• Korean Journal of Microbiology
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• v.42 no.1
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• pp.67-72
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• 2006

The nucleotide sequence of the cloned cellulolytic xylanase gene (bglBC2) from B. circulans ATCC21367 was determined. bglBC2 consists of an 1,224 bp open reading frame (ORF) coding for a polypeptide of 407 amino acids with a deduced molecular weight of 45 kDa. The Shine-Dalgarno (SD) sequence (5'-AAAGGAG-3') was found 9 bp upstream of the initiation codon, ATG. A promoter region corresponding closely to the B. subtilis consensus sequence (-35: TTGACA,-10: TATAAT) was detected, the putative -35 and -10 sequences of which were TTTACA and TATACT, respectively. The deduced amino acid sequence of the cellulolytic xylanase showed 97% homology with that of the alkaline $endo-\beta-1,4-glucanase$ from B. circulans KSM-N257, 75% homology with that of the $endo-\beta-1,3-1,4-glucanase$ from B. circulans WL-12, and 45% homology with that of the $endo-\beta-1,4-glucanase$ (cellulase) from Bacillus sp. KSM-330. The bglBC2 sequence was deposited in Gen-Bank under the accession number AY269256.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.196-202
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• 2000

A gene for cellulolytic xylanase of Bacillus circulnns ATCC21365 was cloned on pUC 19 in Eschwichia coli. The recombinant plasniid pXLI80 contained an 1.8 id, inselt composed of0.5 kb and 1.3 kb PslI fragments derived from B, circulans. The 0.5 kh fragment in the upstream region of 1.3 kb one was confirmed lo be indispensable for not only expression but also hyperexpression of the cloned gene. The transformant overproduced the xylanase 135 times greater than that produced by the orlginal B circulnns. The optimum pH and temperature of the cloned enzyme we]-e pH 5.2 and $60^{\circ}C$, respectively. Heal pretl-eatment at TEX>$55^{\circ}C$C for 1 Indid not cause inhibition of the activity of this enzyme. The elm.ynie could hydl-olyre CMC and lichenan as well as xylan to produce xylose(or GI), xylohiose(or G2) and xylolnose(or G3) as inah products. Hence We defined the cloned enzyme as a cellulolytic xylanase. The SDS-PAG electrophoretic mobility and zyiiogram of this enzyme derived from whole cell extracts or c~~lture supematants or E. coli(pXL180) indicated a molecular weight of 45,000 and nonprocessing of the enzyme in the peilplasln of E. coli.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.449-452
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• 2002

A filamentous microorganism was isolated from completely rotten wood for the production of cellulolytic enzyme. The Trichoderma sp. FJ1 produced a large amount of cellulolytic enzymes, such as CMC, xylanase, ${\beta}-glucosidase$, and avicelase. For the production of the enzymes, when cellulolsic wastes were used as carbon sources of strain FJ1, rice straw showed higher enzyme activities than sawdust and pulp. The activities of CMC, xylanase, ${\beta}-glucosidase$, and avicelase were 2.95, 5.89, 0.45, and 0.12 U/ml in use of rice straw, respectively. To enhance production of the enzymes, the mixture substrate of rice straw and commercial cellulosic materials was investigated as carbon sources. The highest activities of CMCase, ${\beta}-glucosidase$, and avicelase were found in the mixture of rice straw and avicel, particularly rice straw:avicel (50:50), and the highest xylanase was obtained in the mixture ratio of 71:29. Bacto peptone addition of 0.1% showed enhanced production of the cellulolytic enzymes in which the activities of CMCase, xylanase ${\beta}-glucosidase$, and avicelase were 19.23, 27.18, 1.28, and 0.53 U/ml, respectively. The production of the enzymes using rice straw was efficiently induced in present of avicel and pulp containing high content of cellulose. Consequently, the filamentous microorganism, strain FJ1 utilized various cellulosic wastes as carbon sources and cellulases productivities were excellent compared to those of others strains reported previously, suggesting that the strain FJ1 will be expected as a favorable candidate for biological saccharification of cellulosic wastes in further.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1604-1609
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• 2008

A series of in vitro and in vivo experiments were conducted to investigate the effects of the mixture of Tween 80 and cellulolytic enzymes (xylanase and cellulase) on total tract nutrient digestibility and rumen cellulolytic bacterial adhesion rates in Holstein steers. Ground timothy hay sprayed with various levels of Tween 80 and cellulolytic enzymes was used as substrates in an in vitro experiment to find out the best combinations for DM degradation. The application level of 2.5% (v/w) Tween 80 and the combination of 5 U xylanase and 2.5 U cellulase per gram of ground timothy hay (DM basis) resulted in the highest in vitro dry matter degradation rate (p<0.05). Feeding the same timothy hay to Holstein steers also improved in vivo nutrient (DM, CP, CF, NDF and ADF) digesibilities compared to non-treated hay (p<0.05). Moreover, Tween 80 and enzyme combination treatment increased total ruminal VFA and concentrations of propionic acid and isovaleric acid with decreased acetate to propionate ratio (p<0.001). However, adhesion rates of Fibrobacter succinogenes and Ruminococcus flavefaciens determined by Real Time PCR were not influenced by the treatment while that of Ruminococcus albus was decreased (p<0.05). The present results indicate that a mixture of Tween 80 and cellulolytic enzymes can improve rumen environment and feed digestibility with variable influence on cellulolytic bacterial adhesion on feed.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.893-903
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• 2010

A cellulolytic and xylanolytic enzyme complex-producing alkalothermoanaerobacterium strain, Tepidimicrobium xylanilyticum BT14, is described. The cell was Grampositive, rod-shaped, and endospore-forming. Based on 16S rRNA gene analysis and various lines of biochemical and physiological properties, the strain BT14 is a new member of the genus Tepidimicrobium. The strain BT14 cells had the ability to bind to Avicel, xylan, and corn hull. The pH and temperature optima for growth were 9.0 and $60^{\circ}C$, respectively. The strain BT14 was able to use a variety of carbon sources. When the bacterium was grown on corn hulls under an anaerobic condition, a cellulolytic and xylanolytic enzyme complex was produced. Crude enzyme containing cellulase and xylanase of the strain BT14 was active in broad ranges of pH and temperature. The optimum conditions for cellulase and xylanase activities were pH 8.0 and 9.0 at $60^{\circ}C$, respectively. The crude enzyme had the ability to bind to Avicel and xylan. The analysis of native-PAGE and native-zymograms indicated the cellulosebinding protein showing both cellulase and xylanase activities, whereas SDS-PAGE zymograms showed 4 bands of cellulases and 5 bands of xylanases. Evidence of a cohesinlike amino acid sequence seemed to indicate that the protein complex shared a direct relationship with the cellulosome of Clostridium thermocellum. The crude enzyme from the strain BT14 showed effective degradation of plant biomass. When grown on corn hulls at pH 9.0 and $60^{\circ}C$ under anaerobic conditions, the strain BT14 produced ethanol and acetate as the main fermentation products.

• Journal of Applied Biological Chemistry
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• v.61 no.1
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• pp.59-67
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• 2018

This study was conducted to isolate the cellulolytic bacteria able to grow on LB- Carboxymethyl cellulose (CMC) agar trypan blue medium from the mixed forest and Larix leptolepis stands. Three bacterial strains with high activity against both CMC and xylan were isolated. Both API kit test and 16S rRNA gene sequence analysis revealed that the three different isolates belong to the gene Bacillus. Therefore, the isolates named as Bacillus sp. EFL1, Bacillus sp. EFL2, and Bacillus sp. EFP3. The optimum growth temperature of Bacillus sp. EFL1, EFL2, and EFP3 were $37^{\circ}C$. The optimum temperature for CMCase and xylanase from Bacillus sp. EFL1 were $50^{\circ}C$. The optimum pH of Bacillus sp. EFL1 xylanase was pH 5.0 but the optimum pH of CMCase from Bacillus sp. EFL1 was pH 6.0. The optimum temperature of CMCase and xylanase from Bacillus sp. EFL2 was $60^{\circ}C$, respectively. The optimum pH of CMCase of Bacillus sp. EFL2 was 5.0, whereas xylanase showed high activity at pH 3.0-9.0. The optimum temperature for CMCase and xylanase of Bacillus sp. EFP3 was $50^{\circ}C$. The optimum pH for CMCase and xylanse was 5.0 and 4.0, respectively. CMCases from Bacillus sp. EFL1, EFL2, and EFP3 were thermally unstable. Although xylanase from Bacillus sp. EFL1 and EFP3 showed to be thermally unstable, xylanase from Bacillus sp. EFL2 showed to be thermally stable. Therefore, Bacillus sp. EFL2 has great potential for animal feed, biofuels, and food industry applications.

• Microbiology and Biotechnology Letters
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• v.30 no.2
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• pp.172-176
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• 2002

A filamentous microorganism, strain FJ1, was isolated from completely rotten wood for the production of cellulolytic enzymes. For the production of the enzymes, cellulolsic wastes were used as carbon sources of strain FJ1 and rice straw showed higher enzyme activities than sawdust and pulp. The activities of CMCase, xylanase, $\beta$-glucosidase, and avicelase were 2.95, 5.89, 0.45, and 0.12 unit/ml by use of rice straw, respectively. To enhance production of the enzymes, the mixture substrate of rice straw and cellulosic materials were investigated as carbon sources. The highest activities of CMCase, $\beta$-glucosidase, and avicelase were found in the mixture of rice straw (0.5%, w/v) and avicel (0.5%, w/v), and the highest xylanase was obtained at the mixture ratio of 0.71%(w/v) and 0.29%(w/v). Addition of 0.1%(w/v) peptone showed enhanced production of the cellulolytic enzymes in which the activities of CMCase, xylanase, $\beta$-glucosidase, and avicelase were 19.23, 27.18, 1.28, and 0.53 unit/ml, respectively. The production of the enzymes using rice straw was efficiently induced in the presence of avicel and pulp containing cellulose. In particular, a medium composed of rice straw (0.5%, w/v) and pulp (0.5%, w/v) yielded larger cellulolytic enzymes: CMCase 24.3 unit/ml, xylanase 38.7 unit/ml, $\beta$-glucosidase 1.5 unit/ml, and avicelase 0.6 unit/ml. The filamentous microorganism, strain FJ1 utilized various cellulosic wastes as carbon sources and will be expected as a favorable candidate for biological saccharification of cellulosic wastes.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.1
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• pp.50-58
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• 2013

A facultative bacterium producing cellulolytic and hemicellulolytic enzymes was isolated from the rumen of a native Korean goat. The bacterium was identified as a Bacillus licheniformis on the basis of biochemical and morphological characteristics and 16S rDNA sequences, and has been designated Bacillus licheniformis JK7. Endoglucanase activities were higher than those of ${\beta}$-glucosidase and xylanase at all temperatures. Xylanase had the lowest activity among the three enzymes examined. The optimum temperature for the enzymes of Bacillus licheniformis JK7 was $70^{\circ}C$ for endoglucanase (0.75 U/ml) and $50^{\circ}C$ for ${\beta}$-glucosidase and xylanase (0.63 U/ml, 0.44 U/ml, respectively). All three enzymes were stable at a temperature range of 20 to $50^{\circ}C$. At $50^{\circ}C$, endoglucanse, ${\beta}$-glucosidase, and xylanase had 90.29, 94.80, and 88.69% residual activity, respectively. The optimal pH for the three enzymes was 5.0, at which their activity was 1.46, 1.10, and 1.08 U/ml, respectively. The activity of all three enzymes was stable in the pH range of 3.0 to 6.0. Endoglucanase activity was increased 113% by $K^+$, while $K^+$, $Zn^+$, and tween 20 enhanced ${\beta}$-glucosidase activity. Xylanase showed considerable activity even in presence of selected chemical additives, with the exception of $Mn^{2+}$ and $Cu^{2+}$. The broad range of optimum temperatures (20 to $40^{\circ}C$) and the stability under acidic pH (4 to 6) suggest that the cellulolytic enzymes of Bacillus licheniformis JK7 may be good candidates for use in the biofuel industry.

• KSBB Journal
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• v.17 no.2
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• pp.195-202
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• 2002

The production conditions of cellulolytic enzymes by a fungus, strain FJ1, were optimized using response surface analysis. The culture factors which largely affected the production of enzymes such as cultivation time, carbon source concentration, nitrogen source concentration, and composition ratio of carbon sources were employed. Optimizedconditions of the factors above corresponding to each cellulolytic enzyme production were as fellowing: CMCase production was obtained in the conditions of cultivation time of 5.4 days, carbon source concentration of 3.5%, nitrogen source concentration of 0.6%, and composition ratio of carbon sources of 52:48 (avicel:CMC), xylanase appeared in the conditions of 5.3 days, 3.5%, 0.8%, and 54:46, respectively, and $\beta$-glucosidase were 7.0 days, 5.0%, 1.0%, and 83:17, respectively, and avicelase were 6.5 days, 4.0%, 0.9%, and 64:36, respectively. The activities of CMCase, xylanase, p-glucosidase, and avicelase predicted by the response surface methodology were 33.5, 52.6, 2.88, and 1.84 U/mL, respectively, and $\beta$-glucosidase activity was enhanced up to 74% when compared to that obtained in the experimental conditions.

• Journal of Animal Science and Technology
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• v.45 no.6
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• pp.1019-1030
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• 2003

The study was conducted to isolate and identify highly fibrolytic anaerobic fungi from the guts of a Hanwoo steer and a Korean native goat, and then investigate the characterization of cellulolytic activity of an anaerobic fungus. Twenty-one anaerobic fungal colonies were isolated in the study, in which 16 colonies were isolated from the rumen contents of the Hanwoo steer and 5 colonies from the duodenal fluids of the Korean native goat. Four anaerobic fungi were selected based on higher cellulolytic enzyme activities to identify under a optical microscope. NLRI-M003 and -T004 belong to Neocallimastix genus and NLRI-M014 belongs to Piromyces genus based on the morphology of their thallus, sporangia, rhizoid and the number of flagella. NLRI-M001 appeared to be an unknown strain of anaerobic fungi due to its different morphology from existing types of anaerobic fungi, though the morpholgoy is similar to Orpinomyces sp. Supplementation of 2% anaerobic fungal culture(NLRI-M003) in rumen-mixed microorganisms increased in vitro DM degradability of rice straw and filter paper up to 4 and 11%, respectively, compared with non-supplementation(control). CMCase and xylanase activities in in vitro culture were also higher in 2% fungal supplementation than controls in both rice straw and filter paper substrates.