KR101594995B1 - Bacillus pumilus strain derived from Ceratotherium simum and xylanase produced from it - Google Patents
Bacillus pumilus strain derived from Ceratotherium simum and xylanase produced from it Download PDFInfo
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Abstract
본 발명은 바실러스 푸밀러스 H10-1 균주의 자일라나제에 관한 것으로, 더욱 구체적으로는 흰코뿔소 배설물에서 분리된 바실러스 푸밀러스 H10-1 균주가 생산하는 자일라나제가 우수한 섬유소 분해 활성을 가지기 때문에, 섬유질계 바이오매스 당화에 이용 가능하며, 이를 사료첨가용 효소로 이용하면 가축이 영양소를 이용하는 효율을 높이는 효과가 있다.The present invention relates to a xylanase of a strain of Bacillus subtilis H10-1, more specifically, xylanase produced by a strain of Bacillus subtilis H10-1 isolated from a white rhinoceros excrement has excellent fibrinolytic activity, It can be used for biomass saccharification, and it is effective to increase the efficiency of using nutrients in livestock.
Description
흰코뿔소 배설물에서 분리된 바실러스 푸밀러스 H10-1 균주의 자일라나제의 당화 용도에 관한 것이다.
The present invention relates to the glycosylation of xylanase of Bacillus subtilis H10-1 isolated from white rhinoceros.
최근 지구 온난화뿐 아니라 한정된 원유 자원과 그에 따른 유가 급등에 따라 전 세계적으로 새로운 대체 에너지의 개발이 요구되고 있다. 이에 따라 재생가능한 식물자원으로부터 생산할 수 있는 바이오 에탄올 및 바이오연료가 크게 각광을 받고 있다(Lynd LR et al., 2008. Nature Biotechnology 26(2):169-172). 수송용 연료로서의 바이오에탄올은 현재 브라질에서는 사탕수수와 같은 당질계 자원으로부터, 미국에서는 옥수수와 같은 전분질계 자원으로부터 글루코스를 얻어 효모를 이용하여 발효하여 생산되고 있다 (Cassman KG et al., 2007. Biofuels Bioproducts & Biorefining-Biofpr 1(1):18-23; Mabee WE. 2007. Biofuels 108:329-357). 그러나 이들 당질 및 전분질계 자원은 식량자원으로서 연관되어 장기적인 관점에서 볼 때에 원료 수급에 문제를 갖고있어 식량자원이 아닌 리그노셀룰로오스 즉 농림산 부산물을 이용하고자 연구가 이루어지고 있다 (Bak JS et al., 2009. Bioresource Technology 100(3):1285-1290; Purwadi R, Brandberg T, Taherzadeh MJ. 2007. International Journal of Molecular Sciences 8:920-932; Sakai S et al., 2007. Applied and Environmental Microbiology 73(7):2349-2353; Zaldivar J et al., 2001. Applied Microbiology and Biotechnology 56(1-2):17-34).In recent years, not only global warming but also limited oil resources and soaring oil prices have demanded the development of new alternative energy globally. Bioethanol and biofuels, which can be produced from renewable plant resources, have been attracting much attention (Lynd LR et al., 2008. Nature Biotechnology 26 (2): 169-172). Bioethanol as transport fuel is currently produced from sugar-based sources such as sugarcane in Brazil and fermented from yeast using glucose from starchy sources such as corn (Cassman KG et al., 2007. Biofuels Bioproducts & Biorefining-Biofpr 1 (1): 18-23; Mabee WE. 2007. Biofuels 108: 329-357). However, these carbohydrate and starchy resources have been linked to food resources and have long been a problem in raw material supply and demand, and research has been conducted to utilize lignocellulose (ie, agricultural and forestry byproducts) rather than food resources (Bak JS et al. , 2009. Bioresource Technology 100 (3): 1285-1290; Purwadi R, Brandberg T, Taherzadeh MJ 2007. International Journal of Molecular Sciences 8: 920-932; Sakai S et al., 2007. Applied and Environmental Microbiology 73 7): 2349-2353; Zaldivar J et al., 2001. Applied Microbiology and Biotechnology 56 (1-2): 17-34).
일반적으로 리그노셀룰로오스(lignocellulose)는 지구상에서 가장 풍부한 재생 가능한 생물자원으로서 주로 식물체를 구성하는 주성분이다. 리그노셀룰로오스는 탄수화물인 셀룰로오스(cellulose)와 헤미셀룰로오스(hemicellulose), 방향족 화합물의 중합체인 리그닌(lignin) 등이 주성분으로 구성되어 있다 (Reddy N et al., 2005. Trends in Biotechnology 23(1):22-27;Stocker M. 2008. Angewandte Chemie-International Edition 47(48):9200-9211; Zhang YHP. 2008. Journal of Industrial Microbiology & Biotechnology 35(5):367-375). 셀룰로오스를 셀룰라제로 가수분해하면 미생물에 의해 발효가 잘 되는 글루코스를 얻을 수 있기 때문에 리그노셀룰로오스는 글루코스를 얻어 에탄올과 같은 바이오연료를 생산할 수 있는 원료가 될 수 있다 (McKendry P. 2002. Bioresource Technology 83(1):PII S0960-8524(01)00118-3; Stocker M. 2008. Angewandte Chemie-International Edition 47(48):9200-9211;Zhang YHP. 2008. Journal of Industrial Microbiology & Biotechnology 35(5):367-375). 또 다른 탄수화물인 헤미셀룰로오스는 자일로오스(xylose), 아라비노스(arabinose), 갈락토스(galactose), 맨노스(mannose), 글리코오스 등의 다양한 당으로 구성된 다당류인데 이 중에서도 자일로오스가 가장 많이 존재하며, 자일란이 주된 중합체로 알려져 있다 (Krull LH et al., 1980. Journal of Agricultural and Food Chemistry 28(5):917-919; Ren JL, Peng F, Sun RC. 2008. Carbohydrate Research 343(16):2776-2782; Saha BC, Bothast RJ, editors. 1999. Enzymology of xylan degradation. Washington: American Chemical Society. 167-194 p.).
Generally, lignocellulose is the most abundant renewable biomass on the planet and is the main constituent of plants. Lignocellulose is composed mainly of carbohydrate cellulose, hemicellulose, and lignin, a polymer of aromatic compounds (Reddy N et al., 2005. Trends in Biotechnology 23 (1): 22 -27; Stocker M. 2008. Angewandte Chemie-International Edition 47 (48): 9200-9211; Zhang YHP 2008. Journal of Industrial Microbiology & Biotechnology 35 (5): 367-375). Hydrolysis of cellulose into celluloses can result in glucose that can be fermented by microorganisms, so lignocellulose can be used as a raw material to produce glucose and produce biofuels such as ethanol (McKendry P. 2002. Bioresource Technology 83 (1): PII S0960-8524 (01) 00118-3; Stocker M. 2008. Angewandte Chemie-International Edition 47 (48): 9200-9211; Zhang YHP 2008. Journal of Industrial Microbiology & Biotechnology 35 (5): 367-375). Another carbohydrate, hemicellulose, is a polysaccharide composed of various sugars such as xylose, arabinose, galactose, mannose, and glycose, among which xylose is the most abundant , Jilan is known as the main polymer (Krull LH et al., 1980. Journal of Agricultural and Food Chemistry 28 (5): 917-919; Ren JL, Peng F, Sun RC. 2008. Carbohydrate Research 343 (16): 2776-2782; Saha BC, Bothast RJ, editors 1999. Enzymology of xylan degradation, Washington: American Chemical Society 167-194 p.).
자일라나제는 미생물이 생산하는 다양한 효소 중의 하나로써, 현재 그 이용성이 매우 높으며 앞으로도 다양한 용도로 개발될 것으로 기대되는 효소이다. 자일라나제는 지구상에서 가장 풍부하고 태양에너지에 의해 재생 가능한 생물자원인 식물성 섬유물질을 분해·당화하여 에너지 자원화하는 것에 이용할 수 있으며, 섬유소 분해가 어려운 단위동물의 사료이용 증대를 위해 이용될 수 있다. 자일라나제(xylanase)는 농가 부산물인 볏짚, 옥수수 줄기, 보리 짚, 기타 식물체에 존재하는 헤미셀룰로오스의 주요 성분인 β-1,4-D-자일란을 임의로 가수분해하여 자일로비오스(xylobiose)나 자일로트리오스(xylotriose) 등의 자일로올리고당의 생성반응을 촉매하는 효소이다. 이 효소는 맥아나 아스퍼질러스(Aspergillus sp.), 트리코데르마(Trichoderma sp.) 등의 곰팡이와 스트렙토마이세스(Streptomyces sp.), 바실러스(Bacillus sp.) 등의 세균으로부터 생산된다.
Xylanase is one of the various enzymes produced by microorganisms and is currently highly available and is expected to be developed in various applications in the future. Xylanase can be used to decompose, sacrifice and metabolize vegetable fiber materials, the most abundant and renewable biomass on the planet, and can be used to increase the feed utilization of unit animals that are difficult to decompose cellulose . Xylanase hydrolyzes β-1,4-D-xylan, which is a major component of hemicellulose present in agricultural products such as rice straw, corn stalks, barley straw, and other plants, to hydrolyze xylobiose, It is an enzyme that catalyzes the production reaction of xyloglycosides such as xylotriose. This enzyme is produced from fungi such as Aspergillus sp., Trichoderma sp., And bacteria such as Streptomyces sp. And Bacillus sp.
최근 들어 사료첨가용 효소로서 자일라나제의 유용성이 증대되고 있다. 주로 동물의 에너지 대사에 필요한 탄수화물과 소량의 단백질을 공급하는 사료용 곡물들은 대부분의 단위동물이 이용하지 못하는 섬유질을 함유하고 있다. 곡물중의 섬유질은 비전분성 다당류와 리그닌으로 구성되며 식물의 세포벽은 세포의 제일 바깥쪽의 셀룰로즈 층과 그 내부의 알곡 전체를 감싸는 헤미셀룰로즈 및 베타-글루칸으로 구성된 비전분성 다당류와 리그닌이 합쳐져서 이루어진다. 이 비전분성 다당류는 가축의 소화효소에 의해 분해되지도 않을 뿐만 아니라 곡물중의 전분이나 단백질을 감싸고 있기 때문에 가축이 영양소를 이용하는 효율을 떨어뜨리는 문제점이 있다. 또한 소장에서는 비전분성 다당류가 걸쭉한 겔 상태로 끈적끈적하게 녹아 있어 소화효소가 영양소로 접근하는 것을 방해하고 소화관 내 소화물의 흐름을 저해한다. 따라서 미생물이 소화물에 부착하여 이상발효를 일으켜 영양분의 손실을 가져오는 문제점이 있다. 자일라나제 (xylanase)를 곡물 사료에 첨가하면 알곡을 감싸고 있는 헤미셀룰로즈 층을 가수분해하여 알곡내에 있는 영양분의 이용성을 높일 뿐만 아니라 가축의 장내 소화물의 상태가 개선될 수 있다. 특히 헤미셀룰로즈 함량이 높은 소맥제품을 사료로 사용할 경우 가축에 연변이나 적리 현상이 일어나기 쉬운데 자일라나제는 이러한 현상을 예방할 수 있고 사료적 가치를 올려줄 뿐만 아니라 소맥제품의 품질의 편차를 개선하여 소맥제품을 안정적인 사료원료로 사용할 수 있게 하여 사료첨가용 효소로 그 가치가 높다.
Recently, the usefulness of xylanase as an enzyme for feed addition has been increasing. Feed grains, which supply mainly carbohydrates and small amounts of protein for the energy metabolism of animals, contain fiber that most animal species can not use. The fiber in the grains is composed of non-electrolytic polysaccharides and lignin. The cell wall of the plant is composed of the non-electrolytic polysaccharide composed of hemicellulose and beta-glucan and lignin, which surround the cellulosic layer on the outermost side of the cell and the whole inside of the cell. This nonspecific polysaccharide is not only decomposed by the digestive enzymes of the livestock but also contains the starch and protein in the cereal, which lowers the efficiency of using the nutrients. In the small intestine, the non-electrolytic polysaccharide is sticky and melted in a thick gel state, preventing digestive enzymes from accessing nutrients and inhibiting the flow of digestive enzymes. Therefore, there is a problem that microorganisms adhere to the digestive tract and cause abnormal fermentation, resulting in loss of nutrients. The addition of xylanase to grain feeds hydrolyzes the hemicellulose layer surrounding the grain to improve the availability of nutrients in the algae as well as improve the condition of intestinal digestion of livestock. Especially, when wheat products with high hemicellulose content are used as feeds, it is easy for the livestock to cause Yanbian or Fertilizing phenomenon. Xylanase can prevent this phenomenon and not only increase the feed value, but also improve the quality deviation of the wheat product, It is possible to use the product as a stable feed material, so it is highly valuable as an enzyme for feed additive.
현재 알려진 국내의 자일라나제 관련 특허로는 자일라나제를 생산하는 신규한 스트렙토마이세스 속 ＷＬ-2 균주에 대한 특허(대한민국 공개특허 2001-0111986), 고초균 유래 엔도자일라나제의 신호서열 유전자를 포함한 재조합 플라스미드 및 그를 이용한 외래단백질의 제조방법에 관한 특허(대한민국 공개특허 2000-0034279), 바실러스 속 AMX-4(Bacillus sp. AMX-4) 균주의 자일라나제(xylanase)를 코드하는 신규한 자일라나제 유전자(대한민국 공개특허 2003-0085679) 및 신규한 패니바실러스 ｓｐ. ＨＹ-8 균주의 자일라나제(대한민국 공개특허 2007-0082329) 등이 있으나 실제로 국내에서 여러 분야에 사용되지 못하고 있는 실정이다.
A known patent for xylanase in domestic market includes a patent for a novel strain of Streptomyces strain WL-2 producing a xylanase (Korean Patent Laid-Open No. 2001-0111986) and a gene encoding a signal sequence gene of endosyllanase derived from Bacillus subtilis (Korean Patent Laid-Open Publication No. 2000-0034279), a novel gene encoding a xylanase of a Bacillus sp. AMX-4 strain of the genus Bacillus sp. AMX-4 Lanase gene (Korea Patent Publication No. 2003-0085679) and novel Fanny Bacillus sp. And the xylanase of HY-8 strain (Korea Patent Publication 2007-0082329), but it is not actually used in various fields in Korea.
이에, 본 발명자들은 초식동물인 흰코뿔소의 배설물에서 분리된 자일라나제를 생산하는 신규한 미생물인 바실러스 푸밀러스 H10-1 균주를 선별하여 이로부터 분리된 자일라나제의 섬유소 분해능 및 당화 활성을 확인함으로써 본 발명을 완성하였다.
Thus, the inventors of the present invention selected a strain of Bacillus fumillus H10-1, a novel microorganism producing xylanase, isolated from the excrement of white rhinoceros, which is a herbivorous animal, to determine the fibrinolytic activity and glycosylation activity of xylanase isolated therefrom Thereby completing the present invention.
본 발명의 목적은 기탁번호 KACC91761P로 수탁된, 자일라나제를 생산하는 흰코뿔소 유래의 바실러스 푸밀러스 H10-1 (Bacillus pumilus H10-1) 균주를 제공하는 것이다.The object of the present invention is to provide a bacterium belonging to the genus Bacillus fumilus H10-1 ( Bacillus spp.) Derived from a white rhino producing xyloranase, entrusted with accession number KACC91761P pumilus H10-1).
또한, 본 발명의 또 다른 목적은 상기 균주로부터 수득한 자일라나제를 포함하는 당화 조성물을 제공하는 것이다.Still another object of the present invention is to provide a saccharifying composition comprising the xylanase obtained from the strain.
또한, 본 발명의 또 다른 목적은 상기 균주로부터 수득한 자일라나제를 포함하는 사료 첨가용 조성물을 제공하는 것이다.It is still another object of the present invention to provide a feed composition comprising the xylanase obtained from the strain.
또한, 본 발명의 또 다른 목적은 상기 균주를 이용한 당화 방법을 제공하는 것이다.Still another object of the present invention is to provide a saccharification method using the strain.
아울러, 본 발명의 또 다른 목적은 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주의 자일라나제 제조방법을 제공하는 것이다.
In addition, another object of the present invention is to provide a method for producing xylanase of a white rhino derived Bacillus subtilis strain H10-1.
상기 목적을 달성하기 위하여, 본 발명은 기탁번호 KACC91761P로 수탁된, 자일라나제를 생산하는 흰코뿔소 유래의 바실러스 푸밀러스 H10-1 (Bacillus pumilus H10-1) 균주를 제공한다.In order to achieve the above object, the present invention relates to a white rhino-derived Bacillus fumilus H10-1 ( Bacillus sp. pumilus H10-1).
또한, 본 발명은 상기 균주로부터 수득한 자일라나제를 포함하는 당화 조성물을 제공한다.The present invention also provides a saccharifying composition comprising the xylanase obtained from the strain.
또한, 본 발명은 상기 균주로부터 수득한 자일라나제를 포함하는 사료 첨가용 조성물을 제공한다.In addition, the present invention provides a composition for feed addition comprising the xylanase obtained from the strain.
또한, 본 발명은 상기 균주를 이용한 당화 방법을 제공한다.The present invention also provides a saccharification method using the strain.
아울러, 본 발명은 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주의 자일라나제 제조방법을 제공한다.
In addition, the present invention provides a method for producing xylanase of a white rhino derived Bacillus subtilis H10-1 strain.
본 발명의 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주의 자일라나제는 당화 활성이 우수하므로, 바이오에탄올 생산을 위한 섬유질계 바이오매스 당화에 적합하며, 섬유소 분해 활성이 우수하므로 이를 가축의 섬유소 소화를 돕는 사료 첨가제로서 유용하게 이용할 수 있다.
The xylanase of the white rhino derived Bacillus subtilis strain H10-1 of the present invention is excellent in the saccharifying activity and is suitable for the biomass-based fibrosis production for bioethanol production and has excellent fibrinolytic activity, It can be usefully used as a feed additive to help.
도 1은 흰코뿔소 배설물로부터 분리된 바실러스 푸밀러스 H10-1 균주의 자일라나제 활성을 나타낸 그래프이다.
도 2는 흰코뿔소 배설물로부터 분리된 바실러스 푸밀러스 H10-1 균주의 형태학적 특성을 확인한 사진이다.
도 3은 흰코뿔소 배설물로부터 분리된 바실러스 푸밀러스 H10-1 균주의 16S rDNA 염기 서열 분석에 의한 계통수를 나타낸 도이다.
도 4는 흰코뿔소 배설물로부터 분리된 바실러스 푸밀러스 H10-1 균주로부터 자일라나제를 분리하여 그의 활성을 확인한 과정 및 HPLC 결과이다.FIG. 1 is a graph showing the xylanase activity of Bacillus subtilis H10-1 isolated from white rhinoceros.
2 is a photograph showing the morphological characteristics of Bacillus fumillus H10-1 isolated from white rhinoceros.
FIG. 3 is a diagram showing the phylogeny of 16S rDNA sequence analysis of Bacillus fumillatus H10-1 strain isolated from white rhinoceros.
FIG. 4 shows the process and HPLC results of isolating xylanase from the Bacillus fumillatus H10-1 strain isolated from white rhinoceros waste and confirming its activity.
이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 기탁번호 KACC91761P로 수탁된, 자일라나제를 생산하는 흰코뿔소 유래의 바실러스 푸밀러스 H10-1 (Bacillus pumilus H10-1) 균주를 제공한다.
The present invention relates to a bacterium belonging to the genus Bacillus fumilus H10-1 ( Bacillus spp.) Derived from white rhinoceros producing xylanase, entrusted with accession number KACC91761P pumilus H10-1).
또한, 본 발명은 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주로부터 수득한 자일라나제를 포함하는 당화 조성물을 제공한다.
The present invention also provides a saccharifying composition comprising xylanase obtained from a white rhino derived Bacillus subtilis H10-1 strain.
또한, 본 발명은 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주로부터 수득한 자일라나제를 포함하는 사료 첨가용 조성물을 제공한다.
The present invention also provides a composition for feed addition comprising a xylanase obtained from a white rhino-derived Bacillus subtilis H10-1 strain.
또한, 본 발명은 In addition,
1) 자일란(Xylan)을 포함하는 배지에서 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주를 배양한 후 원심분리하여 상층액을 수득하는 단계;1) culturing a white rhino-derived Bacillus subtilis H10-1 strain in a medium containing xylan, and then centrifuging to obtain a supernatant;
2) 상기 단계 1)의 상층액을 동결건조하여 효소를 농축하는 단계; 및2) lyophilizing the supernatant of step 1) to concentrate the enzyme; And
3) 상기 단계 2)의 농축된 효소를 섬유소에 처리하여 섬유소를 분해시키는 단계를 포함하는 당화 방법을 제공한다.3) treating the fibrin with the concentrated enzyme of step 2) to decompose the fibrin.
상기 단계 3)의 섬유소는 섬유질계 바이오매스인 것이 바람직하나, 이에 한정되지 않는다.
The fiber of step 3) is preferably fibrous biomass, but is not limited thereto.
아울러, 본 발명은 In addition,
1) 자일란(Xylan)을 포함하는 배지에서 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주를 배양한 후 원심분리하여 상층액을 수득하는 단계;1) culturing a white rhino-derived Bacillus subtilis H10-1 strain in a medium containing xylan, and then centrifuging to obtain a supernatant;
2) 상기 단계 1)의 상층액을 동결건조하여 효소를 농축하는 단계를 포함하는 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주의 자일라나제 제조방법을 제공한다.
2) lyophilization of the supernatant of step 1) to concentrate the enzyme. The present invention also provides a method for producing xylanase from a white rhino derived Bacillus subtilis strain H10-1.
본 발명의 구체적인 실시예에서, 본 발명자들은 59종의 초식동물의 배설물로부터 섬유소 분해 활성을 가지는 균들을 분리하였으며 (표 2 참조), 분리한 균주들 중 자일라나제 활성이 가장 우수한 균주의 효소 활성을 확인하였고 (도 1 참조), 상기 균주의 형태학적 특성 및 염기서열을 확인하여 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주를 동정하였다 (도 2 참조). 또한, 바실러스 푸밀러스 H10-1 균주가 생산한 효소를 볏짚에 처리한 뒤 HPLC로 볏짚의 자일란이 자일로오스로 당화되는 것을 확인하였다 (도 4).In a specific example of the present invention, the present inventors isolated fungi having fibrinolytic activity from feces of 59 herbivores (see Table 2), and found that the enzyme activity of the strain with the best xylanase activity among the isolated strains (See FIG. 1), and the morphological characteristics and nucleotide sequence of the strain were confirmed to identify Bacillus fumillus H10-1 strain derived from white rhinoceros (see FIG. 2). In addition, it was confirmed that the enzyme produced by Bacillus subtilis strain H10-1 was treated with rice straw, followed by saccharification of xylenes of rice straw into xylose by HPLC (Fig. 4).
따라서, 본 발명의 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주의 자일라나제에 의해 섬유소가 당화되어 분해되므로, 이를 섬유소 분해를 증진시키기 위한 사료 첨가제 및 섬유질계 바이오매스 당화에 이용할 수 있음을 알 수 있다.
Therefore, it can be seen that the flaxseed of Bacillus subtilis strain H10-1 derived from the white rhinocerosum of the present invention is glycosylated and degraded by the xylanase, so that it can be used as a feed additive for promoting the degradation of cellulose and for the fibrous biomass saccharification have.
본 발명은 하기의 실시예에 의하여 보다 구체적으로 이해될 수 있으며, 하기의 실시예는 본 발명을 예시하기 위한 것이며, 본 발명의 보호 범위를 제한하고자 하는 것은 아니다.
The present invention can be more specifically understood by the following examples, and the following examples are intended to illustrate the present invention and are not intended to limit the scope of protection of the present invention.
<< 실시예Example 1> 균주 분리 및 선별 1> Strain isolation and selection
<1-1> <1-1> 자일라나제Xylanase (( xylanasexylanase ) 생산 균주 분리) Production strain isolation
본 발명자들은 초식동물의 배설물로부터 자일라나제를 생산하는 균주들을 분리하였다.The present inventors have isolated strains producing xylanase from the feces of herbivorous animals.
구체적으로, 하기 표 1의 소 과(25종), 사슴 과(14종), 낙타 과(4종), 기린 과(1종), 코끼리 과(1종), 말 과(6종), 캥거루 과(4종), 하마 과(2종), 토끼 과(1종), 코뿔소 과(1종)의 총 10과(59종) 초식동물 배설물(입수처: 서울대공원) 각각 1g을 0.85% NaCl 용액에 현탁 희석하여 PCA (plate count agar) 배지에 100 ㎕씩 도말하였다. 그 후 37 ℃에서 1일 또는 2일 동안 배양하였다. 배양된 배지에서 분리된 콜로니들을 변형된 PCA(1% xylan, bacto-peptone, yeast extract) 고체 배지에 계대 배양한 후 congo red 염색을 통해 활성이 있는 균주를 선별하였다. Specifically, the following can be found in Table 1 (25 species), deer (14 species), camel (4 species), giraffe (1 species), elephant (1 species), horse (6 species) 1g of each of 10 (59 species) herbivorous animal excrements (available at Seoul National University Park) were treated with 0.85% NaCl And the cells were plated on PCA (plate count agar) medium in an amount of 100 μl each. And then cultured at 37 ° C for 1 day or 2 days. The colonies isolated from the cultured medium were subcultured in a modified PCA (1% xylan, bacto-peptone, yeast extract) solid medium, and the strains were selected by congo red staining.
그 결과, 자일라나제 활성 균주를 1차 선별하였다 (표 2).As a result, xylanase-active strains were firstly selected (Table 2).
<1-2> 섬유소 분해 활성 우수 균주의 선별≪ 1-2 > Selection of strains having excellent cellulolytic activity
본 발명자들은 1차 선별한 표 2의 균주 중에 섬유소 분해 활성이 우수한 균주를 선별하였다.The present inventors selected strains having excellent fibrinolytic activity among the strains of Table 2 selected first.
구체적으로, 탄소원으로 1% 자일란(xylan)을 함유한 100 mL PCA 액체 배지에 실시예 <1-1>에서 Congo red 염색 활성을 보인 표 2의 분리 균주들을 각각 접종하여 37 ℃, 150 rpm 배양 (37 ℃, 150 rpm)하였다. 배양한 균주들을 4 ℃, 15,000 rpm에서 10분 동안 원심분리한 후 상층액만을 따로 취하여 조효소로 사용하여 효소 화성을 확인하였다. 자일라나제의 효소 활성은 T. K. GHOSE (Ghose TK, 1987. Measurement of cellulase activities. Pure Appl Chem. 59:257-268) 실험을 변형하여 측정하였다. 상기에서 원심분리한 조효소 상층액 0.5 mL을 2% 자일란 0.5 mL에 첨가한 후 50 ℃에서 30분간 효소와 기질을 반응시켰다. 반응이 끝난 후 즉시 DNS (Dinitrosalicylic acid) 3 mL을 첨가하고, 100 ℃에서 5분 동안 반응시켰다. 반응 후 증류수 20 mL을 첨가하여 냉각시키고 540 nm에서 흡광도를 측정하였다. 환원당 정량을 위하여 자일로오스(xylose)를 표준당으로 0.1 g/ml 내지 1.0 g/ml로 정량곡선을 작성하였으며, 양성대조군으로 섬유소 분해활성이 있다고 알려진 Kitasatospora, Streptomyces, Micromonospora, Cellulomonas gelida 및 Cellulomonas uda 균주(농촌진흥청 농업유전자원센터)를 이용하였다. 효소의 활성은 IU (International Unit)로 나타내었으며, 상기 값은 30분 동안 1 μmol의 자일로오스를 생성하는 효소의 양으로 정의하였다. Specifically, 100 mL of PCA liquid medium containing 1% xylenes as a carbon source was inoculated with each of the isolates shown in Table 2, which showed Congo red staining activity in Example <1-1>, and cultured at 37 ° C and 150
그 결과, 자일란을 자일로오스로 분해하는 섬유소 분해활성은 코뿔소 과에서 분리한 H10-1 균주가 가장 우수하였으며, 양성대조군인 Kitasatospora, Streptomyces, Micromonospora, Cellulomonas gelida 및 Cellulomonas uda 균주보다 4배 이상 우수함을 확인하였다 (도 1).
As a result, the fibrinolytic activity of xylenol to xylose was highest in the H10-1 strain isolated from rhinoceros and positive control strains such as Kitasatospora , Streptomyces , Micromonospora , Cellulomonas gelida and Cellulomonas uda strain (Fig. 1).
<< 실시예Example 2> 균주 동정 2> Identification of strain
본 발명자들은 섬유소 분해 활성으로 선별한 흰코뿔소 배설물 유래 H10-1 균주를 동정하기 위하여 형태학적 특성 및 16S rDNA 염기 서열을 분석하여 서열번호 1로 나타내었다.The present inventors analyzed the morphological characteristics and the 16S rDNA nucleotide sequence in order to identify the H10-1 strain derived from white rhinos excreta selected by the fibrinolytic activity, and are shown in SEQ ID NO: 1.
구체적으로, <실시예 1>에서 선별한 섬유소 분해 활성이 우수한 H10-1 균주를 PCA 배지에 접종 배양한 후 형태학적 특성을 관찰하였다 (표 3 및 도 2). Specifically, the morphological characteristics were observed after inoculation of the H10-1 strain having excellent fibrinolytic activity selected in Example 1 on the PCA medium (Table 3 and FIG. 2).
또한, 유전자 염기서열 분석을 위해 H10-1 균주를 액체 배양한 후 게놈 DNA 추출 키트 (DyneBio, Korea)로 게놈(genomic) DNA를 추출하였다. 추출한 DNA를 16S rDNA universal 프라이머를 이용하여 PCR을 수행하였다. PCR 산물을 T-easy 벡터에 라이게이션한 후 형질전환 재조합 균주를 선발하였다. 선발된 균주에서 플라스미드를 추출한 후 제한효소 HindⅢ 및 BglⅡ를 이용하여 DNA크기를 확인하고 ㈜마크로젠(Daejeon, Korea)에 서열 분석을 의뢰하였다. 분석된 염기서열은 NCBI (National Center for Biotechnology Information)에서 상동성을 분석하여 Clustal X software version 1.81을 이용해 계통수(phylogenetic tree)를 작성하였다 (도 3). For gene sequencing, H10-1 strain was cultured in liquid medium and genomic DNA was extracted with a genomic DNA extraction kit (DyneBio, Korea). The extracted DNA was subjected to PCR using 16S rDNA universal primer. The PCR product was ligated to T-easy vector and transformed recombinant strains were selected. The plasmid was extracted from the selected strains, and the size of the DNA was confirmed using restriction enzymes HindIII and BglII, and sequencing was requested from Daejeon, Korea. The analyzed nucleotide sequences were analyzed for homology in National Center for Biotechnology Information (NCBI) and a phylogenetic tree was created using Clustal X software version 1.81 (FIG. 3).
그 결과, H10-1 균주는 Bacillus pumilus와 99%의 상동성을 보였으며, 이를 Bacillus pumilus H10-1 균주로 명명하였다.As a result, the strain H10-1 was identified as Bacillus It showed a homology of 99% with pumilus, and named it as a Bacillus pumilus strain H10-1.
<< 실시예Example 3> 3> BacillusBacillus pumiluspumilus H10H10 -1 균주의 -1 strain 자일라나제Xylanase 활성 확인 Verify Active
본 발명자들은 상기 <실시예 2>에서 동정한 Bacillus pumilus H10-1 균주가 생산한 자일라나제 효소 활성을 확인하였다.The present inventors have found that Bacillus < RTI ID = 0.0 > The activity of xylanase enzyme produced by pumilus strain H10-1 was confirmed.
구체적으로, Bacillus pumilus H10-1 균주를 PCA 배지 100 ml에 접종한 뒤 37 ℃, 150 rpm에서 24시간 동안 배양하였다. 그 후, 이를 200 ml 자일란 배지로 옮겨 2일 동안 추가 배양하였다. 배양 후 4 ℃에서 8000 rpm으로 20분 동안 원심분리한 뒤, 상층액을 동결건조하여 Bacillus pumilus H10-1 균주가 생산한 자일라나제를 준비하였다. 상기 효소를 볏짚에 적용한 뒤, 자일라나제에의해 당화되어 생산된 자일로오스를 알칼리로 전처리한 볏짚의 자일란의 분해에 사용하였다. 볏짚의 효소 당화 실험은 미국 에너지부 산하의 재생에너지연구소(NREL) 분석법에 따라 수행하였다. 50 mL의 뚜껑이 있는 삼각플라스크에 알칼리로 전처리한 볏짚 (hemcellulose 20% 함유) 0.7 g과 동결 건조한 조효소 2 g을 넣은 후 증류수를 첨가하여 최종 부피를 10 mL로 맞추었다. 기질과 효소의 반응을 위해 반응액을 50 ℃에서 3일 동안 180 rpm으로 교반 반응시켰다. 반응이 완료된 시료를 0.2 ㎛ 멤브레인 필터(membrane filter)로 여과한 후 HPLC (Waters 2414, Aminex HPX-87H Ion Exclusion column, mobile phase: 5 mM H2SO4)를 이용하여 생성된 자일로오스(xylose)를 분석하였다. Specifically, Bacillus The pumilus H10-1 strain was inoculated in 100 ml of PCA medium and cultured at 37 ° C and 150 rpm for 24 hours. Then, it was transferred to 200 ml of xylan medium and further cultured for 2 days. After incubation, the cells were centrifuged at 8000 rpm for 20 minutes at 4 ° C, and the supernatant was lyophilized to obtain Bacillus The xylanase produced by the pumilus strain H10-1 was prepared. After the enzymes were applied to rice straw, xylan produced by saccharification with xylanase was used for digesting xylan of rice straw pretreated with alkali. The enzymatic saccharification test of rice straw was carried out according to the NREL method of the US Department of Energy. 0.7 g of rice straw (containing 20% hemcellulose) pretreated with alkali and 2 g of lyophilized coenzyme were added to an Erlenmeyer flask with a 50-mL lid, and distilled water was added to the final volume to 10 mL. The reaction mixture was stirred at 50 rpm for 3 days at 180 rpm for the reaction of substrate and enzyme. After completion of the reaction, the sample was filtered through a 0.2 쨉 m membrane filter and then washed with xylose (produced by using Waters 2414, Aminex HPX-87H Ion Exclusion column, mobile phase: 5 mM H 2 SO 4 ) ) Were analyzed.
그 결과, Bacillus pumilus H10-1 균주의 효소로 전처리한 볏짚의 자일란이 Bacillus pumilus H10-1 균주가 생산한 자일라나제에 의해 자일로오스로 당화됨을 확인할 수 있었다 (도 4).As a result, Bacillus The xylan of rice straw pre-treated with the enzyme of H10-1 pumilus strain was confirmed that the glycosylation by Giles in agarose by Bacillus pumilus H10-1 xylene Rana the strain is produced (FIG. 4).
<110> REPUBLIC OF KOREA <120> Bacillus pumilus strain derived from Ceratotherium simum and xylanase produced from it <130> p121242 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1513 <212> DNA <213> Bacillus pumilus <400> 1 agagtttgga tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60 cggacagaag ggagcttgct cccggatgtt agcggcggac gggtgagtaa cacgtgggta 120 acctgcctgt aagactggga taactccggg aaaccggagc taataccgga tagttccttg 180 aaccgcatgg ttcaaggatg aaagacggtt tcggctgtca cttacagatg gacccgcggc 240 gcattagcta gttggtgggg taatggctca ccaaggcgac gatgcgtagc cgacctgaga 300 gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag 360 ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt 420 tcggatcgta aagctctgtt gttagggaag aacaagtgcg agagtaactg ctcgcacctt 480 gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 600 gatgtgaaag cccccggctc aaccggggag ggtcattgga aactgggaaa cttgagtgca 660 gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720 agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 780 aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 840 tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 900 aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaacc ctagagatag 1020 ggctttccct tcggggacag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080 agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1140 tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggctgca 1260 agaccgcaag gtttagccaa tcccataaat ctgttctcag ttcggatcgc agtctgcaac 1320 tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgcaacacc cgaagtcggt 1440 gaggtaacct ttatggagcc agccgccgaa ggtgttaaag gggattgggg tgaagtcgta 1500 acaaggtagc cgt 1513 <110> REPUBLIC OF KOREA ≪ 120 > Bacillus pumilus strain derived from Ceratotherium simum and xylanase produced from it <130> p121242 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1513 <212> DNA <213> Bacillus pumilus <400> 1 agagtttgga tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60 cggacagaag ggagcttgct cccggatgtt agcggcggac gggtgagtaa cacgtgggta 120 acctgcctgt aagactggga taactccggg aaaccggagc taataccgga tagttccttg 180 aaccgcatgg ttcaaggatg aaagacggtt tcggctgtca cttacagatg gacccgcggc 240 gcattagcta gttggtgggg taatggctca ccaaggcgac gatgcgtagc cgacctgaga 300 gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag 360 ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt 420 tcggatcgta aagctctgtt gttagggaag aacaagtgcg agagtaactg ctcgcacctt 480 gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 600 gatgtgaaag cccccggctc aaccggggag ggtcattgga aactgggaaa cttgagtgca 660 gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720 agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 780 aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 840 tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 900 aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaacc ctagagatag 1020 ggctttccct tcggggacag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080 agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1140 tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggctgca 1260 agaccgcaag gtttagccaa tcccataaat ctgttctcag ttcggatcgc agtctgcaac 1320 tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgcaacacc cgaagtcggt 1440 gaggtaacct ttatggagcc agccgccgaa ggtgttaaag gggattgggg tgaagtcgta 1500 acaaggtagc cgt 1513
Claims (5)
Bacillus fumillus H10-1 derived from white rhinoceros producing xylanase, entrusted with accession number KACC91761PBacillus pumilus H10-1) strain.
A saccharifying composition comprising xylanase obtained from the white rhino derived Bacillus subtilis strain H10-1 of claim 1.
A composition for feed addition comprising a xylanase obtained from the white rhino derived Bacillus subtilis strain H10-1 of claim 1.
2) 상기 단계 1)의 상층액을 동결건조하여 효소를 농축하는 단계를 포함하는 흰코뿔소 유래 바실러스 푸밀러스 H10-1 균주의 자일라나제 제조방법.
1) culturing the white rhino-derived Bacillus subtilis strain H10-1 of claim 1 in a culture medium containing xylan, and then centrifuging to obtain a supernatant; And
2) lyophilization of the supernatant of step 1) to concentrate the enzyme.
2) 상기 단계 1)의 상층액을 동결건조하여 효소를 농축하는 단계; 및
3) 상기 단계 2)의 농축된 효소를 섬유소에 처리하여 섬유소를 분해시키는 단계를 포함하는 당화 방법.
1) culturing the white rhino-derived Bacillus subtilis strain H10-1 of claim 1 in a culture medium containing xylan, and then centrifuging to obtain a supernatant;
2) lyophilizing the supernatant of step 1) to concentrate the enzyme; And
3) A method of saccharifying comprising treating cellulose with the concentrated enzyme of step 2) to decompose cellulose.
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