• 방사선산업학회지
• /
• 제8권2호
• /
• pp.65-69
• /
• 2014

The cellobiohydrolase gene (cbhI), a key component of a cellulolytic system, of a mutant PfCM4 (Pleurotus florida), developed through gamma ray radiation mutagenesis, was isolated and cloned. The deduced amino acid sequence was closely related to the glycoside hydrolase family 7 (GH7). The molecular weight of the deduced amino acid sequence of cbhI gene was found to be 22.4 kDa. Though the percent identity was found to be much less (35.61%) between the wild type and mutant, the cellulolytic activity of PfCM4 was 17.24% higher than that of the wild type. This shows that the catalytic domain of the cbhI gene was conserved in the mutant PfCM4.

• Mycobiology
• /
• 제40권2호
• /
• pp.107-110
• /
• 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
• /
• 제20권12호
• /
• pp.1681-1688
• /
• 2010

A screening for cellobiohydrolase (CBH) activity was performed and Fomitopsis pinicola KMJ812 was selected for further characterization as it produced a high level of CBH activity. An extracellular CBH was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants. The molecular mass of the F. pinicola CBH was determined to be 64 kDa by SDS-PAGE and by size-exclusion chromatography, indicating that the enzyme is a monomer. The F. pinicola CBH showed a $t_{1/2}$ value of 42 h at $70^{\circ}C$ and catalytic efficiency of $15.8mM^{-1}s^{-1}(k_{cat}/K_m)$ for p-nitrophenyl-${\beta}$-D-cellobioside, one of the highest levels seen for CBH-producing microorganisms. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, the F. pinicola CBH is distinguished from other CBHs by its high catalytic efficiency and thermostability.

• Journal of Microbiology and Biotechnology
• /
• 제21권7호
• /
• pp.711-718
• /
• 2011

A highly efficient cellobiohydrolase (CBH)-secreting basidiomycetous fungus, Agaricus arvensis KMJ623, was isolated and identified based on its morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular CBH was purified to homogeneity from A. arvencis culture supernatant using sequential chromatography. The relative molecular mass of A. arvencis CBH was determined to be 65 kDa by SDSPAGE and 130 kDa by size-exclusion chromatography, indicating that the enzyme is a dimer. A. arvencis CBH showed a catalytic efficiency ($k_{cat}/K_m$) of 31.8 $mM^{-1}\;s^{-1}$ for p-nitrophenyl-${\beta}$-D-cellobioside, the highest level seen for CBH-producing microorganisms. Its internal amino acid sequences showed significant homology with CBHs from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, A. arvencis CBH is distinguished from other CBHs by its high catalytic efficiency.

• Journal of Microbiology
• /
• 제43권6호
• /
• pp.487-492
• /
• 2005

This study demonstrated that the brown rot basidiomycete Fomitopsis palustris was able to degrade crystalline cellulose (Avicel). This fungus could also produce the three major cellulases (exoglucanases, endoglucanases, and $\beta-glucosidase$) when the cells were grown on $2.0\%$ Avicel. Avicel degraded by F. palustris showed a decrease in relative crystallinity from $83\%\;to\;78.5\%$ after 14 days of incubation. The characterization study indicated that optimum pH was 4.5 and optimum temperature was $70^{\circ}C$ for exoglucanase (cellobiohydrolase) activity. Hydrolysis of Avicel by the crude enzyme from F. palustris yielded 1.6 mg/ml of glucose after 43 h, which corresponded to a cellulose conversion degree of $3.2\%$. Therefore, this study revealed for the first time that the brown rot basidiomycete F. palustris produces cellulases capable of yielding soluble sugars from crystalline cellulose.

• 농업생명과학연구
• /
• 제45권1호
• /
• pp.119-124
• /
• 2011

Trichoderma spp.는 white biotechnology에서 이용되는 대표적인 미생물로서 이들이 강력하게 분비 생산하는 효소들은 산업적으로 매우 중요하다. 본 연구에서는 amylase, pectinase, cellobiohydrolase 및 xylanase 분비활성이 높은 것으로 밝혀진 Trichoderma sp. KACC 40541균주에 대한 Agrobacterium이용 형질전환을 수행하였으며 균주개량을 위한 효율적인 유전자도입 방법을 제시하였다. 특히 형질전환을 위하여서는 균사체에 대한 적정 농도의 NaOH처리가 매우 효과적임을 보여주었다.

• 유기물자원화
• /
• 제7권2호
• /
• pp.73-82
• /
• 1999

사과박의 퇴비화에서 효소활성도가 퇴비의 안정성 혹은 부숙도를 나타내는 지표로서의 사용가능성이 있는지를 검증하기 위해 배양계수법에 의한 미생물군집의 천이와, 무형광상태로 기질에 결합되어있던 형광물질(MUF)이 분해되면서 띠게 되는 형광정도가 해당효소의 활성도에 비례하여 높아지는 것을 이용하여 ${\beta}$-glucosidase와 cellobiohydrolase 활성도를 측정하였다. 탄수화물의 분해에 관여하는 두 효소의 활성도는 시간변화에 따라 점점 낮아졌다. 미생물개체군과 효소활성도간의 상관성은 균류군을 제외하고는 없는 것으로 나타났고 또 균류가 높은 개체수를 나타내는 것으로 보아 균류가 사과박의 퇴비화에 지대한 역할을 담당할 것으로 사료된다.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XIII)
• /
• pp.468-472
• /
• 2003

Recently, genetic engineering techniques have been used to display various heterologous peptides and proteins (enzyme, antibody, antigen, receptor and fluorescence protein, etc.) on the yeast cell surface. Living cells displaying various enzymes on their surface could be used repeatedly as 'whole cell biocatalysts' like immobilized enzymes. We constructed a yeast based whole cell biocatalyst displaying T. reesei cellobiohydrolase I (CBH I ) on the cell surface and endowed the yeast-cells with the ability to degrade cellulose. By using a cell surface engineering system based on ${\alpha}-agglutinin,$ CBH I was displayed on the cell surface as a fusion protein containing the N-terminal leader peptide encoding a Gly-Ser linker and the $Xpress^{TM}$ epitope. Localization of the fusion protein on the cell surface was confirmed by confocal microscopy. In this study, we report on the genetic immobilization of T. reesei CBH I on the S. cerevisiae and hydrolytic activity of cell surface displayed CBH I.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XIII)
• /
• pp.499-503
• /
• 2003

본 연구에서는 Trichorderma reerri의 cellobiohydrolase (CBH1) gene을 이용하여 분자진화 방법을 이용한 cellulose분해 활성이 증가된 cellulase 변이체를 선별하고자 하였다. 재조합된 벡터가 도입된 균주의 발현을 SDS-PAGE로 확인한 후, 분자 진화에 의한 cellulase 변이체를 trypan blue staining법으로 cellulase 변이체 228-G2를 선별하였으며, cellulose분해 활성을 DNS법으로도 다시 확인 할 수 있었다. Cellulase 변이체 228-G2의 분해활성 증가율은 original CBH I에 비해 약 300%증가 된 것을 확인하였고, DNA sequence를 확인할 결과 1542bp 중 17개의 염기서열이 바뀐 것을 알 수 있었다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제6권2호
• /
• pp.89-94
• /
• 2001

The binding of cellobiohydrolases to cullulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding changes during hydrolysis is still needed. The adsorption of purified two cellobiohydrolases (Ce17A and Ce16A) from Trichoderma reesei cellulase to microcrystalline cellulose has been studied. Cellobiohydrolase II (Ce16A) does not affect the adsorption of cellobiohydrolase I (Ce17A) significantly, and there are specific binding sites for both Ce17A and Ce16A. The adsorption affinity and tightness of the cullulase binding domain (CBD) for Ce17A are larger than those of the CBD for Ce16A. The CBD for Ce17A binds more rapidly and tightly to Avicel than the CBD for Ce16A. The decrease in adsorption observed when the two cellobihydrolases are studied together would appear to be the result of competition for binding sites on the cellulose. Ce17A competes more efficiently for binding sites than Ce16A. Competition for binding sites is the dominating factor when the two enzymes are acting together, furthermore adsorption to sites specific for Ce17A and Ce16A, also contributes to the total adsorption.