• Asian-Australasian Journal of Animal Sciences
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• 제19권5호
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• pp.684-689
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• 2006

The present study examined the effects of Tween 80 on the attachment and hydrolytic activity of a cellulase enzyme against ball-milled cellulose (BMC), using the whole component (native CBH I) and the catalysis module (core CBH I) of carbohydrolase I purified from Trichoderma viride (Meicelase, Meiji Seika, Tokyo, Japan). The effects were evaluated as protein concentrations in the supernatant after mixing enzyme and substrate with Tween 80 at room temperature. Tween 80 decreased the adsorption of native CBH I and core CBH I onto BMC (p<0.001) and increased the amount of reducing sugars released from BMC by native CBH I (p<0.001). However, Tween 80 did not enhance the hydrolytic activity of core CBH I. Observations using SEM revealed that Tween 80 caused cellulose filter paper to swell and enhanced surface cracks and filaments caused by native CBH I but not by core CBH I. These results suggested that Tween 80 decreases enzyme adsorption to its substrate but enhances enzymatic activity.

• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1681-1688
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• 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
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• 제21권7호
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• pp.711-718
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• 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.

• KSBB Journal
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• 제22권1호
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• pp.16-21
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• 2007

Cellobiohyolase (CBH) I 유전자의 확보는 CBH I 유전자 cellulase 생산 균주인 Trichoderma reesei를 배양, 수거하고 액체 질소를 이용하여 세포를 파쇄 후 RT-PCR kit를 이용하여 CBH I gene을 합성하였다. 그 후 발현벡터인 pYGAL에 cloning하였다. CBH I 유전자 앞부분에는 CBH I 단백질이 cell 외부로 분비할 수 있도록 하는 ppL이 포함되었다. CBH I 단백질 발현은 Protein gel 결과를 통하여 발현을 확인하였다. Cellulase 활성을 증대시키기 위한 분자진화 방법 개발은 error prone PCR과 DNA shuffling을 수행하였다. 얻은 CBH I 유전자를 발현 벡터에 삽입하고 효모에 transformation하여 이것을 다시 screening하였다. 1차 screening 후 confirm test하기 위해 DNS (Dinitrosalicylic Acid) 환원당 측정법을 이용하였으며, 이 결과 121-D8, 228-G2, 389-E3, 412-B4, 456-D2의 cellulase 변이체를 획득할 수 있으며, 456-D2의 경우 original CBH I과 비교하여 약 510%의 활성이 증가된 것을 확인할 수 있었다. 분자 진화 된 cellulase sequence 분석결과 CBH I wild type과 비교하였을 때 121-D8의 경우 변경된 9개의 염기 중 아미노산의 변화에 영향을 준 염기는 6개, 228-G2의 경우 변경된 7개의 바뀐 염기 중 아미노산의 변화에 영향을 준 염기는 4개, 389-E3의 경우 변경된 13개의 바뀐 염기 중 아미노산의 변화에 영향을 준 염기는 9개, 412-B4의 경우 변경된 9개의 바뀐 염기 중 아미노산의 변화에 영향을 준 염기는 6개, 456-D2의 경우 변경된 10개의 바뀐 염기 중 아미노산의 변화에 영향을 준 염기는 7개이었다. Cellulase 생산 공정 최적화는 5 L 발효기를 이용하여 고농도 배양을 실험한 결과 최종 O.D. 120까지 진행할 수 있었으며, 약 1.2 g/L의 cellulase를 얻을 수 있었다. Cellulase 생산 공정 scale-up 5 L 규모에서 확립된 최적 fed-batch 발효 공정을 300 L로 scale-up하여 실험하였다. 최종 O.D.는 약 280 정도이며 cellulase의 발현양은 약 3.2 g/L 수준임을 확인하였다. Cellulase 정제 공정 최적화 결과 80%의 수율과 95%의 순도를 확보하였다.

• 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.

• 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.

• 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.

• Bulletin of the Korean Chemical Society
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• 제22권7호
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• pp.716-720
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• 2001

To test the metal ion effect, hydrolysis experiments for two cellobiohydrolases (CBHⅠ and CBH Ⅱ) from Trichoderma reesei have been carried out in the presence of 10 mM metal ions, such as Cu++, Mn++, Ca++, Hg++, Ba++, Pb++, and Cd++. The addition of Mn++, Ba++, and Ca++(10 mM) during the hydrolysis of Avicel PH 101 caused an increase in the total reducing sugar (TRS) for CBH Ⅰ by 142, 135, and 114 percent, respectively. Those for CBH Ⅱ increased by 177, 175, and 115 percent, respectively. The Mn++ was the most stimulatory metal ion, whereas Hg++ was the most inhibitory metal ion. The adsorption experiments were performed to investigate how the influence of Mn++ and Hg++ on the hydrolysis is related to the adsorption of cellobiohydrolases on cellulose. The increase in TRS during hydrolysis by adding Mn++ caused an increase in adsorption affinity (Kad) and tightness (ΔHa). While, the decrease of TRS during hydrolysis by adding Hg++ caused a decrease in the adsorption affinity (Kad) and tightness (ΔHa). These results indicate the changes in the tightness and affinity of adsorption by adding metal ions play a crucial role in the degradation of the microcrystalline cellulose.

• KSBB Journal
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• 제8권1호
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• pp.75-82
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• 1993

The inhibitory effect of cacao bean husk (CBH) extract on glucosyltransferase(GTasc) from Streptococcus mutans B13 was investigated. Water solube extract from CBH showeda sarong inhibitory effect (88-89%) on GTase from Streptococcus mutans Bl3. GTase inhibitors from sequential extraction by hot water or water-methanol had the strongest inhibition. Sources, fermentation, and types of solvents and fumigation processes did not influence the effect. These active compounds proved to be polyphones through acid hydrolytic analysis of the precipitates by ammonium sulfate or ethanol and proteinase K. It was also confirmed by additional column chromatography of Sephadex G-50, Sephadex LH-20 and DEAE-Sephdex A-50.