KR101402659B1 - Actinobacillus succinogenes AND MANUFACTURING METHOD OF SUCCINIC ACID USING THE SAME - Google Patents
Actinobacillus succinogenes AND MANUFACTURING METHOD OF SUCCINIC ACID USING THE SAME Download PDFInfo
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Abstract
본 발명은 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP 및 이를 이용한 숙신산의 제조 방법에 관한 것으로, 구체적으로 숙신산의 생산성, 수율 및 공정 안정성이 우수하고, 대량 생산공정에 적용 가능한 신규주인 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP 및 이를 이용한 숙신산 제조 방법에 관한 것이다.The present invention relates to Actinobacillus < RTI ID = 0.0 > The present invention relates to a process for the production of succinic acid, which is excellent in the productivity, yield and process stability of succinic acid, and which can be applied to mass production processes, such as Actinobacillus succinogenes ) KCTC 12233BP and a process for producing succinic acid using the same.
Description
본 발명은 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP 및 이를 이용한 숙신산의 제조 방법에 관한 것이다. The present invention relates to Actinobacillus < RTI ID = 0.0 > succinogenes KCTC 12233BP and a process for preparing succinic acid using the same.
숙신산(succinic acid)은 C4계 다이액시드(diacid)로 식료품, 의류, 의약, 플라스틱 등 다양한 용도를 지닌 화합물로서, 최근 바이오매스(biomass)로부터 생산 가능한 대표적인 화합물의 하나로 주목 받고 있다 (Technology development for the production of biobased products from biorefinery carbohydrates-the US Department of Energy's Top 10 revisited. Green Chemistry, (2010), 12, 539-554). 또한, 미생물을 이용한 바이오매스 유래 당류(sugar)의 발효를 통해 숙신산을 생산하는 것이 가능하다.Succinic acid is a C4-based diacid that has been used for a variety of purposes such as food, clothing, medicines, and plastics. Recently, it has attracted attention as one of the representative compounds that can be produced from biomass (Technology development for The production of biobased products from the biorefinery carbohydrates-the US Department of Energy's Top 10 revisited. Green Chemistry, (2010), 12, 539-554). In addition, it is possible to produce succinic acid through fermentation of biomass-derived sugars using microorganisms.
악티노바실러스 숙시노게네스(Actinobacillus succinogenes)는 숙신산을 고농도로 생산하는 것으로 알려진 미생물이다. 상기 미생물에서, 숙신산 등 C4 대사물은 탄소 중심대사의 특성상 해당작용의 결과물인 포스포에놀피루브산(phosphoenolpyruvic acid, C3)의 이산화탄소고정화(CO2 fixation) 경로를 통해 생성되므로, 이론적으로는 생성되는 C4 대사물과 동일수의 이산화탄소가 반응물로 소비되어 이산화탄소 소비를 공업적으로 촉진할 수 있다. Actinobacillus Actinobacillus succinogenes ) are microorganisms known to produce succinic acid at high concentrations. In the microorganism, the C4 metabolites such as succinic acid are produced through the CO 2 fixation pathway of phosphoenolpyruvic acid (C3), which is a result of the action of the carbon central metabolism, The same number of carbon dioxide as the C4 metabolite can be consumed as a reactant to promote the carbon dioxide consumption industrially.
한편, Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen(International Journal of Systematic and Evolutionary Microbiology, (1999), 49, 207-216) 등에서는 숙신산을 제조하기 위한 미생물로, 악티노바실러스 숙시노게네스(Actinobacillus succinogenes) 등을 개시하고 있지만, 숙신산의 생산성 및 수율이 낮고, 숙신산의 대량 생산시 수율이 저하될 수 있는 문제점이 있다.Meanwhile, Actinobacillus succinogenes sp. (1999), 49, 207-216) discloses a microorganism for the production of succinic acid, which is a microorganism belonging to the genus Actinobacillus suisinoge Actinobacillus succinogenes ). However, there is a problem in that productivity and yield of succinic acid are low, and the yield of succinic acid is reduced during mass production.
본 발명의 목적은, 종래 숙신산을 생산하는 균주에 비하여 숙신산 생산성이 우수한, 새로운 균주를 제공하는 것이다. An object of the present invention is to provide a novel strain excellent in productivity of succinic acid compared with a strain producing succinic acid in the past.
또한 본 발명의 목적은 높은 생산성으로 숙신산을 생산하는 균주를 이용하여, 높은 수율로 숙신산을 제조하는 방법을 제공하는 것이다. It is another object of the present invention to provide a method for producing succinic acid at a high yield using a strain producing succinic acid with high productivity.
상기 목적을 달성하기 위하여, 본 발명은 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 제공한다.In order to achieve the above object, the present invention provides Actinobacillus succinogenes KCTC 12233BP.
또한, 본 발명은 A) 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 배양하는 단계; 및 B) 상기 배양액으로부터 숙신산을 수득하는 단계를 포함하는 숙신산의 제조 방법을 제공한다.The present invention also relates to a process for the preparation of A) Actinobacillus culturing succinogenes KCTC 12233BP; And B) obtaining succinic acid from the culture broth.
본 발명은 숙신산의 생산성, 수율 및 공정 안정성이 우수한 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 제공할 수 있다.The present invention relates to a process for producing actinobacillus ( Actinobacillus) which is excellent in the productivity, yield and process stability of succinic acid succinogenes KCTC 12233BP.
또한, 본 발명의 숙신산 제조 방법은, 실험실 규모의 숙신산 생산 공정뿐만 아니라, 산업화를 위한 대량 생산에서도 우수한 숙신산 생산성, 수율 및 안정성을 나타내어, 석유화학 유도체, 의약, 식품산업 등 다양한 분야에 적용할 수 있다.The succinic acid production method of the present invention shows excellent succinic acid productivity, yield and stability not only in the production process of succinic acid on the laboratory scale but also in the mass production for industrialization and can be applied to various fields such as petrochemical derivatives, have.
도 1은 비교예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27에 대한 에틸 메탄설포네이트(EMS)의 처리 농도별 생육 곡선을 나타낸 그래프이다.
도 2는 비교예 1(붉은 대조군 선그래프) 및 실시예 1, 비교예 2 내지 48(막대그래프)의 균체 성장을 나타낸 그래프이다.
도 3은 비교예 1(붉은 대조군 선그래프) 및 실시예 1, 비교예 2 내지 48(막대그래프)의 생산성 분포도를 나타낸 그래프이다.
도 4는 발효조 배양 시 비교예 1의 균주와 실시예 1의 균주의 숙신산 생산량을 나타낸 그래프이다.
도 5는 비교예 1의 균주와 실시예 1의 균주의 시간당 숙신산 생산량을 나타낸 그래프이다.
도 6은 이론 수율의 숙신산 양에서 비교예 1의 균주와 실시예 1의 균주가 생산하는 숙신산을 퍼센트로 나타낸 그래프이다.
도 7은 실시예 1, 비교예 2 내지 48의 균주에 대한 숙신산 생산성에 따른 분포도를 나타내는 히스토그램이다.Figure 1 is a graph showing the effect of Actinobacillus < RTI ID = 0.0 > ( Actinobacillus < / RTI > Actinobacillus < / RTI > derived from succinogenes ATCC 55618 succinogenes ) WD27. < tb >< TABLE > Columns = 2 < tb >
2 is a graph showing cell growth of Comparative Example 1 (red control line graph) and Example 1 and Comparative Examples 2 to 48 (bar graph).
3 is a graph showing the productivity distribution chart of Comparative Example 1 (red control line graph) and Example 1 and Comparative Examples 2 to 48 (bar graph).
4 is a graph showing the production of succinic acid by the strain of Comparative Example 1 and the strain of Example 1 during fermentation.
5 is a graph showing production of succinic acid per hour of the strain of Comparative Example 1 and the strain of Example 1. Fig.
6 is a graph showing the percentage of succinic acid produced by the strain of Comparative Example 1 and the strain of Example 1 in the amount of succinic acid of the theoretical yield.
7 is a histogram showing the distributions according to succinic acid productivity of the strains of Example 1 and Comparative Examples 2 to 48. Fig.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 제공한다. 상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP는 숙신산을 생산하는 균주로서, 숙신산 생산성 및 안정성이 우수하여, 숙신산을 제조하는데 유용하게 이용될 수 있다. 또한, 숙신산 제조에 있어서, 소규모 생산 시설뿐만 아니라, 대규모 생산 시설에 적용이 가능하여, 숙신산을 제조하는 다양한 산업분야에 이용될 수 있다. The present invention relates to Actinobacillus < RTI ID = 0.0 > succinogenes KCTC 12233BP. Actinobacillus succinogenes KCTC 12233BP is a strain that produces succinic acid and is excellent in succinic acid productivity and stability and can be usefully used for producing succinic acid. Further, in the production of succinic acid, it can be applied not only to a small scale production facility but also to a large scale production facility, and can be used in various industrial fields for producing succinic acid.
상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP는 기존에 개발되어 있는 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27을 친균주로 하여 균체를 새로운 화학적 돌연변이원인 에틸 메틸설포네이트(EMS)를 처리하여 제조할 수 있다.The Actinobacillus < RTI ID = 0.0 > succinogenes KCTC 12233BP was prepared by using Actinobacillus succinogenes WD27 derived from Actinobacillus succinogenes ATCC 55618, which was previously developed, as a host strain and transforming the microbial cells into a new chemical mutant ethyl Methylsulfonate (EMS). ≪ / RTI >
숙신산의 생산 특성상 균체 성장이 빠른 균주가 생산성도 높으므로 돌연변이원 처리 후 상대적으로 높은 성장 속도를 나타내는 균주를 선별함으로써 숙신산 생산성이 높은 균주를 수득하는 것이 가능하다. 균주의 상대적인 성장 속도는 액체 배양에서 시간에 따른 탁도의 변화를 측정함으로써 비교, 분석 가능하다.It is possible to obtain a strain having a high productivity of succinic acid by selecting strains showing a relatively high growth rate after the mutagen treatment because the strains with high cell growth are highly productive due to the production characteristics of succinic acid. The relative growth rate of the strain can be compared and analyzed by measuring the change in turbidity over time in liquid culture.
상기 방법을 통해 수득한 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP는 타 돌연변이 균주에 비하여 성장 속도가 높을 뿐만 아니라 30 mL serum bottle 배양에서 숙신산을 약 107 g/L 까지 생산하며 수율도 이론 수율 대비 75%에 이르는 것으로 확인되었다. Actinobacillus succinogenes KCTC 12233BP obtained by the above method has a higher growth rate than other mutant strains and also produces succinic acid up to about 107 g / L in a 30 mL serum bottle culture, It was confirmed that the yield was 75%.
상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP는 한국생명공학연구원 미생물자원센터(KCTC)에 기탁되었다. The Actinobacillus < RTI ID = 0.0 > succinogenes ) KCTC 12233BP was deposited with the Center for Microbial Resources (KCTC) of the Korea Biotechnology Research Institute.
본 발명은 A) 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 배양하는 단계; 및 B) 상기 배양액으로부터 숙신산을 수득하는 단계를 포함하는 숙신산의 제조 방법을 제공한다.The present invention relates to A) Actinobacillus ( Actinobacillus < RTI ID = 0.0 > culturing succinogenes KCTC 12233BP; And B) obtaining succinic acid from the culture broth.
상기 숙신산의 제조 방법은 신규한 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 이용하여 제조하는 것으로, 종래의 숙신산을 생산하는 균주로 알려진, 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27 등에 비하여 숙신산의 생산성을 향상시키고, 숙신산의 대량 생산에 있어서도 높은 생산성을 나타낸다. Actinobacillus succinogenes ATCC ( Actinobacillus succinogenes ), which is known as a strain producing conventional succinic acid, is prepared by using Actinobacillus succinogenes KCTC 12233BP. Actinobacillus succinogenes WD27, which is derived from the strain 55618, and exhibits high productivity in the mass production of succinic acid.
상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP는 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618을 화학적 돌연변이원으로 처리하여 준비되는 것이 바람직하며, 상기 화학적 돌연변이원은 에틸 메탄설포네이트(Ethyl methanesulfonate)인 것이 바람직하지만, 이에 한정되는 것은 아니다. 상기 에틸 메탄설포네이트는 유전자 염기서열 내의 염기서열에 무작위로 알킬화 반응을 유도하여 단백질 발현을 억제하는 돌연변이를 일으키는 화학적 돌연변이원으로 작용한다.The Actinobacillus < RTI ID = 0.0 > succinogenes) KCTC 12233BP Evil Tino Bacillus succinonitrile's Ness (Actinobacillus succinogenes ATCC 55618 as a chemical mutagen. Preferably, the chemical mutagen is ethyl methanesulfonate, but is not limited thereto. The ethyl methanesulfonate acts as a chemical mutagen that induces a mutation in the protein sequence by inducing a random alkylation reaction on the base sequence in the gene base sequence.
본 발명의 숙신산의 제조 방법에서, 상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 배양하는 단계는, 포도당, 효모 추출물, 옥수수 침지액 고형분, 탄산수소나트륨(NaHCO3) 및 탄산마그네슘(MgCO3)을 포함하는 액체 배지에서 배양하는 것이 바람직하지만, 이에 한정되지 않고, 필요에 따라 적절히 선택할 수 있다. In the method for producing succinic acid according to the present invention, the step of culturing Actinobacillus succinogenes KCTC 12233BP comprises culturing glucose, yeast extract, corn steep liquor, sodium hydrogencarbonate (NaHCO 3 ) and magnesium carbonate MgCO 3 ), but the present invention is not limited thereto and can be appropriately selected according to need.
상기 액체 배지는 포도당 90 내지 120 g/L, 효모 추출물 10 내지 30 g/L, 옥수수 침지액 고형분 15 내지 30 g/L, 탄산수소나트륨 5 내지 15 g/L 및 탄산마그네슘 50 내지 120 g/L를 포함하는 액체 배지인 것이 바람직하고, 보다 바람직하게는 포도당 100 내지 110 g/L, 효모 추출물 10 내지 15 g/L, 옥수수 침지액 고형분 15 내지 20 g/L, 탄산수소나트륨 7 내지 12 g/L 및 탄산마그네슘 90 내지 110 g/L를 포함하고, 가장 바람직하게는 포도당 109 g/L, 효모 추출물 13 g/L, 옥수수 침지액 고형분 19 g/L, 탄산수소나트륨 10 g/L 및 탄산마그네슘 100 g/L을 포함하는 액체 배지인 것이 좋다. 상기 액체 배지의 조성물이 상술한 범위를 만족할 경우, 상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP에 의한 숙신산의 생산성이 높아지고, 수율이 향상되며, 경제적인 측면에서 유리하다.The liquid medium contains 90 to 120 g / L of glucose, 10 to 30 g / L of yeast extract, 15 to 30 g / L of corn steep liquor, 5 to 15 g / L of sodium hydrogencarbonate and 50 to 120 g / , More preferably 100 to 110 g / L of glucose, 10 to 15 g / L of yeast extract, 15 to 20 g / L of corn steep liquor, 7 to 12 g / l of sodium hydrogencarbonate, L and 90 to 110 g / L of magnesium carbonate and most preferably 109 g / L of glucose, 13 g / L of yeast extract, 19 g / L of corn steep liquor solid, 10 g / L of sodium hydrogencarbonate, 100 g / L. When the composition of the liquid medium satisfies the above-mentioned range, the actinobacillus succinogenes ) The productivity of succinic acid by KCTC 12233BP is increased, yield is improved, and it is advantageous from the economical point of view.
상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 배양하는 단계에서, 배양 방법은 특별히 한정되지 않으며, 당업자에 의하여 필요에 따라 적절히 선택될 수 있다.
The Actinobacillus < RTI ID = 0.0 > succinogenes ) In the step of culturing KCTC 12233BP, the culture method is not particularly limited and can be suitably selected according to need by a person skilled in the art.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
<< 실시예Example 1: One: 악티노바실러스Actino Bacillus 숙시노게네스SuShinogenes ( ( ActinobacillusActinobacillus succinogenessuccinogenes ) KCTC 12233) KCTC 12233 BPBP >>
악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27를 친균주로 하여 숙신산 생산성이 향상된 돌연변이주를 얻기 위해 친균주의 균체를 수거하여 변이처리에 이용하였다. Actinobacillus Actinobacillus Actinobacillus < / RTI > derived from succinogenes ATCC 55618 succinogenes ) WD27 was used as a host strain to obtain mutant strains with improved productivity of succinic acid.
상기 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27에 0~240ppm의 에틸 메탄설포네이트(Ethyl methanesulfonate, EMS) 처리한 결과 90%의 사멸율을 나타내는 농도인 160ppm을 돌연변이원 처리 농도로 결정하였다 (도 1).The Actinobacillus < RTI ID = 0.0 > succinogenes) After a
균주의 돌연변이 유도를 위해 EMS를 160ppm의 농도로 10분간 처리한 후 세척하고 연속 희석(serial dilution)을 통해 플레이트당 약 1 × 102개 콜로니(colony) 성장이 가능한 조건으로 10개의 한천 플레이트(agar plate)에 도말하였다. 38℃ 에서 24시간 배양 후 10개의 한천 플레이트로부터 약 103개의 단일 콜로니(single colony)를 얻을 수 있었으며, 이 중 단일 콜로니 형태로 명확히 구분되며 성장이 빠른 콜로니 144개를 선정하여 24웰 마이크로플레이트(24 well microplate)에서 액상배양을 수행하였다. 콜로니의 성장 정도는 단일콜로니의 크기로 판단하였다. 액상 배양을 위해 웰 당 하나의 콜로니를 접종하였으며 각 웰의 배양액 부피는 1.25 mL였다. 액상 성장배양은 2회에 걸쳐 실시하였으며 2차 배양시 콜로니별 접종량을 동일하게 조정하여 접종량 차이에 따른 영향을 배제하였다. 성장이 빠른 균주의 스크리닝을 위해 배양 중 각 콜로니별로 균체량을 측정하였다. 배양액의 탁도(파장 660 nm에서의 흡광도) 측정을 통해 상대적인 균체량을 파악하였으며, 탁도 측정은 1차, 2차 성장배양 개시 후 각각 12시간 시점에서 1회씩 실시하였다. 해당 측정치를 바탕으로 성장 속도가 빠른 균주 48개를 선정하였으며 각각 UK1~48로 명명하였다. 상기 UK1~48 중에서, 균체의 성장이 가장 뛰어난 13번 돌연변이주(UK13)인 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 수득하였다.Washing after the EMS for the mutation induced in the strain for 10 minutes at a concentration of 160ppm, and serially diluted (serial dilution) to about 1 × 10 2 gae colonies (colony) growth is the possible conditions of 10 agar plates (agar per plate over plate. At 38
<< 비교예Comparative Example 1: One: 악티노바실러스Actino Bacillus 숙시노게네스SuShinogenes ( ( ActinobacillusActinobacillus succinogenessuccinogenes ) ) WD27WD27 >>
악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27를 비교예 1의 균주로 사용하였다. WD27 균주의 수득 방법을 구체적으로 설명하면 ATCC 55618 균주 스탁(stock)을 증류수 및 20% 글리세롤(glycerol0 용액을 이용하여 적절히 희석하여 플레이트당 약 100개의 단일 콜로니가 형성되도록 한천 플레이트에 도말하였다. 배양 후 5 내지 10개의 한천 플레이트에서 단일 콜로니를 무작위적으로 수득하여 38도의 항온 조건, 1.25 mL의 조업부피로 한 웰당 하나의 콜로니를 접종하여 24웰 마이크로 플레이트에서 성장배양을 수행하였으며 이후 같은 배양 조건에서 생산배지를 사용하여 48시간 동안 배양하였다. 해당 과정을 거쳐 선별된 숙신산 생산성이 가장 높은 균주를 친균주로 하여 상기 서술한 균주 선별과정을 다시 수행하였다. 기술한 과정을 총 5회 수행하여 얻어진 숙신산 생산성이 가장 높은 균주를 WD27 균주로 명명하였다. Actinobacillus Actinobacillus Actinobacillus < / RTI > derived from succinogenes ATCC 55618 succinogenes ) WD27 was used as the strain of Comparative Example 1. To obtain the WD27 strain, ATCC 55618 strain stock was appropriately diluted with distilled water and 20% glycerol solution, and was plated on an agar plate to form about 100 single colonies per plate. A single colony was randomly obtained from 5 to 10 agar plates and inoculated with one colony per well at a constant temperature of 38 DEG C and a working volume of 1.25 mL to perform growth culture on a 24 well microplate and then cultured under the same culture conditions And the culture was continued for 48 hours using the culture medium. [0060] The strain selection process described above was repeated with the strain having the highest productivity of succinic acid selected as the host strain, The highest strain was named WD27 strain.
<< 비교예Comparative Example 2 내지 48> 2 to 48>
상기 실시예 1에 있어서, 상기 한천플레이트에서 자라난 콜로니를 회수하여 144개의 균체 중 성장이 우수한 48개의 균주를 선택하였으며, 균체 성장이 가장 뛰어난 13번 돌연변이주(UK13, 실시예 1)인 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 제외하고, 나머지 47개의 균주(UK1~12, UK14~48)를 각각 비교예 2 내지 48로 사용하였다.In Example 1, the colonies grown on the agar plates were collected to select 48 strains of 144 cells, which were excellent in growth. The strains of Actino (SEQ ID NO: 13), which is the 13th mutant strain (UK13, Example 1) The remaining 47 strains (UK1-12, UK14-48) were used as Comparative Examples 2 to 48, respectively, except for Actinobacillus succinogenes KCTC 12233BP.
<< 참조예Reference Example 1: 균주의 보관> 1: Storing the strain>
상기 실시예 1 및 비교예 1 내지 48의 균주 보관은 액상배양을 통해 얻은 균체를 4℃에 보관하거나 20% 글리세롤 stock을 만들어 -80℃에 보관하였고, 필요시 마다 보관된 stock을 꺼내어 고체 계대배양 배지에 접종 후 배양하여 사용하였다.For the storage of the strains of Example 1 and Comparative Examples 1 to 48, the cells obtained from the liquid culture were stored at 4 ° C or 20% glycerol stock and stored at -80 ° C. When necessary, the stored stocks were taken out, And cultured after inoculation on a medium.
<< 참조예Reference Example 2: 배지 및 배양 조건> 2: Medium and culture condition>
고체배양은 pancreatic digest of casein 15 g/L, papaic digest of soybean 5 g/L, NaCl 5 g/L, 한천(agar) 15 g/L를 증류수에 녹여 제조한 TSA(tryptic soy agar) 배지를 이용하였다. 연속희석법을 통하여 단일콜로니가 배양되게 하였으며 30ml의 TSA한천 배지를 만든 후 희석된 균체 용액을 100 μl 접종하였다. 접종된 한천플레이트는 38도의 항온조건에서 24시간 배양하였다.The solid culture was carried out using TSA (tryptic soy agar) medium prepared by dissolving 15 g / L of pancreatic digest of casein, 5 g / L of papaic digest of soybean, 5 g / L of NaCl and 15 g / L of agar in distilled water Respectively. A single colony was allowed to grow through serial dilution and 30 ml of TSA agar medium was inoculated and 100 μl of the diluted bacterial solution was inoculated. The inoculated agar plates were incubated at 38 ° C for 24 hours.
생산배양에 앞서 균체를 늘리기 위한 성장배양은 pancreatic digest of casein 17 g/L, papaic digest of soybean 3 g/L, 덱스트로스 2.5 g/L, NaCl 5 g/L, K2HPO4(potassium phosphate dibasic) 2.5 g/L를 증류수에 녹여 TSB(tryptic soy broth) 배지를 사용하였다. The growth culture for the growth of the cells prior to the culture was pancreatic digest of casein 17 g / L, papaic digest of soybean 3 g / L, dextrose 2.5 g / L, NaCl 5 g / L, K 2 HPO 4 (potassium phosphate dibasic ) 2.5 g / L was dissolved in distilled water and TSB (tryptic soy broth) medium was used.
숙신산 생산을 위하여는 포도당 109 g/L, 효모 추출물 13 g/L, 옥수수 침지액 고형분 19 g/L, 탄산수소나트륨(NaHCO3) 10 g/L, 탄산마그네슘(MgCO3) 100 g/L를 증류수에 녹여 제조한 pH 7.0의 액상배지를 사용하였다.For the production of succinic acid, 10 g / L of glucose, 13 g / L of yeast extract, 19 g / L of corn steep liquor, 10 g / L of sodium hydrogencarbonate (NaHCO 3 ) and 100 g / L of magnesium carbonate (MgCO 3 ) A liquid medium of pH 7.0 prepared by dissolving in distilled water was used.
배양은 24 웰 마이크로플레이트(well microplate)에서 1.25 mL의 배양 부피로 배양되거나, 100 mL serum bottle에서 30 mL의 배양부피로 진행되었다. 균체의 액상배양은 진탕 배양기에서 38℃, 200rpm으로 1 내지 2일간 배양하거나, 9시간 배양한 후 다른 플라스크로 계대 배양하였다.The cultures were cultured in a 24-well microplate at a culture volume of 1.25 mL, or in a 100 mL serum bottle at a volume of 30 mL. Liquid culture of the cells was incubated for 1 to 2 days at 38 DEG C and 200 rpm in a shaking incubator, or cultured for 9 hours and subcultured in another flask.
발효조 배양은 상기 생산배지 성분과 일치하게 수행하였으며 이산화탄소를 0.6 vvm으로 폭기 시키고 300rpm의 속도로 교반시켜 주었다. pH는 6.8 내지 7.2로 일정하게 유지시켜 주며 배양하였다.The fermenter was cultivated in accordance with the production medium, aerated with carbon dioxide at 0.6 vvm, and stirred at a speed of 300 rpm. The pH was kept constant at 6.8 to 7.2 and cultured.
<< 참조예Reference Example 3: 균체의 농도 측정> 3: Measurement of cell density>
균체의 농도 측정은 균체의 건조중량(D.C.W)을 이용하였다. 배양액으로부터 균일하게 균체를 회수한 다음 1 mL의 세포를 10,000 rpm에서 10분간 원심분리 하였다. 상등액을 회수하여 1.5 mL 마이크로 튜브에 옮겨 남아 있는 당의 측정에 사용하였고, 나머지 상등액을 제거한 후 3번의 세척과정을 통해서 남아 있는 당, 불용성 물질이나 염류들을 제거하였다. The dry weight (D.C.W) of the cells was used to determine the concentration of the cells. After uniformly recovering the cells from the culture, 1 mL of cells were centrifuged at 10,000 rpm for 10 minutes. The supernatant was recovered and transferred to a 1.5 mL microtube for use in the determination of the remaining sugars. The remaining supernatant was removed and the remaining sugars, insoluble matter and salts were removed through three washes.
이후 무게측정용 접시에 담아 90℃에서 12시간 건조하여 건조 균체 농도의 무게에 변화가 없는 것을 확인한 후 건조중량을 측정하여 1L 당 세포농도로 환산하여 나타내었다.Thereafter, the resultant was dried in a weight measuring dish at 90 ° C for 12 hours to confirm that there was no change in the weight of the dried cell mass, and the dry weight was measured and expressed in terms of the cell concentration per 1 L.
<< 참조예Reference Example 4: 숙신산의 정량분석> 4: Quantitative analysis of succinic acid>
악티노바실러스 숙시노겐(Actinobacillus succinogenes)의 배양액에서 숙신산의 정량분석을 위해 1.5 mL 마이크로 튜브에 시료를 채취하고 연속 희석(serial dilution)을 통하여 희석한 다음 0.45 ㎛ 여과지를 이용하여 2번의 여과 과정을 수행한 후, HPLC를 이용하여 다음의 조건으로 분석하였다.For the quantitative analysis of succinic acid in the cultures of Actinobacillus succinogenes , samples were taken in 1.5 ml microtube, diluted by serial dilution, and then filtered twice using 0.45 ㎛ filter paper. And analyzed by HPLC under the following conditions.
분석 온도: 25℃Analysis temperature: 25 ° C
유속: 0.8 ㎖/min Flow rate: 0.8 ml / min
이동상: 0.01N H2SO4 Mobile phase: 0.01NH 2 SO 4
분석시간: 20분Analysis time: 20 minutes
시료 주입양: 20 ㎕Sample injection: 20 μl
칼럼: organic acid column (Bio-Rad, Aminex HPX 87H, 125-0140) Column: organic acid column (Bio-Rad, Aminex HPX 87H, 125-0140)
검출기: UV detector Detector: UV detector
<< 참조예Reference Example 5: 당 분석> 5: sugar analysis>
배양액 중의 당 분석은, 배양액을 12,000rpm에서 10분간 원심분리한 후 상등액을 취하여 12,000rpm에서 10분간 3회 원심분리를 반복 수행하여 상등액 만을 취한 다음, 0.45 ㎛ HPLC용 여과지를 이용하여 여과하여 수행하였다. HPLC를 이용한 당분석 조건은 다음과 같았다:The supernatant was centrifuged at 12,000 rpm for 10 minutes, centrifuged three times for 10 minutes at 12,000 rpm, and the supernatant was collected, followed by filtration using a 0.45 쨉 m filter paper for HPLC . The sugar assay conditions using HPLC were as follows:
분석 온도: 40℃Analysis temperature: 40 ° C
유속: 1.2 ㎖/minFlow rate: 1.2 ml / min
이동상 : 75 : 25 (아세토니트릴 : 물)Mobile phase: 75: 25 (acetonitrile: water)
분석 시간: 15분Analysis time: 15 minutes
시료 주입양: 20 ㎕Sample injection: 20 μl
컬럼: Amine column (RS tech, NH2 column (250 ㎜ × 46 ㎜))Column: Amine column (RS tech, NH2 column (250 mm x 46 mm))
검출기: RI detector
Detector: RI detector
<< 실험예Experimental Example 1: 숙신산 생산성 및 수율 실험> 1: Succinic acid productivity and yield test>
상기 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP 및 상기 비교예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27 및 상기 비교예 2 내지 48의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) 돌연변이주 (UK1~12, UK14~48)를 각각 serum bottle에서 30ml의 부피로 생산배양을 실시하였고, 도 3에 나타낸 것과 같이 균체성장이 가장 뛰어났던 실시예 1의 13번 돌연변이주(UK13)인 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP가 약 107 g/L의 숙신산 생산성을 나타내는 것을 확인하였다.To bad Martino Bacillus succinonitrile in Example 1 Ness (Actinobacillus succinogenes) KCTC 12233BP and evil Martino Bacillus of Comparative Example 1 succinonitrile to Ness (Actinobacillus succinogenes) an evil Martino Bacillus succinonitrile to Ness (Actinobacillus derived from ATCC 55618 succinogenes WD27 and Actinobacillus strains of the above Comparative Examples 2 to 48 succinogenes) mutants (UK1 ~ 12, UK14 ~ 48 ) each were in the serum bottle subjected to the production culture in a volume of 30ml, No. 13 mutant of Example 1 over, cell growth is the most excellent, as shown in Figure 3 weeks (UK13 Actinobacillus < / RTI > ( Actinobacillus < RTI ID = 0.0 & succinogenes KCTC 12233BP showed succinic acid productivity of about 107 g / L.
상기 표 1에서 볼 수 있듯이, 친균주인 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27 (비교예 1)에 비하여, 상기 실시예 1의 변이 균주(UK13)인 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP의 숙신산 생산성은 약 1.34배, 수율은 1.34배 향상된 것을 확인 할 수 있었다. 또한, 상기 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP은 주어진 포도당에서 이론수율에 75% 수준의 숙신산을 생산해내고 있는 것을 확인할 수 있다.
As can be seen from Table 1, the parent strain Bacillus evil Martino succinonitrile to Ness (Actinobacillus succinogenes) an evil Martino Bacillus succinonitrile to Ness (Actinobacillus derived from ATCC 55618 succinogenes ) In comparison with WD27 (Comparative Example 1), Actinobacillus strain < RTI ID = 0.0 > succinic acid productivity succinogenes) KCTC 12233BP is approximately 1.34 times, the yield could be confirmed that increased 1.34 times. In addition, Actinobacillus succinogenes KCTC 12233BP of Example 1 produced 75% of succinic acid at a given yield in a given glucose.
한편, 비교예 2, 6, 9, 14, 16, 20, 21, 22, 29, 37, 38, 39 및 48의 균주의 경우, 비교예 1에 비하여 높은 숙신산 생산성을 나타내었지만(도 3), 균체성장이 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP에 비하여 2배 이상 낮게 나타내고 있는 것을 확인할 수 있으며, (도 2), 이를 제외한 비교예의 균주의 경우, 비교예 1의 균주에 비하여 낮은 숙신산 생산성을 나타냄을 알 수 있다. On the other hand, strains of Comparative Examples 2, 6, 9, 14, 16, 20, 21, 22, 29, 37, 38, 39 and 48 exhibited higher succinic acid productivity than those of Comparative Example 1 (FIG. 3) When the cell growth was compared with that of Actinobacillus < RTI ID = 0.0 > ( Actinobacillus < succinogenes ) KCTC 12233BP (FIG. 2). In the case of the strains of the comparative examples, the productivity of the strains of Comparative Example 1 is lower than that of the strains of Comparative Example 1.
또한, 숙신산 생산성에 따른 분포도를 나타내는 히스토그램을 나타내는 도 7을 통해, 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP가, 비교예 2 내지 48의 균주에 비하여 현저히 우수한 숙신산 생산성을 나타내고 있음을 알 수 있다. 7 showing a histogram showing the degree of distribution according to the productivity of succinic acid, Actinobacillus < RTI ID = 0.0 > ( Actinobacillus < succinogenes KCTC 12233BP showed significantly higher succinic acid productivity than the strains of Comparative Examples 2 to 48. [
<< 실험예Experimental Example 2: 대량 생산 가능성 실험> 2: Mass production possibility experiment>
상기 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP 균주를 발효조 배양을 통하여 scale-up에 적합한 균주인지 실험하였다. Actinobacillus < RTI ID = 0.0 > ( Actinobacillus < / RTI & the succinogenes) KCTC 12233BP strain was tested whether the strains suitable for scale-up through the fermentor culture.
포도당 109 g/L, 효모 추출물 13 g/L, 옥수수 침지액 고형분 19 g/L, 탄산수소나트륨 10 g/L, 탄산마그네슘 100 g/L의 배지 성분을 이용하여 상기 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP의 배양을 수행 하였고 serum bottle의 100배 증가된 3L를 조업부피로 정하고 300rpm의 교반속도 하에서 배양하였다. The culture medium of Actinobacillus strain 1 of Example 1 was cultured in the same manner as in Example 1 using the medium components of 109 g / L of glucose, 13 g / L of yeast extract, 19 g / L of corn steep liquor, 10 g / L of sodium hydrogencarbonate and 100 g / Actinobacillus succinogenes ) KCTC 12233BP were cultured and the volume of 3 L, which was 100 times higher than that of the serum bottle, was determined as the operation volume and cultured at a stirring speed of 300 rpm.
5L 발효조를 통하여 상기 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP 및 상기 비교예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) ATCC 55618에서 유래된 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) WD27를 48시간 배양해 본 결과 비교예 1의 숙신산 생산량은 약 60 g/L 이었고, 실시예 1의 숙신산 생산량은 약 110 g/L인 것을 확인 하였다(도 4). Ill Martino Bacillus in Example 1 through the 5L fermenter succinonitrile to Ness (Actinobacillus succinogenes) KCTC 12233BP and Ill to Martino Bacillus succinonitrile in Comparative Example 1 Ness (Actinobacillus Actinobacillus < / RTI > derived from succinogenes ATCC 55618 succinogenes ) WD27 was cultured for 48 hours. As a result, the succinic acid production amount in Comparative Example 1 was about 60 g / L, and the succinic acid production amount in Example 1 was about 110 g / L (FIG.
이를 통하여, 상기 실시예 1의 악티노바실러스 숙시노게네스 (Actinobacillus succinogenes) KCTC 12233BP를 이용하는 경우, 100배 증가된 scale에서도 훌륭히 발효가 수행되는 것을 알게 되었고 산업화 적용에도 우수한 성공 가능성을 갖는 균주임을 확인 하였다. As a result, when Actinobacillus succinogenes KCTC 12233BP of Example 1 was used, it was found that the fermentation was excellently performed even at a scale of 100 times, and it was confirmed that the strain had excellent success potential in industrial application Respectively.
시간당 생산성의 변화를 나타낸 도 5를 보면 10~30 시간 사이에 집중적으로 숙신산이 생산 된 것을 알 수 있으며 이론수율에 대한 숙신산의 생산량이 76.4% 수준으로 매우 높은 수준으로 생산되고 있음을 알 수 있다(도 6).FIG. 5 shows changes in productivity per hour. It can be seen that succinic acid was intensively produced within 10 to 30 hours, and the production amount of succinic acid to the theoretical yield was as high as 76.4% 6).
Claims (7)
B) 상기 배양액으로부터 숙신산을 수득하는 단계를 포함하는 숙신산의 제조 방법.A) culturing Actinobacillus succinogenes UK13 KCTC 12233BP; And
B) A step of obtaining succinic acid from the culture solution.
상기 화학적 돌연변이원은 에틸 메탄설포네이트(Ethyl methanesulfonate)인 것을 특징으로 하는 숙신산의 제조 방법.The actinobacillus succinogenes UK13 KCTC 12233BP is prepared by treating Actinobacillus succinogenes ATCC 55618 as a chemical mutagen. The Actinobacillus succinogenes UK13 KCTC 12233BP is prepared by treating actinobacillus succinogenes ATCC 55618 as a chemical mutagen.
Wherein the chemical mutagen is ethyl methanesulfonate. ≪ RTI ID = 0.0 > 8. < / RTI >
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