KR20230120932A - Degradation method for cellulose acetate using Penicillium griseofulvum - Google Patents
Degradation method for cellulose acetate using Penicillium griseofulvum Download PDFInfo
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- KR20230120932A KR20230120932A KR1020220017793A KR20220017793A KR20230120932A KR 20230120932 A KR20230120932 A KR 20230120932A KR 1020220017793 A KR1020220017793 A KR 1020220017793A KR 20220017793 A KR20220017793 A KR 20220017793A KR 20230120932 A KR20230120932 A KR 20230120932A
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- South Korea
- Prior art keywords
- cellulose acetate
- penicillium
- medium
- strain
- gene
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- 229920002301 cellulose acetate Polymers 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 24
- 241000228127 Penicillium griseofulvum Species 0.000 title claims abstract description 19
- 230000015556 catabolic process Effects 0.000 title description 2
- 238000006731 degradation reaction Methods 0.000 title description 2
- 239000002609 medium Substances 0.000 claims description 32
- 241000228143 Penicillium Species 0.000 claims description 18
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Abstract
Description
본 발명은 페니실리움 그리세오풀범을 이용한 셀룰로오스 아세테이트의 분해 방법에 관한 것이다.The present invention relates to a method for decomposing cellulose acetate using Penicillium griseoful bum.
플라스틱(Plastic)이라는 말은 그리스어 "Plastikos"라는 말에서 유래하였으며, 이 말은 "성형이 가능한"이라는 뜻이다. 열을 가하면 쉽게 모양을 만들 수 있는 플라스틱의 성질 때문에 이러한 단어가 붙은 것으로 보인다. 인류는 돌, 금속, 유리, 목재와 같은 자연에서 구할 수 있는 소재를 넘어서 인간에게 유익한 소재를 구하기 위해 끊임없이 노력하였으며, 이러한 노력의 결과로 가볍고, 단단하고, 쉽게 변하지 않으면서 쉽게 원하는 모양대로 성형이 가능한 플라스틱을 만들어 낸 것이다. 플라스틱은 크게 석유화학 물질을 기본으로 한 "난분해성 플라스틱(고성능 엔지니어링 플라스틱)" 소재와 식물유래 자원(Biomass) 등 재생 가능한 물질을 포함하는 원료를 이용하여 화학적 또는 생물학적 공정을 거쳐 생산되는 "바이오플라스틱(Bioplastic)"으로 나누어지는데, 최근 생분해성, 식물기반 제품에 대한 높은 소비 트렌드와 석유기반 제품보다는 바이오 기반 제품을 사용하도록 하는 제도적인 환경이 바이오플라스틱 개발 필요성을 더욱 높이고 있다.The word plastic comes from the Greek word "plastikos", which means "to be molded". It seems that the word came about because of the plastic's ability to be easily shaped when heated. Humanity has made ceaseless efforts to find materials that are beneficial to humans beyond materials that can be obtained from nature such as stone, metal, glass, and wood. It made possible plastics. Plastics are largely classified as “bioplastics” produced through chemical or biological processes using raw materials including renewable materials such as “non-degradable plastics (high-performance engineering plastics)” based on petrochemical materials and plant-derived resources (Biomass). It is divided into “Bioplastic”, and the recent high consumption trend for biodegradable, plant-based products and the institutional environment that encourages the use of bio-based products rather than petroleum-based products are further increasing the need for bioplastic development.
바이오플라스틱에는 주로 셀롤로오스(Cellulose)가 사용되는데, 셀룰로오스는 지구상의 가장 다량으로 존재하는 천연 고분자 물질로 주로 목재나 목화의 주성분을 이루고 있으며, 재생성과 생분해성이 우수하여 종이, 방직용 섬유로 대량 사용되고 있다. 일반적으로 천연 셀룰로오스는 약 70%의 결정 부분을 함유하고 있어 매우 우수한 기계적 성질을 가지고 있으나 말단의 -OH 그룹 사이의 수소결합으로 분자간 강한 결합력과 결정화도가 높아 가공하기 어렵고, 융점에 도달하기 전에 분해되기 때문에 성형이 어려운 단점을 가지고 있다(Polym(Korea). 2006;30(1):70-74).Cellulose is mainly used for bioplastics. Cellulose is a natural polymeric substance that exists in the largest amount on earth and is a major component of wood or cotton. are used in large quantities. In general, natural cellulose contains about 70% of crystal parts and has excellent mechanical properties. Therefore, molding is difficult (Polym (Korea). 2006;30(1):70-74).
따라서 이러한 단점을 보완하기 위하여 셀룰로오스 유도체로 전환하여 용액 또는 용융 가공 방법으로 응용되고 있다. 여기서 셀룰로오스 유도체는 목질계 소재의 산화, 치환 및 이 외의 화학적 처리에 의하여 얻어지는 생성물을 말하며 치환되는 관능기의 종류에 따라 다양한 화학공업의 원료로 사용되고 있다(Top Curr Chem. 2010;294:117-28). 하지만 이렇게 단점이 보완된 셀룰로오스 유도체 등을 포함하는 플라스틱은 현재 전 세계적으로 소모되고 있는 양이 연간 200만톤 이상으로 매년 약 5%의 신장율을 보이고 있으나, 최근 산업사회에 환경문제가 크게 대두되면서, 재생 가능한 자원이나, 생분해가 가능한 자원에 많은 관심이 고조되고 있다. 특히 해양에서 발견되는 해양쓰레기는 해양생태계를 위협하고 있으며, 플라스틱 사용을 줄이기 위해 다양한 정책을 시행하고 있다.Therefore, in order to compensate for these disadvantages, it is converted into a cellulose derivative and applied as a solution or melt processing method. Here, cellulose derivative refers to a product obtained by oxidation, substitution, and other chemical treatment of wood-based materials, and is used as a raw material for various chemical industries depending on the type of functional group to be substituted (Top Curr Chem. 2010;294:117-28). . However, plastics including cellulose derivatives, etc., whose disadvantages are compensated for in this way, are currently consumed worldwide at more than 2 million tons per year, showing an annual growth rate of about 5%. There is a lot of interest in possible or biodegradable resources. In particular, marine debris found in the ocean threatens the marine ecosystem, and various policies are being implemented to reduce the use of plastic.
실제로 플라스틱 봉지, 플라스틱 병 이외에 담배필터가 해양생태계를 위협하고 있는 것으로 나타났으며, 지난 30년간 해변에서 수거한 쓰레기 중에서 가장 큰 비중을 차지하는 것이 담배필터로 확인이 되었다. 담배필터의 소재로는 셀룰로오스 유도체 중 하나인 셀룰로오스 아세테이트(CA; Cellulose acetate)가 주로 이용되고 있으며, CA가 주요 성분인 담배필터의 자연상태에서의 분해에 대한 연구결과를 확인한 결과, 토양 매립 혹은 지표면 노출 시 담배필터의 부식 현상은 관찰되었으나 필터형태는 그대로 유지되고 있는 것이 확인되었으며, 또한 환경부가 발표한 자료에서 지표면에 노출 시 담배필터가 완전히 분해되는데는 약 10~12년이 소요되는 것으로 알려져 있다.In fact, cigarette filters in addition to plastic bags and plastic bottles were found to be threatening the marine ecosystem, and it was confirmed that cigarette filters accounted for the largest portion of the garbage collected from the beach over the past 30 years. Cellulose acetate (CA), one of the cellulose derivatives, is mainly used as a material for cigarette filters, and as a result of research on the decomposition of cigarette filters, the main component of which is CA, in the natural state, soil landfill or surface Corrosion of the cigarette filter was observed upon exposure, but it was confirmed that the filter shape was maintained as it was. Also, it is known from data announced by the Ministry of Environment that it takes about 10 to 12 years for the cigarette filter to completely decompose when exposed to the ground. .
국외에서는 네이세리아 시카(Neisseria sicca), 리조비움 멜리로티(Rhizobium meliloti), 알칼리제네스 자일록소시단스(Alcaligenes xylosoxidans) 등과 같은 세균과 페스탈로티옵시스 웨스테르디키 큐엠 381(Pestalotiopsis westerdijkii QM 381), 효모(Yeast)와 같은 미생물이 분비하는 효소를 이용해서 CA를 분해할 수 있는 가능성에 대해 보고된 바 있으나 국내에서 이와 관련한 연구는 전무한 실정이다.In foreign countries, bacteria such as Neisseria sicca , Rhizobium meliloti , and Alcaligenes xylosoxidans and Pestalothiopsis westerdijkii QM 381, The possibility of decomposing CA using enzymes secreted by microorganisms such as yeast has been reported, but there is no research related to this in Korea.
본 발명은 국내 토양에 분리·동정한 페니실리움 그리세오풀범(Penicillium griseofulvum) 균주의 CA 분해능을 개시한다.The present invention discloses the CA resolution of Penicillium griseofulvum strains isolated and identified in domestic soil.
본 발명의 목적은 페니실리움 그리세오풀범 균주를 이용한 셀룰로오스 아세테이트의 분해 방법을 제공하는데 있다.An object of the present invention is to provide a method for decomposing cellulose acetate using a strain of Penicillium griseofull.
본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.Other objects or specific objects of the present invention will be presented below.
본 발명은 아래의 실시예 및 실험예에서 확인되는 바와 같이, 국내 토양에서 균류(fungus)인 페니실리움 그리세오풀범(Penicillium griseofulvum) 균주를 분리·동정하고, 이 분리·동정된 균주가 셀룰로오스 아세테이트(Cellulose acetate)의 분해능을 가짐을 확인함으로서 완성된 것이다.As confirmed in the following examples and experimental examples, the present invention isolates and identifies a fungus, Penicillium griseofulvum strain, from domestic soil, and the isolated and identified strain is cellulose acetate. It was completed by confirming that it has the resolution of (Cellulose acetate).
전술한 바를 고려할 때, 본 발명의 셀룰로오스 아세테이트의 분해 방법은 셀룰로오스 아세테이트에 페니실리움 그리세오풀범을 접촉시켜 배양하는 단계를 포함하는 것으로 이해될 수 있다.Considering the foregoing, it can be understood that the decomposition method of cellulose acetate of the present invention includes the step of culturing Penicillium griseofullum in contact with cellulose acetate.
본 발명의 방법에서, 셀룰로오스 아세테이트는 아래 화학식 1로 표현되는 셀룰로오스의 임의의 아세테이트 에스테르(any acetate ester of cellulose) 고분자를 의미하며, 바람직하게는 셀룰로오스 디아세테이트(cellulose diacetate) 고분자를 의미한다.In the method of the present invention, cellulose acetate means any acetate ester of cellulose polymer represented by Formula 1 below, and preferably means a cellulose diacetate polymer.
<화학식 1> <Formula 1>
또 본 발명의 방법에서, 페니실리움 그리세오풀범의 배양과 증식을 촉진시킴으로써 페니실리움 그리세오풀범에 의한 셀룰로오스 아세테이트의 분해능을 높이기 위하여, 페니실리움 그리세오풀범를 배지에서 배양하여 얻은 배양액 형태로 셀룰로오스 아세테이트 필름과 접촉시키거나, 셀룰로오스 아세테이트 필름 접촉 전후에 페니실리움 그리세오풀범의 배양과 증식을 촉진하기 위한 배지가 셀룰로오스 아세테이트 필름에 첨가되어 혼합될 수 있다.In addition, in the method of the present invention, in order to increase the decomposition of cellulose acetate by Penicillium griseofulvum by promoting the culture and growth of Penicillium griseofulvum, the culture medium obtained by culturing Penicillium griseofulvum in a medium A medium for promoting the culture and growth of Penicillium griseofulbum before or after contact with the raw cellulose acetate film or before and after contact with the cellulose acetate film may be added to and mixed with the cellulose acetate film.
그러한 배지는 일반적으로 탄소원, 질소원, 미량원소, 성장 촉진제 등을 포함한다.Such media generally contain a carbon source, a nitrogen source, trace elements, growth promoters, and the like.
탄소원은 바람직하게는 단당류, 이당류 또는 다당류와 같은 당이다. 예컨대 글루코오스, 프럭토오스, 만노오스, 갈락토오스, 리보오스, 소르보오스, 리불로오스, 락토오스, 말토오스, 수크로오스, 라피노오스, 전분, 전분 가수분해물 등이 사용할 수 있다. 당밀이나 당 정제 과정의 부산물 등 복합 화합물이 또한 사용될 수 있다. 경우에 따라서는 대두유, 해바라기유 등 오일이나 아세트산 등의 유기산, 글리세롤 등이 탄소원으로서 바람직할 수 있다. 이들 탄소원은 단독으로 또는 2종 이상의 혼합물로 상기 배지에 포함될 수 있으며, 단독으로 배지에 포함되든, 2종 이상의 혼합물로 배지에 포함되든 2%(w/w) 내지 40%(w/w)의 범위로 배지에 포함될 수 있다. 전분이나 전분 가수분해물 등 천연물 또는 천연물 가공물을 탄소원으로 사용할 경우 타 미생물에 의한 원치 않는 발효를 방지하기 위해서 이들 탄소원을 멸균하여 사용할 수 있다. 멸균은 고온·고압 멸균법, 자외선 조사 등 당업계에 공지된 방법을 사용하여 수행될 수 있다.The carbon source is preferably a sugar such as a monosaccharide, disaccharide or polysaccharide. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, starch hydrolyzate and the like can be used. Complex compounds, such as molasses or by-products of sugar refining processes, may also be used. In some cases, oils such as soybean oil and sunflower oil, organic acids such as acetic acid, and glycerol may be preferred as the carbon source. These carbon sources may be included in the medium alone or in a mixture of two or more, and whether included in the medium alone or in a mixture of two or more, 2% (w / w) to 40% (w / w) It can be included in the medium as a range. When natural products or processed products of natural products, such as starch or starch hydrolyzate, are used as carbon sources, these carbon sources may be sterilized to prevent unwanted fermentation by other microorganisms. Sterilization may be performed using methods known in the art, such as high-temperature and high-pressure sterilization and ultraviolet irradiation.
질소원으로서는 통상적으로 무기 질소 화합물, 유기 질소 화합물 또는 이들 화합물들을 포함하는 복합 화합물일 수 있다. 예컨대 무기 질소 화합물로서는 암모니아, 암모늄염(예컨대, 암모늄 술페이트, 암모늄 클로라이드, 암모늄 포스페이트, 암모늄 카르보네이트, 암모늄 니트레이트 등), 니트레이트 등을 들 수 있고, 유기 질소 화합물로서는 우레아, 아미노산 등을 들 수 있으며, 복합 화합물로서는 효모 추출물(yeast extract), 소이톤(soytone), 펩톤(peptone), 트립톤(tryptone), 맥아 추출물(malt extract), 육즙, 콩 분말, 콩 비지 분말, 청국장 분말, 된장 분말 등의 천연 질소원 등을 들 수 있다. 이들 질소원도 1종 이상 혼합하여 사용될 수 있으며, 배지에 0.1%(w/w) 내지 8.0%(w/w)의 범위로 포함될 수 있다. 천연 질소원을 사용할 경우 천연 탄소원을 사용할 경우와 관련하여 설명한 바와 같은 이유에서 멸균하여 사용하는 것이 바람직하다.As the nitrogen source, an inorganic nitrogen compound, an organic nitrogen compound, or a complex compound containing these compounds may be used. Examples of inorganic nitrogen compounds include ammonia, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.), nitrate, and the like, and examples of organic nitrogen compounds include urea, amino acids, and the like. Complex compounds include yeast extract, soytone, peptone, tryptone, malt extract, broth, soybean powder, soybean okara powder, cheonggukjang powder, soybean paste Natural nitrogen sources, such as powder, etc. are mentioned. One or more of these nitrogen sources may also be used in combination, and may be included in the medium in the range of 0.1% (w/w) to 8.0% (w/w). When using a natural nitrogen source, it is preferable to sterilize and use it for the same reason as described in relation to the case of using a natural carbon source.
미량원소로서는 칼슘, 마그네슘, 나트륨, 코발트, 몰리브덴, 칼륨, 망간, 아연, 구리, 철, 인, 황 등을 포함하며 이들 미량원소는 염 화합물 형태로 배지에 첨가될 수 있으며, 이들 미량 원소의 배지에서의 용해도를 높이고 용액 상태를 유지하도록 하기 위해 킬레이트제 예컨대 카테콜, 시트르산 등이 함께 첨가될 수 있다. 상기 염 화합물 형태로서는 인산수소나트륨, 황산마그네슘, 염화철, 칼슘염, 망간염, 코발트염, 몰리브텐산염, 킬레이트금속염 등을 들 수 있다. 이들 미량원소는 1종 이상 혼합하여 사용될 수 있으며, 0.001%(w/w) 내지 3.0%(w/w)의 범위로 배지에 포함될 수 있다. Trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus, sulfur, etc. These trace elements may be added to the medium in the form of salt compounds, and the medium of these trace elements A chelating agent such as catechol, citric acid, or the like may be added together to increase solubility in and maintain a solution state. Examples of the salt compound form include sodium hydrogen phosphate, magnesium sulfate, iron chloride, calcium salt, manganese salt, cobalt salt, molybthenate salt, chelate metal salt and the like. These trace elements may be used in combination of one or more, and may be included in the medium in the range of 0.001% (w/w) to 3.0% (w/w).
성장 촉진제로서는 비오틴, 리보플라빈, 티아민, 폴산, 니코틴산, 판토테네이트, 피리독신 등이 사용될 수 있으며, 0.0001%(w/w) 내지 1.0%(w/w)의 범위로 배지에 포함될 수 있다. Biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate, pyridoxine, etc. may be used as growth promoters, and may be included in the medium in the range of 0.0001% (w/w) to 1.0% (w/w).
배지는 전술한 바의 탄소원, 질소원, 미량원소, 성장 촉진제 등을 혼합하여 제조하여 사용할 수 있으나, 당업계에서 균류의 배양에 일반적으로 이용되는 공지된 조성의 배지, 예컨대 BHI 액체 배지(Brain-heart Infusion Broth Medium), Czapek 액체 배지(Czapek Brotho Medium), PDB 배지(Potato Dextrose Broth Medium), SD 배지(Sabouraud Dextrose Medium), SM 배지(Sabouraud Maltose Medium) 등을 사용할 수도 있다. 배지 조성의 최적화에 대해서는 문헌(Applied Microbiol. Physiology, A Practical Approach (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3)을 포함하여 당업계에 많은 문헌이 공지되어 있으며, 이들 공지된 문헌을 참조할 수 있다. The medium may be prepared by mixing the above-described carbon source, nitrogen source, trace element, growth promoter, etc., but a medium of known composition commonly used in the culture of fungi in the art, such as BHI liquid medium (Brain-heart Infusion Broth Medium), Czapek Brotho Medium, PDB Medium (Potato Dextrose Broth Medium), SD Medium (Sabouraud Dextrose Medium), SM Medium (Sabouraud Maltose Medium) may also be used. There is much literature in the art on optimization of medium composition, including Applied Microbiol. Physiology, A Practical Approach (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is known, and reference can be made to these known documents.
배지는 불필요한 미생물의 증식을 방지하기 위하여 멸균하여 사용하는 것이 바람직한데, 배지의 멸균 방법은 당업계에 공지되어 있으며, 공지된 멸균 방법 중 적합한 방법을 선택하여 사용할 수 있다. 예컨대 고압 증기 멸균, 자외선을 이용한 멸균 방법 등이 적합할 수 있다.The medium is preferably used after being sterilized to prevent the proliferation of unnecessary microorganisms. The sterilization method of the medium is known in the art, and a suitable method may be selected and used among known sterilization methods. For example, high-pressure steam sterilization, a sterilization method using ultraviolet rays, and the like may be suitable.
배양 온도는 일반적으로 15℃ 내지 45℃, 바람직하게는 25℃ 내지 40℃, 더 바람직하게는 25℃ 내지 30℃이다. 배지의 pH는 4 내지 8.5, 바람직하게는 4.0 내지 7.0이다. 배지 pH는 배양이 진행되는 동안 수산화나트륨, 수산화칼륨, 수성 암모니아 등의 염기성 화합물이나, 인산, 황산 등의 산성 화합물을 첨가하여 조절될 수 있다. The culture temperature is generally 15°C to 45°C, preferably 25°C to 40°C, more preferably 25°C to 30°C. The pH of the medium is 4 to 8.5, preferably 4.0 to 7.0. The pH of the medium can be adjusted by adding a basic compound such as sodium hydroxide, potassium hydroxide, aqueous ammonia, or an acidic compound such as phosphoric acid or sulfuric acid while the culture is in progress.
배양 중 형성되는 거품의 제거를 위해서 지방산 폴리글리콜 에스테르와 같은 소포제가 첨가될 수 있다. Antifoaming agents such as fatty acid polyglycol esters may be added to remove foam formed during cultivation.
배양은 증식 특성에 따라 호기 조건 또는 혐기 조건을 만들어 주는 것이 유리할 수 있다.Cultivation may be advantageous to create aerobic conditions or anaerobic conditions depending on the growth characteristics.
배양은 회분식(batchwise), 반-회분식(semi-batchwise) 또는 연속식으로 이루어질 수 있다. 적합한 구체적인 배양법은 예컨대 문헌(Bioprozeβtechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991), 문헌(Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994), 미국세균학회(the American Society for Bacteriology)가 발행한 문헌(Manual of Methods for General Bacteriology"(Washington D.C., USA, 1981) 등을 참조할 수 있다. Cultivation can be batchwise, semi-batchwise or continuous. Suitable specific culture methods are described, for example, in Bioprozeβtechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991), Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994), the American Society for Bacteriology (Manual of Methods for General Bacteriology" (Washington D.C., USA, 1981), etc. may be referred to.
다른 측면에 있어서, 본 발명은 페니실리움 그리세오풀범 NIBRFGC000508019 균주(수탁번호: KACC 93376P)에 관한 것이다.In another aspect, the present invention relates to the Penicillium griseofulvum NIBRFGC000508019 strain (Accession Number: KACC 93376P).
본 발명에 따른 페니실리움 그리세오풀범 NIBRFGC000508019 균주(수탁번호: KACC 93376P)는 그 ITS(Internal Transcribed Spacer) 유전자, BTU(Beta-tubulin) 유전자, CaM(Calmodulin) 유전자, RPB2(RNA polymerase II subunit) 유전자가 각각 서열번호 9, 10, 11, 12의 서열을 갖는 균주로 이해될 수 있다.The Penicillium griseofulbum NIBRFGC000508019 strain (accession number: KACC 93376P) according to the present invention has an internal transcribed spacer (ITS) gene, beta-tubulin (BTU) gene, calmodulin (CaM) gene, RNA polymerase II subunit (RPB2) It can be understood as a strain whose genes have sequences of SEQ ID NOs: 9, 10, 11, and 12, respectively.
전술한 바와 같이, 본 발명에 따르면 페니실리움 그리세오풀범을 이용한 셀룰로오스 아세테이트의 분해 방법을 제공할 수 있다. As described above, according to the present invention, it is possible to provide a method for decomposing cellulose acetate using Penicillium griseofulbum.
본 발명의 방법은 자연상태에서 분해되기 어려운 셀룰로오스 아세테이트의 분해에 유용하게 적용될 수 있다.The method of the present invention can be usefully applied to the decomposition of cellulose acetate, which is difficult to decompose in nature.
도 1은 페니실리움 그리세오풀범 NIBRFGC000508019 균주를 PDA 배지에서 7일간 배양한 사진이다.
도 2는 페니실리움 그리세오풀범 NIBRFGC000508019 균주의 계통수이다.
도 3은 셀롤로오스 아세테이트의 분해 매커니즘을 보여주는 반응식이다.
도 4는 페니실리움 그리세오풀범 NIBRFGC000508019 균주의 셀룰로오스 아세테이트 분해능을 X-ray 회절분석법을 이용하여 확인한 결과이다.
도 5는 페니실리움 그리세오풀범 NIBRFGC000508019 균주의 셀룰로오스 아세테이트 분해능을 적외선 분광분석법을 이용하여 확인한 결과이다.Figure 1 is a photograph of the culture of Penicillium griseopulbum NIBRFGC000508019 strain in PDA medium for 7 days.
Figure 2 is a phylogenetic tree of Penicillium griseofullbum NIBRFGC000508019 strain.
Figure 3 is a reaction scheme showing the decomposition mechanism of cellulose acetate.
Figure 4 is the result of confirming the cellulose acetate decomposition ability of the Penicillium griseopulbum NIBRFGC000508019 strain using X-ray diffraction analysis.
Figure 5 is the result of confirming the cellulose acetate decomposition ability of the Penicillium griseofullbum NIBRFGC000508019 strain using infrared spectroscopy.
이하 본 발명을 실시예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.
<실시예 1> 페니실리움 그리세오풀범 NIBRFGC000508019 균주의 분리<Example 1> Separation of strain Penicillium griseofulbum NIBRFGC000508019
페니실리움 그리세오풀범(Penicillium griseofulvum) NIBRFGC000508019 균주의 분리를 위하여, 국내 경북 의성군 쓰레기산에서 채취한 토양시료 1 g에 멸균수 10 mL를 넣고 진탕배양기에서 30분간 150 rpm으로 진탕하였다. 이 후 진탕된 현탁액을 멸균수에 10배씩 희석하여 희석액을 만들고, 감자한천배지(Potato dextrose agar; PDA, Difco, USA)에 희석액 100 μL를 도말하여 25℃에서 암배양하였다. 배양 1-2일 후 관찰되는 균사를 새로운 PDA에 계대 배양하여 NIBRFGC000508019로 명기하고 공시균주로 이용하였다.For the isolation of Penicillium griseofulvum NIBRFGC000508019 strain, 10 mL of sterile water was added to 1 g of a soil sample collected from Mt. Trash, Uiseong-gun, Gyeongsangbuk-do, Korea, and shaken at 150 rpm for 30 minutes in a shaking incubator. Thereafter, the shaken suspension was diluted 10-fold in sterile water to make a diluted solution, and 100 μL of the diluted solution was spread on potato dextrose agar (PDA, Difco, USA) and cultured in the dark at 25 ° C. Mycelia observed after 1-2 days of culture were subcultured to a new PDA, designated as NIBRFGC000508019, and used as a test strain.
NIBRFGC000508019 균주를 PDA 배지에서 7일간 배양한 결과를 도 1에 나타내었는데(도 1의 A는 앞면 사진이고 도 1의 B는 뒷면 사진이며 도 1의 C는 셀룰로오스 에틸아세테이트에서 균주 배양 40일 후의 사진임), 균체의 중심부는 약간 융기되며, 균체는 흰색-녹색과 혼합형태이고, 가장자리는 흰색이었다. 뒷면은 연한 갈색 가장자리를 가지며, 중심부는 진한 갈색을 나타내었다.The results of culturing the strain NIBRFGC000508019 for 7 days in PDA medium are shown in Figure 1 (A in Figure 1 is a front picture, B in Figure 1 is a picture of the back side, C in Figure 1 is a picture after 40 days of culturing the strain in cellulose ethyl acetate) ), the center of the cells was slightly raised, the cells were white-green and mixed, and the edges were white. The back has a light brown edge and the center has a dark brown color.
<실시예 2> 페니실리움 그리세오풀범 NIBRFGC000508019 균주의 동정<Example 2> Identification of Penicillium griseoful bum NIBRFGC000508019 strain
페니실리움 그리세오풀범(Penicillium griseofulvum) NIBRFGC000508019 균주의 분자생물학적 동정을 위해, 공시균주의 genomic DNA는 HiGeneTM Genomic DNA prep kit (BIOFACT, Daejeon, Korea)를 이용하여 추출하고, 염기서열 분석을 위해 ITS region, Beta-tubulin(BTU), Calmodulin(CaM), RNA polymerase II subunit(RPB 2)의 유전자에 대해서 PCR 수행하고 염기서열 분석을 수행하였다.For the molecular biological identification of the strain Penicillium griseofulvum NIBRFGC000508019, the genomic DNA of the strain was extracted using HiGeneTM Genomic DNA prep kit (BIOFACT, Daejeon, Korea), and the ITS region for sequencing analysis , Beta-tubulin (BTU), Calmodulin (CaM), and RNA polymerase II subunit (RPB 2) genes were subjected to PCR and sequencing.
ITS region 유전자 증폭에서는 서열번호 1과 2의 정방향/역방향 프라이머(ITS1(5’-TCC GTA GGT GAA CCT GCG G-3’), ITS4(5’-TCC TCC GCT TAT TGA TAT GC-3’)), BTU 유전자 증폭에는 서열번호 3과 4의 정방향/역방향 프라이머(Bt2a(5’-GGT AAC CAA ATC GGT GCT GCT TTC-3’), Bt2b(5’-ACC CTC AGT GTA GTG ACC CTT GGC-3’)), CaM 유전자 증폭에는 서열번호 5와 6의 정방향/역방향 프라이머(CL1(5’-GAR TWC AAG GAG GCC TTC TC-3’), CL2A(5’-TTT TTG CAT CAT GAG TTG GAC-3’)), RPB2 유전자 증폭에는 서열번호 7과 8의 정방향/역방향 프라이머(5F2(5’-GGG GWG AYC AGA AGA AGG C-3’), 7cR(5’-CCC ATR GCT TGY TTR CCC AT-3’))를 이용하여 PCR를 수행하고, 증폭된 산물은 EXOSAP-IT(Thermo Fisher Scientific, Waltham, MA, USA)를 이용하여 정제하였다. 정제된 PCR 산물은 (주)바이오니아(Daejeon, Korea)에 염기서열 분석을 의뢰하고, 이 후 수령한 데이터는 SeqMan Lasergene software (DNAStar Inc., Madison, Wisconsin, USA)를 이용하여 정리된 염기서열을 확보하였다(ITS region 유전자는 서열번호 9, BTU 유전자는 서열번호 10, CaM 유전자는 서열번호 11, RPB2 유전자는 서열번호 12에 개시되어 있음).For ITS region gene amplification, forward/reverse primers of SEQ ID NOs: 1 and 2 (ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3'), ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3')) For BTU gene amplification, forward/reverse primers of SEQ ID NOs: 3 and 4 (Bt2a (5'-GGT AAC CAA ATC GGT GCT GCT TTC-3'), Bt2b (5'-ACC CTC AGT GTA GTG ACC CTT GGC-3') )), forward/reverse primers of SEQ ID NOs: 5 and 6 (CL1 (5'-GAR TWC AAG GAG GCC TTC TC-3'), CL2A (5'-TTT TTG CAT CAT GAG TTG GAC-3') for CaM gene amplification )), forward/reverse primers of SEQ ID NOs: 7 and 8 (5F2 (5'-GGG GWG AYC AGA AGA AGG C-3'), 7cR (5'-CCC ATR GCT TGY TTR CCC AT-3') for RPB2 gene amplification )) was used to perform PCR, and the amplified product was purified using EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA). The purified PCR product was sent to Bioneer Co., Ltd. (Daejeon, Korea) for sequencing, and the received data was sequenced using SeqMan Lasergene software (DNAStar Inc., Madison, Wisconsin, USA). It was obtained (SEQ ID NO: 9 for the ITS region gene, SEQ ID NO: 10 for the BTU gene, SEQ ID NO: 11 for the CaM gene, and SEQ ID NO: 12 for the RPB2 gene).
공시균주에서 확보된 ITS region 유전자, BTU 유전자, CaM 유전자, RPB2 유전자 염기서열을 기반으로 하여, 계통학적 유연관계를 분석하기 위해 미국국립생물공학정보센터(National Center for Biotechnology Information)의 Blast 프로그램(Basic Local Alignment Search Tool)을 사용하여 기존 보고된 균주와 서열 상동성 비교하였다. 계통학적 유연관계는 MEGA7.0 소프트웨어를 이용하여 작성하였고, 작성한 계통수를 도 2에 나타내었다.The National Center for Biotechnology Information's Blast program (Basic Local Alignment Search Tool) was used to compare sequence homology with previously reported strains. The phylogenetic relationship was created using MEGA7.0 software, and the phylogenetic tree created is shown in FIG.
분리된 균주는 NCBI에서 blast 수행한 결과, BTU 유전자는 페니실리움 그리세오풀범(Penicillium griseofulvum) NRRL 2300 균주와 100% 상동성을 보이고, CaM 유전자는 페니실리움 그리세오풀범(Penicillium griseofulvum) DTO 072A5 균주와 99.8% 상동성을 보이며, RPB2 유전자는 페니실리움 그리세오풀범(Penicillium griseofulvum) DTO 072A5 균주와 99.8%, 페니실리움 그리세오풀범(Penicillium griseofulvum) Dierchx 1901 균주와 100%의 상동성을 보임이 확인되었으며, 페니실리움 그리세오풀범(Penicillium griseofulvum) NRRL 2300 균주와 계통수 상에서 동일한 그룹을 형성하여 페니실리움 그리세오풀범(Penicillium griseofulvum)으로 동정하였고, 국립농업과학원 농업미생물은행(Korean Agricultural Culture Collection, KACC)에 기탁하고 수탁번호 KACC 93376P를 부여받았다.As a result of blasting the isolated strain at NCBI, the BTU gene showed 100% homology with the strain Penicillium griseofulvum NRRL 2300, and the CaM gene showed Penicillium griseofulvum DTO 072A5. Shows 99.8% homology with the strain, and the RPB2 gene shows 99.8% homology with Penicillium griseofulvum DTO 072A5 strain and 100% homology with Dierchx 1901 strain. This was confirmed, and it was identified as Penicillium griseofulvum by forming the same group on the phylogenetic tree with Penicillium griseofulvum NRRL 2300 strain, and it was identified as Penicillium griseofulvum , and the Korean Agricultural Culture Collection , KACC) and assigned accession number KACC 93376P.
<실시예 3> X-선 회절분석법과 적외선 분광분석법을 이용한 CA의 분해능 확인<Example 3> Confirmation of resolution of CA using X-ray diffraction analysis and infrared spectroscopy
실험에 사용한 CA 필름은 셀룰로스 아세테이트 필름(Cellulose Acetate, Film)(Sigma-aldrich GF68004772, thickness 0.025mm)를 사용하였으며, 표면의 오염원을 제거하기 위하여 70% EtOH을 이용하여 2-3회 닦아내고, 5×6 cm로 잘라 실험재료로 사용하였다.Cellulose Acetate (Film) (Sigma-aldrich GF68004772, thickness 0.025mm) was used as the CA film used in the experiment, and in order to remove contaminants on the surface, it was wiped 2-3 times with 70% EtOH, and 5× It was cut into 6 cm and used as an experimental material.
배양 배지는 Mao 등의 방법을 이용하여 다음과 같이 제작하였다(Mao et al., 2015). 탄소원이 없는 최소배지(MgSO4·7H2O 0.5 g/L, NH4Cl 1.0 g/L, KH2PO4 5.54 g/L, Na2HPO4 4.78 g/L, CaCl2·7H2O 0.005 g/L, Agar powder 15 g/L)를 제작하여 준비된 CA 필름을 올려두고 필름 표면에 공시균주를 치상하여 25℃ 인큐베이터에 배양하였다.The culture medium was prepared as follows using the method of Mao et al. (Mao et al., 2015). Minimum medium without carbon source (MgSO4 7H 2 O 0.5 g/L, NH 4 Cl 1.0 g/L, KH 2 PO 4 5.54 g/L, Na 2 HPO 4 4.78 g/L, CaCl 2 7H 2 O 0.005 g / L, Agar powder 15 g / L) was prepared and put on the prepared CA film, and the test strain was plated on the surface of the film and cultured in a 25 ℃ incubator.
CA 필름은 도 3에 보이는 바와 같이 산화반응(Oxidation)과 가수분해반응(Hydrolysis)를 통하여 메인 사슬과 아세틸기(R=COCH3)의 분해가 이루어 질 수가 있다. CA 필름에 대한 해당 균주의 영향을 확인하기 위하여, 배양 40일 후 CA 필름(도 1의 C)을 X-선 회절분석법(X-ray Diffraction; XRD)과 적외선 분광분석법(Infrared Spectrophotometer; IR)을 이용하여, 균주 배양 전후의 CA 필름의 화학구조와 결정구조 변화에 대하여 분석하였다.As shown in FIG. 3, the CA film can undergo decomposition of the main chain and the acetyl group (R=COCH3) through oxidation and hydrolysis. In order to confirm the effect of the strain on the CA film, after 40 days of cultivation, the CA film (C in FIG. 1) was analyzed by X-ray diffraction (XRD) and infrared spectrophotometer (IR). Using this, the chemical structure and crystal structure changes of the CA film before and after culturing the strain were analyzed.
XRD 분석을 통한 CA 분해능을 확인한 결과를 도 4에 나타내었는데, 고분자의 분해과정에서 상대적으로 비결정영역에서 분해가 먼저 일어나게 되며, 분해가 진행됨에 따라 결정구조의 변화가 나타난다. 또한 아세틸기가 분해되며 수산화기가 생성되면 수소결합의 증가로 인해 결정구조에 영향을 미치게 된다. 일반적인 CA 필름의 경우, 2θ=11.7o, 20.1o, 21.7o에서 회절 피크가 나타나게 되는데 XRD 분석 결과 균주 배양 전후에 CA 필름의 회절 패턴이 다르게 나타나는 것을 확인하였다. 이를 통해 CA 필름의 미세구조에 변화가 있음을 확인할 수 있었으며, CA 분자사슬의 분해로 인하여 필름의 아세틸기로 인한 결정구조의 변화(disorder)와 Iβ 결정형태에 영향을 미치기 때문인 것으로 보인다.The results of confirming the CA resolution through XRD analysis are shown in FIG. 4, and in the process of decomposition of the polymer, decomposition occurs first in the amorphous region, and as the decomposition progresses, the crystal structure changes. In addition, when an acetyl group is decomposed and a hydroxyl group is generated, the crystal structure is affected due to an increase in hydrogen bonding. In the case of a general CA film, diffraction peaks appear at 2θ = 11.7 o , 20.1 o , and 21.7 o . As a result of XRD analysis, it was confirmed that the diffraction pattern of the CA film appeared differently before and after culturing the strain. Through this, it was confirmed that there was a change in the microstructure of the CA film, and it seems to be because the decomposition of the CA molecular chain affects the crystal structure disorder and I β crystal form due to the acetyl group of the film.
IR 분석을 통한 CA 분해능을 확인한 결과를 도 5에 나타내었는데, IR 스펙트럼의 경우 작용기의 종류가 달라지면 밴드 면적을 바로 비교할 수는 없지만, 두 밴드의 상대적인 크기로부터 정성적인 비교는 가능하다. 도 5에서 CA 필름은 1750cm-1에서 아세틸기의 카르보닐기(C=O), 3470cm-1에서의 브로드한 수산화기(-OH), 1370cm-1에서 메틸기(CH3), 1040cm-1에서 Pyranoid ring의 C-O-C의 작용기들의 밴드가 나타난다. CA 필름이 가수분해 됨에 따라 아세틸기는 수산화기로 전환되기 때문에, 공시균주 배양 전에 비하여 아세틸기의 C=O, C-H 밴드는 감소하며 -OH 밴드는 점차 커지고 있다. 이를 통하여 곰팡이 배양 후의 샘플들의 각 작용기들의 비율을 계산하였을 때, 균주 배양 후 CA 필름에서 아세틸 그룹이 분해되었음을 알 수 있다.The results of confirming the CA resolution through IR analysis are shown in FIG. 5. In the case of the IR spectrum, if the type of functional group is different, the band area cannot be directly compared, but a qualitative comparison can be made from the relative size of the two bands. In FIG. 5, the CA film has a carbonyl group (C=O) of an acetyl group at 1750 cm -1 , a broad hydroxyl group (-OH) at 3470 cm -1 , a methyl group (CH3) at 1370 cm -1 , and a COC of a Pyranoid ring at 1040 cm -1 A band of functional groups appears. Since the acetyl group is converted to a hydroxyl group as the CA film is hydrolyzed, the C=O and CH bands of the acetyl group decrease and the -OH band gradually increases compared to before culturing the strain. Through this, when the ratio of each functional group of the samples after mold culture was calculated, it can be seen that the acetyl group was decomposed in the CA film after strain culture.
<110> National Institute of Biological Resources <120> Degradation method for cellulose acetate using Penicillium griseofulvum <130> P21-037 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Foward primer of Beta-tubulin <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Foward primer of Beta-tubulin <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Foward primer of Beta-tubulin <400> 3 ggtaaccaaa tcggtgctgc tttc 24 <210> 4 <211> 916 <212> DNA <213> Artificial Sequence <220> <223> Backward primer of Beta-tubulin <400> 4 tggtatgtcc caggtgctga gtcgttacac atttgcctct tcattgtctc atctgcgccg 60 cacgaacacc cccattggca gagatggaaa gattgccaaa ccccgtcaac tccataatac 120 tcactggggt ctggtctgcc cggctgagac tcccgaaggt caagcttgtg gtctggtcaa 180 gaacttggca ttgatgtgct acatcactgt cggtacacct gcagagccta tcgtggactt 240 tatgattcag cgcaacatgg aagtcctcga ggagttcgaa ccccaagtga cgcccaatgc 300 gacaaaggtg tttgttaatg gtgtttgggt gggtattcac cgggaccctt cgcatctcgt 360 tactacgatg cagaacctgc gtcgacgaaa catgatctca cacgaagtca gtttgattcg 420 tgacatccgt gaacgggaat tcaaaatctt cacggatact ggacgtgtct gccggccact 480 cttcgtcatc gataatgatc ccaagagtga aaactcgggt ggattggtcc ttaataagga 540 acacattcgg aagctcgagg ccgacaaaga cttgccagta gacttggccc cagaagaacg 600 tcgcgaacaa tacttcggat gggatggtct ggtgcgttct ggagcagtcg agtatgtcga 660 tgccgaagaa gaggaaacca tcatgattgt catgaccccc gaggatctcg agatctctcg 720 acagcttcag gccggttacg ctctgccaga tgacgaagcc ggcgacccca ataaacgtgt 780 tcgatcgatt ctcagccagc gtgcccacac ctggacgcac tgtgaaatcc accctagtat 840 gatcttgggt gtttgcgcta gtattattcc tttcccggat cacaaccagt cgccccgtaa 900 cacctaccag tctgcc 916 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Calmodulin <400> 5 gartwcaagg aggccttctc 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Backward primer of Calmodulin <400> 6 tttttgcatc atgagttgga c 21 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of RNA polymerase II subunit <400> 7 ggggwgayca gaagaaggc 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Backward primer of RNA polymerase II subunit <400> 8 cccatrgctt gyttrcccat 20 <210> 9 <211> 552 <212> DNA <213> Artificial Sequence <220> <223> ITS region <400> 9 cctgcggaag gatcattact gagtgagggc cctctgggtc caacctccca cccgtgttta 60 tttaccttgt tgcttcggcg ggcccgcctt aactggccgc cggggggctt acgcccccgg 120 gcccgcgccc gccgaagaca ccctcgaact ctgtctgaag attgtagtct gagtgaaaat 180 ataaattatt taaaactttc aacaacggat ctcttggttc cggcatcgat gaagaacgca 240 gcgaaatgcg atacgtaatg tgaattgcaa attcagtgaa tcatcgagtc tttgaacgca 300 cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc tgccctcaag 360 cacggcttgt gtgttgggcc ccgtcctccg attccggggg acgggcccga aaggcagcgg 420 cggcaccgcg tccggtcctc gagcgtatgg ggctttgtca cccgctctgt aggcccggcc 480 ggcgcttgcc gatcaaccca aatttttatc caggttgacc tcggatcagg tagggatacc 540 cgctgaactt aa 552 <210> 10 <211> 432 <212> DNA <213> Artificial Sequence <220> <223> Beta-tubulin gene <400> 10 tggtacgtgc ctagcttttt ttttcccgcg ttgggtatca attgacaagt tactaactgg 60 attacaggca aaccatttcc ggcgagcacg gtctcgatgg tgatggacag taagttaatg 120 gcgacgaatt ctccagtgga tggcatgtct gatatcttgt taggtacaat ggtacctccg 180 acctccagct cgagcgtatg aacgtctact tcaaccatgt gagtatagcc gactgggaac 240 cgaataatcg tgcatcaact gatcgggtct ttttctttga tcatctaggc tagcggtgat 300 aagtacgttc cccgtgccgt tctggtcgat ttggagcccg gtaccatgga cgctgtccgc 360 tccggcccct tcggcaagct tttccgcccc gacaacttcg tcttcggtca gtccggtgct 420 ggtaacaact gg 432 <210> 11 <211> 664 <212> DNA <213> Artificial Sequence <220> <223> Calmodulin gene <400> 11 cctgtttgtg agtgattcct acagattgaa gatattgaag atatgctgac cgggagtttg 60 ttttgcgaaa taggacaagg atggcgatgg tacgtgtggt tgcgcccgac agctcagttg 120 agcccacagc agcgtcctct gctttcgaat attaagagca aggtattcta acatacaatc 180 ctaccaatag gacaaatcac caccaaggag cttggtaccg tcatgcgctc gctgggccag 240 aacccctccg agtccgaact gcaggatatg atcaacgagg tcgacgccga caacaacggc 300 accattgact tccccggtac ttgctccata ccccactgat ataaataaga gacggttatt 360 gacgtgcgat agaattcctg accatgatgg ctcgtaagat gaaggatact gattccgagg 420 aggagatccg tgaggcattc aaggttttcg atcgcgacaa caacggattc atttccgctg 480 ccgagctgcg ccacgtcatg acttcaatcg gtgagaagct gaccgacgac gaggttgacg 540 agatgatccg cgaggcggac caggatggtg atggccggat cgactgtatg ttttggaatc 600 ttggagccta ccggcacttg ccttcccgaa tgctaacctc cccatttata cagacaacga 660 gttc 664 <210> 12 <211> 916 <212> DNA <213> Artificial Sequence <220> <223> RNA polymerase II (RPB2) gene <400> 12 tggtatgtcc caggtgctga gtcgttacac atttgcctct tcattgtctc atctgcgccg 60 cacgaacacc cccattggca gagatggaaa gattgccaaa ccccgtcaac tccataatac 120 tcactggggt ctggtctgcc cggctgagac tcccgaaggt caagcttgtg gtctggtcaa 180 gaacttggca ttgatgtgct acatcactgt cggtacacct gcagagccta tcgtggactt 240 tatgattcag cgcaacatgg aagtcctcga ggagttcgaa ccccaagtga cgcccaatgc 300 gacaaaggtg tttgttaatg gtgtttgggt gggtattcac cgggaccctt cgcatctcgt 360 tactacgatg cagaacctgc gtcgacgaaa catgatctca cacgaagtca gtttgattcg 420 tgacatccgt gaacgggaat tcaaaatctt cacggatact ggacgtgtct gccggccact 480 cttcgtcatc gataatgatc ccaagagtga aaactcgggt ggattggtcc ttaataagga 540 acacattcgg aagctcgagg ccgacaaaga cttgccagta gacttggccc cagaagaacg 600 tcgcgaacaa tacttcggat gggatggtct ggtgcgttct ggagcagtcg agtatgtcga 660 tgccgaagaa gaggaaacca tcatgattgt catgaccccc gaggatctcg agatctctcg 720 acagcttcag gccggttacg ctctgccaga tgacgaagcc ggcgacccca ataaacgtgt 780 tcgatcgatt ctcagccagc gtgcccacac ctggacgcac tgtgaaatcc accctagtat 840 gatcttgggt gtttgcgcta gtattattcc tttcccggat cacaaccagt cgccccgtaa 900 cacctaccag tctgcc 916 <110> National Institute of Biological Resources <120> Degradation method for cellulose acetate using Penicillium griseofulvum <130> P21-037 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> artificial sequence <220> <223> Forward primer of beta-tubulin <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer of beta-tubulin <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 24 <212> DNA <213> artificial sequence <220> <223> Forward primer of beta-tubulin <400> 3 ggtaaccaaa tcggtgctgc tttc 24 <210> 4 <211> 916 <212> DNA <213> artificial sequence <220> <223> Backward primer of beta-tubulin <400> 4 tggtatgtcc caggtgctga gtcgttacac atttgcctct tcattgtctc atctgcgccg 60 cacgaacacc cccattggca gagatggaaa gattgccaaa ccccgtcaac tccataatac 120 tcactggggt ctggtctgcc cggctgagac tcccgaaggt caagcttgtg gtctggtcaa 180 gaacttggca ttgatgtgct acatcactgt cggtacacct gcagagccta tcgtggactt 240 tatgattcag cgcaacatgg aagtcctcga ggagttcgaa ccccaagtga cgcccaatgc 300 gacaaaggtg tttgttaatg gtgtttgggt gggtattcac cgggaccctt cgcatctcgt 360 tactacgatg cagaacctgc gtcgacgaaa catgatctca cacgaagtca gtttgattcg 420 tgacatccgt gaacgggaat tcaaaatctt cacggatact ggacgtgtct gccggccact 480 cttcgtcatc gataatgatc ccaagagtga aaactcgggt ggattggtcc ttaataagga 540 acacattcgg aagctcgagg ccgacaaaga cttgccagta gacttggccc cagaagaacg 600 tcgcgaacaa tacttcggat gggatggtct ggtgcgttct ggagcagtcg agtatgtcga 660 tgccgaagaa gaggaaacca tcatgattgt catgaccccc gaggatctcg agatctctcg 720 acagcttcag gccggttacg ctctgccaga tgacgaagcc ggcgacccca ataaacgtgt 780 tcgatcgatt ctcagccagc gtgcccacac ctggacgcac tgtgaaatcc accctagtat 840 gatcttgggt gtttgcgcta gtattattcc tttcccggat cacaaccagt cgccccgtaa 900 cacctaccag tctgcc 916 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer of Calmodulin <400> 5 gartwcaagg aggccttctc 20 <210> 6 <211> 21 <212> DNA <213> artificial sequence <220> <223> Backward primer of Calmodulin <400> 6 tttttgcatc atgagttgga c 21 <210> 7 <211> 19 <212> DNA <213> artificial sequence <220> <223> Forward primer of RNA polymerase II subunit <400> 7 ggggwgayca gaagaaggc 19 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> Backward primer of RNA polymerase II subunit <400> 8 cccatrgctt gyttrcccat 20 <210> 9 <211> 552 <212> DNA <213> artificial sequence <220> <223> ITS region <400> 9 cctgcggaag gatcattact gagtgagggc cctctgggtc caacctccca cccgtgttta 60 tttaccttgt tgcttcggcg ggcccgcctt aactggccgc cggggggctt acgcccccgg 120 gcccgcgccc gccgaagaca ccctcgaact ctgtctgaag attgtagtct gagtgaaaat 180 ataaattatt taaaactttc aacaacggat ctcttggttc cggcatcgat gaagaacgca 240 gcgaaatgcg atacgtaatg tgaattgcaa attcagtgaa tcatcgagtc tttgaacgca 300 cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc tgccctcaag 360 cacggcttgt gtgttgggcc ccgtcctccg attccggggg acgggcccga aaggcagcgg 420 cggcaccgcg tccggtcctc gagcgtatgg ggctttgtca cccgctctgt aggcccggcc 480 ggcgcttgcc gatcaaccca aatttttatc caggttgacc tcggatcagg tagggatacc 540 cgctgaactt aa 552 <210> 10 <211> 432 <212> DNA <213> artificial sequence <220> <223> Beta-tubulin gene <400> 10 tggtacgtgc ctagcttttt ttttcccgcg ttgggtatca attgacaagt tactaactgg 60 attacaggca aaccatttcc ggcgagcacg gtctcgatgg tgatggacag taagttaatg 120 gcgacgaatt ctccagtgga tggcatgtct gatatcttgt taggtacaat ggtacctccg 180 acctccagct cgagcgtatg aacgtctact tcaaccatgt gagtatagcc gactgggaac 240 cgaataatcg tgcatcaact gatcgggtct ttttctttga tcatctaggc tagcggtgat 300 aagtacgttc cccgtgccgt tctggtcgat ttggagcccg gtaccatgga cgctgtccgc 360 tccggcccct tcggcaagct tttccgcccc gacaacttcg tcttcggtca gtccggtgct 420 ggtaacaact gg 432 <210> 11 <211> 664 <212> DNA <213> artificial sequence <220> <223> Calmodulin gene <400> 11 cctgtttgg agtgattcct acagattgaa gatattgaag atatgctgac cgggagtttg 60 ttttgcgaaa taggacaagg atggcgatgg tacgtgtggt tgcgcccgac agctcagttg 120 agcccacagc agcgtcctct gctttcgaat attaagagca aggtattcta acatacaatc 180 ctaccaatag gacaaatcac caccaaggag cttggtaccg tcatgcgctc gctgggccag 240 aacccctccg agtccgaact gcaggatatg atcaacgagg tcgacgccga caacaacggc 300 accattgact tccccggtac ttgctccata ccccactgat ataaataaga gacggttat 360 gacgtgcgat agaattcctg accatgatgg ctcgtaagat gaaggatact gattccgagg 420 aggagatccg tgaggcattc aaggttttcg atcgcgacaa caacggattc atttccgctg 480 ccgagctgcg ccacgtcatg acttcaatcg gtgagaagct gaccgacgac gaggttgacg 540 agatgatccg cgaggcggac caggatggtg atggccggat cgactgtatg ttttggaatc 600 ttggagccta ccggcacttg ccttcccgaa tgctaacctc cccatttata cagacaacga 660 gttc 664 <210> 12 <211> 916 <212> DNA <213> artificial sequence <220> <223> RNA polymerase II (RPB2) gene <400> 12 tggtatgtcc caggtgctga gtcgttacac atttgcctct tcattgtctc atctgcgccg 60 cacgaacacc cccattggca gagatggaaa gattgccaaa ccccgtcaac tccataatac 120 tcactggggt ctggtctgcc cggctgagac tcccgaaggt caagcttgtg gtctggtcaa 180 gaacttggca ttgatgtgct acatcactgt cggtacacct gcagagccta tcgtggactt 240 tatgattcag cgcaacatgg aagtcctcga ggagttcgaa ccccaagtga cgcccaatgc 300 gacaaaggtg tttgttaatg gtgtttgggt gggtattcac cgggaccctt cgcatctcgt 360 tactacgatg cagaacctgc gtcgacgaaa catgatctca cacgaagtca gtttgattcg 420 tgacatccgt gaacgggaat tcaaaatctt cacggatact ggacgtgtct gccggccact 480 cttcgtcatc gataatgatc ccaagagtga aaactcgggt ggattggtcc ttaataagga 540 acacattcgg aagctcgagg ccgacaaaga cttgccagta gacttggccc cagaagaacg 600 tcgcgaacaa tacttcggat gggatggtct ggtgcgttct ggagcagtcg agtatgtcga 660 tgccgaagaa gaggaaacca tcatgattgt catgaccccc gaggatctcg agatctctcg 720 acagcttcag gccggttacg ctctgccaga tgacgaagcc ggcgacccca ataaacgtgt 780 tcgatcgatt ctcagccagc gtgcccacac ctggacgcac tgtgaaatcc accctagtat 840 gatcttgggt gtttgcgcta gtattattcc tttcccggat cacaaccagt cgccccgtaa 900 cacctaccag tctgcc 916
Claims (5)
A method of decomposing cellulose acetate comprising the step of contacting and culturing cellulose acetate with Penicillium griseofulvum .
상기 페니실리움 그리세오풀범은 ITS(Internal Transcribed Spacer) 유전자, BTU(Beta-tubulin) 유전자, CaM(Calmodulin) 유전자, RPB2(RNA polymerase II subunit) 유전자가 각각 서열번호 9, 10, 11, 12의 서열을 갖는 균주인 것을 특징으로 하는 방법.
According to claim 1,
The Penicillium griseofulbum has an internal transcribed spacer (ITS) gene, a beta-tubulin (BTU) gene, a calmodulin (CaM) gene, and an RNA polymerase II subunit (RPB2) gene of SEQ ID NOs: 9, 10, 11, and 12, respectively. A method characterized in that it is a strain having a sequence.
상기 셀룰로오스 아세테이트에 페니실리움 그리세오풀범의 접촉은 페니실리움 그리세오풀범을 배지에서 배양하여 얻은 배양액 형태로 셀룰로오스 아세테이트와 접촉시켜 이루어지고,
상기 배지는 탄소원, 질소원, 미량원소 및 성장 촉진제 중 하나 이상을 포함하는 것을 특징으로 하는 방법.
According to claim 1,
The contact of the Penicillium griseofulbum with the cellulose acetate is made by contacting the cellulose acetate in the form of a culture medium obtained by culturing the Penicillium griseofullbum in a medium,
The method characterized in that the medium contains at least one of a carbon source, a nitrogen source, a trace element and a growth promoter.
상기 셀룰로오스 아세테이트와 페니실리움 그리세오풀범의 접촉 전후에 페니실리움 그리세오풀범의 배양과 증식을 촉진하기 위한 배지가 셀룰로오스 아세테이트에 첨가되어 혼합되고,
상기 배지는 탄소원, 질소원, 미량원소 및 성장 촉진제 중 하나 이상을 포함하는 것을 특징으로 하는 방법.
According to claim 1,
Before and after the contact of the cellulose acetate and Penicillium griseofulbum, a medium for promoting the culture and growth of Penicillium griseofullbum is added to the cellulose acetate and mixed,
The method characterized in that the medium contains at least one of a carbon source, a nitrogen source, a trace element and a growth promoter.
Penicillium griseofulbum NIBRFGC000508019 strain (accession number: KACC 93376P).
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