• Journal of Animal Science and Technology
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• 제48권3호
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• pp.415-424
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• 2006

한국 재래산양에서 분리된 Neocallimastix sp NLRI-3을 이용하여 기질의 종류에 따른 미생물의 성장 특성 및 효소단백질의 발현양상을 비교하고자 본 연구를 수행하였다. 혐기배지에 glucose, starch, 볏짚, filter paper, CMC, sigmacell, xylan 및 xylose를 0.2% 수준으로 첨가하여 배양하였다. 배양결과 가스 발생량은 96시간대에서 filter paper 배지에서 가장 높았고 CMC배지에서 가장 낮게 조사되었다(P<0.05). 곰팡이 포자 발생량은 72시간대에서 살펴보면 처리구들 중 볏짚 배지에서 가장 높게 나타났다 (P<0.05). 곰팡이가 분비하는 enzyme activity는 보통 72시간대 이후부터 급격히 증가함을 보이는데 CMCase의 경우에 filter paper disc, sigmacell, xylose에서 활성이 높게 나타났다. xylanase 역가는 starch, rice straw, sigmacell에서 높게 나타났다. Zymogram을 이용하여 각 효소단백질의 발현을 분석한 결과 효소단백질의 발현양상은 전 처리구에 걸쳐 유사하였으나 발현정도는 기질의 종류에 따라 차이가 있음을 알 수 있었다. 또한, zymogram 결과에 의해 6개의 CMCase와 4개의 xylanase가 검출 되었다. 본 연구의 결과로 혐기성 곰팡이의 효소단백질은 배양액에 존재하는 기질의 종류에 의해 발현정도가 영향을 받음을 알 수 있었다. 이러한 결과는 산업적으로 이용 가능한 혐기곰팡이 유래 효소제의 개발에 필요한 배양조건의 결정에 이용될 수 있을 것으로 사료된다.

• Journal of Animal Science and Technology
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• 제46권5호
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• pp.763-772
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• 2004

Neocallimastix frontalis SA에게 에너지원으로 filter paper 또는 볏짚만을 공급하여 반추위 곰팡이를 배양하는 동안 섬유소 분해 양상을 현미경으로 관찰하고 cellulase와 xylanase 생산에 미치는 영향을 비교하였다. 혐기성 반추위 곰팡이를 접종한 후 filter paper를 광학 현미경으로 관찰하였을 때 filter paper의 표면과 모서리에 유주자의 부착, 포자낭의 발달 그리고 복잡한 그물망의 균사 엽상체의 형성이 관찰되었으며, 배양 7일 후에는 filter paper의 소화 그리고 섬유사의 결착성 및 견고성의 감소 현상이 나타났다. 또한 분쇄한 볏짚 표면에서도 미성숙 및 성숙한 포자낭들이 관찰되었으며, 일반적으로 이들 균사들은 볏짚의 부스러진 부분이나 잘리어진 모서리에서 많이 발견되었다. cellulase와 xylanase는 배양기간 동안 filter paper와 볏짚 기질 모두에서 빠르게 그 농도가 증가하였으며, 볏짚 첨가시에 비해 filter paper 첨가시가 더 높은 경향을 보였다. 특히 두 가질간의 cellulase와 xylanase 효소 활성은 각각 48 그리고 96시간 배양 이후에 큰 차이를 보였다(P<0.05). 따라서 filter paper는 복합 구조를 갖는 볏짚에 비하여 cellulase와 xylanase 생산을 위한 더 좋은 유도 물질임을 발견하였다. 이상의 결과들을 N. frontalis에 대한 에너지원으로서 단일 복합체인 filter paper가 복합 구조의 볏짚에 비해 더 우수하였으며, 물리적 및 화학적으로 섬유소를 분해하는 혐기성 반추위 곰팡이라 할 지라도, 리그닌화된 견고한 섬유소 구조를 파괴시킬 수 있는 물리적 처리는 반추위 곰팡이의 분해 작용 및 성장에 도움을 줄 수 있음을 시사한다.

• Asian-Australasian Journal of Animal Sciences
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• 제2권3호
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• pp.473-474
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• 1989

• Asian-Australasian Journal of Animal Sciences
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• 제22권11호
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• pp.1555-1565
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• 2009

Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastix frontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five $\beta$-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis.

• Asian-Australasian Journal of Animal Sciences
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• 제29권11호
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• pp.1576-1584
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• 2016

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

• Asian-Australasian Journal of Animal Sciences
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• 제12권5호
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• pp.752-757
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• 1999

Studies were made of digestion of timothy (Pheleum pretense) hay, tall fescue (Festuca elatior) hay, and rice (Oryza sativa) straw in pure cultures of rumen anaerobic fungus, Neocallimastix frontails MCH3. The fungus was inoculated on ground forages (1%, w/v) in an anaerobic medium and incubated at $39^{\circ}C$. Incubation was continued for 24, 48, 72 and 96 h. The losses of dry matter, xylose and glucose of forage during incubation were determined at the end of these incubation periods. Xylose and glucose were considered to be released from xylan and cellulose, respectively. The digested xylan to digested cellulose (X/C) ratios of the substrate were calculated. Xylanase and carboxymethyl cellulose (CMCase) of culture supernatant and residual substrate was measured at the same time. The X/C ratios in the cultures on timothy hay and rice straw were greater than 0.5 in the first 24-h incubation period. The values were smaller than 0.3 in tall fesque. The ratio of xylanase activity to that of CMCase in the first 24-h incubation period correlated well with the traits in X/C ratio. However xylanase activity was still superior to CMCase in the following incubation period (48 to 96 h), although the glucose (designated as cellulose) was more intensively digested than xylose (designated as xylan). The production of these polysaccharidases appeared to correlate with substrate cell-wall sugar composition, xylose to glucose ratios, at the beginning of fast growing period.

• Journal of Animal Science and Technology
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• 제62권2호
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• pp.121-140
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• 2020

Anaerobic fungi habitat in the gastrointestinal tract of foregut fermenters or hindgut fermenters and degrade fibrous plant biomass through the hydrolysis reactions with a wide variety of cellulolytic enzymes and physical penetration through fiber matrix with their rhizoids. To date, seventeen genera have been described in family Neocallimasticaceae, class Neocallimastigomycetes, phylum Neocallimastigomycota and one genus has been described in phylum Neocallimastigomycota. In National Center for Biotechnology Information (NCBI) database (DB), 23,830 nucleotide sequences and 59,512 protein sequences have been deposited and most of them were originated from Piromyces, Neocallimastix and Anaeromyces. Most of protein sequences (44,025) were acquired with PacBio next generation sequencing system. The whole genome sequences of Anaeromyces robustus, Neocallimastix californiae, Pecoramyces ruminantium, Piromyces finnis and Piromyces sp. E2 are available in Joint Genome Institute (JGI) database. According to the results of protein prediction, average Isoelectric points (pIs) were ranged from 5.88 (Anaeromyces) to 6.57 (Piromyces) and average molecular weights were ranged from 38.7 kDa (Orpinomyces) to 56.6 kDa (Piromyces). In Carbohydrate-Active enZYmes (CAZY) database, glycoside hydrolases (36), carbohydrate binding module (11), carbohydrate esterases (8), glycosyltransferase (5) and polysaccharide lyases (3) from anaerobic fungi were registered. During four decades, 1,031 research articles about anaerobic fungi were published and 444 and 719 articles were available in PubMed (PM) and PubMed Central (PMC) DB.

• Journal of Animal Science and Technology
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• 제45권6호
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• pp.1019-1030
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• 2003

본 연구는 국내의 재래 반추동물인 재래산양과 한우의 장내에 서식하며 강력한 섬유소를 분해하는 혐기 곰팡이를 탐색하고 분리하여 섬유소 분해 특성을 구명하고자 실시되었다. 산양의 반추위로부터 16종과 한우의 십이지장 소화물로부터 5종의 혐기 곰팡이를 분리하여 총 21종의 혐기성 곰팡이가 분리되었다. 섬유소 분해효소의 활력을 측정하여 그 중 섬유소 분해력이 높은 4종의 곰팡이에 대하여 광학현미경에 의한 형태학적 관찰을 기초로 동정 작업을 수행하였다. NLRI-M003은 monocentric 성장형태, 구형의 포자낭, filamentous rhizoid 및 유주자의 flagella가 다수인 Neocallimastix sp., NLRI-M014는 monocentric 성장형태, 방추형의 포자낭, filamentous rhizoid 및 유주자의 flagella가 단수인 Piromyces sp.로, NLRI-T004는 monocentric 성장형태, 난형의 포자낭, filamentous rhizoid 및 유주자의 fagella 수가 다수인 Neocallimastix sp.로 각각 확인되었다. NLRI-M001은 Orpinomyces sp. 와 유사한 것으로 추측되나 지금까지 밝혀진 곰팡이 이외에 다른 밝혀지지 않은 곰팡이가 존재할 가능성이 있을 것으로 평가되어 더욱 더 세부적인 조사가 필요하다고 사료되었다. 혐기 곰팡이의 섬유소 분해 특성을 조사하기 위해 산양의 반추위로부터 분리된 NLRI-M003 혐기 곰팡이 배양액을 2% 첨가하여 혼합 반추위 미생물의 in vitro 건물 분해율을 볏짚과 filter paper를 기질로 하여 조사하였다. 모든 처리구에서 혐기 곰팡이 배양액을 첨가한 첨가구가 무첨가구에 비하여 볏짚의 경우 약 4%이상(p〈0.05) 그리고 filtre paper를 기질로 사용시 11% 이상(p〈0.001)의 분해율이 증가하였다. 또한 CMCase와 xylanase 효소의 활력도 첨가구에서 증가하였으며 특히 반추위 곰팡이는 강력한 xylanase 효소활력이 높음을 보여주었다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권8호
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• pp.1110-1117
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• 2001

Responses of the rumen fungus, Neocallimastix frontalis RE1, to long chain fatty acid (LCFA) were evaluated by measuring gas production, filter paper (FP) cellulose digestion and polysaccharidase enzyme activities. LCFA (stearic acid, $C_{18:0}$; oleic acid, $C_{18:1}$; linoleic acid, $C_{18:2}$ and linolenic acid, $C_{18:3}$) were emulsitied by ultrasonication under anaerobic condition, and added to the medium. When N frontalis RE1 was grown in culture with stearic, oleic and linoleic acid, the cumulative gas production, gas pool size, FP cellulose digestion and enzymes activities significantly (p<0.05) increased at some incubation times(especially, exponential phases of fungal growth, 48~120 h of incubation) relative to that for control cultures. However, the addition of linolenic acid strongly inhibited all of the investigated parameters up to 120 h incubation, but not after 168 and 216 h of incubation. These results indicated that stearic, oleic and linoleic acids tended to have great stimulatory effects on fungal cellulolysis, whereas linolenic acid caused a significant (p<0.05) inhibitory effects on the cellulolysis by the rumen fungus. These results are the first report of the effect of LCFAs on the ruminal fungi. Further research is needed to identify the mode of action of LCFAs on fungal strains and to verify whether or not ruminal fungi have ability to hydrate unsaturated LCFAs to saturated FAs. There was high correlation between cumulative in vitro gas production and fungal growth (94.78%), FP cellulose degradation (96.34%), CMCase activity(90.86%) or xylanase activity (87.67%). Thus measuring of cumulative gas production could be a useful tool for evaluating fungal growth and/or enzyme production by ruminal fungi.

• Asian-Australasian Journal of Animal Sciences
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• 제24권3호
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• pp.379-385
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• 2011

Ferulic acid esterase (FAE) and acetyl esterase (AE) cleave feruloyl groups substituted at the 5'-OH group of arabinosyl residues and acetyl groups substituted at O-2/O-3 of the xylan backbone, respectively, of arabinoxylans in the cell wall of grasses. In this study, the enzyme profiles of FAE, AE and polysaccharide hydrolases of the anaerobic rumen fungus Neocallimastix sp. YQ1 grown on Chinese wildrye grass hay (CW) or alfalfa hay (AH) were investigated by two $2{\times}4$ factorial experiments, each in 10-day pure cultures. The treatments consisted of two glucose levels ($G^+$: glucose at 1.0 g/L, $G^-$: no glucose) and four N sources (N1: 1.0 g/L yeast extract, 1.0 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N2: 2.8 g/L yeast extract and 0.5 g/L $(NH_4)_2SO_4$; N3: 1.6 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N4: 1.4 g/L tryptone and 1.7 g/L yeast extract) in defined media. The optimal combinations of glucose level and N source for the fungus on CW, instead of AH, were $G^-N4$ and $G^-N3$ for maximum production of FAE and AE, respectively. Xylanase activity peaked on day 4 and day 6 for the fungus grown on CW and AH, respectively. The activities of esterases were positively correlated with those of xylanase and carboxymethyl cellulase. The fungus grown on CW exhibited a greater volatile fatty acid production than on AH with a greater release of ferulic acid from plant cell wall.