• 한국미생물·생명공학회지
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• 제45권2호
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• pp.155-161
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• 2017

Paenibacillus woosongensis의 유전체 부분 염기서열로부터 유추된 xylanase 유전자를 PCR 증폭하여 클로닝하고 염기서열을 결정하였다. 클로닝된 xylanase 유전자는 xyn11B로 명명되었으며, 356 아미노산으로 구성된 단백질을 코드하는 1,071 뉴클레오티드로 이루어졌다. Xyn11B의 아미노산 배열을 분석한 결과 glycosyl hydrolase family 11에 속하는 xylanase와 상동성이 높은 활성영역과 탄수화물 결합영역을 포함하고 있는 다영역 효소로 확인되었다. SignalP4.1 server로부터 아미노 말단의 26개 잔기가 signal peptide로 예측되었다. DEAE-Sepharose와 Phenyl-Separose 컬럼 크로마토그래피 과정을 통해 xyn11B 유전자를 함유한 재조합 대장균의 균체 파쇄상등액으로부터 Xyn11B를 부분 정제하였다. 부분 정제된 Xyn11B의 반응특성을 조사한 결과 pH 6.5와 $50^{\circ}C$에서 최대 반응활성을 보였고 birchwood xylan이나 oat spelt xylan보다 arabinoxylan에 대한 활성이 높았으며 셀룰로스, 만난과 para-nitrophenyl-${\beta}$-xylopyranoside에 대해서는 분해활성이 없었다. Xyn11B의 활성은 $Ca^{2+}$과 $Mg^{2+}$에 의해서는 약간 증가한 반면에 $Cu^{2+}$, $Ni^{2+}$, $Fe^{3+}$, $Mn^{2+}$에 의해서는 크게 저해되었고 SDS에 의해서 완전히 저해되었다.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1514-1519
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• 2009

A gene encoding the xylanase of Bacillus subtilis AMX-4 isolated from soil was cloned into Escherichia coli and the gene product was purified from the cell-free extract of the recombinant strain. The gene, designated xylA, consisted of 639 nucleotides encoding a polypeptide of 213 residues. The deduced amino acid sequence was highly homologous to those of xylanases belonging to glycosyl hydrolase family 11. The molecular mass of the purified xylanase was 23 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum of 6.0-7.0 and a temperature optimum of $50-55^{\circ}C$. Xylanase activity was significantly inhibited by 5 mM $Cu^{2+}$ and 5 mM $Mn^{2+}$, and noticeably enhanced by 5 mM $Fe^{2+}$. The enzyme was active on xylans including arabinoxylan, birchwood xylan, and oat spelt xylan, but it did not exhibit activity toward carboxymethylcellulose or p-nitrophenyl-$\beta$-xylopyranoside. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylobiose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylotriose.

• 미생물학회지
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• 제31권1호
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• pp.63-71
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• 1993

A xylanase (xylanase I) was purified 11.9-fold from the culture filtrate of Trichoderma koningii ATCC 26113 by the column chromatography on Sephadex G-75, SP-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-50 with an overall yield of 8.2%. The molecular mass determined by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to be a monomeric polypeptide of ca. 35 kDa. The isoelectric point of the enzyme was estimated to be 9.3. The optimal reaction pH and temperature are 5.8 and 55.deg.C, respectively. The enzyme is stable up to 60.deg.C, while 78% of its activity is lost after the incubation for 10 min at 70.deg.C. The enzyme hydrolyzes sylan with relatively high activity, as well as carboxymethyl cellulose and avicel. The $K_{m}$ values of the enzyme for oat-spelf sylan, larchwood xylan and Avicel were 3.5, 1.6 and 10. 1 mg/ml, respectively. The enzyme hydrolyzed oat-spelt sylan to sylose, sylobiose, sylotriose and arabionoxylobiose, while it degraded larchwood xylan to xylose, xylobiose, xylotriose and arabionoxylobiose, while it degraded larchwood xylan to xylose, xylobiose and xylotriose as the major products. The hydrolysis patterns indicate that xylanase I is endo-enzyme.e.

• 한국미생물·생명공학회지
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• 제29권4호
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• pp.221-226
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• 2001

S. thermocyaneoviolaceus 가 생산하는 xylanases를 황산암모늄에 의한 염석 및 투석에 의해 조제한 효소를 이용하여 자일로오리고당을 생산하기 위한 최적 반응조건을 설정하였다. 자알로올리고당 생산을 위한 기질로는 oat spelt xyaln 보다 birchwood xylan을 사용하는 것이 더 좋았다. 반응온도의 영향을 조사한 결과 효소의 열 안정성과 자일로올리고당의 생선량 등을 고려할 때 $60^{\circ}C$에서 반응하는 것이 가장 좋았다 효소의 농도 pH의 영향및 반응시간의 영향을 조사한 결과 10 unit/ml xylanases를 첨가하여 pH 6.0에서 12 시간 반응시켰을 때 가장 많은 양의 자일로올리고당을 생산할 수 있었다. 이러한 최적 반응 조건에서 10% birchwood xylan 으로 부터 생산할 수 있는 자일로올리고당의 조성는 X2, X3, X4, X5 및 X6가 각각 20.1, 8.9 , 4.5, 16.2 및 9.1 g/I 이었으며 이때 생산되는 총 자일로올리고당 (X2~X6)은 58.8 g/I 이었다.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1132-1138
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• 2006

Three types of xylanases (EC 3.2.1.8) were detected in the strain Aspergillus niger A-25, one of which, designated as XynIII, also displayed ${\beta}-(l,3-1,4)-glucanase$ (EC 3.2.1.73) activity, as determined by a zymogram analysis. XynIII was purified by ultrafiltration and ion-exchange chromatography methods. Its apparent molecular weight was about 27.9 kDa, as estimated by SDS-PAGE. The purified XynIII could hydrolyze birchwood xylan, oat spelt xylan, lichenin, and barley ${\beta}-glucan$, but not CMC, avicel cellulose, or soluble starch under the assay conditions in this study. The xylanase and ${\beta}-(l,3-1,4)-glucanase$ activities of XynIII both had a similar optimal pH and pH stability, as well as a similar optimal temperature and temperature stability. Moreover, the effects of metal ions on the two enzymatic activities were also similar. The overall hydrolytic rates of XynIII in different mixtures of xylan and lichenin coincided with those calculated using the Michaelis-Menten model when assuming the two substrates were competing for the same active site in the enzyme. Accordingly, the results indicated that XynIII is a novel bifunctional enzyme and its xylanase and ${\beta}-(l,3-1,4)-glucanase$ activities are catalyzed by the same active center.

• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.397-404
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• 2013

Paenibacillus xylanilyticus KJ-03 was isolated from soil samples obtained from a field with Amorphophallus konjac plants. A gene encoding xylanase was isolated from KJ-03 and cloned using a fosmid library. The xynA gene encodes xylanase; it consists of 1,035 bp and encodes 345 amino acids. The amino acid sequence deduced from the P. xylanilyticus KJ-03 xylanase showed 81% and 69% identities with those deduced from the P. polymyxa E681 and Paenibacillus sp. HPL-001 xylanases, respectively. The xynA gene comprises a single domain, consisting of a catalytic domain of the glycosyl hydrolase (GH) 10 family. The xynA gene was expressed in Escherichia coli BL21 (trxB), and the recombinant xylanase was purified by Niaffinity chromatography. The purified xylanase showed optimum activity with birchwood xylan as a substrate at $40^{\circ}C$ and pH 7.4. Treatment with $Mg^{2+}$ and $Li^+$ showed a slight decrease in XynA activity; however, treatment with 5 mM $Cu^{2+}$ completely inhibited its activity. The results of the thin layer chromatography analysis indicated that the major hydrolysis product was xylobiose and small amounts of xylose and xylotriose. XynA showed increased activity with oat spelt xylan and birchwood xylan, but showed only slight activity with locust bean gum.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.251-259
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• 2013

An endoxylanase gene (PoxynA) that belongs to the glycoside hydrolase (GH) family 11 was cloned from a xylanolytic strain, Penicillium oxalicum B3-11(2). PoxynA was overexpressed in Trichoderma reesei QM9414 by using a constitutive strong promoter of the encoding pyruvate decarboxylase (pdc). The high extracellular xylanase activities in the fermentation liquid of the transformants were maintained 29~35-fold higher compared with the wild strain. The recombinant POXYNA was purified to homogeneity, and its characters were analyzed. Its optimal temperature and pH value were $50^{\circ}C$ and 5.0, respectively. The enzyme was stable at a pH range of 2.0 to 7.0. Using beechwood as the substrate, POXYNA had a high specific activity of $1,856{\pm}53.5$ IU/mg. In the presence of metal ions, such as $Cu^{2+}$, and $Mg^{2+}$, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of $Mn^{2+}$ and $Fe^{2+}$. The recombinant POXYNA hydrolyzed birchwood xylan, beechwood xylan, and oat spelt xylan to produce short-chain xylooligosaccharides, xylopentaose, xylotriose, and xylobiose as the main products. This is the first report on the expression properties of a recombinant endoxylanase gene from Penicillium oxalicum. The properties of this endoxylanase make it promising for applications in the food and feed industries.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.236-244
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• 2014

The gene encoding ${\alpha}-\small{L}$-arabinofuranosidase (AFase) from Paenibacillus sp. DG-22 was cloned, sequenced, and expressed in Escherichia coli. The AFase gene (abfA) comprises a 1,509 bp open reading frame encoding 502 amino acids with a molecular mass of 56,520 daltons. The deduced amino acid sequence of the gene shows that AbfA is an enzyme consisting of only a catalytic domain, and that the enzyme has significant similarity to AFases classified into the family 51 of the glycosyl hydrolases. abfA was subcloned into the pQE60 expression vector to fuse it with a six-histidine tag and the recombinant AFase (rAbfA) was purified to homogeneity. The specific activity of the recombinant enzyme was 96.7 U/mg protein. Determination of the apparent molecular mass by gel-filtration chromatography indicated that AbfA has a tetrameric structure. The optimal pH and temperature of the enzyme were 6.0 and $60^{\circ}C$, respectively. The enzyme activity was completely inhibited by 1 mM $HgCl_2$. rAbfA was active only towards p-nitrophephenyl ${\alpha}-\small{L}$-arabinofuranoside and exhibited $K_m$ and $V_{max}$ values of 3.5 mM and 306.1 U/mg, respectively. rAbfA showed a synergistic effect in combination with endoxylanase on the degradation of oat spelt xylan and wheat arabinoxylan.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.410-416
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• 2008

The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-${\beta}$-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and $60^{\circ}C$, respectively. The enzyme showed stability over a pH range of 4.0-10.0. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by $Fe^{2+}$ and strongly inhibited by $Hg^{2+}$ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.414-419
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• 1996

The xylnX gene encoding a xylanase from Clostridium thermocellum ATCC27405 was cloned in the plasmid pJH27, an E. coli-Bacillus shuttle vector and the resultant recombinant plasmid, pJX18 was transformed into E. coli HB101. The overexpressed xylanase was found to be secreted into the periplasmic space of the recombinant E. coli cells. The crude enzyme was obtained by treating the E. coli cells with lysozyme, and purified by DEAE-Sepharose column chromatography. Molecular wieght of the xylanase was estimated to be 53 kDa by gel filtration. The pI value was determined to be pH 8.8. The N-terminal sequence of the enzyme protein was Asp-Asp-Asn-Asn-Ala-Asn-Leu-Val-Ser-Asn which was considered to be the sequence of that of the mature form protein. The Km value of the enzyme for oat spelt xylan was calculated to be 2.63 mg/ml and the Vmax value was $0.47 {\mu}mole/min$. The xylanase had a pH optimum for its activity at pH 5.4 and a temperature optimum at $60^{\circ}C$. The enzyme hydrolyzed xylan into xylooligosaccharides which were composed mainly of xylobiose (40%) and xyloltriose (12%) after 5 hour reaction. This result indicates that the xylanase from C. thermocellum ATCC27405 is an endo-acting enzyme.