• Journal of Microbiology and Biotechnology
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• 제27권10호
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• pp.1783-1789
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• 2017

Thermostability is an important property of xylanase because high temperature is required for its applications, such as wood pulp bleaching, baking, and animal feedstuff processing. In this study, XynB from Thermobacillus composti, a moderately thermophilic gram-negative bacterium, was modified via site-directed mutagenesis (based on its 3D structure) to obtain thermostable xylanase, and the properties of this enzyme were analyzed. Results revealed that the half-life of xylanase at $65^{\circ}C$ increased from 10 to 50 min after a disulfide bridge was introduced between the ${\alpha}$-helix and its adjacent ${\beta}$-sheet at S98 and N145. Further mutation at the side of A153E named XynB-CE in the C-terminal of this ${\alpha}$-helix enhanced the half-life of xylanase for 60 min at $65^{\circ}C$. Therefore, the mutant may be utilized for industrial applications.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.775-784
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• 2017

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase ($CcXyn-{\Delta}5C$) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but $CcXyn-{\Delta}5C$ contained less ${\alpha}-helices$ (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature ($45^{\circ}C$) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and $CcXyn-{\Delta}5C$, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, $CcXyn-{\Delta}5C$ exhibited a much lower $K_m$ value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of $CcXyn-{\Delta}5C$ was 2.4-times higher than that of CcXyn. These properties make $CcXyn-{\Delta}5C$ a good model for the structure-function study of $({\alpha}/{\beta})_8$-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

• BMB Reports
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• 제38권1호
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• pp.17-23
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• 2005

During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.

• 산업기술연구
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• 제29권A호
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• pp.161-167
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• 2009

The bifunctional Xylanase-Cellulase hybrid protein was constructed by gene fusion. Two genes corresponding to endoxylanase gene (xylS) and endocellulase gene (celA) were amplified by PCR from Bacillus licleniformis NBL420. It was then linked through splicing by overlap extension (SOE) by PCR method. The two resulting fused hybrids, xyl/cel and cel/xyl, which differ by its orientation, were confirmed by its nucleotide sequencings. One of two fusion genes, xyl/cel was successfully expressed into pET22b(+) vector (pxyl/cel) with bifunctional xylanase-cellulase activity. On the contrary, the other cel/xyl fusion protein showed only cellulase activity with much decreased xylanase activity. Enzymatic properties of Xyl/Cel fusion protein were investigated regarding optimum pH, optimum temp, thermostability, and pH stability. It was revealed that Xyl/Cel fusion protein retained the bifunctional xylanase-cellulase activities eventhough two enzymes were connected with each other directly. These informations could be useful for construction of other hybrid proteins as well as increased range of substrate utilization.

• 산업기술연구
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• 제29권A호
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• pp.169-176
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• 2009

The gene encoding endoxylanase (xylS) was isolated from a genomic library of Bacillus licheniformis NBL420. Two positive clones, which harbor 1.5 kb and 0.8 kb inserts respectively, were screened on RBB dyed-xylan plates and the recombinant plasmids were named as pBX3 and pBX5. The nucleotide sequencings of two inserts revealed the existence of common 639 bp of open reading frame which encode 232 amino acids. The xylS gene was successfully subcloned into pET22b(+) vector and overexpressed. Enzymatic properties including optimum pH, optimum temp, thermostability and pH stability were investigated. Activity staining of XylS was identical with that of original Bacillus licheniformis NBL420.

• BMB Reports
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• 제39권1호
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• pp.105-110
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• 2006

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as $90^{\circ}C$. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of $90^{\circ}C$. When using xylan from birchwood as substrate, it exhibits $K_m$ and $V_{max}$ values of $2.6{\pm}0.6\;mg/ml$ and $428{\pm}26\;U/mg$, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to $70^{\circ}C$. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at $70^{\circ}C$ for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1415-1423
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• 2010

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular mass of the F. pinicola xylanase was determined to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size-exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of $70^{\circ}C$. The enzyme showed a $t_{1/2}$ value of 33 h at $70^{\circ}C$ and catalytic efficiency ($k_{cat}=77.4\;s^{-1}$, $k_{cat}/K_m$=22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.

• Journal of Microbiology and Biotechnology
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• 제22권10호
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• pp.1388-1394
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• 2012

A xylanase-producing thermophilic strain, Geobacillus sp. TC-W7, was isolated from a hot spring in Yongtai (Fuzhou, China). Subsequently, the xylanase gene that encoded 407 amino acids was cloned and expressed. The recombinant xylanase was purified by GST affinity chromatography and exhibited maximum activity at $75^{\circ}C$ and a pH of 8.2. The enzyme was active up to $95^{\circ}C$ and showed activity over a wide pH range of 5.2 to 10.2. Additionally, the recombinant xylanase showed high thermostability and pH stability. More than 85% of the enzyme's activity was retained after incubation at $70^{\circ}C$ for 90 min at a pH of 8.2. The activity of the recombinant xylanase was enhanced by treatment with 10 mM enzyme inhibitors (DDT, Tween-20, 2-Me, or TritonX-100) and was inhibited by EDTA or PMSF. Its functionality was stable in the presence of $Li^+$, $Na^+$, and $K^+$, but inhibited by $Hg^{2+}$, $Ni^{2+}$, $Co^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Pb^{2+}$, $Fe^{3+}$, and $Al^{3+}$. The functionality of the crude xylanase had similar properties to the recombinant xylanase except for when it was treated with $Al^{2+}$ or $Fe^{2+}$. The enzyme might be a promising candidate for various industrial applications such as the biofuel, food, and paper and pulp industries.

• 한국균학회지
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• 제39권3호
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• pp.198-204
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• 2011

팔만대장경판과 이를 보존하고 있는 건물인 장경판전으로부터 미생물조사를 실시하였고 조사를 통해 분리된 미생물들이 생산하는 xylanase의 특성을 조사하였다. 조사 당시에 장경판전의 내부 온도는 $20^{\circ}C$ 안팎으로 적절하게 유지되고 있었지만 상대습도는 최대 80%에 이르는 곳도 있어 미생물과 같은 생물적 열화원의 발생으로 문화재의 손상 가능성이 있을 것으로 생각된다. 분리된 5종류의 미생물인 Cladosporium cladosporioides H1, Penicillium citreonigrum H3, Penicillium toxicarium H4, Aspergillus versicolor H6, Acremonium alternatum H7은 모두 재질의 표면을 오염시키고 유기질 (cellulose, xylan, lignin)을 분해시킬 수 있는 곰팡이였다. 이들은 $20^{\circ}C$의 저온에서도 성장이 활발하여 xylanase의 생산도 활발히 이루어졌다. 이들에 의해 생산되는 xylanase는 균주의 종류에 따라 생산 속도의 차이가 있었으며 대부분 산성인 조건에서 xylan을 효과적으로 분해하는 것으로 조사되었다. 또한, Cladosporium cladosporioides H1와 Penicillium toxicarium H4의 xylanase는 산성 조건에서 열에 안정한 것으로 나타났지만 그 외 균주들이 생산한 xylanase는 열에 대한 안정성은 높지 않은 것으로 조사되었다. 이러한 조사 결과는 문화재의 보존 환경의 조절이나 보존제 개발을 하는데 기초자료로 활용될 수 있을 것으로 보이며 생물학적 열화를 일으키는 미생물을 제어할 수 있는 대안을 마련하는데 도움을 줄 것으로 기대된다.

• Mycobiology
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• 제33권2호
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• pp.84-89
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• 2005

Five types of agricultural wastes were used for the production of xylanolytic enzyme by Aspergillus flavus K-03. All wastes materials supported high levels of xylanase and ${\beta}-xylosidase$ production. A high level of proteolytic activity was observed in barley and rice bran cultures, while only a weak proteolytic activity was detected in corn cob, barley and rice straw cultures. Maximum production of xylanase was achieved in basal liquid medium containing rice barn as carbon source for 5 days of culture at pH 6.5 and $25^{\circ}C$. The xylanolytic enzyme of A. flavus K-03 showed low thermostability. The times required for 50% reduction of the initial enzyme activity were 90 min at $40^{\circ}C$, 13 min at $50^{\circ}C$, and 3 min at $60^{\circ}C$. Xylanolytic activity showed the highest level at pH $5.5{\sim}10.5$ and more than 70% of the original activity was retained at pH 6.5 and 7.0. The higher stability of xylanolytic enzymes in the broad range of alkaline pH is useful for utilization of the enzymes in industrial process requiring in alkaline conditions. Moreover, the highest production of xylanolytic enzyme was obtained when 0.5% of rice bran was supplied in basal liquid medium. SDS-PAGE analysis revealed a single xylanase band of approximately 28.5 kDa from the culture filtrates.