획득하고, 이를 pCutisLal이라 명명하였다.In order to improve the L-carnosine production ability of the BaBacD enzyme identified in Example 3, target amino acids were selected as shown in Table 6 below through three-dimensional structural analysis, and the BaBacD variant obtained through PCR was analyzed using Gibson One type of recombinant plasmid was obtained by introducing it into the pET28a vector using the assembly method, and named it pCutisLal.
아래의 표 6은 CutisLal의 아미노산 서열이며, 변이된 아미노산은 밑줄 및 굵은 글씨로 표시되었다.Table 6 below shows the amino acid sequence of CutisLal, with mutated amino acids underlined and in bold.
번호order
number
YFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTN E ELFIAPMAKAAE
RLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPT
YLASSIGVTLFHDKAGSDDLFLQVQSYLETIPVPDAVTYEAPFVAETYLEGAYEDWYEDE
GYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEG
LGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKA
VLPKQLLSGHTFYVADC K LYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVSQSF
PTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKGMSKKTVLVIADLGGCPPHMFYESVAASYHIVSYIPRPFAITKGHAELIEKYSIAVIKDRD
YFETHPSFEHPDSIYWAHDDYPKSEEEVVEDFIRVASFFKADAITTN E ELFIAPMAKAAE
RLGLRGAGVKAAEMARDKSQMRAAFNASGVKAVKTQPVTTLSDFQKAIESIGTPLILKPT
YLASSIGVTLFHDKAGSDDLFLQVQSYLETIPVPDAVTYEAPFVAETYLEGAYEDWYEDE
GYADYVSVEGLVVEGEYLPFVIHDKTPQIGFTETAHITPTILDNEAKQIIIEAARKANEG
LGLEHCATHTEIKLMKNRETGLIESAARFAGWNMIPNIKKVFGVDMAKLLIDVLVDGKKA
VLPKQLLSGHTFYVADC K LYPQHFKESGHIPPEATHITIDHVSIPEEAFVGDTAIVSQSF
PTKGTMVDLALFEAFNGIVSLELKGSSSQDVAASIRNIQKQATIQLMDELVKG
중합효소는 KOD OneTM 폴리머라제를 사용하였으며, PCR 증폭 조건은 98℃에서 1 분간 변성 후, 98℃ 10초 변성, 55℃ 5초 어닐링, 68℃ 1kb 당 5초로 중합을 29회 반복한 후, 72℃에서 1 분간 중합 반응을 수행하였다. 깁슨 어셈블리를 이용한 클로닝은 깁슨 어셈블리 시약과 정제된 각 유전자 단편들을 혼합한 후 50℃에서 1시간 반응하여 수행하였다.The polymerase used was KOD One TM polymerase, and the PCR amplification conditions were denaturation at 98°C for 1 minute, denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and polymerization at 68°C for 5 seconds per 1 kb repeated 29 times. The polymerization reaction was performed at 72°C for 1 minute. Cloning using Gibson assembly was performed by mixing Gibson assembly reagent and each purified gene fragment and reacting at 50°C for 1 hour.
이렇게 수득한 CutisLal의 L-카르노신 생산 능을 평가하기 위하여, 상기 구축된 pCutisLal을 상기 실시예 1에서 제작한 β-알라닌 생산 균주 CE01EC006에 형질 전환하여 L-카르노신 생산 균주 1종을 획득하고, 이를 Ec_CutisLal로 명명하였다.In order to evaluate the L-carnosine production ability of CutisLal thus obtained, pCutisLal constructed above was transformed into the β-alanine producing strain CE01EC006 prepared in Example 1 to obtain one L-carnosine producing strain, This was named Ec_CutisLal.
실시예 5. Ec_CutisLal의 L-카르노신 생산 능 평가Example 5. Evaluation of L-carnosine production ability of Ec_CutisLal
상기 실시예 4에서 제작한 Ec_CutisLal 균주의 L-카르노신 생산 능을 확인하고자, 아래와 같은 방법으로 배양하여 평가하였다.To confirm the L-carnosine production ability of the Ec_CutisLal strain prepared in Example 4, it was cultured and evaluated in the following manner.
먼저, 50 ㎍/㎖ 농도의 카나마이신(kanamycin)이 첨가된 LB 배지(Luria-Bertani broth) 5 ml을 함유하는 15 ml 배양 튜브에 균주를 접종하고, 37℃에서 18시간 동안 200 rpm으로 진탕 배양하였다. 그 다음, 50 ㎍/㎖ 농도의 카나마이신이 첨가된 생산 배지 25 ml을 함유하는 250 ml 코너-바플 플라스크에 0.25 ml의 종 배양액을 접종하고 O.D.600 값이 0.4~0.6이 되었을 때 IPTG (Isopropyl-β-D-thio-galactoside) 0.1 mM를 첨가하여 재조합 단백질의 발현을 유도하였다. 37℃에서 18 시간 동안 200 rpm에서 진탕 배양하였으며, 배양 종료 후, HPLC를 이용하여 L-카르노신의 생산량을 측정하였다. 모든 실험은 세트 당 2회 반복되었다.First, the strain was inoculated into a 15 ml culture tube containing 5 ml of LB medium (Luria-Bertani broth) supplemented with kanamycin at a concentration of 50 μg/ml, and cultured with shaking at 200 rpm for 18 hours at 37°C. . Then, 0.25 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 25 ml of production medium supplemented with kanamycin at a concentration of 50 ㎍/㎖, and when the O.D.600 value reached 0.4~0.6, IPTG (Isopropyl-β -D-thio-galactoside) 0.1 mM was added to induce expression of the recombinant protein. The culture was performed with shaking at 200 rpm for 18 hours at 37°C, and after completion of the culture, the production amount of L-carnosine was measured using HPLC. All experiments were repeated twice per set.
<생산 배지 (pH 7.0)><Production medium (pH 7.0)>
포도당 50 g, KH2PO4 0.3 g, K2HPO4 0.6 g, (NH4)2SO4 15 g, NaCl 2.5 g, 효모추출물 2.5 g, MgSO4ㆍ7H20 1 g, FeSO4ㆍH2O 0.005 g, MnSO4ㆍH20 0.005 g, L-히스티딘 15.5 g, CaCO3 30 g (증류수 1리터 기준) Glucose 50 g, KH 2 PO 4 0.3 g, K 2 HPO 4 0.6 g, (NH 4 ) 2 SO 4 15 g, NaCl 2.5 g, yeast extract 2.5 g, MgSO 4 ㆍ7H 2 0 1 g, FeSO 4 ㆍH 2 O 0.005 g, MnSO 4 ㆍH 2 0 0.005 g, L-histidine 15.5 g, CaCO 3 30 g (based on 1 liter of distilled water)
상기 실험 결과, Ec_CutisLal은 72시간의 배양시간 동안 672.41±7.33 mg/L의 L-카르노신을 생산하였고, 이는 상기 실시예 3에서 확인한 Ec_BaBacD의 생산량(4.77±1.08 mg/L)과 비교하여 141배 향상된 L-카르노신 생산 능에 해당한다(표 7 및 도 2 참조)As a result of the above experiment, Ec_CutisLal produced 672.41 ± 7.33 mg/L of L-carnosine over a 72-hour culture period, which was 141 times improved compared to the production of Ec_BaBacD (4.77 ± 1.08 mg/L) confirmed in Example 3. Corresponds to L-carnosine production ability (see Table 7 and Figure 2)
이상 살펴본 바와 같이, 본 발명에 따른 L-카르노신 합성 활성이 있는 단백질 및 이를 이용한 L-카르노신의 생산 방법은 L-카르노신의 생산 효율이 뛰어나므로 L-카르노신의 생산에 유용하게 이용될 수 있어 산업상 이용가능성이 높다. As discussed above, the protein with L-carnosine synthesis activity and the production method of L-carnosine using the same according to the present invention have excellent production efficiency of L-carnosine and can be usefully used in the production of L-carnosine for industrial purposes. The availability is high.
Claims (12)
An isolated polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
The polypeptide according to claim 1, wherein the polypeptide is L-amino acid ligase (Lal).
A polynucleotide encoding the polypeptide of claim 1.
An expression vector containing the polynucleotide of claim 3.
A host cell containing the expression vector of claim 4.
The host cell according to claim 5, wherein the host cell is E. coli.
The method of claim 6, wherein the E. coli is E. coli BL21 (DE3) in which poxB , pflB , ldhA , adhE , pepB , and pepN are deleted and Cg_panD , Ec_ppc , Cg_aspB , Ec_aspA , Cg_pyc , and Ec_gdh are inserted. host cells.
A composition for producing L-carnosine comprising at least one of the polypeptide according to claim 1 and the host cell according to claims 5 to 7.
(b) 상기 배양물에 i) 글루코스(glucose) 및 베타 알라닌(β-alanine) 중 어느 하나 이상, 및 ii) L-히스티딘(L-histidine)을 혼합하는 단계; 및
(c) L-카르노신(L-carnosine)을 수득하는 단계;
를 포함하는 L-카르노신(L-carnosine)의 생산 방법.
(a) cultivating a recombinant strain expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in a medium under time and conditions suitable for producing the protein;
(b) mixing i) one or more of glucose and beta-alanine, and ii) L-histidine into the culture; and
(c) obtaining L-carnosine;
A method of producing L-carnosine comprising.
The method of producing L-carnosine according to claim 9, wherein the protein is L-amino acid ligase (Lal).
The method for producing L-carnosine according to claim 9, wherein the recombinant strain is Escherichia coli.
The method of claim 11, wherein the E. coli is E. coli BL21 (DE3) in which poxB , pflB , ldhA , adhE , pepB , and pepN are deleted and Cg_panD , Ec_ppc , Cg_aspB , Ec_aspA , Cg_pyc , and Ec_gdh are inserted. A method of producing L-carnosine.
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CN117511889A (en) * | 2023-06-19 | 2024-02-06 | 珠海瑞德林生物有限公司 | Enzyme and application thereof in preparation of unnatural amino acid dipeptide |
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