• Journal of Microbiology and Biotechnology
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• v.19 no.8
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• pp.823-828
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• 2009

An endo-${\beta}-1$,4-xylanase (${\beta}$-xylanase) from Trichoderma harzianum C4 was purified without cellulase activity by sequential chromatographies. The specific activity of the purified enzyme preparation was 430 units/mg protein on D-xylan. The complementary DNA (cDNA) encoding ${\beta}$-xylanase (xynII) was amplified by PCR and isolated from cDNA PCR libraries constructed from T. harzianum C4. The nucleotide sequence of the cDNA fragment contained an open reading frame of 663 bp that encodes 221 amino acids, of which the mature protein is homologous to several ${\beta}$-xylanases II. An intron of 63 bp was identified in the genomic DNA sequence of xynII. This gene was expressed in Saccharomyces cerevisiae strains under the control of adh1 (alcohol dehydrogenase I) and pgk1 (phosphoglycerate kinase I) promoters in 2 ${\mu}$-based plasmids, which could render recombinants able to secrete ${\beta}$-xylanase into the media.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.254-264
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• 2010

To obtain an efficient natural lignocellulolytic complex enzyme, we screened an efficient lignocellulose-degrading composite microbial system (XDC-2) from composted agricultural and animal wastes amended soil following a long-term directed acclimation. Not only could the XDC-2 degrade natural lignocelluloses, but it could also secrete extracellular xylanase efficiently in liquid culture under static conditions at room temperature. The XDC-2 degraded rice straw by 60.3% after fermentation for 15 days. Hemicelluloses were decomposed effectively, whereas the extracellular xylanase activity was dominant with an activity of 8.357 U/ml on day 6 of the fermentation period. The extracellular crude enzyme noticeably hydrolyzed natural lignocelluloses. The optimum temperature and pH for the xylanase activity were $40^{\circ}C$ and 6.0. However, the xylanase was activated in a wide pH range of 3.0-10.0, and retained more than 80% of its activity at $25-35^{\circ}C$ and pH 5.0-8.0 after three days of incubation in liquid culture under static conditions. PCR-DGGE analysis of successive subcultures indicated that the XDC-2 was structurally stable over long-term restricted and directed cultivation. Analysis of the 168 rRNA gene clone library showed that the XDC-2 was mainly composed of mesophilic bacteria related to the genera Clostridium, Bacteroides, Alcaligenes, Pseudomonas, etc. Our results offer a new approach to exploring efficient lignocellulolytic enzymes by constructing a high-performance composite microbial system with synergistic complex enzymes.

• 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 Chemical Engineering Research
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• v.54 no.6
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• pp.822-829
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• 2016

Our aim was to optimize the production of cellulase-free thermoactive xylanase by Aureobasidium pullulans CBS 135684 with statistical methodology based on experimental designs. Among eleven variables, the nutrient sources that had significant effect on xylanase production were corncob, $(NH_4)_2SO_4$, xylose, $KH_2PO_4$ and tween 80, identified by the initial screening method of Plackett-Burman. The optimum concentrations of these five components were subsequently investigated using response surface methodology. The optimal concentrations ($g{\cdot}l^{-1}$) for maximum production of xylanase were corncob, 39.0; $(NH_4)_2SO_4$, 3.0; xylose, 1.8; $KH_2PO_4$ 1.4; and tween 80, 1.4, respectively. An improved xylanase yield of $8.74{\pm}0.84U{\cdot}ml^{-1}$ was obtained with optimized medium which is 2.1-fold higher production than previously obtained results ($4.10{\pm}0.10U{\cdot}ml^{-1}$) after 48 h of cultivation. In addition, the xylanase production under optimal condition reached $10.09{\pm}0.27U{\cdot}ml^{-1}$ after 72 h of cultivation.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.316-325
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• 2012

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.8
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• pp.1141-1146
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• 2005

A trial was conducted to evaluate the level of wheat substituted for corn in a traditional corn-soy diet and the xylanase supplementation effect on the growth performance and nutrient digestion of broiler chickens. This experiment was a randomized design with a 4${\times}$2 factorial arrangement with four levels of wheat substitution and two levels of enzyme inclusion in the diet. Wheat replaced 0, 25, 50 or 100% corn with or without 1 g/kg xylanase supplementation in iso-nitrogenous and iso-calorific experimental diets. The results showed that in the growing period, broilers attained the highest (p<0.05) body weight gain, feed intake, and relative small intestine weight when wheat was substituted at 25% for corn. The relative caecum weight increased (p<0.05) linearly with increasing levels of wheat substitution for corn. However, during the finishing period and entire experimental period from 0 to 6 weeks, no significant difference was shown in the growth performance among all treatments. Xylanase inclusion significantly improved the body weight gain, fat availability (p<0.01) and diet metabolisable energy (p<0.1) but decreased (p<0.05) the relative GI tract weight during the growing period. The digesta viscosity of 6-week old broilers was also decreased (p<0.05). It appears that wheat substituted for corn did not affect the growth performance, nutrient digestion, and the digesta viscosity of chickens. It is acceptable to completely substitute wheat for corn. Xylanase supplementation improved performance.

• Food Engineering Progress
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• v.14 no.3
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• pp.263-270
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• 2010

An alkalophilic microorganism named DK-2386, which produces xylanase, was isolated from soil of Taejo-mountain, Cheonan-si, Chungnam, Korea. The isolated strain was characterized as Gram-positive, with size of 0.4${\times}$2.5 ${\mu}$m, spore forming, anaerobic, catalase positive, possessed with hydrolysis abilities of casein, starch, sodium carboxy methyl cellulose, and xylan, reduction of nitrate to nitrite, resistant against lysozyme, urease positive, and motility positive. The color of culture broth was reddish yellow. The strain DK-2386 was identified as Bacillus agaradhaerens by whole cell fatty-acid composition analysis and 16S rDNA sequence analysis. However, it was not identical to Bacillus agaradhaerens 40952 obtained from the Korean Culture Center of Microorganism in its colour of culture broth. Therefore, we have named the newly isolated strain as Bacillus agaradhaerens DK-2386.

• Journal of Applied Biological Chemistry
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• v.60 no.1
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• pp.79-86
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• 2017

Microbes in forest are very important due to not only to enhance soil fertility but also maintain a healthy ecosystem by supplying the energy available to living organisms by producing various kinds of enzymes related to degradation of lignocellulosic biomass. In order to isolate a lignocellulosic biomass degrading bacterial strain from the Jurassic park located in Gyeongnam National University of Science and Technology, We used the Luria-Bertani-Carboxymethyl cellulose (CMC) agar trypan blue method containing 0.4 % carboxymethyl cellulose and 0.01 % trypan blue. As a result, we isolated a bacterial strain showing both activity on the CMC and xylan. To identify the isolated strain, 16S rRNA sequencing and API kit analysis were used. The isolated strain turned out to belong to Bacillus species and then named Bacillus sp. GJY. In the CMC zymogram analysis, it showed that one active band of about 28kDa in size is present. Xylan zymogram analysis also showed to have one active band of about 25kDa in size. The optimal growth temperature of Bacillus sp. GJY was $37^{\circ}C$. The maximal activities of CMCase and xylanase were 12 hour after incubation. The optimal pH and temperature for CMCase were 5.0 and $40^{\circ}C$, respectively, whereas the optimal pH and temperature for xylanase was 4.0 and $40^{\circ}C$. Both activities for CMCase and xylanase showed to be thermally stable at 40and $50^{\circ}C$, while both activities rapidly decreased at over $60^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.370-375
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• 2016

Two xylanase genes were cloned into Escherichia coli from Bacillus sp. YB-1401 and B. amyloliquefaciens YB-1402, which had been isolated as mannanase producer from home-made doenjang, respectively, and their nucleotide sequences were determined. Both xylanase genes consisted of 642 nucleotides, encoding polypeptides of 213 amino acid residues. The deduced amino acid sequences of the YB-1401 and YB-1402 xylanase, designated Xyn1401 and Xyn1402, differed from each other by single amino acid residue, Asn for Xyn1401 and Lys for Xyn1402, corresponding to amino acid position of 127. Their amino acid sequences were highly homologous to those of xylanases belonging to the glycosyl hydrolase family 11. The 28 amino acid stretch in the N-terminus of both enzymes was predicted as signal peptide by SignalP4.1 server. Both xylanases were localized at the level of 91−94% in culture filtrate of the recombinant E. coli cells, suggesting they were secreted efficiently in E. coli cells. The optimal reaction conditions were 50℃ and pH 6.0 for Xyn1401, and 55℃ and pH 6.5 for Xyn1402, respectively, indicating one amino acid difference from each other affected pH and temperature profiles of their activities. In addition, their thermostabilities were somewhat different from each other.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.512-521
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• 2016

A bacterial strain was isolated from a soil sample collected on Jeju Island, Korea. The strain, designated WJ-2, exhibited a high xylanase activity, whereas cellulase activity was not detected. The 16S rRNA gene sequence of WJ-2 was highly similar to type strains of the genus Streptomyces. A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain WJ-2 is phylogenetically related to Streptomyces atrovirens. Furthermore, DNA-DNA hybridization analysis confirmed that strain WJ-2 is a novel subspecies of Streptomyces atrovirens. The genomic DNA G+C content was 73.98 mol% and the major fatty acid present was anteiso-C15:0 (36.19%). The growth and xylanase production of strain WJ-2 were significantly enhanced by using soytone and xylan as nitrogen and carbon sources, respectively. Crude enzyme preparations from the culture broth of strain WJ-2 exhibited maximal total xylanase activities at pH 7.0 and $55^{\circ}C$. Thin-layer chromatography analysis revealed that the crude enzyme degrades beechwood xylan to yield xylobiose and xylotriose as the principal hydrolyzed end products.