KR102120670B1 - Method for preparing antioxidants using mutant strain - Google Patents
Method for preparing antioxidants using mutant strain Download PDFInfo
- Publication number
- KR102120670B1 KR102120670B1 KR1020190000915A KR20190000915A KR102120670B1 KR 102120670 B1 KR102120670 B1 KR 102120670B1 KR 1020190000915 A KR1020190000915 A KR 1020190000915A KR 20190000915 A KR20190000915 A KR 20190000915A KR 102120670 B1 KR102120670 B1 KR 102120670B1
- Authority
- KR
- South Korea
- Prior art keywords
- pqq
- quinone
- strain
- culture
- methanol
- Prior art date
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- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 17
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 16
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 137
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Abstract
Description
본 발명은 고기능성 항산화 물질을 고농도로 생산하는 하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주를 이용한 항산화 물질 생산 방법에 관한 것이다.The present invention relates to a method for producing an antioxidant using a strain of Hyphomicrobium sp. that produces a high concentration of a highly functional antioxidant.
항산화 물질은 산화를 방지하는 물질을 총칭하여 일컫는 말인데, 생체 내에서 대사 과정 중 생산되는 유리 라디칼을 효율적으로 처리 및 제거할 수 있는 물질이다. 이러한 항산화 물질에는 루테인, 제아잔틴, 알리신, 플라보노이드, 커규민, 글루타티온, NADH, 비타민 C, 알파-토코페롤, 유비퀴논, 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ) 등 외에 많은 물질들이 알려져 있다. Antioxidant refers to a substance that prevents oxidation, and is a substance capable of efficiently treating and removing free radicals produced during metabolic processes in vivo. In addition to these antioxidants, lutein, zeaxanthin, allicin, flavonoids, curcumin, glutathione, NADH, vitamin C, alpha-tocopherol, ubiquinone, pyrroquinoline quinone (PQQ), and many other substances are known.
상기 항산화 물질 중 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ)은 1967년 메탄올을 포름알데하이드로 전환하는 미생물의 메탄올 디하이드로게나제(methanol dehydrogenase)로부터 최초로 분리되어 메토잔틴(methoxantin)으로 명명되었다. 1979년 X선 회절분석법(X-ray diffraction, XRD)에 의해 구조가 밝혀지면서 NAD(nicotinamide adenine dinucleotide), FAD(flavin adenine dinucleotide)에 이은 제3의 산화환원 조효소로 알려지기 시작했다. PQQ를 조효소로 하는 퀴노단백질에는 메틸로박터(Methylobacter)의 메탄올 디하이드로게나제(dehydrogenase), 초산균(Acetobacter)의 알코올 디하이드로게나제 및 글루코노박터(Gluconobacter)의 글루코오스 디하이드로게나제 등이 있다. Among the antioxidants, pyrroquinoline quinone (PQQ) was first isolated from methanol dehydrogenase of a microorganism that converts methanol to formaldehyde in 1967 and was named metoxanthin. When the structure was revealed by X-ray diffraction (XRD) in 1979, it began to be known as a third redox coenzyme following NAD (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). Quinoproteins using PQQ as a coenzyme include methanol dehydrogenase of methylobacter, alcohol dehydrogenase of acetobacter, and glucose dehydrogenase of gluconobacter. .
PQQ는 박테리아, 곰팡이, 효모에서 발견되고 있고 파슬리, 콩, 피망, 감자, 키위, 시금치, 파파야 등 다양한 식품뿐만 아니라 사람의 모유를 포함한 쥐의 생체 내에도 존재한다고 보고되고 있다. PQQ is found in bacteria, fungi, and yeast, and has been reported to exist in a variety of foods such as parsley, soybeans, green peppers, potatoes, kiwi, spinach, and papaya, as well as in humans, including human breast milk.
PQQ는 대사과정이나 효소 반응을 촉진하는 조효소이기 때문에 다양한 분야에서 그 중요성이 부각되고 있다. PQQ의 생리활성 기능으로는 극한 환경에서 미토콘드리아 활성화(미토콘드리아 에너지 대사 기능 강화, 미토콘드리아 수 증가), 전자 전달뿐만 아니라 활성 산소를 제거하는 항산화 효과, 역전사 효소를 억제하는 효과, 멜라닌 합성을 억제하는 효과, 식물의 생육을 활성화하는 효과 등이 있다. Since PQQ is a coenzyme that promotes metabolic processes or enzyme reactions, its importance is highlighted in various fields. The bioactive functions of PQQ include mitochondrial activation in extreme environments (strengthening mitochondrial energy metabolism, increased mitochondrial number), antioxidant effect to remove free radicals as well as electron transfer, inhibitory effect on reverse transcriptase, inhibitory effect on melanin synthesis, It has the effect of activating the growth of plants.
최근 PQQ에 대한 초기 임상 실험 결과에 의하면 뇌기능, 생리기능의 다양한 부분에서 효과를 보이는 것으로 보고되고 있다. 구체적으로 노화, 뇌졸중, 신경기능 저하로 인한 인지 저하 방지, 기억력 개선, 뇌세포, 수은, 글루탐산, 옥시도파민 등 독성물질로 야기되는 신경독성으로부터 신경세포의 보호, 신경 성장인자의 생산 및 분비기능 촉진, 파킨슨병의 유전자 산화 기작의 방어, 독성 단백질 생성 저해, 뇌손상을 일으키는 활성 질소화합물 합성 저해 및 알츠하이머 질환에서 신경세포의 손상을 저해하는 기능 등이 알려지고 있다. According to the results of recent clinical trials on PQQ, it has been reported to show effects in various parts of brain function and physiological function. Specifically, it prevents cognitive decline due to aging, stroke, and decreased neurological function, improves memory, protects nerve cells from neurotoxicity caused by toxic substances such as brain cells, mercury, glutamic acid, and oxidopamine, and promotes the production and secretion functions of nerve growth factors. , Parkinson's disease has been known to defend the mechanism of gene oxidation, inhibit toxic protein production, inhibit the synthesis of active nitrogen compounds that cause brain damage, and inhibit neuronal damage in Alzheimer's disease.
상기와 같이 PQQ는 세포 기능에 관여하는 효소와 결합하여 세포의 성장을 촉진시키고, 비타민 C에 비해 약 5,000 배의 산화환원 반응을 촉진하는 뛰어난 항산화 효과 및 뇌기능, 생리기능의 다양한 부분에서 효과를 보이는 것으로 알려져 있어서, 식품, 화장품, 의약 및 농업분야에서 사용이 늘어나고 있다. As described above, PQQ promotes cell growth by combining with enzymes involved in cell function, and has an excellent antioxidant effect that promotes a redox reaction of about 5,000 times that of vitamin C, and effects in various parts of brain function and physiological function. As it is known to be seen, its use is increasing in food, cosmetics, medicine and agriculture.
PQQ는 그 자체로 에너지 대사를 증진시키는 기능 외에 CoQ10과 함께 작용하여 뇌신경 기능을 향상시킨다고 알려져 있다. 또한 이를 영양분으로 섭취하지 않을 경우 생육 부진, 면역기능 손상, 기억력 저하 등의 문제를 야기할 수 있다는 주장에 따라 PQQ는 필수 영양원으로 여겨져 새로운 기능성 물질로 각광을 받고 있으며 14번째 새로운 비타민으로 기대되고 있다.It is known that PQQ improves cranial nerve function by working with CoQ10 in addition to its ability to enhance energy metabolism by itself. In addition, PQQ is regarded as an essential nutrient and is in the spotlight as a new functional substance, and is expected to be the 14th new vitamin, according to the claim that it can cause problems such as poor growth, impaired immune function, and decreased memory if not consumed as nutrients. .
이처럼 다양한 기능적 유용성을 가진 PQQ는 유기 화학적 방법과 세균을 이용한 발효에 의한 방법으로 생산되고 있다.PQQ with various functional usefulness is produced by organic chemical method and fermentation method using bacteria.
유기 화학적 합성법은 다단계의 합성반응으로 구성되어, 제조에 장시간을 필요로 하며, 이성질체를 비롯한 부산물의 제거를 위해 복잡한 조작을 필요로 하고, PQQ 회수율도 낮은 문제가 있다. The organic chemical synthesis method is composed of a multi-step synthesis reaction, requires a long time to manufacture, requires complicated manipulation to remove by-products including isomers, and has a problem of low PQQ recovery.
세균을 이용한 발효에 의한 방법은 발효에 의해 얻어지는 PQQ의 농도가 통상 0.4 g/L 이하로 수율이 낮고, 세균의 배양기간도 약 15일 정도로 길다. 또한 메탄올을 에너지원으로 이용하는 이들 메탄올 자화균들은 메탄올의 농도가 너무 낮거나 높은 경우 균주의 생육이 저해되어 균주의 생육 안정성이 낮아 배양 과정 중 메탄올 농도를 유지하기가 어렵다. 그리고, 정제 공정에 사용되는 이온교환수지의 비용부담이 큰데다 정제 과정 중 PQQ의 손실이 커서 최종 회수율이 10 % 정도에 그치는 경우가 많고, 최종 정제된 결정의 순도가 기준인 93 %에 미치지 못하는 등 세균을 이용한 PQQ 생산에도 극복하여야 할 과제가 많다. In the method by fermentation using bacteria, the concentration of PQQ obtained by fermentation is usually 0.4 g/L or less, and the yield is low, and the culture period of bacteria is long, about 15 days. In addition, these methanol magnetization bacteria using methanol as an energy source are difficult to maintain the methanol concentration during the cultivation process because the growth of the strain is inhibited because the concentration of methanol is too low or too high. In addition, the cost of ion exchange resin used in the purification process is high, and the loss of PQQ during the purification process is large, so the final recovery rate is often only 10%, and the purity of the final purified crystal does not reach the 93% standard. There are many challenges to overcome in the production of PQQ using bacteria.
이에 본 발명자는 고기능성 항산화 물질인 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ)을 고농도로 생산할 수 있는 하이포마이크로비움 데니트리피칸즈(Hyphomicrobium denitrificans)의 돌연변이 균주를 제조하여 세균을 이용한 PQQ 합성시의 문제점으로 지적되는 메탄올 농도에 따른 균주의 낮은 생육 안정성, 장시간의 세균 배양 및 낮은 PQQ 회수율 등의 문제를 해결하고자 한다. Accordingly, the present inventor prepared a mutant strain of Hypomicrobium denitrificans capable of producing high-performance antioxidant pyroquinoline quinone (PQQ) at a high concentration and synthesized PQQ using bacteria. To solve the problems such as low growth stability, long-term bacterial culture and low PQQ recovery of the strain according to the methanol concentration indicated as a problem.
또한, 고순도 정제를 위해 전처리 단계를 추가한 공정을 개발하여 PQQ 합성의 다른 문제점으로 지적되는 정제 공정의 비용 부담, PQQ의 낮은 회수율 및 낮은 순도 문제를 해결하고자 한다. In addition, a process in which a pre-treatment step was added for high-purity purification was developed to solve the cost burden of the purification process, a low recovery rate of PQQ, and a low purity problem, which are pointed out as another problem of PQQ synthesis.
상기의 문제점들을 해결하기 위해 본 발명은 고기능성 항산화 물질인 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ)을 고농도로 생산할 수 있는 하이포마이크로비움 속(Hyphomicrobium sp.) NPP-230(KCTC13718BP) 변이 균주를 제공한다.In order to solve the above problems, the present invention is a hypermicrobium sp.NPP-230 (KCTC13718BP) mutant strain capable of producing high-performance antioxidant pyroquinoline quinone (PQQ) at a high concentration. Provides
본 발명의 다른 양태는 하이포마이크로비움 데니트리피칸즈 균주에 자외선을 조사하여 돌연변이를 유발하는 단계; 및 상기 돌연변이된 균주를 고농도의 메탄올 배지에서 배양하여 피로로퀴놀린 퀴논을 생산하는 변이주를 선별하는 단계; 를 포함하는 하이포마이크로비움 속(Hyphomicrobium sp.) NPP-230(KCTC13718BP) 변이 균주 제조방법을 제공한다.Another aspect of the present invention is a step of inducing a mutation by irradiating ultraviolet rays to the hypomicrobium denititripcans strain; And culturing the mutated strain in a high concentration of methanol medium to select strains producing pyroquinoline quinone; It provides a method for producing a strain strain of NPP-230 (KCTC13718BP) of the genus Hypomicrobium containing (Hyphomicrobium sp.).
본 발명의 또 다른 양태는, 하이포마이크로비움 속 NPP-230(KCTC13718BP) 변이주를 배양하는 단계; 상기 배양 발효액으로부터 흡착수지를 이용하여 발효액 내의 피로로퀴놀린 퀴논을 흡착하는 단계; 및 용리액을 사용하여 흡착 수지로부터 피로로퀴놀린 퀴논을 회수하는 단계; 를 포함하는 항산화 물질인 피로로퀴놀린 퀴논의 생산 방법을 제공한다.Another aspect of the present invention, culturing NPP-230 (KCTC13718BP) mutant strains of the hypomicrobium; Adsorbing pyroquinoline quinone in the fermentation broth using an adsorption resin from the culture fermentation broth; And recovering pyroquinoline quinone from the adsorbent resin using an eluent. It provides a method for producing a pyroquinoline quinone, an antioxidant that includes.
본 발명의 또 다른 양태는, 상기 생산 방법을 통해 얻어진 pH 6.0 내지 8.0의 피로로퀴놀린 퀴논 용액에 산을 첨가하여 상기 용액의 pH를 4.0 내지 7.0으로 조절하는 단계; 및 상기 용액에 염을 포함하는 결정화 용매를 첨가하고 수득된 용액을 냉각시켜 피로로퀴놀린 퀴논염을 석출시키는 과정을 포함하는 피로로퀴놀린 퀴논염 수화물의 제조방법을 제공한다. In another aspect of the present invention, the pH of the solution is adjusted to 4.0 to 7.0 by adding an acid to a pyroquinoline quinone solution having a pH of 6.0 to 8.0 obtained through the production method; And adding a crystallization solvent containing a salt to the solution and cooling the obtained solution to precipitate a pyrroloquinoline quinone salt.
본 발명의 또 다른 양태는, 분말 X-ray 회절 분석에서 주요 피크(peak)의 2θ 값이 바람직하게는 6.28 ± 0.4°, 8.74 ± 0.4°, 17.58 ± 0.4°, 18.28 ± 0.4°, 18.70 ± 0.4°, 22.96 ± 0.4°, 25.48 ± 0.4°, 26.56 ± 0.4°, 33.72 ± 0.4°, 36.28 ± 0.4°인 피로로퀴놀린 퀴논 나트륨염 수화물 결정을 제공한다. In another aspect of the invention, the 2θ value of the main peak in the powder X-ray diffraction analysis is preferably 6.28±0.4°, 8.74±0.4°, 17.58±0.4°, 18.28±0.4°, 18.70±0.4 Provided are pyrroloquinoline quinone sodium salt hydrate crystals of 22.96 ± 0.4°, 25.48 ± 0.4°, 26.56 ± 0.4°, 33.72 ± 0.4°, 36.28 ± 0.4°.
본 발명은 또한 하이포마이크로비움 속 NPP-230(KCTC13718BP) 변이 균주를 이용해 생산된 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ), 상기 정제 과정을 통해 얻어진 피로로퀴놀린 퀴논, 및 피로로퀴놀린 퀴논 나트륨염 수화물 중 적어도 어느 하나를 포함하는 항산화 기능의 제품으로 식품, 화장품 및 의약품을 제공한다.The present invention is also Pyrorroloquinoline quinone (PQQ) produced using the strain NPP-230 (KCTC13718BP) of the genus Hypomicrobium (Pyrrolo-quinoline quinone, PQQ), pyroquinoline quinone obtained through the purification process, and pyroquinoline quinone sodium It provides food, cosmetics, and medicines as an antioxidant function product containing at least one of salt hydrates.
본 발명의 하이포마이크로비움 속 변이 균주 NPP-230(KCTC13718BP)은 고농도의 메탄올에서도 성장이 가능하고 단기간에 대량의 피로로퀴놀린 퀴논을 생산할 수 있는 효과를 제공하는 신균주이다. 또한 본 발명의 변이 균주에 의한 고기능성 항산화 물질인 피로로퀴놀린 퀴논 생산 방법은 경제적이며 고순도의 피로로퀴놀린 퀴논 나트륨염 수화물을 제공한다.The mutant strain NPP-230 (KCTC13718BP) in the genus Hypomicrobium of the present invention is a new strain that can grow in a high concentration of methanol and provides the effect of producing a large amount of pyrroloquinoline quinone in a short period of time. In addition, the method for producing pyroquinoline quinone, which is a highly functional antioxidant by the mutant strain of the present invention, provides an economical and highly pure pyroquinoline quinone sodium salt hydrate.
도1은 PQQ의 화학 구조, 퀴노단백질에 PQQ가 조효소로 결합한 모습 및 PQQ의 원료 형상을 나타내는 참고 도면이다.
도2는 하이포마이크로비움 데니트리피칸즈(ATCC 51888)에 자외선을 조사했을 때, 자외선 조사 시간에 따른 상기 균주의 치사율을 나타내는 그래프이다.
도3은 자외선 조사 후, AMS 배지에서 메탄올 농도에 따른 균주의 생존율을 보여주는 사진이다.
도4는 하이포마이크로비움 데니트리피칸즈 변이 균주 NPP-230을 30 L에서 배양했을 때 균체의 증가, PQQ 생산량 및 메탄올 농도를 동시에 나타내는 그래프이다.
도5은 하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주 NPP-230을 3000 L 배양기를 이용해 배양했을 때 균체의 증가, PQQ 생산량 및 메탄올 농도를 동시에 나타내는 그래프이다.
도6은 전처리 공정을 추가한 후 정제된 PQQ의 HPLC 분석 결과를 나타내는 그래프이다.
도7는 30 L 규모로 하이포마이크로비움 속 변이 균주 NPP-230을 배양한 후 본 명세서에 기재된 전처리 과정과 결정화 과정을 진행한 뒤, 수득된 PQQ 나트륨염 수화물 결정의 순도를 HPLC로 분석한 결과를 나타내는 그래프이다.
도8은 3000 L 규모로 하이포마이크로비움 속 변이 균주 NPP-230을 배양한 후 본 명세서에 기재된 전처리 과정과 결정화 과정을 진행한 뒤, 수득된 PQQ 나트륨염 수화물 결정의 순도를 HPLC로 분석한 결과를 나타내는 그래프이다.
도9a는 PQQ 디소디움염의 표준품에 대한 질량 분석 그래프이다.
도9b는 본 발명에 따른 PQQ 디소디움염에 대한 질량 분석 그래프이다.
도10는 본 발명에 따른 PQQ 디소디움염에 대한 DSC & TGA분석을 나타내는 그패프이다.
도11은 수득된 PQQ 나트륨염 수화물 결정의 분말 X-ray 회절 분석 결과를 나타내는 그래프이다.
도12는 표준품들의 X-ray 회절 분석 패턴과 본 발명에 따른 PQQ 나트륨염 수화물 결정의 X-ray 회절 분석 패턴을 비교한 그래프이다.1 is a reference diagram showing the chemical structure of PQQ, the appearance of PQQ as a coenzyme bound to quinoprotein, and the raw material shape of PQQ.
Figure 2 is a graph showing the lethality of the strain according to the ultraviolet irradiation time, when irradiated with ultraviolet rays to Hypomicrobial denititripcans (ATCC 51888).
Figure 3 is a photograph showing the survival rate of the strain according to the methanol concentration in the AMS medium after ultraviolet irradiation.
Figure 4 is a graph showing the increase in cell growth, PQQ production and methanol concentration at the same time when cultured at 30 L of the Hypomicrobial denititripcans mutant strain NPP-230.
5 is a graph showing the growth of cells, PQQ production and methanol concentration at the same time when the strain of NPP-230, a strain of Hyphomicrobium sp. , is cultured using a 3000 L incubator.
6 is a graph showing the results of HPLC analysis of purified PQQ after adding a pretreatment process.
FIG. 7 shows the results of analyzing the purity of the obtained PQQ sodium salt hydrate crystals by HPLC after culturing the strain strain NPP-230 in the hypomicrobium on a 30 L scale and performing the pretreatment and crystallization processes described herein. It is a graph to show.
FIG. 8 shows the results of analyzing the purity of the obtained PQQ sodium salt hydrate crystals by HPLC after culturing the strain strain NPP-230 in hypomicrobium on a 3000 L scale and performing the pretreatment and crystallization processes described herein. It is a graph to show.
9A is a mass spectrometry graph for a standard product of PQQ disodium salt.
9B is a mass spectrometry graph for PQQ disodium salt according to the present invention.
10 is a graph showing DSC & TGA analysis for PQQ disodium salt according to the present invention.
11 is a graph showing the results of powder X-ray diffraction analysis of the obtained PQQ sodium salt hydrate crystal.
12 is a graph comparing X-ray diffraction analysis patterns of standard products and X-ray diffraction analysis patterns of PQQ sodium salt hydrate crystals according to the present invention.
이하, 본원 발명에 대하여 보다 상세히 설명한다. 그러나 이들 실시예는 본 발명에 대한 이해를 돕기 위한 것일 뿐, 어떤 의미로든 본 발명의 범위가 이들에 의해 제한되는 것은 아니다. 또한, 본원 명세서에 기재되지 않은 내용은 본원 발명의 기술 분야 또는 유사 분야에서 숙련된 자이면 충분히 인식하고 유추할 수 있는 것이므로 그 설명을 생략한다.Hereinafter, the present invention will be described in more detail. However, these examples are only to aid understanding of the present invention, and the scope of the present invention is not limited by them in any sense. In addition, the contents that are not described in the present specification will be sufficiently recognized and inferred by those skilled in the technical field or similar field of the present invention, and the description thereof will be omitted.
본 발명의 용어, “유가식 배양”은 회분식 배양과 달리 배양액을 빼내는 일 없이 배지를 연속 혹은 간헐적으로 공급하는 배양법을 말하며, 용어 "회분식 배양"은 초기에 한번 배지를 채운 후 더 이상의 영양 물질의 공급이나 제거 없이 반응기에서 세포를 배양하는 방법을 말한다. 용어 “불순물”은 PQQ를 제외한 모든 물질을 말한다. Unlike the batch culture, the term “fed-batch culture” of the present invention refers to a culture method in which the medium is continuously or intermittently supplied without removing the culture medium, and the term “batch culture” refers to the culture of more nutrients after the medium is initially filled once. It refers to a method of culturing cells in a reactor without supply or removal. The term “impurity” refers to all substances except PQQ.
본 발명은 고기능성 항산화 물질인 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ)을 고농도로 생산할 수 있는 변이 균주를 제공한다. The present invention provides a mutant strain capable of producing a high functional antioxidant pyroquinoline quinone (Pyrrolo-quinoline quinone, PQQ) at a high concentration.
본 발명은 모균주인 하이포마이크로비움 데니트리피칸즈(Hyphomicrobium denitrificans)에 비해 PQQ를 2 배 이상, 바람직하게는 3 배 이상, 보다 바람직하게는 4 배 이상 생산하는 변이 균주를 제공한다. The present invention provides a mutant strain that produces
본 발명은 고농도의 메탄올에 내성을 갖는 변이 균주를 제공한다.The present invention provides a mutant strain resistant to high concentrations of methanol.
상기 변이 균주는 0.5 중량% 내지 5 중량%의 메탄올에 내성을 갖는 변이 균주일 수 있다. 메탄올을 에너지원으로 이용하는 균주들은 메탄올을 흡수하여 포름알데하이드 산화물을 합성하는데 이는 세포 내 글리신과 함께 탄소원 동화 기작인 세린 회로에 의해 이용된다. 그러나 배지 내 메탄올 농도가 높아질 경우 이로부터 생산된 과량의 포름알데하이드는 세포 내 독성 성분으로 작용하여 균주의 생육을 저해한다. 그러므로 배지의 메탄올 농도가 5 중량% 이상인 경우 균주가 성장하기 어려울 수 있다.The mutant strain may be a mutant strain resistant to 0.5% to 5% by weight of methanol. Strains using methanol as an energy source absorb methanol and synthesize formaldehyde oxide, which is used by the serine cycle, a carbon source assimilation mechanism with glycine in the cell. However, when the methanol concentration in the medium increases, the excess formaldehyde produced therefrom acts as a toxic component in the cell, thereby inhibiting the growth of the strain. Therefore, when the methanol concentration of the medium is 5% by weight or more, the strain may be difficult to grow.
본 발명의 상기 변이 균주는 하이포마이크로비움 속 NPP-230(KCTC13718BP) 균주일 수 있다.The mutant strain of the present invention may be NPP-230 (KCTC13718BP) strain of the genus Hypomicrobium.
상기의 돌연 변이 균주를 수득함으로써, 미생물 발효에 의한 PQQ 생산의 문제점 중의 하나로 지적되는 발효 배양물 내 PQQ 농도가 낮은 문제를 해결할 수 있다.By obtaining the above mutant strain, it is possible to solve the problem of low PQQ concentration in the fermentation culture pointed out as one of the problems of PQQ production by microbial fermentation.
본 발명은 또한 하이포마이크로비움 데니트리피칸즈(Hyphomicrobium denitrificans) 균주에 자외선을 조사하여 돌연변이를 유발하는 단계; 및 상기 돌연변이된 균주를 고농도의 메탄올 배지에서 배양하여 피로로퀴놀린 퀴논을 생산하는 변이주를 선별하는 단계; 를 포함하는 하이포마이크로비움 속 변이 균주의 제조방법을 제공한다.The present invention is also Hyphomicrobium denitrificans Irradiating the strain with ultraviolet rays to cause mutation; And culturing the mutated strain in a high concentration of methanol medium to select strains producing pyroquinoline quinone; It provides a method for producing a strain strain of hypomicrobium containing.
상기 하이포마이크로비움 속 변이 균주는 NPP-230(KCTC13718BP)일 수 있고, 상기 배양 배지의 메탄올 농도는 바람직하게는 0.5 내지 5 중량%일 수 있다. The mutant strain of the hypomicrobium may be NPP-230 (KCTC13718BP), and the methanol concentration of the culture medium may be preferably 0.5 to 5% by weight.
본 발명은 또한, 하이포마이크로비움 속 변이주를 배양하는 단계; 상기 배양 발효액으로부터 흡착수지를 이용하여 발효액 내의 피로로퀴놀린 퀴논을 흡착하는 단계; 및 용리액을 사용하여 흡착 수지로부터 피로로퀴놀린 퀴논을 회수하는 단계; 를 포함하는 피로로퀴놀린 퀴논의 대량 생산 방법을 제공한다.The present invention also, the step of culturing the strains of the hypomicrobium; Adsorbing pyroquinoline quinone in the fermentation broth using an adsorption resin from the culture fermentation broth; And recovering pyroquinoline quinone from the adsorbent resin using an eluent. It provides a method for mass production of pyroquinoline quinone containing.
상기 피로로퀴놀린 퀴논의 대량 생산 방법에서 하이포마이크로비움 속 변이주는 하이포마이크로비움 데니트리피칸즈(Hyphomicrobium denitrificans)에 비해 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ)을 2 배 이상, 바람직하게는 3 배 이상, 보다 바람직하게는 4 배 이상 생산하는 변이 균주일 수 있다.In the method of mass production of the pyrroloquinoline quinone, the variation in the hypomicrobium is more than 2 times, preferably 3, the pyrrolo-quinoline quinone (PQQ) compared to the hypomicrobium denitrificans. It may be a mutant strain that produces more than 5 times, more preferably 4 times or more.
상기 피로로퀴놀린 퀴논의 대량 생산 방법에서 하이포마이크로비움 속 변이주는 0.5 중량% 내지 5 중량%의 메탄올에 내성을 갖는 변이 균주일 수 있다. In the mass production method of the pyrroloquinoline quinone, the strain in the hypomicrobium may be a mutant strain resistant to 0.5% to 5% by weight of methanol.
상기 피로로퀴놀린 퀴논의 대량 생산 방법에서 하이포마이크로비움 속 변이주는 NPP-230(KCTC13718BP) 균주일 수 있다. In the method for mass production of the pyrroloquinoline quinone, the strain of hypomicrobium may be a strain of NPP-230 (KCTC13718BP).
상기 피로로퀴놀린 퀴논의 대량 생산 방법에서 하이포마이크로비움 속 변이주를 배양하는 상기 배양은 유가식 배양일 수 있고 상기 배양 단계의 pH 조절제 및 질소원으로 바람직하게는 암모니아수를 사용할 수 있다. In the mass production method of the pyrroloquinoline quinone, the culture of culturing a mutant strain in hypomicrobium may be fed-batch culture, and preferably ammonia water may be used as a pH regulator and a nitrogen source in the culture step.
상기 유가식 배양에서 배양액의 메탄올의 농도는 바람직하게는 0.01 내지 0.5 중량%로 유지시킬 수 있다.In the fed-batch culture, the concentration of methanol in the culture medium can be preferably maintained at 0.01 to 0.5% by weight.
상기 하이포마이크로비움 속 변이 균주 NPP-230에 의해 피로로퀴놀린 퀴논을 대량 생산하기 위해 배양 중 6 시간 마다 배양액 시료를 채취하여 잔류 메탄올 농도를 가스 크로마토그래피 방법으로 분석하여 모니터링할 수 있다. 이것으로 배지 내 메탄올 농도를 0.01 내지 0.5 중량%로 일정하게 유지시켜 메탄올 농도가 너무 낮아 균주의 생육이 저조해지거나 메탄올 농도가 너무 높아 PQQ 생산이 저조해지지 않도록 함으로서 미생물 발효에 의한 PQQ 생산의 문제점 중의 하나로 지적되는 배양 공정을 개선할 수 있다.In order to mass-produce pyrroquinoline quinones by the mutant strain NPP-230 in the hypomicrobium, a culture medium sample is taken every 6 hours during the culture, and the residual methanol concentration can be analyzed and monitored by gas chromatography. As a result, the concentration of methanol in the medium is kept constant at 0.01 to 0.5% by weight, so that the concentration of methanol is too low to prevent the growth of the strain or the concentration of methanol is too high to prevent the production of PQQ, resulting in PQQ production by microorganism fermentation. It is possible to improve the culture process indicated as one.
피로로퀴놀린 퀴논의 대량 생산 방법에서 상기 용리액은 바람직하게는 염화나트륨 용액일 수 있다.In the method for mass production of pyrroquinoline quinone, the eluent may preferably be a sodium chloride solution.
미생물 발효에 의한 PQQ 생산시의 문제점 중 정제 과정에서의 문제점을 극복하기 위하여 음이온교환 컬럼이 장착된 FPLC(fast protein liquid chromatography)를 이용한 정제 조건을 확립하였다. 본 발명에서 확립된 FPLC를 이용한 정제 조건은 균체가 제거된 여과액을 이용함으로서 균주로부터 PQQ를 경제적이면서 고수율로 수득하는 것이 가능하다. 상기 음이온 교환칼럼은 바람직하게는 DEAE 또는 ZCQ-6250 수지일 수 있다.Among the problems of PQQ production by microbial fermentation, purification conditions using fast protein liquid chromatography (FPLC) equipped with an anion exchange column were established to overcome problems in the purification process. Purification conditions using FPLC established in the present invention, it is possible to obtain PQQ from the strain in an economical and high yield by using the filtrate from which the cells have been removed. The anion exchange column may be preferably DEAE or ZCQ-6250 resin.
또한, 피로로퀴놀린 퀴논을 대량 생산하기 위해 대규모 배양 시 불순물도 더 많이 발생하여 추가 연구를 통해 불순물 제거를 위한 전처리 공정을 개발하였다. 상기의 전처리 공정은 하이포마이크로비움 속 변이주의 배양 단계 후에 산 침전으로 염기성 단백질, DNA 및 세포질 등의 불순물을 1차로 제거한 후, 염추출 처리하여 불순물을 2차로 제거하는 단계를 포함할 수 있고 이와 같은 전처리 단계는 pH 4 이하의 등전점(pI값)을 갖는 용액을 회수하는 단계를 포함할 수 있다. 상기와 같은 전처리 단계의 도입으로 대부분의 불순물을 효과적으로 제거할 수 있을 뿐만 아니라 이온교환 수지의 사용을 줄이고도 고순도의 정제가 가능하다. 또한 이로 인해 약 30 %의 경비 절감이 가능하다. 상기 전처리 단계를 거쳐 정제된 PQQ의 순도는 93 % 이상, 바람직하게는 95 % 이상, 보다 바람직하게는 99 % 이상일 수 있다. In addition, in order to mass-produce pyroquinoline quinone, more impurities are generated during large-scale cultivation, and further studies have developed a pretreatment process for removing impurities. The pre-treatment process may include a step of first removing impurities such as basic protein, DNA, and cytoplasm by acid precipitation after the culture step of the mutant strain in Hypomicrobium, and then subjecting the salt extraction treatment to secondly removing the impurities. The pretreatment step may include recovering a solution having an isoelectric point (pI value) of
본 발명은 상기 생산 방법을 통해 얻어진 pH 6.0 내지 8.0의 피로로퀴놀린 퀴논 용액에 산을 첨가하여 상기 용액의 pH를 4.0 내지 7.0으로 조절하는 단계; 및 상기 용액에 염을 포함하는 결정화 용매를 첨가하고 수득된 용액을 냉각시켜 피로로퀴놀린 퀴논염을 석출시키는 과정을 포함하는 피로로퀴놀린 퀴논염 수화물의 제조방법을 제공한다. The present invention comprises the steps of adjusting the pH of the solution to 4.0 to 7.0 by adding an acid to a pyroquinoline quinone solution of pH 6.0 to 8.0 obtained through the production method; And adding a crystallization solvent containing a salt to the solution and cooling the obtained solution to precipitate a pyrroloquinoline quinone salt.
상기 피로로퀴놀린 퀴논염 수화물의 제조방법에서, pH를 조절하기 위해 첨가되는 상기 산의 종류로는 염산, 브롬화수소산, 요오드화수소산, 질산, 황산 또는 과염소산 등이 있고, 이에 제한되지 않으며, 바람직하게는 염산일 수 있다.In the method of manufacturing the pyrroloquinoline quinone salt hydrate, the type of the acid added to adjust the pH includes hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid or perchloric acid, but is not limited thereto, preferably It may be hydrochloric acid.
상기 피로로퀴놀린 퀴논염 수화물의 제조방법에서, 상기 피로로퀴놀린 퀴논 용액에 바람직하게는 염화나트륨(NaCl) 용액을 결정화 용매로 첨가하여 석출시키는 방법을 사용할 수 있다. 상기 결정화를 위한 염화나트륨 용액의 농도는 바람직하게는 0.6 M 내지 5 M이고 더욱 바람직하게는 1 M일 수 있다.In the manufacturing method of the pyrroloquinoline quinone salt hydrate, a method of precipitating by adding a sodium chloride (NaCl) solution as a crystallization solvent to the pyrroloquinoline quinone solution may be used. The concentration of the sodium chloride solution for the crystallization is preferably 0.6 M to 5 M and more preferably 1 M.
상기 피로로퀴놀린 퀴논염 수화물의 제조 방법에서, 상기 석출시키는 과정의 냉각 온도가 4 내지 20 ℃, 바람직하게는 4 내지 15 ℃, 보다 바람직하게는 5 내지 15 ℃일 수 있다.In the manufacturing method of the pyrroloquinoline quinone salt hydrate, the cooling temperature of the precipitation process may be 4 to 20°C, preferably 4 to 15°C, more preferably 5 to 15°C.
상기 피로로퀴놀린 퀴논염 수화물은 바람직하게는 하기 화학식을 가지는 피로로퀴놀린 퀴논염 수화물일 수 있다. The pyrroloquinoline quinone salt hydrate may be a pyrroloquinoline quinone salt hydrate having the following formula.
상기 피로로퀴놀린 퀴논염 수화물에서 바람직하게는 n은 2, M은 나트륨염이고 피로로퀴놀린 퀴논염 수화물을 건조시켜 얻은 m 값은 1.5일 수 있다. In the pyrroloquinoline quinone salt hydrate, n is preferably 2 and M is a sodium salt, and the m value obtained by drying the pyrroloquinoline quinone salt hydrate may be 1.5.
상기 제조방법에 의한 피로로퀴놀린 퀴논염 수화물의 순도는 93 % 이상, 바람직하게는 95 % 이상, 보다 바람직하게는 99 % 이상일 수 있다. Purity of the pyroquinoline quinone salt hydrate by the above manufacturing method may be 93% or more, preferably 95% or more, and more preferably 99% or more.
본 발명은 Cu-K을 이용한 분말 X-ray 회절 분석에서 주요 피크(peak)의 2θ 값이 바람직하게는 6.28 ± 0.4°, 8.74 ± 0.4°, 17.58 ± 0.4°, 18.28 ± 0.4°, 18.70 ± 0.4°, 22.96 ± 0.4°, 25.48 ± 0.4°, 26.56 ± 0.4°, 33.72 ± 0.4°, 36.28 ± 0.4°인 PQQ 나트륨염 수화물 결정을 제공한다. In the present invention, the 2θ value of the main peak in powder X-ray diffraction analysis using Cu-K is preferably 6.28 ± 0.4°, 8.74 ± 0.4°, 17.58 ± 0.4°, 18.28 ± 0.4°, 18.70 ± 0.4 PQQ sodium salt hydrate crystals of 22.96 ± 0.4°, 25.48 ± 0.4°, 26.56 ± 0.4°, 33.72 ± 0.4°, 36.28 ± 0.4° are provided.
본 발명은 또한 하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주 NPP-230(KCTC13718BP)을 이용해 생산된 피로로퀴놀린 퀴논(Pyrrolo-quinoline quinone, PQQ), 상기 정제 과정을 통해 얻어진 피로로퀴놀린 퀴논, 및 피로로퀴놀린 퀴논염 수화물의 제조방법으로 얻어진 피로로퀴놀린 퀴논 나트륨염 수화물 중 적어도 어느 하나를 포함하는 항산화 기능 제품인 식품, 화장품 및 의약품을 제공할 수 있다.The present invention is also a hypomicrobium (Hyphomicrobium sp. ) Pyrorrholine quinone (Pyrrolo-quinoline quinone) produced using the mutant strain NPP-230 (KCTC13718BP), pyroquinoline quinone obtained through the purification process, and It is possible to provide foods, cosmetics, and pharmaceuticals, which are antioxidant functional products containing at least one of pyroquinoline quinone sodium hydrate obtained by a method for producing pyroquinoline quinone salt hydrate.
본 발명에 따른 피로로퀴놀린 퀴논 나트륨염 수화물은 건강 기능 식품 조성물에 유효성분으로서 함유되는 것도 가능하고, 비타민 C와 함께 혼합하여 조성물 형태로 제조할 수 있으며, 식품학적으로 허용 가능한 식품 보조 첨가제를 추가로 포함할 수 있다. 본 발명의 건강 기능 식품 조성물은 기능성 식품, 영양 보조제, 건강 식품 및 식품 첨가제 등의 모든 형태의 식품에 대한 조성물을 포함한다. The pyroquinoline quinone sodium salt hydrate according to the present invention can also be contained as an active ingredient in a health functional food composition, and can be prepared in a composition form by mixing with vitamin C, and adds a food additive that is food-pharmaceutically acceptable. It can contain as. The health functional food composition of the present invention includes a composition for all types of foods such as functional foods, nutritional supplements, health foods, and food additives.
상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 예를 들면, 건강식품으로는 정제, 환약, 분말, 캡슐, 껌, 비타민 복합체, 쥬스 및 드링크 등의 다양한 형태로 제조될 수 있으며, 본 발명에 따른 피로로퀴놀린 퀴논 나트륨염 수화물을 유효성분으로 하는 식품 조성물을 과립화, 캡슐화 또는 분말화하여 섭취할 수 있다. 본 발명의 식품 조성물은 식품 제조 시에 통상적으로 첨가되는 성분을 포함할 수 있으며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함할 수 있다. 예컨대, 드링크제로 제조되는 경우에는 본 발명의 피로로퀴놀린 퀴논 나트륨염 수화물 외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 대추 추출액, 감초 추출액 등을 추가로 포함시킬 수 있다. 또한 식품 보조 첨가제로서, 당업계에 통상적인 식품 첨가제, 예를 들어 향미제, 풍미제, 착색제, 충진제, 안정화제 등을 포함시킬 수 있다. 또한, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수도 있다. 천연 탄수화물의 구체적인 예로는 예를 들어, 포도당, 과당 등의 단당류, 말토스, 슈크로스 등의 이당류, 덱스트린, 시클로덱스트린 등의 다당류 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어, 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 포함시킬 수도 있다. Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, as a health food, tablets, pills, powders, capsules, chewing gum, vitamin complexes, juices and drinks can be prepared in various forms, and pyroquinoline quinone sodium salt hydrate according to the present invention as an active ingredient The food composition may be consumed by granulating, encapsulating or powdering. The food composition of the present invention may include ingredients that are commonly added during food preparation, and may include, for example, proteins, carbohydrates, fats, nutrients, and flavoring agents. For example, when prepared as a drink agent, in addition to the pyroquinoline quinone sodium salt hydrate of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, jujube extract, licorice extract, etc. may be further included. In addition, as food supplement additives, food additives conventional in the art may be included, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. In addition, it can also contain various flavoring agents or natural carbohydrates, etc., as additional components, as in conventional beverages. Specific examples of natural carbohydrates include, for example, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may also be included. have.
더 나아가, 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수도 있다. 이러한 성분들은 독립적으로 또는 조합하여 사용할 수 있다. Furthermore, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and It may also contain salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. These ingredients can be used independently or in combination.
더 나아가, 본 발명에 따른 피로로퀴놀린 퀴논 나트륨염 수화물은 기능성 화장품 조성물에 유효성분으로 함유되는 것도 가능한데, 이는 개별 화장품으로 활용하거나 다른 기능성 화장품과 병용하여 사용될 수 있다. 본 발명에서 기능성 화장품 조성물은 당업계에서 통상적으로 제조되는 어떠한 형태의 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면 활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 스프레이, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포움, 클렌징 워터, 팩 또는 파우더 등과 같은 다양한 제형으로 제조될 수 있다. Furthermore, the pyroquinoline quinone sodium salt hydrate according to the present invention may also be contained as an active ingredient in the functional cosmetic composition, which may be used as an individual cosmetic or in combination with other functional cosmetics. The functional cosmetic composition in the present invention may be prepared in any form of a formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactants -Contains various cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, soft lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack or powder, etc. It can be prepared in a formulation.
또한 본 발명은 고기능성 항산화 물질인 피로로퀴놀린 퀴논 나트륨염 수화물을 유효성분으로 포함하는, 산화관련 질환의 예방 또는 치료용 약제학적 조성물을 제공할 수 있다.In addition, the present invention can provide a pharmaceutical composition for the prevention or treatment of oxidation-related diseases, which includes pyroquinoline quinone sodium salt hydrate, a highly functional antioxidant, as an active ingredient.
본 발명에서 용어 “예방” 또는 “치료”는 본 발명에 따른 피로로퀴놀린 퀴논 나트륨염 수화물 또는 이를 유효성분으로 포함하는 약제학적 조성물의 투여로 산화 관련 질환의 발병을 늦추거나 질환을 전개할 가능성을 줄이거나 또는 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.In the present invention, the term "prevention" or "treatment" refers to the possibility of delaying the onset of oxidative-related diseases or developing the disease by administering a pyroquinoline quinone sodium salt hydrate according to the present invention or a pharmaceutical composition comprising it as an active ingredient. Any action that reduces or improves or changes beneficially.
본 발명에서 용어 “투여”는 임의의 적절한 방법으로 대상에게 소정의 물질, 즉 본 발명에 따른 피로로퀴놀린 퀴논 나트륨염 수화물 또는 이를 포함하는 약제학적 조성물을 도입하는 것을 의미하고 이의 투여 경로는 약제학적 조성물이 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여서든지 투여될 수 있다. 예를 들어, 투여 경로는 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비내 투여, 폐내 투여, 직장내 투여 등이 될 수 있으나 이에 제한되지 않는다. 바람직하게는 경구로 투여될 수 있다. In the present invention, the term “administration” means to introduce a predetermined substance to a subject, a pyroquinoline quinone sodium salt hydrate or a pharmaceutical composition comprising the same, to a subject in any suitable way, and the route of administration thereof is pharmaceutical. The composition can be administered via any general route as long as it can reach the target tissue. For example, the route of administration may be, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. Preferably it can be administered orally.
본 발명에 따른 피로로퀴놀린 퀴논 나트륨염 수화물 또는 이를 포함하는 약제학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며 단일 또는 다중 투여될 수 있다. The pyroquinoline quinone sodium salt hydrate or pharmaceutical composition comprising the same according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple Can be.
본 발명에 따른 약제학적 조성물은 본 발명에 따른 피로로퀴놀린 퀴논 나트륨염 수화물이 유효 성분으로 함유되는 한, 약학적으로 허용가능한 다양한 담체를 포함할 수 있다. 약학적으로 허용되는 담체는 경구 투여용의 경우에는 결합제, 활택제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장화제, 안정화제 등을 혼합하여 사용할 수 있고, 국소 투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 약제학적 조성물의 제형은 상술한 바와 같은 약학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여용의 경우에는 정제, 트로키, 캡슐, 엘릭서,서스펜션, 시럽, 웨이퍼 등의 형태로 제조될 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타 용액, 현탁액, 정제, 환약, 캡슐, 서방형 제제 등으로 제형화할 수도 있다. 제제화에 적합한 담체, 부형제 및 희석제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀롤로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 스테아르산 마그네슘 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 방부제 등을 추가로 포함할 수 있다. The pharmaceutical composition according to the present invention may include various pharmaceutically acceptable carriers, as long as the pyrroloquinoline quinone sodium salt hydrate according to the present invention is contained as an active ingredient. Pharmaceutically acceptable carriers can be used for oral administration, including binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc., and in the case of injections, buffers, preservatives , Painless agent, solubilizer, isotonic agent, stabilizer, etc. can be used in combination. For topical administration, a base, excipient, lubricant, preservative, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it may be prepared in unit dosage ampoules or multiple dosage forms. . Other solutions, suspensions, tablets, pills, capsules, or sustained-release preparations may also be formulated. Examples of carriers, excipients and diluents suitable for formulation are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, preservatives and the like may be further included.
이하, 실시예를 기술함으로써 본원 발명을 보다 상세히 설명한다. 다만, 하기의 실시예는 본원 발명의 일 예시에 불과하며, 본원 발명의 내용이 이에 한정되는 것으로 해석되어서는 아니된다.Hereinafter, the present invention will be described in more detail by describing examples. However, the following examples are only examples of the present invention, and the contents of the present invention should not be construed as being limited thereto.
실시예 1 <돌연변이 균주 제조> Example 1 < Preparation of mutant strains>
하이포마이크로비움 데니트리피칸즈(Hyphomicrobium denitrificans) ATCC 51888을 모균주로 자외선 조사 실험과 메탄올에 대한 내성 실험을 진행하였다. 15W 자외선 램프를 사용하여 253 nm 파장으로 30 cm 떨어진 곳에서 0에서 60 초까지 5 초 단위로 조사하였다(도2). 자외선 조사 후, AMS(Ammonium Mineral Salt) 배지의 메탄올 농도를 각각 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 %로 하여, 10일 동안 배양하였다(도3). 배양 결과, 3 % 메탄올에서 자란 균주를 다시 4 % 메탄올 및 5 % 메탄올 배지로 계대 배양하여 4 % 메탄올 배지에서 성장하는 균주를 얻었다. 4 % 메탄올 배지에서 자란 균주를 현탁 배양하여, 생육 속도가 가장 우수하고 피로로퀴놀린 퀴논을 가장 많이 생산하는 돌연변이 균주를 선별하여 분리하였다. Hyphomicrobium denitrificans ATCC 51888 were tested for UV irradiation and methanol resistance to the parent strain. Using a 15W ultraviolet lamp, irradiation was performed in 5 second increments from 0 to 60 seconds at a distance of 30 cm at a wavelength of 253 nm (FIG. 2). After UV irradiation, the methanol concentration of AMS (Ammonium Mineral Salt) medium was 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5%, respectively, and cultured for 10 days (FIG. 3). As a result of the culture, the strain grown in 3% methanol was again subcultured with 4% methanol and 5% methanol medium to obtain a strain grown in 4% methanol medium. The strains grown in 4% methanol medium were suspended and cultured to select and isolate the mutant strain having the best growth rate and producing the most pyroquinoline quinone.
자외선 조사 및 고농도의 메탄올 내성 균주 선별을 통해 얻은 돌연변이 균주 중, NPP-230 균주의 수율은 모균주보다 피로로퀴놀린 퀴논을 4.79 배 많은 1.2 g/L를 생산하는 고생산성 균주였다. PQQ 수율은 안정적이었고 균주의 돌연변이 상태도 안정적으로 유지됨을 확인하였다. Among the mutant strains obtained through UV irradiation and high-concentration methanol-resistant strain selection, the yield of the NPP-230 strain was a high-productivity strain producing 1.2 g/L of pyrroquinoline quinone 4.79 times more than the parent strain. It was confirmed that the PQQ yield was stable and that the mutant state of the strain was maintained stably.
상기의 PQQ를 고농도로 생산하고 자외선 및 고농도의 메탄올에 대한 내성을 보이는 변이 균주를 하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주 NPP-230으로 명명하였다. 하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주 NPP-230은 2018년 11월 19일자로 생물 자원 센터(Korean Collection for Type Culture, 전라북도 정읍시 입신길 181 한국생명공학연구원 생물자원센터)에 기탁번호 KCTC13718BP로 기탁되었다.The above-mentioned PQQ was produced at a high concentration and the mutant strain showing resistance to ultraviolet rays and high concentration of methanol was named as Hyphomicrobium sp. mutant strain NPP-230. Hypomicrobium sp. mutant strain NPP-230 was deposited as KCTC13718BP on November 19, 2018, at the Bioresource Center (Korean Collection for Type Culture, 181 Biosin Center, Jeongeup-si, Jeollabuk-do, Korea Research Institute of Bioscience and Biotechnology) Have been deposited.
실시예 2 <피로로퀴놀린 퀴논의 대량 생산> Example 2 < mass production of pyroquinoline quinone>
하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주 NPP-230(KCTC13718BP)을 플라스크에 현탁 배양하여 단계적으로 일정 비율 증가시켜 30 L 배양을 진행하였다. 하기 표2의 배지 조성에 따라 배양을 3회 진행하였다. Hyphomicrobium sp (Hyphomicrobium sp. ) mutant strain NPP-230 (KCTC13718BP) was suspended and cultured in a flask to increase the ratio in a stepwise manner to perform 30 L culture. The culture was performed three times according to the composition of the medium in Table 2 below.
암모니아 25 % ~ 28 %를 이용하여 멸균된 배양액의 pH를 7.0으로 조절하고, 멸균 여과된 50 % 메탄올로 초기 농도를 1 g/L가 되게 한 뒤, 실시예 1에서 제조된 하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주 NPP-230(KCTC13718BP)을 접종하였다. The pH of the sterilized culture solution was adjusted to 7.0 using 25% to 28% of ammonia, and the initial concentration was adjusted to 1 g/L with 50% methanol filtered through sterilization, followed by the hypomicrobium prepared in Example 1 ( Hyphomicrobium sp. ) mutant strain NPP-230 (KCTC13718BP) was inoculated.
배양조건은 다음과 같았다.The culture conditions were as follows.
1) 메탄올 첨가1) Methanol addition
- 메탄올 주입 농도: 50 %-Methanol injection concentration: 50%
- 초기 발효조 배양액의 메탄올 농도: 1,000 ppm (1 g/L)-Methanol concentration of the initial fermenter culture medium: 1,000 ppm (1 g/L)
- 접종 후 6 시간 마다 배양액 시료를 채취하여 잔류 메탄올 농도를 가스 크로마토그래피 방법으로 분석하여 모니터링했다. -After inoculation, a culture sample was taken every 6 hours, and the residual methanol concentration was analyzed and monitored by a gas chromatography method.
- 배양액 내 메탄올 농도 유지: 500 ~ 1,000 ppm (급격한 메탄올 투입 금지)-Maintain methanol concentration in the culture medium: 500 ~ 1,000 ppm (prohibition of rapid methanol input)
- 1일 메탄올 소비량: 평균 1 ~ 1.5 % (순수한 메탄올 기준으로 계산된 값)-Daily methanol consumption: 1 to 1.5% on average (calculated based on pure methanol)
2) pH 조절2) pH adjustment
- 25 % 암모니아를 이용하여 0 ~ 4 일까지의 로그기에서는 pH를 6.8로 유지하였고, 4 일 이후의 PQQ 생성 단계에서는 pH를 7.0으로 유지하였다.-The pH was maintained at 6.8 in the log phase from 0 to 4 days using 25% ammonia, and the pH was maintained at 7.0 in the PQQ generation step after 4 days.
3) 거품 조절3) Foam control
- 배양 중반과 후반에 거품이 발생하면, 20 % PPG2000 거품 억제제를 사용하여 제거하였다.-If foaming occurred in the middle and the second half of culture, it was removed using a 20% PPG2000 foam inhibitor.
4) 용존 산소 (DO) 조절4) Dissolved oxygen (DO) control
- 로그기 (0 ~ 4일): 0.08 MPa-Log phase (0 ~ 4 days): 0.08 MPa
- PQQ 생성 단계 (4일 후): 0.05 MPa-PQQ generation stage (after 4 days): 0.05 MPa
- 공기 유량: 0.3 ~ 1.0 vvm-Air flow: 0.3 ~ 1.0 vvm
- 교반 속도: DO가 10 % 이상이 되도록 유지-Stirring speed: DO is maintained to be more than 10%
상기와 같은 조건에서 1차 배양은 30 L에서 218 시간 동안 진행하여, 균체는 O.D 50까지 증가하였으며, PQQ는 최대 1.1 g/L까지 생산되었다.Under the above conditions, the primary culture proceeded at 30 L for 218 hours, and the cells were increased to O.D 50 and PQQ was produced up to 1.1 g/L.
상기와 같은 조건에서 2차 30 L 배양은 210 시간 동안 진행하였다. 균체는 O.D 100까지 증가하였으며, PQQ 생산량은 최대 1.26 g/L까지 생산되었다(도4). Under the above conditions, the second 30 L culture was performed for 210 hours. The cells were increased to O.D 100, and the PQQ production was up to 1.26 g/L (Fig. 4).
상기와 같은 조건에서 3차 30 L 배양은 210 시간 동안 진행하였다. 균체는 O.D 100까지 증가하였으며, PQQ 생산량은 최대 1.3 g/L까지 생산되었다.In the above conditions, the third 30 L culture was performed for 210 hours. Cells increased to O.D 100, and PQQ production was up to 1.3 g/L.
<3000 L 배양기를 이용한 균주의 배양조건 확인><Confirmation of culture conditions of strain using 3000 L incubator>
상기와 동일한 배지 조성과 배양 조건을 이용하여 3000 L 배양공정을 진행하였다. 3000 L 배양은 180시간 동안 진행하였으며, 균체는 O.D 110까지 증가하였고 PQQ의 최대 함량은 1.28 g/L 였다(도5). 3000 L culture process was performed using the same medium composition and culture conditions as above. 3000 L incubation was carried out for 180 hours, the cells were increased to O.D 110 and the maximum content of PQQ was 1.28 g/L (FIG. 5).
실시예 3 <피로로퀴놀린 퀴논의 정제> Example 3 < Pyroroquinoline quinone tablets>
실시예 3-1 <DEAE-세파로오스 수지를 이용한 피로로퀴놀린 퀴논의 정제> Example 3-1 <Refining of pyrroloquinoline quinone using DEAE-Sepharose resin>
1) 정제조건1) Purification conditions
- 장치: AKTA Explorer-Device: AKTA Explorer
- 컬럼: 1 mL HiTrap DEAE FF-Column: 1 mL HiTrap DEAE FF
- 시료: 10 mL (0.22 um 필터로 여과)-Sample: 10 mL (filtered with 0.22 um filter)
- 평형 버퍼: 50 mM 인산나트륨(Sodium phosphate) (pH 6.9) -Equilibration buffer: 50 mM sodium phosphate (pH 6.9)
- 용리 버퍼: 50 mM 인산나트륨 및 2 M 염화나트륨 (pH 6.9)-Elution buffer: 50 mM sodium phosphate and 2 M sodium chloride (pH 6.9)
- 진행순서 -Procedure
(1)DEAE-세파로오스 수지를 이용하여 여과액의 PQQ를 흡착 (1) Adsorption of PQQ of filtrate using DEAE-Sepharose resin
(2)시료 적재 후, 인산나트륨 버퍼로 세척 (2) After loading the sample, wash it with sodium phosphate buffer
(3)100 mM, 300 mM, 1 M, 2 M 농도차 염화나트륨으로 용리 (3) Elution with sodium chloride at a concentration difference of 100 mM, 300 mM, 1 M, 2 M
- PQQ는 300 mM 염화나트륨부터 용출됨-PQQ elutes from 300 mM sodium chloride
2) 정제 결과 2) Purification result
통과액 구간에서는 PQQ가 확인되지 않았고, 0.3 M 염화나트륨부터 PQQ가 용출되기 시작하였다. HPLC 분석결과 0.3 M 염화나트륨 용출 영역에서 91.7 % 수율을 보였다. PQQ was not observed in the flow-through section, and PQQ began to elute from 0.3 M sodium chloride. The HPLC analysis showed 91.7% yield in the 0.3 M sodium chloride elution region.
실시예 3-2 <ZCQ-6250 수지를 이용한 피로로퀴놀린 퀴논의 정제 1> Example 3-2 <
1) 정제조건1) Purification conditions
- 장치: AKTA Explorer -Device: AKTA Explorer
- 컬럼: 28 mL XK16_14cmH-Column: 28 mL XK16_14cmH
- 시료: 150 mL (0.22 um 필터로 여과)-Sample: 150 mL (filtered with 0.22 um filter)
- 평형 버퍼: 50 mM 인산나트륨 (pH 6.9) -Equilibration buffer: 50 mM sodium phosphate (pH 6.9)
- 용리 버퍼: 50 mM 인산나트륨 및 2 M 염화나트륨 (pH 6.9)-Elution buffer: 50 mM sodium phosphate and 2 M sodium chloride (pH 6.9)
- 진행순서 -Procedure
(1) ZCQ-6250 수지를 이용하여 여과액의 PQQ를 흡착 (1) Adsorption of PQQ of filtrate using ZCQ-6250 resin
(2) 시료 적재 후, 인산나트륨 버퍼로 세척 (2) After loading the sample, wash it with sodium phosphate buffer
(3) 100 mM, 300 mM, 1 M, 2 M 농도차 염화나트륨으로 용리 (3) Elution with sodium chloride at a concentration difference of 100 mM, 300 mM, 1 M, 2 M
- PQQ는 300 mM 염화나트륨부터 용출됨-PQQ elutes from 300 mM sodium chloride
2) 정제 결과2) Purification result
통과액 구간에서는 PQQ가 확인되지 않았고, 0.3 M 염화나트륨부터 PQQ가 용출되기 시작하였다. 상기 HPLC 조건에서 HPLC 분석결과 0.3 M 염화나트륨 용출 영역에서 74.1 %의 수율을 보였다.PQQ was not observed in the flow-through section, and PQQ began to elute from 0.3 M sodium chloride. HPLC analysis under the above HPLC conditions showed a yield of 74.1% in the 0.3 M sodium chloride elution region.
실시예 3-3 <ZCQ-6250 수지를 이용한 피로로퀴놀린 퀴논의 정제 2> Example 3-3 <
1) 정제조건1) Purification conditions
- 장치: AKTA Explorer -Device: AKTA Explorer
- 컬럼: 28 mL XK16_14cmH-Column: 28 mL XK16_14cmH
- 평형 버퍼: 50 mM 인산나트륨 (pH 6.9) -Equilibration buffer: 50 mM sodium phosphate (pH 6.9)
- 용리 버퍼: 50 mM 인산나트륨 및 2 M 염화나트륨 (pH 6.9)-Elution buffer: 50 mM sodium phosphate and 2 M sodium chloride (pH 6.9)
- 진행순서 -Procedure
(1) ZCQ-6250 수지를 이용하여 여과액의 PQQ를 흡착 (1) Adsorption of PQQ of filtrate using ZCQ-6250 resin
(2) 시료 적재 후, 인산나트륨 버퍼로 세척 (2) After loading the sample, wash it with sodium phosphate buffer
(3) 600 mM, 1.3 M, 2 M 염화나트륨으로 용리 (3) Elution with 600 mM, 1.3 M, 2 M sodium chloride
- PQQ는 600 mM 염화나트륨부터 용출됨-PQQ elutes from 600 mM sodium chloride
2) 정제 결과2) Purification result
0.6 M 염화나트륨을 이용한 용출로 오염물질이 제거되었다. 1.3 M 염화나트륨으로 용출시 1 M 염화나트륨 용출에 비해 테일링이 줄어들었고 HPLC 분석결과 1.3 M 염화나트륨을 이용한 용출로 75.9 %의 수율을 보였다. Contaminants were removed by elution with 0.6 M sodium chloride. When elution with 1.3 M sodium chloride, the tailing was reduced compared to the elution with 1 M sodium chloride, and HPLC analysis showed a yield of 75.9% by elution with 1.3 M sodium chloride.
실시예 4 <전처리 공정을 추가한 피로로퀴놀린 퀴논의 정제> Example 4 < Refining of pyrroloquinoline quinone with a pretreatment process>
실시예 3에서 정제된 PQQ의 HPLC 분석 결과 불순물 피크가 함께 검출되었다. 대규모 배양 시 불순물이 더 많이 발생할 것으로 예상되어 불순물을 효과적으로 제거하기 위한 전처리 공정을 추가하였다. HPLC analysis of the purified PQQ in Example 3 detected impurity peaks together. It is expected that more impurities will be generated during large-scale cultivation, so a pretreatment process for effectively removing impurities has been added.
산 침전 방법으로 단백질 등의 불순물을 1차로 제거한 후, 염 추출 처리하여 불순물을 2차로 제거하는 상기의 전처리 공정은 하기와 같이 진행되었다. After the impurities such as proteins were first removed by an acid precipitation method, the above pretreatment process of removing impurities by salt extraction treatment was performed as follows.
균주를 배양한 후 여과한 여과액(pH 7)의 pH를 10 % 황산(H2SO4)으로 pH 4로 조절하여 염기성 단백질, DNA 및 세포질 등의 불순물을 제거하였다. 이어 0.45μm 필터로 여과하여 PQQ가 포함된 pH 4 이하의 등전점(pI) 값을 갖는 용액을 회수하였다. 회수된 용액에 1 M 염화나트륨을 투입하여 염추출을 진행하였다. 10 % 황산으로 pH 3.5로 조절하여 PQQ의 1차 등전점(pI) 값 근처로 낮추었다. 이것을 규조토와 혼합 여과하여 PQQ 규조토 케이크를 얻었다. PQQ 규조토 케이크를 50 mM 인산염 버퍼(phosphate buffer)로 용해한 후, 10 % 수산화나트륨을 이용하여 pH 7로 조절하였다. 0.6 g/L의 활성탄을 용해조에 투입하고, 15 ℃에서 1시간 동안 교반한 후, 5 μm, 1 μm, 0.45 μm 여과막을 이용하여 순차적으로 여과를 진행했다. After culturing the strain, the pH of the filtered filtrate (pH 7) was adjusted to
상기와 같은 전처리 공정을 거친 후, ZCQ-6250 수지를 이용한 정제조건은 하기와 같았다. After the pre-treatment process as described above, purification conditions using ZCQ-6250 resin were as follows.
1) 정제조건1) Purification conditions
- 컬럼: XK16/20_ZCQ-6250 12.5 mL-Column: XK16/20_ZCQ-6250 12.5 mL
- 평형 버퍼: 50 mM 인산나트륨 (pH 6.9) -Equilibration buffer: 50 mM sodium phosphate (pH 6.9)
- 용리 버퍼: 50 mM 인산나트륨 및 2 M 염화나트륨 (pH 6.9)-Elution buffer: 50 mM sodium phosphate and 2 M sodium chloride (pH 6.9)
- 진행순서 -Procedure
(1) ZCQ-6250 수지를 이용하여 여과액의 PQQ를 흡착(1) Adsorption of PQQ of filtrate using ZCQ-6250 resin
(2) 시료 적재 후, 50 mM 인산나트륨 버퍼 및 300 mM 염화나트륨(pH 6.9)로 세척(2) After loading the sample, wash with 50 mM sodium phosphate buffer and 300 mM sodium chloride (pH 6.9)
(3) 50 mM 인산나트륨 버퍼 및 400 mM 염화나트륨(pH 6.9)로 용리(3) Elution with 50 mM sodium phosphate buffer and 400 mM sodium chloride (pH 6.9)
(4) 50 mM 인산나트륨 버퍼 및 800 mM 염화나트륨(pH 6.9)로 용리(4) Elution with 50 mM sodium phosphate buffer and 800 mM sodium chloride (pH 6.9)
(5) 50 mM 인산나트륨 버퍼 및 1 M 염화나트륨(pH 6.9)로 폴리싱(5) Polishing with 50 mM sodium phosphate buffer and 1 M sodium chloride (pH 6.9)
(6) 50 mM 인산나트륨 버퍼 및 200 mM 염화나트륨(pH 6.9)로 재평형(6) Re-equilibration with 50 mM sodium phosphate buffer and 200 mM sodium chloride (pH 6.9)
2) 정제 결과 2) Purification result
배양액을 전처리한 후 0.8 M 염화나트륨으로 단계적 용리 시, 순도 99 %까지 정제가 가능하였다. 이로 인하여 염화나트륨으로 용리하는 단계의 농도와 용리액의 양을 줄여 PQQ를 보다 경제적으로 생산하는 것이 가능해졌다. 상기와 같은 전처리 공정을 통해 대부분의 불순물이 제거되었으며, 하기 분석 조건에서 HPLC에 의한 분석 결과 99 % 이상의 순도가 확인되었다(도 6).When the culture was pre-treated and eluted stepwise with 0.8 M sodium chloride, it was possible to purify to 99% purity. This made it possible to produce PQQ more economically by reducing the concentration of elution with sodium chloride and the amount of eluent. Most impurities were removed through the pre-treatment process as described above, and purity of 99% or more was confirmed as a result of analysis by HPLC under the following analysis conditions (FIG. 6).
<HPLC 분석 조건><HPLC analysis conditions>
정제된 PQQ의 순도를 확인하기 위해 HPLC 분석을 수행하였다. HPLC analysis was performed to confirm the purity of the purified PQQ.
1) HPLC 시스템의 조건은 하기와 같았다.1) The conditions of the HPLC system were as follows.
이동상: 0.1 M CH3COOH : 0.1 M CH3COONH4 = 30 : 70 (pH 5.1)Mobile phase: 0.1 M CH 3 COOH: 0.1 M CH 3 COONH 4 = 30: 70 (pH 5.1)
파장: 259 nmWavelength: 259 nm
용리 시간: 10 분Elution time: 10 minutes
흐름 속도: 1.5 mL/minFlow rate: 1.5 mL/min
주입 부피: 20 μLInjection volume: 20 μL
검출기: UV-검출기Detector: UV-detector
컬럼: YMC-Pack A-302 ODS (4.6 mm×150 mm, 5μm)Column: YMC-Pack A-302 ODS (4.6 mm×150 mm, 5 μm)
컬럼 온도: 40 ℃Column temperature: 40 ℃
2) HPLC에 의한 PQQ 검출2) PQQ detection by HPLC
PQQ 표준품 10 mg, 15 mg, 20 mg, 25 mg, 30 mg을 정확하게 재서 각각의 50 mL 용량 측정 병에 이동상과 함께 녹여 표준 용액을 제조하였다. 상기 표준 용액 각각을 3회 주입하여 피크의 면적을 계산하고 선형 회귀 곡선을 그려 계산 식을 얻었다. 정제된 샘플을 순수한 물이나 이동상으로 희석했다. 이 때, 희석된 샘플의 PQQ 농도가 선형 회귀 곡선의 범위를 벗어나지 않도록 조절했다. A standard solution was prepared by accurately weighing 10 mg, 15 mg, 20 mg, 25 mg, and 30 mg of the PQQ standard and dissolving it with the mobile phase in each 50 mL volume measuring bottle. Each of the standard solutions was injected three times to calculate the area of the peak and draw a linear regression curve to obtain a calculation formula. The purified sample was diluted with pure water or mobile phase. At this time, the PQQ concentration of the diluted sample was adjusted so as not to fall outside the range of the linear regression curve.
실시예 5 <PQQ 결정화> Example 5 < PQQ crystallization>
실시예 4에서 얻은 PQQ 용액을 0.2 μm 필터로 여과한 후, 하기와 같이 PQQ 결정화 과정을 진행하였다.After filtering the PQQ solution obtained in Example 4 with a 0.2 μm filter, the PQQ crystallization process was performed as follows.
0.2 μm 필터로 여과한 뒤 회수된 PQQ 용액에 0.6 M 염화나트륨을 첨가하였다. 5 M 염화나트륨이 함유된 인산염 버퍼(phosphate buffer)를 반응기에 1 L첨가하고, 10 % 염산으로 pH를 5로 조절하였다. 상기의 PQQ 용액 9 L을 직경 3 mm의 유연한 관을 통해 15 ℃에서 6 ml/min의 속도로 반응기에 투입하였다. 10 % 염산으로 pH를 5로 조절하고, 15 ℃에서 18 시간 동안 천천히 교반하여 결정화를 진행시켰다. PQQ 용액에 포함된 과량의 나트륨을 제거하기 위해 50 % 에탄올을 1:35(PQQ:에탄올)가 되도록 PQQ 용액과 섞어준 후, 10 % 염산으로 pH를 3.5로 조절하였다. 15 ℃에서 18 시간 동안 천천히 교반하여 세척을 하였고 이와 같은 세척과정을 3회 반복하였다. 세척이 완료된 PQQ 나트륨염 수화물을 40 ℃에서 24 시간 동안 진공 조건에서 건조시켰다. After filtering through a 0.2 μm filter, 0.6 M sodium chloride was added to the recovered PQQ solution. A 1 L phosphate buffer containing 5 M sodium chloride was added to the reactor, and the pH was adjusted to 5 with 10% hydrochloric acid. 9 L of the above PQQ solution was introduced into the reactor at a rate of 6 ml/min at 15°C through a flexible tube having a diameter of 3 mm. The pH was adjusted to 5 with 10% hydrochloric acid and crystallization proceeded by stirring slowly at 15° C. for 18 hours. To remove the excess sodium contained in the PQQ solution, 50% ethanol was mixed with the PQQ solution to be 1:35 (PQQ:ethanol), and then the pH was adjusted to 3.5 with 10% hydrochloric acid. The mixture was washed by stirring slowly at 15° C. for 18 hours, and the washing process was repeated three times. The washed PQQ sodium salt hydrate was dried under vacuum at 40° C. for 24 hours.
30 L 규모로 하이포마이크로비움 속(Hyphomicrobium sp.) 변이 균주 NPP-230을 배양한 후, 상기 전처리를 포함한 정제과정과 결정화 과정을 진행한 결과, 순도 99.36 %의 PQQ 나트륨염 수화물 결정을 수득하였다(도 7). 30 in Scientific Micro emptying the L scale (Hyphomicrobium sp.) Variation after culturing the strain NPP-230, a result of proceeding the purification and the crystallization process including the pre-treatment, to obtain a purity of 99.36% of the PQQ disodium salt hydrate crystals ( Fig. 7).
3000 L 규모로 배양하여 상기 전처리를 포함한 정제 과정과 결정화 과정을 진행한 결과, 순도 100 %의 PQQ 나트륨염 수화물 결정을 수득하였다(도 8). After culturing at a scale of 3000 L and performing the purification and crystallization processes including the pretreatment, crystals of PQQ sodium salt hydrate having a purity of 100% were obtained (FIG. 8).
상기 실시예 1 내지 5를 통해 수득된 PQQ의 수율을 공정 단계별로 정리하면 하기 표 3과 같다. 배양 후 회수된 PQQ 2.3 kg으로부터 상기 정제 및 결정화 공정을 통해 최종적으로 99 % 이상의 PQQ 1.09 kg을 수득하였다. 전처리 공정을 추가해 PQQ의 순도를 100 %까지 향상시킬 수 있었으며, FPLC 과정에서 염화나트륨 농도를 높여주어 PQQ를 더욱 쉽고 경제적으로 회수할 수 있었다.The yield of PQQ obtained through Examples 1 to 5 is summarized in Table 3 below. From the 2.3 kg of PQQ recovered after the culture, 1.09 kg of PQQ of 99% or more was finally obtained through the purification and crystallization process. The purity of PQQ could be improved to 100% by adding a pretreatment process, and PQQ could be recovered more easily and economically by increasing the sodium chloride concentration in the FPLC process.
실시예 6 <PQQ 결정의 확인> Example 6 < Confirmation of PQQ decision>
실시예 6-1 < IR에 의한 확인 > Example 6-1 < Confirmation by IR >
수득된 PQQ 나트륨염 수화물 결정을 시판 중인 표준품(미쯔비시(Mitsubishi)사 PQQ 결정)과 IR(Infrared spectroscopy)로 비교했을 때, 동일 물질인 피로로퀴놀린 퀴논으로 확인되었다(표 4).When the obtained PQQ sodium salt hydrate crystals were compared with commercially available standard products (Mitsubishi PQQ crystals) and IR (Infrared spectroscopy), they were confirmed to be the same substance, pyrroloquinoline quinone (Table 4).
실시예 6-2 < MS에 의한 확인 > Example 6-2 < Confirmation by MS >
고해상도 질량분석법(high resolution Mass Spectrometry)에 의한 원소 분석을 수행한 결과, 원소 구성이 PQQ 디소디움염의 분자식(C14H5N2Na2O8)과 일치했다(표 5 및 도 9a 및 9b). As a result of performing elemental analysis by high resolution mass spectrometry, the elemental composition was consistent with the molecular formula (C14H5N2Na2O8) of PQQ disodium salt (Table 5 and FIGS. 9A and 9B).
실시예 6-3 < PQQ 결정의 수화도 확인> Example 6-3 < Confirmation of hydration degree of PQQ crystal >
시차주사열랑계(Differential Scanning Calorimeter, DSC) 및 열중량분석기(Thermogravimetric Analyzer, TGA)에 의한 방법으로 PQQ 결정의 수화도를 확인하였다. 도 10에 도시한 바와 같이, 물분자에 의한 중량 감소는 7.731 %로 추정되고, PQQ의 분자량은 374.1, 물의 분자량은 18이어서 수화도는 (374.1 x 0.07731)/18 = 1.49임으로 PQQ : H2O = 1: 1.5, 즉 PQQ에는 1.5개의 물분자가 존재한다고 볼 수 있다.The hydration degree of PQQ crystals was confirmed by a method using a differential scanning calorimeter (DSC) and a thermogravimetric analyzer (TGA). As shown in FIG. 10, the weight loss due to water molecules is estimated to be 7.731%, the molecular weight of PQQ is 374.1, and the molecular weight of water is 18, so that the degree of hydration is (374.1 x 0.07731)/18 = 1.49, so PQQ: H 2 O = 1: 1.5, that is, there can be 1.5 water molecules in PQQ.
실시예 6-4 < XRD에 의한 확인> Example 6-4 < Confirmation by XRD >
상기 PQQ 나트륨염 수화물 결정의 Cu-K를 이용한 분말 X-ray 회절 분석 결과는 도 11과 같다. 본 발명에 따른 PQQ 결정의 X-ray 회절 분석 결과를 표준품의 X-ray 회절 분석 결과와 비교한 결과가 도12에 도시되어 있다. 도12의 가장 아래 부분의 그래프는 도11과 동일한 본 발명에 따른 PQQ 나트륨염 수화물 결정의 X-ray 회절 분석 패턴이다. 이와 같은 비교로 본 발명의 PQQ 나트륨염 수화물 결정은 공지된 PQQ 나트륨염 수화물 결정과는 다른 입자구조를 가지고 있는 새로운 물질임을 확인할 수 있었다(도12).The results of powder X-ray diffraction analysis using Cu-K of the PQQ sodium salt hydrate crystal are shown in FIG. 11. The result of comparing the X-ray diffraction analysis result of the PQQ crystal according to the present invention with the X-ray diffraction analysis result of a standard product is shown in FIG. 12. The graph at the bottom of FIG. 12 is an X-ray diffraction analysis pattern of the PQQ sodium salt hydrate crystal according to the present invention as in FIG. By this comparison, it was confirmed that the PQQ sodium salt hydrate crystal of the present invention is a new material having a different particle structure from the known PQQ sodium salt hydrate crystal (FIG. 12).
상기 PQQ 나트륨염 수화물 결정의 주요 피크(peak)의 2θ 값은 6.28 ± 0.4°, 8.74 ± 0.4°, 17.58 ± 0.4°, 18.28 ± 0.4°, 18.70 ± 0.4°, 22.96 ± 0.4°, 25.48 ± 0.4°, 26.56 ± 0.4°, 33.72 ± 0.4°, 36.28 ± 0.4°이었다.The 2θ values of the main peaks of the PQQ sodium salt hydrate crystals are 6.28 ± 0.4°, 8.74 ± 0.4°, 17.58 ± 0.4°, 18.28 ± 0.4°, 18.70 ± 0.4°, 22.96 ± 0.4°, 25.48 ± 0.4° , 26.56 ± 0.4°, 33.72 ± 0.4°, 36.28 ± 0.4°.
Claims (19)
상기 화학식에서, n은 2이고 M은 나트륨염이며, m은 건조 후에 1.5이다.The method according to claim 14, wherein the pyroquinoline quinone salt hydrate has the following formula:
In the above formula, n is 2, M is a sodium salt, and m is 1.5 after drying.
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| EP0206471A2 (en) * | 1985-04-24 | 1986-12-30 | Mitsubishi Gas Chemical Company, Inc. | Process for preparation of pyrrolo-quinoline quinone |
| US4994382A (en) | 1984-05-29 | 1991-02-19 | Ube Industries, Ltd. | Process for production of pyrrolo-quinoline quinone |
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| WO2012173217A1 (en) * | 2011-06-16 | 2012-12-20 | 三菱瓦斯化学株式会社 | Crystal of pyrroloquinolinequinone disodium salt, and method for producing same |
| US20130337511A1 (en) | 2011-03-03 | 2013-12-19 | Ajinomoto Co., Inc. | Method for Producing Pyrroloquinoline Quinone Using a Bacterium of the Genus Methylobacterium or Hyphomicrobium |
| KR20160068313A (en) * | 2014-12-05 | 2016-06-15 | 주식회사 성운바이오 | Novel microorganism of hyphomicrobium sp. and method of producing pyrrolo-quinoline quinone using the same |
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| EP0206471A2 (en) * | 1985-04-24 | 1986-12-30 | Mitsubishi Gas Chemical Company, Inc. | Process for preparation of pyrrolo-quinoline quinone |
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