• BMB Reports
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• v.31 no.1
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• pp.53-57
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• 1998

The CMCax gene from Acetobacter xylinum ATCC 23769 was cloned and expressed in E. coli. With this gene, three gene products - mature CMCax, CMCax containing signal peptide(pre-CMCax), and a glutathione-S-transferase(GST)-CMCax fusion enzyme - were expressed. CMCax and pre-CMCax are aggregated to multimeric forms which showed high CMC hydrolysis activity, whereas GST-CMCax was less aggregated and showed lower activity, indicating that oligomerization of CMCax controbutes to the cellulose hydrolysis activity to achieve greater efficiency. The enzyme was identified to be an $\beta$-1,4-endoglucanase, which catalyzes the cleavage of internal $\beta$-1,4-glycosidic bonds of cellulose. The reaction products, cellobiose and cellotriose, from cellopentaose as a substrate, were identified by HPLC. Substrate specificity of cellotetraose by this enzyme was poor, and the reaction products consisted of glucose, cellobiose, and cellotriose in a very low yield. Theses results suggested that cellopentaose might be the oligosaccharide substrate consisting of the lowest number of glucose. The optimum pH of CMCax and pre CMCax was about 4.5, whereas that of GST-CMCas was rather broad at pH 4.5-8. The physiological significance of cellulose-hydrolyzing enzyme, CMCax, having such low $\beta$-1,4-endoglucanase activity and low optimum pH in cellulose-producing A. xylinum is not clearly known yet, but it seems to be closely related to the production of cellulose.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2005.06a
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• pp.245-247
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• 2005

A cellulolytic fungus isolated from Agave plantation in northeastern Thailand was identified as Acrophialophora nainiana. The fungus was capable of growing at pH between 3 - 7 and 25 - 45 $^{\circ}C$, with the optimum conditions at pH 5.0 and 40 $^{\circ}C$. The wild isolate produced cellulases, comprising of exoglucanase (0.019 U/mg protein), endoglucanase (0.366 U/mg protein), and ${\beta}$-glucosidase (0.001 U/mg protein). Mutations with UV and NTG produced the UV 10-2 mutant with cellulases activities including exoglucanase (0.093 U/mg protein), endoglucanase (0.585 U/mg protein), and ${\beta}$-glucosidase (0.013 U/mg protein). Purification of the enzymes with ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography yielded the maximal cellulase specific activities of 2.736 U/mg protein (exoglucanase), 0.235 U/mg protein (endoglucanase), and 0.008 U/mg protein (${\beta}$-glucosidase). The mutant's cellulases were the most active at pH 5.0 and 60 $^{\circ}C$. Ion-exchange chromatography revealed that A. nainiana UV 10-2 cellulases were comprised of two peaks with one peak showing the single endoglucanase activity while the other peak showed a mixture of the three enzyme activities. Production of A. nainiana UV 10-2 cellulases using banana leaf stalk as the sole carbon source gave comparable yields to that of the pure ${\alpha}$-cellulose. The enzymes were used in the simultaneous saccharification and fermentation (SSF) of plant residue (Coix aquatica) along with Kluveromyces marxianus to produce ethanol. Moreover, when the enzymes were used in the bioscouring process of fabric, the desiravle traits of textile processing including immediate water absorbency, increased in whiteness and reduction of yellowness of the treated fabric were observed.

• Korean Journal of Microbiology
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• v.36 no.1
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• pp.20-25
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• 2000

One of endoglucanases(F-II-II) was purified from the culture filtrate of Trichoderma sp. C-4 through two step procedures including chromatography on Sephacryl S-200 and Sephacryl S-100. The molecular weight of the enzyme was determined to be about 26,000 by SDS-PAGE and the isoelectric point as 8.0 by analytical isoelectric focusing. The optimum temperature of the enzyme was $50^{\circ}C$ and the optimum pH was 5.0. No loss of activity was observed when the enzyme was preincubated at $50^{\circ}C$ for 24 hours. The specific activity of the enzyme toward carboxymethylcellulose (CMC) was estimated to be 776.2 U/mg. The internal amino acid sequence was analysed.

• The Korean Journal of Mycology
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• v.17 no.2
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• pp.57-65
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• 1989

The effect of various cellulosic and hemicellulosic substrates on the induction of cellulase and xylanase complexes in Aapergillus nidulans was investigated. The most efficient substrates for the induction of cellulase and xylanase complexes were carboxymethylcellulose for endoglucanase, cellobiose for ${\beta}-glucosidase$, and xylan for endoxylanase and ${\beta}-xylosidase$, respectively. However, the mixtures of these substrates, especially CMC-xylan and CMC-xylan-laminarin mixture, were much more effective not only for the enhancement of the biosynthesis of all the cellulase and xylanase complexes but also for the balanced production of these enzyme components than individual substrate. The polyacrylamide gel electrophoresis followed by activity staining showed the variation in the patterns and relative intensity of ${\beta}-glucosidase$, endoglucanase and endoxylanase components in individual enzyme preparations from A. nidulans cultures grown on different substrates. These results suggest that the biosynthesis is of cellulase and xylanase systems in A. nidulans is regulated in coordination at the level of induction.

• Journal of Microbiology and Biotechnology
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• v.24 no.8
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• pp.1073-1080
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• 2014

A total of 10 cellulase-producing bacteria were isolated from soil samples irrigated with paper and pulp mill effluents. The sequencing of 16S rRNA gene revealed that all isolates belonged to different species of genus Bacillus. Among the different isolates, B. subtilis IARI-SP-1 exhibited a high degree of ${\beta}$-1,4-endoglucanase (2.5 IU/ml), ${\beta}$-1,4-exoglucanase (0.8 IU/ml), and ${\beta}$-glucosidase (0.084 IU/ml) activity, followed by B. amyloliquefaciens IARI-SP-2. CMC was found to be the best carbon source for production of endo/exoglucanase and ${\beta}$-glucosidase. The ${\beta}$-1,4-endoglucanase gene was amplified from all isolates and their deduced amino acid sequences belonged to glycosyl hydrolase family 5. Among the domains of different isolates, the catalytic domains exhibited the highest homology of 93.7%, whereas the regions of signal, leader, linker, and carbohydrate-binding domain indicated low homology (73-74%). These variations in sequence homology are significant and could contribute to the structure and function of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.5 no.1
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• pp.26-30
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• 1995

The cytoplasmic endo-$\beta$-l, 4-glucanase (endoglucanase) was purified from cell extracts of Escherichia coli (pBS1) transformant carrying the Bacillus subtilis endo-$\beta$-l, 4-glucanase gene after full growth, and its molecular weight was found to be 52 kilodaltons (kDa). The endo-$\beta$-l, 4-glucanase isolated from the periplasmic space was smaller than 52-kDa cytoplasmic enzyme. The 52-kDa endoglucanase was found to be cleaved in the periplasm and finally converted to 34.5-kDa protein. Small amounts of both 52-kDa and 34.5-kDa proteins were secreted into the culture broth. The cleavage took place in the C-terminal portion of the enzyme. The N-terminal amino acid residues of both 52-kDa and 34.5-kDa enzymes were determined to be the same, Ala, the 30th residue of the primary translation product. Cleavage of the C-terminal portion showed to have no significant effect on the basic enzyme properties.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.265-270
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• 2010

Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of the cel7B gene in its chromosomal DNA. The expression levels of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that the two genes were coordinately expressed, and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimally active at $60^{\circ}C$ and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, and 5-fold higher enzyme activities toward carboxymethyl cellulose, barley $\beta$-glucan, and PASC, respectively, in comparison with that of Cel5A. However, their activities toward pNPC and Avicel showed minor differences. The results suggested that Cel7B is a strict endoglucanase, whereas Cel5A showed processivity because of its relative higher ability to hydrolyze the crystal cellulose.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.607-615
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• 2018

Objective: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. Methods: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. Results: The maximum activity of recombinant Cel5A (rCel5A) was observed at $50^{\circ}C$ and pH 4.0. The enzyme was constant at the temperature range of $20^{\circ}C$ to $40^{\circ}C$ but also, at the pH range of 3 to 9. The metal ions including $Ca^{2+}$, $K^+$, $Ni^{2+}$,$Mg^{2+}$, and $Fe^{2+}$ increased the endoglucanase activity but the addition of $Mn^{2+}$, $Cu^{2+}$, and $Zn^{2+}$ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and $45.66{\mu}mol/min/mg$. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was $96.69(s^{-1})$ and 6.88 (mL/mg/s), respectively. Conclusion: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

• Bulletin of the Korean Chemical Society
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• v.16 no.8
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• pp.720-725
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• 1995

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.219-225
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• 1988

Two isozymes of cellobiohydrolase and fifteen isozymes of endoglucanase from Trichodema viride QM 9414 were purified by ammonium sulfate fractionation, Sephadex G-100 column chromatography, DEAE-Sephadex A-50 column chromatography and preparative electrophoresis. The purified cellobiohydrolnse had a molecular weight of 71,000 estimated by electrophoresis and amino acid analysis showed its main amino acids to be in the form of aspartic acid and glutamic acid result-ing from its low pI point of 3.81. The optimum pH and temperature were 5.1 and 5$0^{\circ}C$ respectively.