• KSBB Journal
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• 제28권1호
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• pp.42-47
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• 2013

This study was performed to find cellulolytic strain of enzymatic saccharification for bioethanol production. Cellulolytic strains were isolated from 59 different feces of herbivores from Seoul Grand Park located in Gwacheon Gyeonggi-Do. The celluloytic strain was selected by congo red staining and DNS method. Among the isolated strains, H9-1 strain isolated from the feces of rabbit has the highest CMCase activity. H9-1 strain was identified as Bacillus sp. based on 16S rDNA gene sequencing. The optimal conditions for CMCase activity by Bacillus sp. H9-1 were at $40^{\circ}C$ and at initial pH 8.

• 한국균학회지
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• 제18권4호
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• pp.209-217
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• 1990

Aspergillus niger에 있어서 여러가지 섬유질 기질이 세포내외의 섬유질 분해효소계의 생합성 및 동질효소의 양상에 미치는 영향을 조사하였다. Cellulase 및 xylanase 효소계의 생합성은 사용된 기질에 따라 큰 차이가 있었으며, 특히 cellulase 효소계의 CMCase와 ${\beta}-glucosidase$는 혼합기질의 사용시 효소활성이 증진되는 기질의 공조효과를 보였다. 또한 기질에 따라 섬유질분해효소계 동질효소의 생성 양상이 다르게 나타났으나 세포내외간 동질효소 양상의 차이는 발견되지 않았으며 배양시간에 따른 동질효소의 변화도 없었다. 이러한 결과들은 A. niger의 cellulase 및 xylanase효소계의 생합성은 기질에 의한 효소의 유도 수준에서 상호 연관적으로 조절되어짐을 보여주며, 세포외 효소에서 나타나는 multiple-isozyme의 형성이 합성되어진 효소의 post-secretional modification에 의한 결과라기 보다는 각각의 유전자 발현 결과에 의한 것임을 시사한다.

• 한국토양비료학회지
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• 제34권5호
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• pp.333-341
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• 2001

퇴비화 촉진 미생물인 Bacillus subtilis LYH201균주가 분비하는 섬유소 분해효소를 분자생물학적으로 연구한 결과는 다음과 같다. 섬유소를 분해하는 유전자는 유전자은행에 의해 구한 약 5,000개의 clone 중 CMC 배지 상에서 활성을 가지는 clone을 선발하여 bglC(pLYH7-39)로 명명하였다. 섬유소를 분해하는 bglC 유전자는 Pvu II, EcoRI, SspI의 제한효소 site를 가지고 있었으며, BglC는 Clostridium acetobutylicum GUN_CLOAB(P15704)와 57%의 identity와 71%의 homology를 나타내어 상동성이 가장 높았으며, CMC-SDS-PAGE 분석으로 56 kDa의 분자량을 나타냈고, 온도는 $50^{\circ}C$, pH는 7에서 활성이 가장 높았다.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.302-309
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• 2005

Phytopathogenic Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 secretes multiple isozymes of the plant cell wall degrading enzyme endoglucanases. We have cloned a third cel gene encoding CMCase from Pcc LY34. The structural organization of the celC gene (AY188753) consisted of an open reading frame (ORP) of 1,116 bp encoding 371 amino acid residues with a signal peptide of 22 amino acids within the NH$_2$-terminal region of pre-CelC. The predicted amino acid sequence of CelC was similar to that of Peetobaeterium ehrysanthemi Cel8Y (AF282321). The CelC has the conserved region of the glycoside hydrolase family 8. The apparent molecular mass of CelC was calculated to be 39 kDa by CMC-SDS-PAGE. The cellulase­minus mutant of Pee LY34 was as virulent as the wild-type in pathogenicity tests on tubers of potato. The results suggest that the CelC of Pce LY34 is a minor factor for the pathogenesis of soft-rot.

• 한국미생물·생명공학회지
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• 제31권2호
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• pp.124-128
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• 2003

대전광역시 근교의 야산과 들판 등지에서 썩은 나뭇잎, 짚, 흙을 채취하여 각각을 배양한 다음 Congo red test에 의해 cellulase 활성을 보이는 균주를 선별하였다. Genomic DNA를 분리한 후 PCR을 수행하여 DNA sequence를 Gene Bank를 통해 분석한 결과 A. tubingensis로 밝혀졌다. 이것을 배양하여 상등액을 crude enzyme으로 사용하여 온도와 pH를 달리하면서 효소의 활성정도를 측정하였다. 대조균주로 A. oryzae KCTC 6291를 이용하였고, 본 연구를 통하여 분리한 균주인 A. tubingensis가 생산하는 cellulase는 A. oryzae의 cellulase에 비하여 각각 다른 온도와 pH에서 높은 안정성을 보여주었다. A. tubingensis는 각각의 온도에서 활성의 정도가 비슷했으며, 45$^{\circ}C$, 55$^{\circ}C$에서 높은 활성을 나타내고 있지만, 고르게 활성이 나타났다. 또한 pH 12.0에서 가장 높은 활성을 보여 주었고, pH 2.0, 3.0, 4.0에서는 양쪽 모두 거의 활성이 없었으며, 중성, 염기성에 대해서 활성에는 큰 변화가 없었다. 따라서, 분리 동정한 A. tubingensis는 온도와 pH에서 고르게 활성을 나타내므로 생균제로 활용할 수 있는 범위가 클 것으로 여겨진다.

• 한국유기농업학회지
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• 제20권3호
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• pp.327-343
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• 2012

In order to develop a high cellulolytic direct-fed microorganism (DFM) for ruminant productivity improvement, this study isolated cellulolytic bacteria from the rumen of Holstein dairy cows, and compared their cellulolytic abilities via DM degradability, gas production and cellulolytic enzyme activities. Twenty six bacteria were isolated from colonies grown in Dehority's artificial (DA) medium with 2% agar and cultured in DA medium containing filter paper at $39^{\circ}C$ for 24h. 16s rDNA gene sequencing of four strains from isolated bacteria showed that H8, H20 and H25 strains identified as Ruminococcus flavefaciens, and H23 strain identified as Fibrobacter succinogenes. H20 strain had higher degradability of filter paper compared with others during the incubation. H8 (R. flavefaciens), H20 (R. flavefaciens), H23 (F. succinogenes), H25 (R. flavefaciens) and RF (R. flavefaciens sijpesteijn, ATCC 19208) were cultured in DA medium with filter paper as a single carbon source for 0, 1, 2, 3, 4 and 6 days without shaking at $39^{\circ}C$, respectively. Dry matter degradability rates of H20, H23 and H25 were relatively higher than those of H8 and RF since 2 d incubation. The cumulative gas production of isolated cellulolytic bacteria increased with incubation time. At every incubation time, the gas production was highest in H20 strain. The activities of carboxymethylcellulase (CMCase) and Avicelase in the culture supernatant were significantly higher in H20 strain compared with others at every incubation time (p<0.05). Therefore, although further researches are required, the present results suggest that H20 strain could be a candidate of DFM in animal feed due to high cellulolytic ability.

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.