• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1856-1862
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• 2015

In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70℃, much higher than that of FEG, which was approximately 50℃. Moreover, DEG showed 91.1% activity at 65℃ for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65℃, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability.

• 생명과학회지
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• 제17권9호통권89호
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• pp.1248-1254
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• 2007

Kluyveromyces marxianus 유래의 exoinulinase (INU1) 분비 서열과 GAL10 promoter를 이용하여 Clostridium thermocellum endoglucanase A gene (celA)의 과발현과 분비 성능 검증연구를 수행하였다. 자체 분비 서열을 가지는 pYEG- CT1 과 INU1 분비 서열을 가지는 pYInu-CT1, 두개의 plasmid는 S. cer-evisiae SEY2102와 S. cerevisiae 2805에 각각 형질전환시켜 ur-acil이 결핍된 배지에서 선별하였다. celA gene 자체 분비 서열보다 INU1 분비 서열을 사용했을 때 발현량과 분비효율은 각각 약 $18{\sim}22%$ 와 11% 향상되었다. 이 중 361 unit/l의 발현율과 89%의 plasmid 안정성, 그리고 70%의 분비효율을 보인 S. cerevisiae 2805/pYinu-CT1 효모 형질전환체를 cellolose를 효과적으로 분해하는 재조합 효모 생균체로 선별하였다. Galactose 배지내에서 S. cerevisiae 2805/ pYInu-CT1의 발효조 회분배양 결과, 총발현량과 분비효율은 각각 418 unit/l 와 73% 로 나타났다. 또한 분비된 endoglucanase A는 분자량 100kDa 이상에서 활성 밴드를 보여, N-linked 당쇄부가에 의해 상당한 비율의 당쇄가 부가됨을 추측할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2076-2080
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• 2007

The endoxylanase (GenBank Access No. U51675) of Bacillus spp. and endoglucanase (GenBank Access No. AY466436) of Trichoderma spp. were separately inserted downstream of the yeast constitutive ADHI promoter, resulting in three different plasmids (pAGX1, pAGX2, and pAGX3) according to the transcription direction of two genes. When the yeast transformants, S. cerevisiae SEY2102 harboring each expression plasmid, were grown on YPD medium, the total activities of the enzymes were approximately 3.01 unit/ml, 3.24 unit/ml, and 7.56 unit/ml for endoxylanase and 0.60 unit/ml, 0.54 unit/ml, and 0.39 unit/ ml for endoglucanase, in the following order: the pAGX1, pAGX2, and pAGX3. More than 70% of the endoxylanase and endoglucanase activities was found in the extracellular media.

• 한국토양동물학회지
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• 제8권1_2호
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• pp.7-12
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• 2003

지렁이 (Eisenia andrei)의 중장에서 내생의 endoglucanase 유전자의 전체 염기 서열을 동정하였다. ORF의 길이는 1,371 bp이며, 456개의 아미노산으로 번역된다. NCBI에 등록된 가재와 흰개미의 cellulase 및 endo-$\beta$-1, 4-glucanase와 50-51%의 유사성을 보이며, 활성 부위가 잘 보존되어 있었다. 계통수 분석에서는 다른 동물 분류군에서 밝혀진 GHF9 그룹의 cellulase와 근연관계가 없음이 확인되었다.

• Bulletin of the Korean Chemical Society
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• 제15권9호
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• pp.719-724
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• 1994

Major cellulase components, such as three endoglucanases (endoglucanases I, II, and III) and one exoglucanase (exoglucanase II), were isolated from a commercial cellulase (Meicelase TP 60) derived from the fungus Trichoderma viride by a series of chromatography procedures. These procedures were the gel filtration on Bio-Gel, the anion exchange on DEAE-Bio-Gel A, the cation exchange on SP-Sephadex C50, and the affinity chromatography on Avicel cellulose. The average molecular weights determined by SDS-polyacrylamide gel electrophoretic analysis were 51,000, 59,000, 41,000 and 62,000 Da for endoglucanases I, II and III and exoglucanase II, respectively. The extinction coefficients, ${\varepsilon}^{1%}$ 280 nm, of these enzymes were 11.7, 3.3, 7.2 and 11.3, respectively. Among them, the endoglucanase II showed the very low value of the coefficient compared with the others. On the other hand, it was found that endoglucanase II and III were of more random hydrolytic mode on carboxymethylcellulose as compared with those of endoglucanase I and exoglucanase II. Especially, endoglucanase I showed less random action than that of exoglucanase II. In the hydrolysis of insoluble cellulose by the enzyme components, cellobiose was the major product, but glucose was the major product by endoglucanase III.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.659-664
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• 1995

To improve stability of recombinant DNA pLC1 encoding endoglucanase gene and pGL1 encoding $\beta $-glucosidase gene, DNA fragments of genes coding endoglucanase and $\beta $-glucosidase were cloned within the recA gene on a pDR1453, and the pDRE10 and pDRG20 of recombinant plasmids were integrated into the recA gene on the E. coli 1100 chromosomal DNAs. The stability of inheritance was completely maintained in E. coli 1100; Transformants E. coli 1100/pDREIO and pDRG20 were expressed well by recA promoter and increased endoglucanase and $\beta $-glucosidase activities. This method can be used as a model to improve the stability of recombinant plasmid in large scale culture.

• Journal of Plant Biology
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• 제31권3호
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• pp.239-247
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• 1988

Spermidine, spermine and IAA promoted an increase in $\beta$-1, 4-endoglucanase activity in hypocotyls of Glycine max. The optimal concentration for the increase of the enzyme activity was 10-6 M for spermidine, 10-8 M for spermine and 10-6 M for IAA. However, IAA had innocuous effect on arginine decarboxylase and ornithine decarboxylase activites, and the content of polyamine. Such cumulative results suggest that the increase in $\beta$-1, 4-endoglucanase activity by IAA is not attributed by the effect on the biosynthesis of polyamine by IAA but spermidine, spermine and IAA induce cell wall loosening and therefore extension growth of cells.

• Journal of Plant Biology
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• 제32권4호
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• pp.275-283
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• 1989

$\beta$-1, 4-endoglucanase (EC 3, 2, 1, 4) was isolated and purified from Nicotiana tabaccum L. Var. Virginia 115 suspensin cultured cells. The molecular weight as estimated by Sephadex G-100 was about 14, 000. The optimum pH for activity was 5.4. The Km value for carboxymethyl cellulose was 0.18 mg/unit and the enzyme was quite resistant to heating. Polyamine did not affect the activity of $\beta$-1, 4-endoglucanase in vitro but the activity increased drastically when polyamines were added in the suspension culture medium. It is suggested that increase in $\beta$-1, 4-endoglucenase activity due to polyamine might be related to growth of the plant.

• Molecules and Cells
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• 제28권4호
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• pp.369-373
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• 2009

Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and ${\beta}$-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an ${\alpha}$-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and ${\beta}$-glucosidase was able to produce ethanol from ${\beta}$-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.404-409
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• 2008

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and ${\beta}$-glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.