• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1991년도 춘계학술발표대회 논문집
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• pp.187-199
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• 1991

Penicillium verruculosum 배양액으로부터 소단위체 분자량이 60,000(cellobiohydrolase I)과 66,000(cellobiohydrolase II) 및 76,000(cellobiohydrolase III)인 cellobiohydrolase를 분리 정제하여 그들의 일반적 특성을 검토하였다. 이들 세 효소들은 모두 당단백질로서 cellobiohydrolase I, II 그리고 III는 각각 $8.6\%$, $4.2\%$ 그리고 $8.5\%$의 당함량을 나타냈으며 세 효소 모두 pH 4.5 - 5.0, 온도 $50 - 60^{\circ}C$에서 최적조건을 나타냈다. Cellobiohydrolase I, II 및 III는 모두 Avicel, cotton, 여지 등의 결정성 섬유소 뿐만 아니라 carboxymethyl cellulose에도 활성도를 나타냈다. 정제된 cellobiohydrolase I, II 및 III의 Avicel에 대한 비 활성도는 각각 0.07, 0.10, 그리고 0.07 unit per mg. of protein 이었으며 Avicel 분해 생성물은 거의 cellobiose였다. 또한 p-Nitrophenyl-$\beta$-D-cellobioside를 기질로 하였을 때 이들 세 효소 모두 포도당에 의해 활성도가 저해되지 않은 반면 cellobiose에 의해서는 저해되었다. 아미노산조성, 트립신에 의한 펩타이드들의 용출양상 그리고 항체를 이용한 Immunoblotting 결과로부터 cellobiohydrolase II와 III는 동일 유전자산물이거나 1차 구조가 거의 유사한 단백질로 추정된다. Cellobiohydrolase II로부터 분리한 2 개의 펩타이드의 아미노산 서열은 Trichoderma cellobiohydrolase I과 상동성을 보였으며 또한 이 두 호소의 아미노산조성은 매우 유사하였다.

• 한국미생물·생명공학회지
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• 제16권3호
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• pp.219-225
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• 1988

Trichoderma viride가 분비하는 섬유소 분해효소를 황산암모늄침전, Sephadex G-100 및 DEAE Sephadex column을 통과시켜서 5개의 섬유소 분해 아이조자임을 분리하였으며 전기영동 방법에 의해서 2개의 celloblohydrolase와 15개의 endoglucanase로 분리하였다. 분리된 cellobiohydrolase는 분자량이 71,000, 최적 pH5.1, 최적온도 5$0^{\circ}C$, pI가 3.81이었으며 주종아미노산은 aspartric acid, glutamic acid. threonine, serine 및 glycine이었다.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.248-252
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• 1991

Trichoderma viride의 변이주인 QM9123, QM9414, TK041 및 MCG77은 면역학적으로 동일한 cellobiohydrolase을 분비하는 결과로서 변이균주간은 구조 유전자는 동일하나 조절유전자가 변이했음을 알 수 있었고 탄소원의 cellobiohydrolase의 유도효과는 고분자 섬유소인 Alpha-cellulose와 Solk Floc의 가장 좋은 유도체였고, 저분자 유도체인 cellobiose와 CMC에 의해서 유도되는 형식이 다름을 알았다.

• 방사선산업학회지
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• 제8권2호
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• pp.65-69
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• 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.

• 산업기술연구
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• 제36권
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• pp.33-37
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• 2016

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli. Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

• 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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.468-472
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• 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
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.499-503
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• 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.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.808-817
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• 2011

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed ${\alpha}$- helices and ${\beta}$-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of $50^{\circ}C$ with specific activities against Avicel and p-nitrophenyl-${\beta}$-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

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