• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.482-493
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• 2005

Biomass is originally photosynthesized from inorgainic compounds such as $CO_2$, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellullosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria, Clostridium thermocellum, Clostridium stercorarium, and Clostridium josui, were isolated from compost and the chitinolytic Clostridium paraputrificum from beach soil and Ruminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed in Escherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex, i.e., cellulosome, which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced by E. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed in E. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing $CO_2$ reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase from C. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated. C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol $H_2/mol$ glucos) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene in C. paraputrficum using a modified vector of Clostridiu, perfringens. The hydrygen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel(RDF), were also found to be effective in converting biomass waste to hydrogen gas.

• 한국식품과학회지
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• 제18권2호
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• pp.130-136
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• 1986

'신고' 품종의 배를 두께가 다른$(0.04{\sim}0.08mm)$ 폴리에틸렌 필름으로 밀봉하여 KAIST 제작 농가용 저장고에서 MA저장 가능성을 검토하였다. 저장 5개월 동안에 저장고 내의 온도는 $0{\sim}7^{\circ}C$ 범위에서 상대습도는 $86{\sim}89%$가 유지되었다. 저장기간중 포장 내의 산소농도는 $5{\sim}14%$, 이산화탄소 농도는 $3{\sim}5%$ 범위에서 변화되었다. 배의 부패율은 $1.5{\sim}3.0%$로 포장구와 '비포장구간에 차이가 별로 없었으나 중량감소의 경우 비포장구는 저장 5재월 만에 4.1%까지 중량이 감소되어 위조현상이 나타났고 필름 포장구의 배는 중량감소가 약 1%밖에 발생하지 않아서 외관상 좋은 품질이 유지되었다. 한편, 관능경사 결과 0.07mm 두께의 필름으로 포장되었던 배가 풍미, 조직감, 다즙성 면에서 가장 높은 선호도를 나타냈는데 이는 당도가 가장 높고 산도가 가장 낮았던데 기인한 듯하다. 저장기간이 경과함에 따라 과육의 경도는 계속 감소하는 경향을 보였으며 섬유소 가수분해효소의 활성은 증가되었는데 과일 경도의 감소와는 밀접한 상관관계(상관계수는 -0.946)를 나타내었다. 과일의 세포벽은 점차적인 붕괴현상을 나타내었는데 이는 섬유소 가수분해효소가 작용한 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.529-533
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• 1999

In addition to catalyzing the synthesis of dextran from sucrose as a primary reaction, dextransucrase also catalyzes the transfer of glucose from sucrose to other carbohydrates that are present or are added to the reaction digest. We have synthesized new glucans having new structures and new characteristics, by transferring D-glucose of sucrose to $\alpha$-cellulose and by using the constitutive dextransucrase obtained from Leuconostoc mesenteroides B-742CBM. The final reaction products were composed of soluble- and insoluble-glucans. The yields of soluble- and insoluble-glucans were theoretically 21% $\pm$ 2.2 and 68% $\pm$ 5.1, respectively. The remainder of the reaction products was recovered as a mixture of olgiosaccharides that could not be precipitated by 67%(v/v) ethanol. Treating the modified glucans with endo-dextranase and/or cellulase, oligosaccharides were produced that were not formed from the hydrolysis of native cellulose or B-742CBM dextran. The modification of the cellulose was confirmed by methylation and acid hydrolysis of the soluble-and insoluble-glucan. Both (1->4) and(1->6) glycosidic linkages were found in both of the glucans.

• 한국식품과학회지
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• 제48권2호
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• pp.165-171
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• 2016

찰옥수수가루의 제빵 또는 제과 등 식품가공적성 향상을 위한 기초자료로서 활용하고자 열수 처리 및 효소 처리에 따른 물리 화학적 특성을 분석하였다. HMT 처리한 찰옥수수가루의 경우 $L^*$이 감소하고, $a^*$와 $b^*$이 증가하고 조단백질의 함량과 조지방함량이 감소하였다. 또한 HMT 처리 시 수분흡수지수가 낮아지고 수분용해지수가 높아짐에 따라 찰옥수수가루 내의 녹말의 호화현상으로 녹말 입자들의 치밀도가 낮아지고 비결정성부분이 많아지는 것으로 생각된다. HMT 처리 시 높은 열 처리로 인해 DSC를 통해 호화엔탈피(${\Delta}H$)의 감소, RVA를 통해 점도(viscosity)의 감소 및 XRD를 통해 결정성이 낮아짐을 알 수 있었다. 반면 ANN 또는 효소 처리 시 색도의 변화는 없었지만, 조단백질 및 조지방 함량의 변화가 있었고, DSC를 통해 확인한 호화엔탈피값은 대조군보다 약간 증가하거나 비슷하고, RVA를 통해 점도가 증가하는 것을 확인하였다. 또한 XRD 결과 전형적인 A-type의 결정성을 보였다. 전체적인 결과를 볼 때, HMT 처리는 찰옥수수가루 특성이 변화함에 따라 즉석식품으로 활용가능 할 것으로 생각되며 ANN에 의한 찰옥수수가루의 경우에는 점증제로서의 활용이 가능할 것으로 사료된다. 효소 처리한 경우 ANN 처리한 찰옥수수가루와 비교했을 때 큰 차이가 없으므로 효소 처리 가공보다는 ANN 처리하여 사용하는 것이 더 효과적이라고 생각된다.

• Mycobiology
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• 제40권2호
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• pp.107-110
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• 2012

Genes encoding the cellobiohydrolase enzyme (CBHI), designated as cbhI, were isolated from the basidiomycetes Auricularia fuscosuccinea, Pleurotus giganteus, P. eryngii, P. ostreatus, and P. sajor-caju. Initially, the fungal genomic DNA was extracted using a modified cetyltrimethyl ammonium bromide (CTAB) protocol and used as a DNA template. The cbhI genes were then amplified and cloned using the pGEM-T Easy Vector Systems. The sizes of these PCR amplicons were between 700~800 bp. The DNA sequences obtained were similar showing high identity to the cbhI gene family. These cbhI genes were partial consisting of three coding regions and two introns. The deduced amino acid sequences exhibited significant similarity to those of fungal CBHI enzymes belonging to glycosyl hydrolase family 7.

• 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.

• 한국식품과학회지
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• 제37권3호
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• pp.504-507
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• 2005

Trichoderma reesei KCTC 6952는 고분자 섬유소인 cellulose를 분해하는데 관여하는 3종류의 효소로 구성된 cellulase system을 보유하고 있다. 그 중 ${\beta}$-glucosidase는 glucose를 함유한 glycoside 유도체들로부터 glucose를 효율적으로 유리할 수 있는 것으로 알려져 있다. 본 연구에서는 변형된 Mandels의 배지를 사용하여 T. reesei KCTC 6952를 배양하면서 배지로 분비된 ${\beta}$-glucosidase를 조효소액으로 회수하여 조효소액내의 ${\beta}$-glucosidase 활성을 측정하고, 또한 효소활성이 온도와 pH에 대하여 어느 정도 안정한 지를 조사하였다. 그 결과 배양 4일째에 ${\beta}$-glucosidase의 효소활성이 최고(1.33 unit/mL)에 도달하였으며, 조효소액내의 ${\beta}$-glucosidase의 최적 반응 조건은 pH 5과 $70^{\circ}C$에서 10분간 반응하는 것이었으며, 그리고 효소의 안정성을 실험한 결과에 의하면 pH 4-5의 범위에서, $50^{\circ}C$ 이하에서는 안정적이었다. 그러므로 결론적으로 본 연구를 위하여 선택된 ${\beta}$-glucosidase 조효소액은 비교적 높은 온도와 넓은 pH범위에서 촉매반응을 일으킬 수 있었다.

• 농업과학연구
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• 제22권2호
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• pp.151-155
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• 1995

Whatman carboxymethyl cellulose CM 32와 CM 70으로 부터 색소단으로서 trinitrophenyl를 함유하는 섬유소 유도체를 합성하였다. 먼저 diaminoethyl기를 부가하여 DAE-CM-celulose를 얻고 다시 DAE-CM기 부위를 2,4,6-trinitrophenyl기로 치환시켜 TNP-cellulose를 조제하였다. CM 32로 부터 얻은 TNP-cellulose는 입자의 평균 크기가 $44.6{\pm}9.6{\mu}mD{\times}127.9{\pm}22.5{\mu}mL$로서 CM 70의 것에 비하여 3분의 1 수준으로 길이가 짧은 반면 폭은 보다 팽윤된 상태이었으며, ${\varepsilon}$-TNP-lysine의 molar extinction coefficient $1.33{\times}10^4$을 적용하여 측정한 그램당 TNP의 몰분율은 0.68 millimole로서 CM 70의 것보다 5.6배 높았다. 섬유소 분해효소에 의한 TNP-cellulose의 분해산물인 TNP-oligosaccharide의 최대 흡수 파장 ${\lambda}_{max}$는 344 nm이었다. 이들 TNP-cellulose를 기질로 하여 Onozuka R-10 효소액을 반응시킨 결과 반응 생성물의 흡광도가 효소의 농도에 비례하는 관계를 나타내었으며 CM 32의 것을 사용한 경우에 그 기울기가 현저히 커서 낮은 효소 농도 범위에서도 감도가 높음을 확인하였다.

• 한국균학회지
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• 제18권3호
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• pp.115-126
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• 1990

구름버섯은 항종양작용이 인정되었으며 근자에는 AIDS virus에 대한 억제효과가 보고되고 있다. 이와 같은 약효를 인정받고 있는 구름버섯 균주간의 세포융합이나 구름버섯과 타 유효한 버섯과 세포융합을 시행함으로써 더욱 효능이 우수하거나 다양한 균주의 약효를 한 균주내에서 기대할 수 있는 새로운 균주의 개발이 가능하다. 이러한 목적으로 구름버섯의 원형질체융합을 시행하기 위하여, 원형질체분리, 재생 및 두 영양요구주의 융합에 관하여 실험하였다. 균사체를 2.5일간 셀로판지위에서 배양하여 Novozym 234와 cellulase Onozuka R-10이 각각 15 mg/ml와 10 mg/ml 포함되어 있는 0.6M sucrose 용액에 넣어 $30^{\circ}C$에서 3-4.5시간 반응시킬 때 원형질체 수득률이 가장 높았다. 삼투압 안정제로 0.6M sucrose는 원형질체 형성과 재생에서 최적조건이었다. 0.6M sucrose를 포함하는 고체배지에서의 재생빈도는 3.48%이었으며 UV 조사시 생존률은 0.02-7.4%이었다. 원형질체융합은 $30^{\circ}C$에서 10분 동안 polyethylene glycol(M.W. 4,000)을 이용하여 시행하였는데 그 융합빈도는 1.86%이었다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 추계학술대회 및 정기총회
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• pp.16-16
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• 2015

Ectomycorrhizal (ECM) mushrooms play a major role in plant growth promotion through symbiotic association with roots of forest trees. They also provide an economically important food resource to us and therefore they have been studied for their artificial cultivation for decades in Korea. We have secured bio-resources of ECM mushrooms from Korean forests and performed their physiological studies. To investigate the cultural characteristics, the fungi were cultured under different conditions (medium, temperature, pH of the medium, inorganic nitrogen source). More than 90% of total 160 strains grew on three solid media (potato dextrose agar, PDA; sabouraud dextrose agar, SDA; modified Melin-Norkrans medium, MMN). The rate of mycelial growth on malt extract agar (MEA) was lower than those of three media (PDA, SDA, MMN). None of the Tricholomataceae strains grew on MEA. Many strains of ECM mushrooms were able to grow at the temperature range of $15{\sim}25^{\circ}C$ on PDA, while they showed poor growth at $10^{\circ}C$ or $30^{\circ}C$. In particular, the growth rates of both Gomphaceae and Tricholomataceae were significantly lower at $10^{\circ}C$ than at $30^{\circ}C$. The optimal pH of many strains was pH 5.0 when they cultured in potato dextrose broth (PDB). Fifty-seven percent of tested strains grew well on medium containing ammonium source than nitrate source. Many strains of Tricholomataceae showed a notable growth on ammonium medium than nitrate medium. Twenty-three percent of strains preferred nitrate source than ammonium source for their mycelial growth. The production and activity of two enzymes (cellulase and laccase) by ECM fungi were also assayed on the enzyme screening media containing CMC or ABTS. Each strains exhibited different levels of enzymatic activities as well as enzyme production. The number of laccase-producing strains was less than that of cellulase-producing strains. We found that 77% of tested strains produced both cellulase and laccase, whereas 2% of strains did not produce any enzymes. The morphological characteristics of mycelial colony were also examined on four different solid media. Yellow was a dominant color in mycelial colony and followed by white and brown on all culture media. ECM mushrooms formed mycelial colonies with a single or multiple colors within a culture medium depending on the strains and culture media. The most common shape of mycelial colony was a circular form on all media tested. Other families except for Amanitaceae formed an irregular colony on MMN than PDA. All strains of Tricholomataceae did not form a filamentous colony on all media. The pigmentation of culture media by mycelial colonies was observed in more than 50% of strains tested on both PDA and SDA. The degree of pigmentation on PDA or SDA was higher than MMN and brown color was dominant than yellow color. The production of exudates from mycelial colony was higher on PDA than MMN. Brown exudates were mainly produced by many strains on PDA or SDA, whereas transparent exudates were mainly produced by strains on MMN. We observed the mycelial colonies with a single or multiple textures in just one culture plate. Wrinkled or uneven colony surfaces were remarkably observed in many strains on PDA or SDA, while an even colony surface was observed in many strains on MMN. Sixty percent of Tricholomaceae strains formed wrinkled surface on PDA. However, they did not form any wrinkle on MMN plate. Cottony texture was observed in mycelia colonies of many strains. Velvety texture was often observed in the mycelial colonies on SDA than PDA and accounted for 60% of Suillaceae strains on SDA.