• Current Research on Agriculture and Life Sciences
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• 제14권
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• pp.111-122
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• 1996

Streptomyces chibaensis J-59 xylanase는 CSL 배지(1% corn steep liquor, 0.15% glucose, 0.1% $MgSO_4{\cdot}7H_2O$, pH 7.0)에서 1% xylan을 효소생산 유도물질로 첨가하였을 경우에 생산(0.83 unit/ml) 되었으나, xylose를 비롯한 각 종 탄소원을 포함하는 배지에서는 효소가 생산되지 않았다. S. chibaensis로 부터 생산된 세포외 xylanase를 $(NH_4)_2SO_4$ 분획침전, DEAE-Sephadex A-50 column chromatography, Sephadex G-200 gel filtration 과정으로 약 29%의 수율로 20배 정제하였다. 정제한 xylanase는 SDS-PAGE 및 Sephadex G-200 gel filtraion에서 분자량이 모두 25,000 Da으로 나타나 monomenc enzyme으로 확인되었다. 금속이온에 대한 영향은 $Fe^{3+}$, $Hg^{2+}$, $Cu^{2+}$, sodium dodecyl sulfate, N-bromosuccinide 등에서는 아주 강한 저해를 받았고, $Mn^{2+}$, $Zn^{2+}$에서는 약간의 저해를 받았다. 반면에 $Mg^{2+}$는 효소활성을 증가시켰다. Xylanase에 의한 xylooligo 당의 생산양상을 TLC로 조사한 결과 xylobiose, xylotriose 및 xylotetrose가 주 생산물이었으며, 반응 1시간 이후부터 소량의 xylose가 생성되었다. 따라서 본 효소는 전형적인 endotype xylanase로 확인되었으며 새로운 기능성식품인 자일로 올리고당(xylooligo-saccharides)의 제조에 이용할 수 있을 것이다.

• BMB Reports
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• 제28권4호
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• pp.348-352
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• 1995

A thermophilic Bacillus sp. strain KK-1 isolated from soil produced an extracellular xylanase. From the culture supernatant of Bacillus sp., the xylanase was purified to homogeneity by ammonium sulfate precipitation and DEAE-Sephadex A-50 chromatography. The molecular weight of the purified xylanase was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography. The apparent $K_m$ values for xylanase, using oat spelt xylan and birchwood xylan as substrates, were 7.1 mg/ml and 3.2 mg/ml, and $V_{max}$ values were $27.0\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}$ and $29.0\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}$, respectively. The xylanase hydrolyzed oat spelt xylan to mostly xylobiose, xylotriose, and xylose. The amino acid composition indicated that the xylanase contained high amounts of amino add residues of glutamic acid and glutamine (Glx) and aspartic acid and asparagine (Asx).

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.57-63
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• 2006

We have cloned a xylanase gene (xynA) from Streptomyces thermocyaneoviolaceus. The deduced amino acid sequences of the XynA, including the active site sequences of glycosyl hydrolase family 10, showed high sequence homology with several xylanases assigned in this category. The XynA was overexpressed under an IPTG inducible T7 promoter control in E. coli BLR(DE3). The overproduced enzymes were excreted into culture supernatants and periplasmic space. The purified XynA had an apparent molecular mass of near 54 kDa, which corresponds to the molecular mass calculated from its gene. The optimum pH and temperature of the purified XynA were determined to be 5.0 and $65^{\circ}C$, respectively. The XynA retained over $90\%$ its activity after the heat treatment at $65^{\circ}C$ for 30 min. The XynA was highly efficient in producing xylose (X1), xylobiose (X2), xylotriose (X3), and xylotetraose (X4) from xylan.

• 한국미생물·생명공학회지
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• 제19권6호
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• pp.592-597
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• 1991

토양 분리균인 B.stearothermophilus가 생산하는 xylanase를 ammonium sulfate 분획, DEAE-Sepharose CL-6B ion exchange chromatography, Sephadex G-100 gel 여과 및 열처리 등의 과정을 거쳐 단일 단배질로 분리 정제하였다. 170,000의 분자량을 본 정제 xylanase는 pH8과 pH10 사이의 넓은 최적 pH를 보였으며 $55^{\circ}C$에서 최대 활성을 나타내었다. $55^{\circ}C$에서 2시간의 열처리에 의해서도 활서의 손실이 거의 없을 정도로 열에 매우 안정하였으며 $co^{2+}$와 $Mn^{2+}$에 의해서 현저한 활성화 효과를 보였다.

• 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.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.28-33
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• 1998

A gene coding for ${\beta}$-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isloated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-${\beta}$-D xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is ${\beta}$-xylosidase gene. The ${\beta}$-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 ${\beta}$-xylosidase was highly homologous to the ${\beta}$-xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable ${\beta}$-xylosidase from Bacillus stearothermophilus.

• 한국미생물·생명공학회지
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• 제22권6호
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• pp.636-642
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• 1994

Essential amino acids involving in the catalytic mechanism of the $\beta$-D-xylosidase of Bacillus stearothermophilus were determined by chemical modification studies. Among various che- mical modifiers tested N-bromosuccinimide (NBS), $\rho$-hydroxymercurybenzoate (PHMB), N-ethylma- leimide, 1-[3-(di-ethylamino)-propyl]$-3-ethylcarbodi-imide (EDC), and Woodward's Reagent K(WRK)inactivated the enzyme, resulting in the residual activity of less than 20%. WRK reduced the enzyme activity by modifying carboxylic amino acids, and the inactivation reacion proceeded in the form of pseudo-first-order kinetics. The double-lagarithmic plot of the observed pseudo-first- order rate constant against the modifier concentration yielded a reaction order of 2, indicating that two carboxylic amino acids were essential for the enzyme activity. The $\beta$-D-xylosidase was also inactivated by N-ethylmaleimide which specifically modified a cysteine residue with a reaction order of 1, implying that one cysteine residue was important for the enzyme activity. Xylobiose protected the enzyme against inactivation by WRK and N-ethylmaleimide, revealing that carboxylic amino acids and a cysteine residue were present at the substrate-binding site of the enzyme molecule.

• Journal of Microbiology and Biotechnology
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• 제16권4호
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• pp.613-618
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• 2006

Two endoxylanases from an alkaliphilic bacterium, Bacillus halodurans C-1, were purified 3.8- and 7.9- fold with specific activities of 9.4 and 19.8U/mg protein, respectively. The molecular masses of both purified enzymes were 23 and 47 kDa, respectively, and 23 kDa xylanase I (Xyl I) exhibited an optimum pH at 7.0, whereas 47 kDa xylanase II (Xyl II) showed a broad pH range of 5.0 to 9.0. The temperature optima of both xylanases were $60^{\circ}C\;and\;70^{\circ}C$, respectively. Both were stable in the pH range of 6.0 to 9.0 and 5.0 to 10.0, respectively, and they were stable up to $60^{\circ}C\;and\;70^{\circ}C$, respectively. The $K_m\;and\;V_{max}$ of Xyl I were 4.33mg/ml and $63.5{\mu}mol/min/mg$, respectively, whereas Xyl II had a $K_m$ value of 0.30 mg/ml and $V_{max}$ of $210{\mu}mol/min/mg$. Both xylanases hydrolyzed xylans from birchwood, oat spelt, and larchwood. However, they showed different modes of action; a series of xylooligosaccharides larger than xylotriose were released as the major products by Xyl I, whereas xylobiose and xylotriose were the main products by Xyl II. The maximum synergistic action of the two enzymes on hydrolysis of xylan was 2.16 with the ratio of Xyl I to Xyl II at 1:9.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.873-880
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• 2009

A novel xylanase gene, Kxyn, was cloned from Kocuria sp. Mn22, a bacteria isolated from the deep sea of the east Pacific. Kxyn consists of 1,170 bp and encodes a protein of 390 amino acids that shows the highest identity (63%) with a xylanase from Thermohifida fusca YX. The mature protein with a molecular mass of approximately 40 kDa was expressed in Escherichia coli BL21 (DE3). The recombinant Kxyn displayed its maximum activity at $55^{\circ}C$ and at pH 8.5. The $K_m,\;V_{max}$, and $k_{cat}$ values of Kxyn for birchwood xylan were 5.4 mg/ml, $272{\mu}mol/min{\cdot}mg$, and 185.1/s, respectively. Kxyn hydrolyzed birchwood xylan to produce xylobiose and xylotriose as the predominant products. The activity of Kxyn was not affected by $Ca^{2+},\;Mg^{2+},\;Na^+,\;K^+$, ${\beta}$-mercaptoethanol, DTT, or SDS, but was strongly inhibited by $Hg^{2+},\;Cu^{2+},Zn^{2+}$, and $Pb^{2+}$. It was stable over a wide pH range, retaining more than 80% activity after overnight incubation at pH 7.5-12. Kxyn is a cellulase-free xylanase. Therefore, these properties make it a candidate for various industrial applications.

• 미생물학회지
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• 제48권2호
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• pp.141-146
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• 2012

Paenibacillus woosongensis의 유전체 부분 염기서열로부터 유추된 xylanase 유전자를 PCR 증폭하여 대장균에 클로닝하였다. Xylanase 유전자는 211 아미노산으로 구성된 단백질을 코드하며 633 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 xylanase는 glycosyl hydrolase family 11에 속하며 Paenibacillus의 xylanase와 85-89% 상동성을 보였다. Xylanase 유전자를 T7 promoter로 과잉발현한 결과 그 발현량이 높지 않았으며, 균체내 외에서 모두 효소활성이 관찰되었다. 재조합 대장균의 균체파쇄상등액을 사용하여 효소 반응특성을 조사한 결과 pH 5.5와 $60^{\circ}C$에서 최대 반응활성을 보였다. 한편 xylanase의 기질로 xylan과 xylooligosaccharides를 사용하였을 때 xylose와 xylotriose가 주된 최종 반응산물로 관찰되었으며 xylobiose는 분해하지 못하였으나 이보다 중합도가 큰 xylooligosaccharides는 분해하였다.