• 농업과학연구
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• 제25권1호
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• pp.124-130
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• 1998

알칼리성 cellulase를 분비하는 세균을 토양으로 부터 분리하고 그 중 알칼리 환경하에서 생육이 양호하며 CMCase 생성능이 우수한 균주 AC-711를 선발하였다. AC-711 균주의 형태적, 배양적 및 생리학적 특성을 조사한 결과 호알칼리성 Pseudomonas속의 균주로 동정되었으나 염색체 DNA의 G+C함량이 54.43 mol%로서 Pseudomonas속보다 다소 낮은 값이었으며, 세포벽 지방산의 주성분은 15:0 anteiso 및 17:0 anteiso이었다. AC-711 균주의 생육은 $25{\sim}37^{\circ}C$, pH 9.5~10.5에서 좋았으며 효소생산을 위한 최적 조건은 $30^{\circ}C$, pH 10.3 이었다. 또한 효소생산을 위한 배지조성은 $KH_2PO_4$ 0.1%, $CoCl_2$ 0.02%, Tween 80 0.02%, $Na_2CO_3$ 0.5%에 탄소원으로서 CMC 1%, 질소원으로서 yeast extract 0.8%를 사용하였을 때가 가장 좋았다. CMCase의 생산은 3일 배양후 가장 높았으며, 생성된 효소는 filter paer와 avicel과 같은 결정성 섬유소보다는 수용성 섬유소인 CMC에 대한 분해 활성이 현저히 높아서 세제 첨가제로서의 이용 가능성이 주목되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.307-311
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• 2005

해양에서 유용물질을 생산하는 미생물을 분리하기 위하여 경상도 지역의 해안에서 시료를 채취하여 총 371 균주의 해양미생물을 얻었으며 전분, CMC 및 단백질 등과 같은 기질에 대한 분해활성을 측정하여 CMC에 대한 우수한 분해 능력을 가진 30균주를 선별하였다. 이들 30 균주를 배양하여 CMCase의 생산능력이 우수한 12 균주를 선발하였다. 이들 해양미생물을 배양하고 섬유소 분해효소를 생산한다고 알려진 B. amyloliquefaciens DL-3와 CMCase의 활성을 비교하였다. 이 중 다대포에서 분리하여 명명한 A-53 균주가 가장 높은 CMCase 활성을 나타를 생산한다고 알려진 B. amyloliquefaciens DL-3와 CMCase의 활성을 비교하였다. 이 중 다대포에서 분리하여 명명한 A-53 균주가 가장 높은 CMCase 활성을 나타내었다. A-53 균주를 16S rDNA partial sequencing 및 gyrase A partial sequencing하여 동정한 결과, Bacillus subtilis subsp. subtilis로 확인되었으며 B. subtilis subsp. subtilis A-53으로 명명하였다.

• Applied Biological Chemistry
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• 제48권4호
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• pp.322-325
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• 2005

Pseudomonas sp. JH1014는 세제 첨가용으로 사용 가능한 알칼리성 단백질 분해효소를 생산하는 균주로 하천수에서 분리되었다. 이 균주는 LB 배지에서는 CMCase를 생산하지 않았으나, 배지에 기질인 carboxymethyl cellulose를 첨가하면 분해효소인 CMCase의 생산이 유도되었으며, CMC 첨가에 따른 증식 양상의 변화는 없었다. CMC를 첨가한 배지로 $37^{\circ}C$에서 진탕배양 하였을 때 균의 증식은 $9{\sim}12$시간 후에 최대에 도달하였으며, 생산된 효소의 활성은 21시간 후에 최대에 도달하였다. 조효소액의 CMCase 활성 최적 pH는 6.0, 최적 온도는 $55^{\circ}C$였으며, $70^{\circ}C$에서 10분 동안 열처리한 후의 잔존활성은 70%였다. 부분 정제된 효소를 SDS-PAGE 후 활성염색한 결과 분자량이 54와 30 kDa인 두 개의 활성띠가 관찰되어, Pseudomonas sp. JH1014는 적어도 두 종류의 CMCase를 생산하는 것으로 보인다.

• 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.

• 한국환경보건학회지
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• 제22권2호
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• pp.10-18
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• 1996

Experiments were carried out to find the possibility of an economic production of single cell protein(SCP) in mixed culture by Cellulomonas sp. KL-6 and a second organism. The second organism, strain LI-10, was isolated from the large intestines of a mouse. 1. When these strains were mixed, cell growth and carboxymethyl cellulase (CMCase) activity were increased to about 63% and 161%, respectively compared with that of single culture of strain KL-6. We found the mixed culture as a proper method of degradation of cellulose in our study. 2. Strain LI-10 was identified as E. coli. 3. This strain produced trace amounts of cellobiose, but glucose was not found in detectable amounts in the filter paper(FP) medium. 4. $CaCO_3$ injected in the medium at the ratio of 0.1% not only enhanced cell growth but also was effective as an acid neutralizing agent. 5. When this organism was cultured under the optimal medium (glucose 0.1%, $NH_4Cl$ 0.1%, yeast extract 2.0%, $KH_2PO_4$ 0.1%, KCl 0.05%, pH 7.2 and a temperature 30$\circ$C) for 5 days, a cell mass produced 1.18 g/l. The results showed the increase of cell mass up to 300% compared to 0.28 g/l produced in CMC medium.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1178-1188
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• 2014

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.

• Applied Biological Chemistry
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• 제51권4호
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• pp.299-304
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• 2008

A ${\beta}$-1,4-endoglucanase gene from Bacillus subtilis H12 was cloned into Escherichia coli JM109 (pBC8) and sequenced. The endoglucanase gene with an insert DNA of 2.5 kb possessed an open reading frame of 1,500 bp encoding a mature protein of 499 amino acids with a calculated molecular mass of 55 kDa. The deduced amino acid sequence showed similarity to those of the known neutral cellulase genes of B. subtilis PAP115 (99.2%) and BSE616 (97.8%), as well as the alkaline gene of Bacillus sp. N4 (55.1%). The endoglucanase activity expressed by E. coli (pBC8) was localized in the periplasmic fraction (80%) and the cytoplasmic fraction (20%). An endoglucanase was purified from the periplasmic fraction by performing gel filtration and anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 31 kDa by SDS-PAGE, and the maximum activity occurred at pH 7 and $40^{\circ}C$. The enzyme easily hydrolyzed soluble substrates such as carboxymethyl cellulose and barely ${\beta}$-glucan, whereas the sigmacell and xylan, the known insoluble substrates, were not entirely hydrolyzed.

• 환경생물
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• 제22권1호
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• pp.184-191
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• 2004

To investigate bacteria with algalytic activities against Anabaena cylindrica when water blooming occurs and to study enzyme profiles associated with alga-lytic activity, various bacterial strains were isolated from surface waters and sediments in eutrophic lakes or reservoirs in Korea. Among 178 isolates, only nine isolates exhibited lytic abilities against A cylindrica on the agar plates, and then the isolate AK-13 was selected as the strongest in lysing the cyanobacterium A. cytindrica. The strain AK-13 was characterized and identified as Sinorhizobium sp. based on fatty acid methyl ether profiles and 16S rDNA sequence. According to the results of the enzyme assays, in the strain An-13 of Sinorhizobium sp., alginase, amylase, proteinase (caseinase and gelatinase), carboxymethyl-cellulase (CMCase), laminarinase, and lipase was produced, namely CMCase, laminarinase and protease were highly active. None of glycosidase was produced. Therefore, enzyme systems of Sinorhizobium sp. AK-13 were very complex to degrade cell walls of A. cylindrica. The peptidoglycans of A. cylindrica mat be hydrolyzed and metabolized to a range of easily utilizable monosaccharides or other low molecular weight organic substances by Sinorhizobium sp. AK-13.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
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• pp.78-89
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• 2000

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount of growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature-sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive-temperature. Incubation of the mcb mutant at restrictive-temperature results in a three- to five-fold increase in the level of extracellular protein and a 20- fold increase in carboxymethyl cellulase activity relative to a wild-type strain. A mutation in the cr-l gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb; cr-l double mutant was reduced to that of the wild-type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced following a shift of the mcb mutant to restrictive-temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.800-805
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• 2007

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose(Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein(EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein(EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was $100{\mu}g/ml$. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.