KR101135178B1 - Cellulase producing Schizophyllum commune and its use for saccharification - Google Patents

Cellulase producing Schizophyllum commune and its use for saccharification Download PDF

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KR101135178B1
KR101135178B1 KR1020090108828A KR20090108828A KR101135178B1 KR 101135178 B1 KR101135178 B1 KR 101135178B1 KR 1020090108828 A KR1020090108828 A KR 1020090108828A KR 20090108828 A KR20090108828 A KR 20090108828A KR 101135178 B1 KR101135178 B1 KR 101135178B1
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cellulase
cellulose
saccharification
present
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KR20110051984A (en
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이정걸
우장훈
제야 마리무투
노항덕
유호섭
김영숙
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건국대학교 산학협력단
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    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
    • C12N9/2434Glucanases acting on beta-1,4-glucosidic bonds
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    • C12P19/14Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Abstract

본 발명은 셀룰라아제를 생산하는 스키조필럼 코뮨 (Schizophyllum commune) 및 그를 이용한 당화 방법에 관한 것으로, 본 발명의 균주로부터 생산된 셀룰라아제는 종래 당화 효소보다 우수한 당화 수율을 나타내므로 바이오에너지의 생산, 섬유산업, 제지산업, 세제산업, 사료산업 및 식품 산업 등 다양한 용도에 적용될 수 있다.The present invention relates to a Schizophyllum commune producing a cellulase ( Schizophyllum commune ) and a saccharification method using the same, Cellulase produced from the strain of the present invention shows a better glycation yield than the conventional glycosylation enzymes in the production of bioenergy, textile industry It can be applied to various applications such as paper, paper industry, detergent industry, feed industry and food industry.

셀룰라아제, 당화, 바이오에너지 Cellulase, Saccharification, Bioenergy

Description

셀룰라아제를 생산하는 스키조필럼 코뮨 및 당화에의 이용 {Cellulase producing Schizophyllum commune and its use for saccharification}Cellulase producing Schizophyllum commune and its use for saccharification

본 발명은 셀룰라아제를 생산하는 균주 및 셀룰라아제를 이용한 당화방법에 관한 것이다.The present invention relates to a strain for producing cellulase and a saccharification method using cellulase.

셀룰로스(cellulose)는 지구상에 존재하는 가장 풍부한 유기물질로 석유나 석탄처럼 고갈에 대하여 걱정할 필요가 없는 재생 가능한 자원이다. 그러나 대부분의 셀룰로스 자원은 농산 및 임산 폐기물이 범람하고 있는 현재 주요한 환경오염원으로 작용하고 있다. 그러므로 이에 대한 효율적인 당화공정의 개발은 식량문제, 연료문제 그리고 환경문제의 해결에 큰 도움이 될 수 있을 것이다. 전 세계의 농산 및 임산 폐자원은 매년 30억 톤 이상이며, 아시아에서만 8억 톤 이상이 생산된다. 이는 주로 셀룰로스와 헤미셀룰로스로 이루어져있어 이들의 당화에 의한 포도당을 포함한 단당류의 생산은 바이오에너지 자원의 중요한 원료 생산 기술이 된다. 현재 농산 및 임산 폐자원으로부터 단당류를 회수하기 위해서는 대부분의 경우 황산을 첨가하여 고온에서 가압 및 분해 방법을 사용하고 있다. 이 경우 산 및 고온에 견딜 수 있는 고가의 생산장비가 필요하며, 분해산물도 각종 부반응으로 인하여 대단 히 다양하므로 분리 공정이 어렵고, 폐기물 처리 등의 비용으로 인해 생산단가가 높으며 환경 비친화적이라는 문제점을 안고 있다.Cellulose is the most abundant organic substance on earth and is a renewable resource that you don't have to worry about running out like oil or coal. However, most of the cellulose resources are now a major source of environmental pollution, inundated by agricultural and forest wastes. Therefore, the development of an efficient saccharification process can be a great help in solving food, fuel and environmental problems. The world's agricultural and forestry waste is more than 3 billion tonnes annually, with more than 800 million tonnes produced in Asia alone. It consists mainly of cellulose and hemicellulose, so the production of monosaccharides, including glucose by their saccharification, is an important raw material production technology of bioenergy resources. Currently, in order to recover monosaccharides from agricultural and forest waste resources, sulfuric acid is added in most cases to pressurize and decompose at high temperature. In this case, expensive production equipment that can withstand acids and high temperatures is required, and the decomposition products are also diverse due to various side reactions, which makes the separation process difficult and the production cost is high due to the cost of waste disposal and environmentally unfriendly. Holding it.

따라서, 오랫동안 셀룰로스를 고효율로 당화시키기 위한 많은 연구가 있어 왔으며 그 중 특히 당화효소의 개발에 초점을 맞춰 많은 연구가 이루어져 왔다. 그 결과 현재에는 이들 당화효소의 용도가 다양하게 개발되어 여러 분야에서 상업화 되었으며 또한 새로운 응용연구도 활발하게 진행되고 있다. 셀룰라아제는 섬유산업, 제지산업, 세제산업, 사료산업 등에서 많이 이용되고 있으며 이외에도 식품 산업에 있어, 저 칼로리 식품의 제조와 음식물 쓰레기의 발효 등 다양한 용도에 적용되고 있다.Therefore, there have been many studies for glycosylating cellulose with high efficiency for a long time, and a lot of research has been made, especially focusing on the development of glycosylase. As a result, various uses of these glycosylases have been developed and commercialized in various fields, and new applied researches are being actively conducted. Cellulase is widely used in the textile industry, the paper industry, the detergent industry, the feed industry, etc. In addition to the food industry, it is applied to various uses such as the production of low-calorie foods and the fermentation of food waste.

한편, 식물체의 세포벽은 셀룰로스(불용성 β-1,4-글루칸 섬유), 헤미셀룰로스 (hemicellulose, 비셀룰로스계 다당류), 리그닌(lignin, 복잡한 폴리페놀 구조의 다당류)과 같은 중합체로 구성되어 있다. 구성성분 중 셀룰로스가 가장 많이 존재하고 그 다음으로 자일란(xylan)이 주성분인 헤미셀룰로스가 많이 존재하며 이들 두 성분이 전체 식물 바이오매스의 50% 이상을 차지한다. 셀룰로스는 포도당 단위가 β-1,4 결합으로 연결된 동종 중합체로서 이를 단당류로 분해하기 위해서는 엔도-β-1,4-글루칸아제 (endo-β-1,4-glucanase) [EC 3. 2. 1. 4], 엑소-β-1,4-글루칸아제(exo-β-1,4-glucanase) [EC 3. 2. 1. 91], 베타-글루코시다아제 (β-glucosidase) (EC 3. 2. 1. 21) 등 세 종류의 효소가 필요하다. 엔도-글루칸아제는 안쪽에서 β-1,4 포도당 결합을 무작위적으로 절단하고 엑소-글루칸아제가 비환원당 말단에서 포도당 이당체인 셀로바이오스 (cellobiose)로 절단해 나간다. 셀 로바이오스는 베타-글루코시다아제에 의해서 포도당으로 최종 분해된다.On the other hand, the cell wall of the plant is composed of polymers such as cellulose (insoluble β-1,4-glucan fiber), hemicellulose (hemicellulose, non-cellulose polysaccharide), and lignin (lignin, polysaccharide of complex polyphenol structure). Cellulose is the most abundant component, followed by hexylcellulose, the main component of xylan, and these two components make up more than 50% of the total plant biomass. Cellulose is a homopolymer in which glucose units are linked by β-1,4 bonds. In order to decompose it into monosaccharides, cellulose [endo-β-1,4-glucanase] [EC 3. 2. 1 4], exo-β-1,4-glucanase [EC 3. 2. 1. 91], beta-glucosidase (EC 3. 2. 1. 21) Three enzymes are required. Endo-glucanase randomly cleaves β-1,4 glucose bonds from the inside and exo-glucanase cleaves into cellobiose, a glucose disaccharide at the non-reducing sugar end. Cellobiose is finally degraded to glucose by beta-glucosidase.

종래에 이들 효소의 생산은 주로 곰팡이(fungi)를 이용하였으며, 특히 산업적인 측면에서 효소 생산은 아스퍼질러스(Aspergillus)와 트리코더마(Trichoderma)를 이용하였다. 셀룰라아제 생산 균주로는 트리코더마 리제이 ZU-02 (Trichoderma reesei ATCC 56764)가 대표적인 균주로 집중 연구되어 왔으나, 효소의 농도 및 활성이 산업화를 충족시킬 만큼 충분하지 못하다는 문제점이 있다. 예를 들어, 바이오매스로부터 에탄올을 생산하는 공정은 원료 물질의 재생, 생산 연료의 환경 친화성 등 많은 장점을 갖고 있지만 리그노셀룰로스계 물질을 원료로 하여 생산된 에탄올은 휘발유에 비해 생산 단가가 현저히 높아 실용화에 장애가 되고 있다. 이러한 에탄올 생산 공정에 있어 비용의 가장 큰 부분은 당화 효소의 생산비로서 전체 비용의 약 60 %에 해당한다. 그러므로 셀룰라아제를 생산하는 고활성 균주의 개발이 절실히 요구되고 있다.Conventionally, the production of these enzymes mainly used fungi (fungi), particularly in the industrial aspect of the production of enzymes (Aspergillus) and Trichoderma (Trichoderma). As a cellulase producing strain, Tricoderma reese ZU-02 (Trichoderma reesei ATCC 56764) has been intensively studied as a representative strain, but there is a problem that the concentration and activity of the enzyme is not sufficient to meet the industrialization. For example, the process of producing ethanol from biomass has many advantages such as regeneration of raw materials and environmental friendliness of fuel produced. However, ethanol produced from lignocellulosic materials is significantly more expensive than gasoline. It is high and becomes obstacle to practical use. The largest part of the cost of this ethanol production process is about 60% of the total cost of the production of glycosylating enzyme. Therefore, there is an urgent need for the development of highly active strains that produce cellulase.

본 발명은 상기의 문제점을 해결하고, 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 고활성 셀룰라아제를 생산하는 균주를 제공하는 것이다.The present invention solves the above problems, and the object of the present invention as devised by the necessity of the above is to provide a strain for producing high activity cellulase.

본 발명의 다른 목적은 상기 균주를 이용하여 셀룰라아제를 생산하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing cellulase using the strain.

본 발명의 또 다른 목적은 상기 셀룰라아제를 이용한 셀룰로스의 당화방법을 제공하는 것이다.Still another object of the present invention is to provide a method for saccharifying cellulose using the cellulase.

상기의 목적을 달성하기 위하여 본 발명은 셀룰라아제를 생산하는 스키조필럼 코뮨(Schizophyllum commune)를 제공한다.In order to achieve the above object, the present invention provides a Schizophyllum commune producing cellulase.

본 발명의 일 구체예에 있어서, 상기 스키조필럼 코뮨(Schizophyllum commune)은 기탁번호 KMJ820(KACC93084P)인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the Schizophyllum commune ( Schizophyllum commune ) is preferably an accession number KMJ820 (KACC93084P), but is not limited thereto.

또 본 발명은 상기 본 발명의 스키조필럼 코뮨(Schizophyllum commune)을 셀룰라아제 생산배지에서 배양하는 단계를 포함하는 셀룰라아제의 생산방법을 제공한다.In another aspect, the present invention provides a method for producing cellulase comprising culturing the Schizophyllum commune of the present invention in a cellulase production medium.

본 발명의 생산방법은 스키조필럼 코뮨을 배양하고; 배양액으로부터 본 발명의 효소를 채취하는 것을 포함하는, 본 발명의 효소를 생산하는 방법을 제공한다.The production method of the present invention is cultivated Schizofil column; It provides a method for producing the enzyme of the present invention, comprising collecting the enzyme of the present invention from a culture.

본 발명의 일 구체예에 있어서, 상기 셀룰라아제의 생산배지는 탄소원으로 카르복시메칠 셀룰로스, 아비셀,볏짚, 사탕수수 버개스, 귤 껍질, 및 셀룰로스 분말로 구성된 군으로부터 선택된 하나를 사용하는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the production medium of the cellulase is preferably used as a carbon source selected from the group consisting of carboxymethyl cellulose, Avicel, rice straw, sugar cane bagasse, tangerine peel, and cellulose powder, but is not limited thereto. Not.

본 발명의 일 구체예에 있어서, 상기 탄소원은 10~40g/L 농도인 것이 바람직하고, 상기 셀룰라아제의 생산 배지는 효모추출물, 펩톤, 일인산칼륨, 이인산칼륨 및 황산마그네슘으로 구성된 군으로부터 선택된 하나 이상의 물질을 더욱 포함하는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the carbon source is preferably 10 ~ 40g / L concentration, the production medium of the cellulase is one selected from the group consisting of yeast extract, peptone, potassium monophosphate, potassium diphosphate and magnesium sulfate It is preferable to further include the above materials, but is not limited thereto.

또한 본 발명의 바람직한 실시예에 있어서, 상기 배양은 교반 속도 200 ~ 350 rpm, pH 4 ~ 7 또는 배양온도 29 ~ 37℃ 또는 이들의 조합 조건에서 배양시키는 것이 바람직하나 이에 한정되지 아니한다. In addition, in a preferred embodiment of the present invention, the culture is preferably incubated at a stirring speed of 200 to 350 rpm, pH 4 to 7 or a culture temperature of 29 to 37 ℃ or a combination thereof, but is not limited thereto.

또한 본 발명은 상기 본 발명의 균주 또는 그 배양액을 유효성분으로 포함하는 셀룰라아제 생산용 조성물을 제공한다.In another aspect, the present invention provides a composition for producing cellulase comprising the strain of the present invention or a culture solution thereof as an active ingredient.

또한 본 발명은 기질에 본 발명의 스키조필럼 코뮨(Schizophyllum commune)로부터 생산된 셀룰라아제를 처리하는 단계를 포함하는 셀룰로스의 당화방법을 제공한다.The present invention also provides a method for saccharifying cellulose comprising treating a cellulase produced from the Schizophyllum commune of the present invention to a substrate.

본 발명의 바람직한 실시예에 있어서, 상기 셀룰라아제의 농도는 37.5 ~ 87.5 U/(셀룰로스)g이고, 상기 기질 셀룰로스 농도는 3.5 ~ 5.5중량%이며, 상기 반응 pH는 3 ~ 7, 반응온도는 25 ~ 45℃에서 셀룰로스를 당화하는 것이 바람직하나 이에 ㅎ한정되지 아니한다.In a preferred embodiment of the present invention, the concentration of the cellulase is 37.5 ~ 87.5 U / (cellulose) g, the substrate cellulose concentration is 3.5 ~ 5.5% by weight, the reaction pH is 3 ~ 7, the reaction temperature is 25 ~ It is preferred to glycosylate cellulose at 45 ° C. but is not limited thereto.

본 발명의 일 구체예에 있어서, 상기 셀룰로스 공급원 기질은 편백인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the invention, the cellulose source substrate is preferably, but not limited to, one-sided.

본 발명에서 셀룰라아제는 셀룰로오스(cellulose)에서의 β-D-글리코시딕(glycosidic) 결합을 가수분해하는 효소이다. 셀룰로오스 분해 효소는 다음 3가지의 주요한 클래스로 분류될 수 있다: 엔도-베타-1,4-글루카나아제 (endo-β-1,4-Cellulase in the present invention is an enzyme that hydrolyzes β-D-glycosidic bond in cellulose. Cellulolytic enzymes can be classified into three main classes: endo-beta-1,4-glucanase (endo-β-1,4-

glucanase, EC 3.2.1.4, endoglucanase), 엑소글루카나아제(exoglucanase) 또는 셀로비오히드롤라제(cellobiohydrolase, EC 3.2.1.91), 및 베타-글루코시다제(β-glucosidase, EC 3.2.1.21).glucanase, EC 3.2.1.4, endoglucanase, exoglucanase or cellobiohydrolase (EC 3.2.1.91), and beta-glucosidase (EC 3.2.1.21).

본 발명의 균주의 배양방법은, 배치(batch)식, 유동배치식, 연속배양, 리액터형식 등, 통상의 미생물의 배양에 사용하는 어떠한 방법도 사용할 수 있다.As the culturing method of the strain of the present invention, any method used for culturing ordinary microorganisms, such as batch type, flow batch type, continuous culture, reactor type, or the like can be used.

또, 배양온도는 상기 언급한 적온의 범위에서 배양함으로써 셀룰라아제를 균체에 축적시키고, 회수한다. Incidentally, the culturing temperature accumulates and recovers cellulase in the cells by culturing in the above-mentioned temperature range.

본 발명의 효소 단백질은 분리하거나 정제될 수 있다. 그러한 분리 또는 정제는 본 발명의 균주를 배양하여 얻게 되는 배양물 중으로부터 균체 또는 상청액을 원심분리 회수하여, 균체를 파쇄하거나 또는 전체 세포, 세포찌꺼기, 외래 단백질 또는 최종 조성물에서 원하지 않는 단백질을 제거하기 위하여 당업계에 인지된 분리기술 예를 들어 이온 교환크로마토그래피, 친화성 크로마토그래피, 소수성 분리, 투석, 프로테이즈 처리, 황산 암모늄 침천, 크기 배제 크로마토그래피, 여과, 겔 전기영동 또는 구배상의 분리 또는 이들의 적당한 조합에 의하여 성취될 수 있다.Enzyme proteins of the invention can be isolated or purified. Such isolation or purification involves centrifugation of the cells or supernatants from the culture obtained by culturing the strains of the present invention to disrupt the cells or remove unwanted proteins from whole cells, debris, foreign proteins or the final composition. Separation techniques recognized in the art for example ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation, size exclusion chromatography, filtration, gel electrophoresis or gradient phase separation Or a suitable combination thereof.

얻게 된 정제물질이 목적의 효소인 것의 확인은, 통상의 방법, 예를 들면 SDS-폴리아크릴아미드겔 전기영동, 웨스턴블로팅 등에 의해 행할 수 있다.Confirmation that the obtained purified substance is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis, western blotting or the like.

본 발명의 효소는 본 발명의 목적하는 활성을 향상시키기 위하여 예를 들어 활성화제, 항저해제, 바람직한 이온, pH를 조절하는 화합물 또는 다른 효소 등을 포함할 수 있다. Enzymes of the invention may include, for example, activators, anti-inhibitors, preferred ions, compounds that adjust pH or other enzymes, etc. to enhance the desired activity of the invention.

본 발명의 효소는 일반적인 제형기를 사용하여 적당한 크기의 분말, 펠렛 또는 과립 등의 다양한 형태로 제형화 될 수 있다. The enzymes of the present invention can be formulated into a variety of forms, such as powders, pellets, or granules of suitable size using common formulators.

상기에서 제형화된 본 발명의 효소는 그대로 사용되거나 또는 실온에서 풍건하거나 동결 건조 등으로 건조시켜 사용할 수 있다.The enzyme of the present invention formulated above may be used as it is, or it may be dried at room temperature or dried by freeze drying or the like.

본 발명의 효소에 의해 가수분해될 수 있는 대상의 구체적인 예로는 식물의 줄기, 잎, 짚, 속대, 겨, 손상된 곡류, 과일, 야채류 등과 가공 후의 껍질, 종자, 잎, 송이, 껍질, 착즙박 등이 포함된다. 보다 구체적으로 예를 들면, 한정되지는 않으나 옥수수 외피(corn hull), 옥수수 속대(corn cop),사탕수수 착즙박(sugarcane bagasse), 볏짚(rice straw) 쌀겨(rice husk) 및 미강(rice bran) 등이 있다. 상기 대상물을 본 발명의 효소로 가수분해함으로써 다양한 당류로 전환시킬 수 있다. 따라서 환경오염을 방지하면서 이들 농업 잔존물을 재활용할 수 있는 장점이 있다. Specific examples of the object that can be hydrolyzed by the enzyme of the present invention include plant stems, leaves, straw, genus, bran, damaged grains, fruits, vegetables and the like after processing the shells, seeds, leaves, clusters, shells, juices, etc. This includes. More specifically, for example, but not limited to corn hull, corn cop, sugarcane bagasse, rice straw rice husk and rice bran Etc. The object can be converted into various sugars by hydrolysis with the enzyme of the present invention. Therefore, there is an advantage that can be recycled these agricultural residues while preventing environmental pollution.

본 발명의 버섯에서 분리한 스키조필럼 코뮨 KMJ820은 고활성 셀룰라아제를 생산하므로, 셀룰로스 당화에 이용될 수 있고, 본 발명의 균주로부터 생산된 셀룰라아제는 종래 당화 효소보다 우수한 당화수율을 나타내므로 바이오에너지의 생산, 섬유산업, 제지산업, 세제산업, 사료산업 및 식품 산업에 있어 저 칼로리 식품의 제조와 음식물 쓰레기의 발효 등 다양한 용도에 적용될 수 있다.Schizopilum commune KMJ820 isolated from the mushroom of the present invention produces a highly active cellulase, can be used for cellulose glycosylation, cellulase produced from the strain of the present invention shows better glycation yield than conventional glycosylation enzymes of bioenergy In the production, textile, paper, detergent, feed, and food industries, it can be applied to a variety of uses, such as the production of low-calorie foods and the fermentation of food waste.

이하, 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 목적으로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention and the scope of the present invention is not to be construed as limited by the following examples.

실시예Example 1: 셀룰라아제  1: cellulase 생산균의Production 선별 Selection

셀룰라아제를 생산하는 균주를 분리하기 위하여 각종 버섯 균의 배양액 10ul를 생리식염수 10 ml에 현탁하고, 현탁액의 10ul(1x104 cfu ml-1) 취하여 2% 카르복시메칠 셀룰로스가 첨가된 복합한천배지 (potato dextrose agar)에 도말한 후, 27 ℃에서 3일간 배양하였다. 고체 한천배지에서 콜로니가 형성된 후 0.1%의 콩고레드 시약으로 염색하고, 1 M 염화나트륨으로 탈색하여 그 콜로니 주위에 섬유소 분해환이 생성된 균들을 선별하는 방법으로 다양한 버섯 포자로부터 셀룰라아제를 생산하는 버섯균을 다수 탐색하였다.To isolate cellulase-producing strains, 10ul of various cultures of mushrooms were suspended in 10 ml of physiological saline, 10ul (1x10 4 cfu ml -1 ) of the suspension was taken, and the agar medium (potato dextrose) added with 2% carboxymethyl cellulose was added. agar) and incubated at 27 ° C. for 3 days. After colonies were formed on a solid agar medium, stained with 0.1% Congo red reagent, and decolorized with 1 M sodium chloride to screen for bacteria that produced fibrinolytic rings around the colonies. Explored.

위의 탐색과정을 통해 1차 선별된 균주(S1부터 S8까지)를 대조군(C)으로 종래 셀룰라아제 생산균주로 이용되는 트리코더마 리제이 ZU-02를 이용하여, 상기와 같이 카르복시메칠 셀룰로스를 첨가한 고체 한천배지에서 섬유소 분해능을 확인한 후, 섬유소 분해능이 가장 뛰어난 S6 균주를 선별하였다.Solid agar to which carboxymethyl cellulose was added as described above using Tricoderma Reese ZU-02, which was used as a conventional cellulase producing strain as the control (C), the first selected strain (S1 to S8) through the above search process. After confirming the fibrinolytic ability in the medium, the S6 strain having the best fibrin resolution was selected.

실시예Example 2: 균주의 동정  2: Identification of the strain

실시예 1에서 분리한 S6 균주의 동정을 위하여 한국미생물 보존센터에서 ITS-5.8S rDNA 서열을 분석하였다. S6 균주의 ITS-5.8S rDNA 서열은 서열번호 1에 나타내었다.In order to identify the S6 strain isolated in Example 1, the ITS-5.8S rDNA sequence was analyzed at the Korea Microorganism Conservation Center. The ITS-5.8S rDNA sequence of the S6 strain is shown in SEQ ID NO: 1.

상기 S6 균주의 ITS-5.8S rDNA 서열의 유사종과의 유연관계를 분석한 결과 스키조필럼 코뮨으로 동정되었다(도 1).As a result of analyzing the soft relationship with the analogous species of the ITS-5.8S rDNA sequence of the S6 strain, it was identified as Schizofilum coccum (FIG. 1).

상기 S6 균주는 스키조필럼 코뮨(Shizophyllum commune) KMJ820으로 명명하였고, 대한민국 경기도 수원시 권선구 소재 한국농업미생물보존센터에 2009년 11월 9일 기탁번호 KACC93084P호로 기탁하였다. The S6 strain was named as Schizophyllum commune KMJ820, and was deposited with KACC93084P on November 9, 2009 at the Korea Agricultural Microbiological Conservation Center in Gwonseon-gu, Gyeonggi-do, Korea .

실시예Example 3: 균주의 셀룰라아제 생산을 위한 배지 최적화 실험  3: Medium Optimization Experiment for Cellulase Production of Strains

(1)(One) 탄소원의Carbon source 종류에 따른 셀룰라아제 활성 시험 Cellulase Activity Test by Type

7L 발효조에서 탄소원 종류에 따른 본 발명 균주 스키조필럼 코뮨 KMJ820 균주의 셀룰라아제 생산 실험을 수행하였다. 셀룰라아제 활성 측정을 위하여, 베타-글루코시다아제, 엑소-β-1,4-글루칸아제는 파라-니트로페닐 (p-nitro- phenyl, pNP)기가 붙은 기질인 파라-니트로페닐 글루코스 (pNPG), 파라-니트로페닐 셀로바이오스(pNPC)를 각각 이용하였고, 엔도-β-1,4-글루칸아제의 활성은 환원당을 이용한 somogyi-nelson법을 이용하여 측정하였다.Cellulase production experiments of the strain Schizofilum commun KMJ820 strain of the present invention according to the type of carbon source in a 7L fermenter were performed. For measuring cellulase activity, beta-glucosidase and exo-β-1,4-glucanase are para-nitrophenyl glucose (pNPG), para-nitrogen substrates with p-nitrophenyl (pNP) groups. -Nitrophenyl cellobiose (pNPC) was used, respectively, and the activity of endo-β-1,4-glucanase was measured by using a somogyi-nelson method using a reducing sugar.

종 배양: 보관된 스키조필럼 코뮨의 단일 군락을 전 배양 배지 (Potato starch 4g/L, Dextrose 20g/L) 50mL가 들어있는 500mL 플라스크에 접종하여 진탕배양기에서 200rpm, 30℃로 3일간 배양하였다.Species culture: A single colony of stored Sjozofilum commune was inoculated into a 500 mL flask containing 50 mL of preculture medium (Potato starch 4g / L, Dextrose 20g / L) and incubated at 200 rpm, 30 ° C. for 3 days in a shaker.

본 배양: 200mL의 종배양액을 생산배지(탄소원 20g/L, 효모추출액 2g/L, 펩톤 8g/L, 일인산칼륨 5g/L, 이인산칼륨 5g/L, 황산마그네슘 3g/L) 4L가 들어있는 7L 발효조에 접종하여 교반속도 200rpm, 배양온도 33℃, pH 5에서 6일간 본 배양을 수행하였다. Main culture: 200 mL of seed culture medium containing 4L of medium (20g / L carbon source, yeast extract 2g / L, peptone 8g / L, potassium monophosphate 5g / L, potassium diphosphate 5g / L, magnesium sulfate 3g / L) Inoculated in a 7L fermenter in the present culture was carried out for 6 days at a stirring speed of 200rpm, incubation temperature 33 ℃, pH 5.

탄소원
(20g/L)
Carbon source
(20 g / L)
엔도-β-1,4-글루칸아제 (U/mg-단백질)Endo-β-1,4-glucanase (U / mg-protein) 엑소-β-1,4-글루칸아제 (U/mg-단백질) Exo-β-1,4-glucanase (U / mg-protein) β-글루코시다아제 (U/mg-단백질)β-glucosidase (U / mg-protein)
카르복시메칠 셀룰로스Carboxymethyl Cellulose 15.115.1 4.04.0 14.214.2 아비셀Avicel 6.56.5 9.79.7 23.223.2 볏짚Straw 5.15.1 2.22.2 8.98.9 사탕수수 버개스Sugar cane bagasse 4.14.1 3.73.7 7.47.4 귤 껍질Tangerine peel 12.012.0 7.97.9 11.211.2 셀룰로스 분말Cellulose powder 12.712.7 12.012.0 23.523.5

표 1은 여러 탄소원을 이용한 셀룰라아제의 생산을 나타낸 표이다.Table 1 is a table showing the production of cellulase using several carbon sources.

(2) (2) 탄소원(셀룰로스 분말)의Of carbon source (cellulose powder) 농도에 따른 셀룰라아제 활성 시험 Cellulase Activity Test According to Concentration

7L발효조에서 셀룰로스 분말 농도를 5~40g/L로 달리하여 배양한 결과 활성은 표 2와 같았고, 20g/L 셀룰로스 분말에서 최대활성을 나타내었다.The cellulose powder concentration in the 7 L fermentation tank was cultured at different concentrations of 5-40 g / L, and the activity was shown in Table 2, and the maximum activity was shown in 20 g / L cellulose powder.

g/Lg / L 엔도-β-1,4-글루칸아제 (U/mg-단백질)Endo-β-1,4-glucanase (U / mg-protein) 엑소-β-1,4-글루칸아제 (U/mg-단백질) Exo-β-1,4-glucanase (U / mg-protein) β-글루코시다아제 (U/mg-단백질)β-glucosidase (U / mg-protein) 셀룰로스
분말
Cellulose
powder
55 7.27.2 4.54.5 15.415.4
1010 10.010.0 6.26.2 20.120.1 2020 12.412.4 11.811.8 24.124.1 3030 10.310.3 10.110.1 23.223.2 4040 9.69.6 8.88.8 18.118.1

표 2는 여러 셀룰로스 분말 농도에서 셀룰라아제 효소의 활성을 나타낸 표이다.Table 2 is a table showing the activity of the cellulase enzyme at various cellulose powder concentrations.

실시예4Example 4 : 고활성 효소 생산을 위한 최적 배양조건 실험: Experiment of Optimum Culture Condition for Highly Active Enzyme Production

7L 발효조에서 셀룰로스 분말, 효모추출액, 펩톤, 일인산칼륨, 이인산칼륨, 황산 마그네슘의 농도를 각각 20g/L, 2g/L, 8g/L, 5g/L, 5g/L, 3g/L로 하여 배양 환경조건의 최적화 실험을 수행하였다. 교반속도 200-350rpm, pH 4-7 및 배양온도를 29-37℃로 달리하여 셀룰라아제 활성을 비교한 결과 표 3과 같이 교반속도 250rpm, pH 5, 배양온도 35℃에서 최대 셀룰라아제 활성을 나타내었다. 상기 최적조건에서의 배양 시간 별 엔도-β-1,4-글루칸아제, 엑소-β-1,4-글루칸아제 및 베타-글루코시다아제의 시간에 따른 활성 변화를 도 2a, 도 2b 및 도 2c에 각각 나타내었다The concentrations of cellulose powder, yeast extract, peptone, potassium monophosphate, potassium diphosphate, and magnesium sulfate in 7L fermenters were set at 20 g / L, 2 g / L, 8 g / L, 5 g / L, 5 g / L, and 3 g / L, respectively. Optimization experiments of cultivation environmental conditions were performed. As a result of comparing the cellulase activity by varying the stirring speed of 200-350rpm, pH 4-7 and the culture temperature at 29-37 ° C, the maximum cellulase activity was shown at the stirring speed of 250rpm, pH 5 and the culture temperature of 35 ° C. 2A, 2B and 2C show changes in time-dependent activity of endo-β-1,4-glucanase, exo-β-1,4-glucanase and beta-glucosidase at the optimum conditions. Respectively indicated in

엔도-β-1,4-글루칸아제
(U/mg-단백질)
Endo-β-1,4-glucanase
(U / mg-protein)
엑소-β-1,4-글루칸아제
(U/mg-단백질)
Exo-β-1,4-glucanase
(U / mg-protein)
β-글루코시다아제
(U/mg-단백질)
β-glucosidase
(U / mg-protein)
배양 온도 (℃)Incubation temperature (℃) 2929 9.29.2 7.97.9 18.218.2 3131 11.011.0 10.110.1 19.819.8 3333 12.412.4 11.811.8 23.723.7 3535 13.413.4 13.313.3 25.725.7 3737 11.311.3 12.212.2 24.324.3 교반조건(rpm)Stirring Condition (rpm) 200200 13.213.2 13.213.2 25.925.9 250250 14.014.0 14.314.3 27.627.6 300300 12.912.9 12.512.5 27.327.3 350350 11.011.0 10.210.2 26.226.2 pHpH 44 8.18.1 11.211.2 23.523.5 55 14.114.1 14.614.6 27.927.9 66 12.912.9 13.513.5 26.726.7 77 9.49.4 9.49.4 22.922.9

상기 표 3은 효소활성 최적화를 위한 조건 실험 표이다.Table 3 is a table of experimental conditions for the optimization of enzyme activity.

세 효소의 활성 대조균으로 트리코더마 리제이 ZU-02를 이용하여 스키조필럼 코뮨 KMJ820균주의 활성을 비교하였다. 배양조건은 모두 일관되게 적용되었으며, 활성 비교 결과 스키조필럼 코뮨 KMJ820균주가 생산하는 셀룰라아제 효소들의 활성이 각각 엔도-β-1,4-글루칸아제 14.1 U/mg-단백질, 엑소-β-1,4-글루칸아제 14.6 U/mg-단백질, 베타-글루코시다아제 27.9 U/mg-단백질로서 대조군에 비해 현저히 높은 셀룰라아제 활성을 지니는 것을 확인하였다 (표 4).Trichoderma Reese ZU-02 was used as an active control bacterium of three enzymes to compare the activity of Schizopilum commune KMJ820 strain. The culture conditions were consistently applied, and as a result of the activity comparison, the cellulase enzymes produced by the strain Sjozofilum cochlear KMJ820 were found to be endo-β-1,4-glucanase 14.1 U / mg-protein, exo-β-1, 4-glucanase 14.6 U / mg-protein, beta-glucosidase 27.9 U / mg-protein was found to have significantly higher cellulase activity compared to the control (Table 4).

균주Strain 엔도-β-1,4-글루칸아제
(U/mg-단백질)
Endo-β-1,4-glucanase
(U / mg-protein)
엑소-β-1,4-글루칸아제
(U/mg-단백질)
Exo-β-1,4-glucanase
(U / mg-protein)
β-글루코시다아제
(U/mg-단백질)
β-glucosidase
(U / mg-protein)
트리코더마 리제이 ZU-02Tricoderma RJ ZU-02 4.74.7 0.400.40 3.43.4 스키조필럼
코뮨KJM820
Szzopilum
KJM820
14.114.1 14.614.6 27.927.9

실시예Example 5: 균주의  5: strain 당화수율Glycation yield 분석 analysis

일반적으로 식물체가 함유하고 있는 리그노셀룰로스는 효소의 가수분해만으로 높은 당화수율을 얻을 수 없다. 따라서 효소가수분해 과정 전에 전처리 과정을 거치게 되는데 본 발명에서는 2 중량% 수산화나트륨의 알칼리 처리방법을 이용하였다. 이러한 전처리 과정은 리그닌과 헤미셀룰로스의 조각화를 제공함으로써 셀룰라아제 효소의 섬유소 가수분해 효율의 증가를 가져오게 된다. 전처리를 위해 10 g의 편백을 40 ml의 2 중량% 수산화나트륨 용액이 든 플라스크에 넣고 85 ℃에서 1시간 동안 반응시킨 후 0.45 uM 필터에 여과한다. 이와 같이 전 처리 및 여과된 편백을 65 ℃에서 건조시켜 사용하였다.In general, lignocellulosic contained in plants cannot obtain high glycation yields only by hydrolysis of enzymes. Therefore, the enzyme is subjected to a pretreatment before the hydrolysis process. In the present invention, an alkali treatment method of 2% by weight sodium hydroxide was used. This pretreatment process provides fragmentation of lignin and hemicellulose, resulting in an increase in fibrinase efficiency of the cellulase enzyme. For pretreatment, 10 g of the protein is placed in a flask containing 40 ml of 2 wt% sodium hydroxide solution, reacted at 85 ° C. for 1 hour and filtered through a 0.45 uM filter. The pretreated and filtered white patches were dried at 65 ° C. and used.

균주의 최적 당화 조건을 탐색하기 위하여, 효소의 농도, 기질의 농도, 반응온도, 반응 pH 에 대한 실험을 진행하였다.In order to explore the optimum glycosylation conditions of the strain, experiments were carried out for the concentration of enzyme, substrate concentration, reaction temperature, reaction pH.

먼저, 전 처리된 편백을 농도별로 20 ml의 0.1 M 소디움 아세테이트 완충액 (pH 5.0)에 다양한 농도의 셀룰라아제와 함께 첨가하였다. 셀룰라아제가 첨가된 완충액은 25 ~ 45 ℃에서 200 rpm으로 72시간 동안 반응시킨 후 변성된 효소를 제거하기 위하여 반응액을 100℃에서 3분간 끓이고 실온에서 식힌 뒤 4000 rpm에서 15분간 원심분리 하여 침전시켰다. 효소활성 측정은 환원당 측정법으로 그 상등액을 이용하였다. First, the pretreated protein was added to various concentrations of cellulase in 20 ml of 0.1 M sodium acetate buffer (pH 5.0) by concentration. The cellulase-added buffer was reacted at 200 rpm at 25 to 45 ° C. for 72 hours, and then the reaction solution was boiled at 100 ° C. for 3 minutes, cooled at room temperature, and then centrifuged at 4000 rpm for 15 minutes to remove denatured enzyme. . Enzyme activity was measured using the supernatant as a reducing sugar measurement.

당화율 기준은 반응이 끝난 편백을 105 ℃에서 24시간 건조한 후 줄어든 1 g의 편백 무게를 기준으로 다음 식과 같이 측정되었다.The glycosylation rate was determined by the following equation, based on the reduced weight of 1 g of the protein, after the reaction was dried for 24 hours at 105 ℃.

Figure 112009069389468-pat00001
Figure 112009069389468-pat00001

(1)효소 농도에 따른 (1) according to enzyme concentration 당화수율Glycation yield 실험 Experiment

500 ml 삼각플라스크에서 효소 농도에 따른 본 발명 균주 스키조필럼 코뮨 KMJ820의 당화실험을 수행하였다. 여러 농도의 효소를 처리하여 35℃, 200rpm에서 실험한 결과 표 5와 같았으며, 37.5 ~ 87.5 FPU/g-기질, 바람직하게는 50 FPU/g-기질의 효소를 사용하였을 때 최적의 당화수율을 나타내었다.The saccharification experiment of the strain Schizofilum commune KMJ820 of the present invention according to the enzyme concentration in a 500 ml Erlenmeyer flask was performed. Experiments at various concentrations of enzymes at 35 ° C. and 200 rpm were shown in Table 5. The optimum glycation yield was obtained when enzymes of 37.5-87.5 FPU / g-substrate, preferably 50 FPU / g-substrate, were used. Indicated.

효소 농도 (FPU/g-기질)Enzyme Concentration (FPU / g-substrate) 당화수율 (%)Glycation yield (%) 37.537.5 21.221.2 50.050.0 22.522.5 62.562.5 20.020.0 75.075.0 16.816.8 87.587.5 12.512.5

(2)기질 농도에 따른 (2) depending on substrate concentration 당화수율Glycation yield 실험 Experiment

본 발명 균주 스키조필럼 코뮨 KMJ820가 생산한 당화효소에 의한 편백의 당화에 미치는 기질 농도의 영향을 확인하였다. 편백 초기 농도를3.5 중량%, 4중량%, 4.5 중량%, 5 중량%, 5.5중량%로 각각 달리하여 배양한 결과를 표 6에 나타내었다. 5.0 ~ 5.5 중량%에서 당화수율이 우수했고, 5.0 중량% 편백 농도에서 최적의 당화수율이 나타났다.The effect of substrate concentration on the glycosylation of glycoproteins produced by the strain Schizozolum colum KMJ820 of the present invention was confirmed. Table 6 shows the results of incubation with different initial concentrations of 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, and 5.5 wt%. The saccharification yield was excellent at 5.0 to 5.5 wt%, and the optimum saccharification yield was found at 5.0 wt% of the protein concentration.

기질농도(%)Substrate concentration (%) 당화수율(%)Glycation yield (%) 3.53.5 16.316.3 4.04.0 18.218.2 4.54.5 19.619.6 5.05.0 25.725.7 5.55.5 23.523.5

(3)온도에 따른 (3) according to temperature 당화수율Glycation yield 실험 Experiment

본 발명 균주 스키조필럼 코뮨 KMJ820가 생산한 당화효소에 의한 볏짚의 당화에 미치는 온도의 영향을 확인하였다. 반응 온도를 25, 30, 35, 40, 45 ℃로 각각 달리하여 배양한 결과를 표7에 나타내었다. 30 ~ 40 ℃에서 당화수율이 우수하였고, 40 ℃에서 최적의 당화수율이 나타났다.The effect of temperature on the glycosylation of rice straw by the glycosylation enzyme produced by the strain Schizozolum Colum KMJ820 of the present invention was confirmed. Table 7 shows the results of incubation at different reaction temperatures of 25, 30, 35, 40, and 45 ° C. The saccharification yield was excellent at 30 ~ 40 ℃, the optimum saccharification yield was appeared at 40 ℃.

반응온도 (℃)Reaction temperature (℃) 당화수율 (%)Glycation yield (%) 2525 15.415.4 3030 18.318.3 3535 20.720.7 4040 25.325.3 4545 16.316.3

(4)(4) pHpH 에 따른 In accordance 당화실험Glycation Experiment

본 발명 균주 스키조필럼 코뮨 KMJ820가 생산한 당화효소에 의한 편백의 당화에 미치는 pH의 영향을 확인하였다. 반응 pH를 3, 4, 5, 6, 7로 각각 달리하여 배양한 결과를 표 8에 나타내었다. pH 3 ~ 5에서 당화수율이 우수하였고, pH 4에서 최적의 당화수율이 나타났다. The effect of pH on glycosylation of glycoprotein by the glycosylase produced by the strain Schizozolum columbium KMJ820 of the present invention was confirmed. Table 8 shows the results of culturing with different reaction pHs of 3, 4, 5, 6 and 7. The saccharification yield was excellent at pH 3 ~ 5, and the optimum glycation yield was found at pH 4.

pHpH 당화수율 (%)Glycation yield (%) 3.03.0 24.924.9 4.04.0 28.628.6 5.05.0 21.321.3 6.06.0 19.419.4 7.07.0 18.018.0

실시예Example 6: 최적 조건에서의  6: at optimum conditions 당화실험Glycation Experiment

반응기에서 최적화한 조건에서 본 발명 균주 스키조필럼 코뮨 KMJ820의 당화효소를 이용한 당화 실험을 수행하였다. 반응액 내의 효소 농도는 50 FPU/g-기질, 기질 농도는 5.0 %, pH는 4로 조절하였으며 온도는 40℃로 조절하였다. 최적화 된 조건에서 실험한 결과 반응 시간별 당화수율을 도 3에 나타내었다.Glycosylation experiment using the glycosylation enzyme of the present invention Schizopilum commune KMJ820 under conditions optimized in the reactor. Enzyme concentration in the reaction solution was adjusted to 50 FPU / g-substrate, substrate concentration 5.0%, pH was 4 and the temperature was adjusted to 40 ℃. As a result of the experiment under the optimized conditions, the glycation yield according to the reaction time is shown in FIG. 3.

본 발명에서 시행된 반응 시간에 따른 스키조필럼 코뮨 KMJ820의 셀룰라아제에 의한 편백의 당화효율을 조사한 결과 50 FPU/g-기질의 효소농도, 5.0%의 기질을 사용하여, pH 4, 40℃에서 36시간 반응시켰을 때 32.6%의 최대 당화율을 나타내었다. As a result of investigating the glycosylation efficiency of the protein by the cellulase of Schizopilum commune KMJ820 according to the reaction time implemented in the present invention, using an enzyme concentration of 50 FPU / g-substrate, a substrate of 5.0%, pH 4, 40 ℃ When reacted with time, the maximum glycosylation rate was 32.6%.

또한 본 발명에서는 셀룰라아제 생산을 위하여 교반속도는 200~350 rpm, pH 4~7, 배양온도는 29 ~ 37℃에서 배양시키는 것이 바람직하다.In addition, in the present invention, for the production of cellulase, the stirring speed is preferably 200 to 350 rpm, pH 4 to 7, and the incubation temperature is 29 to 37 ° C.

한편 본 발명의 스키조필럼 코뮨 KMJ820균주에서 생산된 셀룰라아제와 노보자임사의 셀룰라아제(상품명 celluclast)을 각각의 최적 조건에서 당화수율을 비교하여 표9에 나타내었다.Meanwhile, the cellulase produced in the Sjozofilum coquette KMJ820 strain of the present invention and the cellulase (trade name celluclast) of Novozyme are shown in Table 9 by comparing the glycation yield under the optimum conditions.

균주Strain 당생산량(mg/g-편백)Sugar output (mg / g-cylinder) 당화수율(%)Glycation yield (%) SchizophyllumSchizophyllum communecommune 295 ± 10295 ± 10 32 ± 1.132 ± 1.1 Novozyme (celluclast)Novozyme (celluclast) 241 ± 8241 ± 8 26 ± 1.026 ± 1.0

도 1은 본 발명 균주의 ITS-5.8S rDNA 서열의 유사종과의 유연관계를 분석한 결과이다.1 is a result of analyzing the soft relationship with the similar species of the ITS-5.8S rDNA sequence of the strain of the present invention.

도 2(a)는 스키조필럼 코뮨 균주의 엔도-β-1,4-글루칸아제 활성을 배양시간에 따라 비교하여 나타낸 그래프이다.Figure 2 (a) is a graph showing the endo-β-1,4-glucanase activity of the Schizopilum comb strain compared to the culture time.

도 2(b)는 스키조필럼 코뮨 균주의 엑소-β-1,4-글루칸아제 활성을 배양시간에 따라 비교하여 나타낸 그래프이다.Figure 2 (b) is a graph showing the comparison of exo-β-1,4-glucanase activity of Schizopilum combs strain according to the culture time.

도 2(c)는 스키조필럼 코뮨 균주의 베타-글루코시다아제 활성을 배양시간에 따라 비교하여 나타낸 그래프이다.Figure 2 (c) is a graph showing the beta-glucosidase activity of Schizopilum comb strain compared to the culture time.

도 3은 스키조필럼 코뮨 균주에 의해 생산된 당화효소를 이용한 최적 조건에서 반응시간 별 편백의 당화율을 나타낸 그래프이다.Figure 3 is a graph showing the glycosylation rate of each protein by reaction time under the optimum conditions using the glycosylation enzyme produced by Schizofilum comb strain.

<110> Konkuk University Industrial Cooperation Corp. <120> Cellulase producing Schizophyllum commune and its use for saccharification <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 615 <212> DNA <213> Schizophyllum commune <400> 1 cctgcggaag gatcattaac gaatcaaaca agttcatctt gttctgatcc tgtgcacctt 60 atgtagtccc aaagccttca cgggcggcgg atgactacgt ctacctcaca ccttaaagta 120 tgttaacgaa tgtaatcatg gtcttgacag accctaaaaa gttaatacaa ctttcgacaa 180 cggatctctt ggctctcgca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat 240 tgcagaattc agtgaatcat cgaatctttg aacgcacctt gcgccctttg gtattccgag 300 gggcatgcct gtttgagtgt cattaaatac catcaaccct cttttgactt cggtctcgag 360 agtggcttgg aagtggaggt ctgctggagc ctaacggagc cagctcctct taaatgtatt 420 agcggatttc ccttgcggga tcgcgtctcc gatgtgataa tttctacgtc gttgaccatc 480 tcggggctga cctagtcagt ttcaatagga gtctgcttct aaccgtctct tgactgagac 540 tagcgacttg tgcgctaact tttgacttga cctcaaatca ggtaggacta cccgctgaac 600 ttaagcatat caata 615 <110> Konkuk University Industrial Cooperation Corp. <120> Cellulase producing Schizophyllum commune and its use for          saccharification <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 615 <212> DNA <213> Schizophyllum commune <400> 1 cctgcggaag gatcattaac gaatcaaaca agttcatctt gttctgatcc tgtgcacctt 60 atgtagtccc aaagccttca cgggcggcgg atgactacgt ctacctcaca ccttaaagta 120 tgttaacgaa tgtaatcatg gtcttgacag accctaaaaa gttaatacaa ctttcgacaa 180 cggatctctt ggctctcgca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat 240 tgcagaattc agtgaatcat cgaatctttg aacgcacctt gcgccctttg gtattccgag 300 gggcatgcct gtttgagtgt cattaaatac catcaaccct cttttgactt cggtctcgag 360 agtggcttgg aagtggaggt ctgctggagc ctaacggagc cagctcctct taaatgtatt 420 agcggatttc ccttgcggga tcgcgtctcc gatgtgataa tttctacgtc gttgaccatc 480 tcggggctga cctagtcagt ttcaatagga gtctgcttct aaccgtctct tgactgagac 540 tagcgacttg tgcgctaact tttgacttga cctcaaatca ggtaggacta cccgctgaac 600 ttaagcatat caata 615  

Claims (12)

셀룰라아제를 생산하는 스키조필럼 코뮨(Schizophyllum commune) KMJ820(기탁번호 KACC93084P). Schizophyllum commune KMJ820 (Accession No. KACC93084P), which produces cellulase. 삭제delete 제 1항의 스키조필럼 코뮨 KMJ820을 셀룰라아제 생산배지에서 배양하는 단계를 포함하는 셀룰라아제의 생산방법.The method of producing a cellulase comprising the step of culturing the skim colum commun KMJ820 of claim 1 in a cellulase production medium. 제 3항에 있어서, 상기 셀룰라아제 생산배지의 탄소원은 카르복시메칠 셀룰로스, 아비셀,볏짚, 사탕수수 버개스, 귤 껍질, 및 셀룰로스 분말로 구성된 군으로부터 선택된 하나인 것을 특징으로 하는 셀룰라아제의 생산방법.The method of claim 3, wherein the carbon source of the cellulase production medium is one selected from the group consisting of carboxymethyl cellulose, Avicel, rice straw, sugar cane bagasse, tangerine peel, and cellulose powder. 제 4항에 있어서, 상기 탄소원은 10~40g/L 농도인 것을 특징으로 하는 셀룰라아제의 생산방법.The method of claim 4, wherein the carbon source is 10 ~ 40g / L concentration method of producing a cellulase. 제 3항에 있어서, 상기 배양은 교반 속도 200 ~ 350 rpm, pH 4 ~ 7 또는 배양온도 29 ~ 37℃ 또는 이들의 조합 조건에서 배양시키는 것을 특징으로 하는 셀룰라아제의 생산방법. The method of claim 3, wherein the culturing is performed at a stirring speed of 200 to 350 rpm, pH 4 to 7 or at a culture temperature of 29 to 37 ° C, or a combination thereof. 제 1항의 균주 또는 그 배양액을 유효성분으로 포함하는 셀룰라아제 생산용 조성물.Cellulase production composition comprising the strain of claim 1 or a culture thereof as an active ingredient. 기질에 제 1항의 스키조필럼 코뮨(Schizophyllum commune)로부터 생산된 셀룰라아제를 처리하는 단계를 포함하는 셀룰로스의 당화방법.A method of saccharification of cellulose comprising treating a cellulase produced from Schizophyllum commune of claim 1 to a substrate. 제 8항에 있어서, 상기 셀룰라아제의 농도는 37.5 ~ 87.5 U/(셀룰로스)그램인 것을 특징으로 하는 셀룰로스의 당화방법.The method of claim 8, wherein the concentration of the cellulase is 37.5 ~ 87.5 U / (cellulose) grams, characterized in that the cellulose saccharification method. 제 8항에 있어서, 상기 기질 셀룰로스 농도는 3.5 ~ 5.5중량%인 것을 특징으로 하는 셀룰로스의 당화방법.9. The method of claim 8, wherein the substrate cellulose concentration is 3.5 to 5.5% by weight of cellulose saccharification method. 제 8항에 있어서, 상기 반응 pH는 3 ~ 7이고, 반응온도는 25 ~ 45℃인 것을 특징으로 하는 셀룰로스의 당화방법.The method of claim 8, wherein the reaction pH is 3 to 7, and the reaction temperature is 25 to 45 ℃ characterized in that the cellulose saccharification method. 제 8 항에 있어서, 상기 셀룰로스 공급원 기질은 편백인 것을 특징으로 하는 셀룰로스의 당화방법.9. The method of claim 8, wherein said cellulose source substrate is one-sided protein.
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