• 한국응용곤충학회지
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• 제46권2호
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• pp.303-311
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• 2007

Xylan이 풍부한 식물체를 먹이로 하는 하늘소의 장내에 존재하는 xylanase 생산 미생물의 탐색 과정에서 털두꺼비하늘소 (Moechotypa diphysis) 성충의 장으로부터 우수한 xylanase 생산균주 Paenibacillus sp. HY-8을 분리하였다. 생화학적, 계통학적 분석결과를 바탕으로 이 분리균은 Paenibacillus 속에 속하는 종으로 분석되었다. HY-8 균주에서 xylanase 생산은 제한배지에 xylan을 첨가함으로써 유도되는 특성을 나타내었고 1% 의 yeast extract와 0.5%의 birchwood xylan이 포함된 M9 배지에서 $25^{\circ}C$, 24시간의 배양에 의해 xylanase의 생산이 최대치에 도달하였다. HY-8 균주가 생산하는 xylanase는 pH6.0에서 여러 가지 식물성 사료의 원료에 대하여 대조구로 사용된 Tricoderma sp. 유래의 xylanase에 비해 우수한 분해능을 나타내었다.

• 한국미생물·생명공학회지
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• 제32권1호
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• pp.22-28
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• 2004

경주지역에 있는 목재 저장소의 토양으로부터 내별성 xylanase를 생산하는 호열성 세균인 DG-22 균주가 분리되었다. 형태적, 생화학적, 계통학적 연구에 근거하여 이 분리균은 Paenibacillus 속하는 종으로 판명되었다. 이 균주에서 xylanase의 생산은 성장배지에 xylan을 첨가함으로서 유도되었고 glucose또는 xylose에 의해서 억제되었다. Cellulase 활성은 탐지되지 많았다. 효소 활성을 위한 최적온도와 pH는 각각 $80^{\circ}C$와 5.0-5.5이었다. 이 조효소는 $60^{\circ}C$매서 안정하였고 $70^{\circ}C$에서는 2시간 추에도 60%의 활성을 유지하였다. 배양상등액의 zymogram 분석을 통해 22 kDa과 30 kDa 크기의 xylanase활성 band를 확인하였다

• 생명과학회지
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• 제13권5호
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• pp.618-625
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• 2003

목재 저장소의 토양에서 분리된 호열성 세균인 Paenibacillus sp. DG-22로부터 xylanase를 생산하기 위한 배양조건을 최적화시키기 위해 연구를 수행하였다. Xylanase생산은 세포의 생장과 연관된 양상을 나타내었다. Xylanase 활성은 배양상청액에서만 발견된 반면 $\beta-xylosidase$활성은 주로 세포와 결합되어 있었다. Xylanase활성의 형성은 자일란에 의해 유도되었고 포도당과 자일로스에 의해서 억제되었다. 여러 상업적 자일란을 이용하여 xylanase의 생산양상을 조사한 결과 0.1-0.5%의 birchwood xylan에서 가장 높은 생산율을 나타내었다. 조사된 여러 질소 원들 중 효모추출물이 xylanase생산을 위하여 최적이었다. xylanase의 활성은 $Co^{2+},\; Cu^{2+},\; Fe^{3+},\; Hg^{2+}\;$ 와$\; Mn^{2+}$ 이온들에 의하여 억제된 반면 $Ca^{2+},\; Mg^{2+},\; Ni^{2+},\; Zn^{2+}$ 이온들과 DTT에 의해서는 촉진되었다. 수은은 5 mM의 농도에서 xylanase 활성을 완전히 파괴하였다. 자일란 가수분해의 주된 산물은 자일로바이오스, 자일로트라이오스 그리고 자일로 올리고당이었고 이것은 이 효소가 endoxylanase라는 것을 나타낸다.

• 한국미생물·생명공학회지
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• 제41권3호
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• pp.263-271
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• 2013

균주 HX-1은 토양샘플로부터 분리된 자일라네이즈 생산 미생물로서 16S rRNA 유전자 염기서열 분석과 이를 이용한 phylogenetic tree 제작을 통하여 Paenibacillus 속의 한 종으로 동정되었다. 그러나 HX-1 균주가 계통발생적 연관관계가 높은 기존에 알려진 표준군주들과는 상당히 다른 생리적-생화학적 특성을 나타내는 사실로부터 HX-1이 신아종일 것으로 판단하고, Paenibacillus sp. HX-1으로 명명하였다. 균주 HX-1로부터의 자일라네이즈 생산을 증가시키는 배지조건을 탐색하여 최적화된 TNX 배지(1% bacto tryptone, 0.7% 자일란, 1% NaCl; pH 7.0)에서 약 7.4배에 달하는 자일라네이즈 생산량의 증가가 가능하였다. 균주 HX-1이 분비하는 자일라네이즈는 pH 7.0과 $45^{\circ}C$에서 최적의 효소활성을 나타냈으며, beechwood 자일란을 기질로 하는 효소반응으로부터 xylobiose를 최종산물로 생산하는 endo-${\beta}$-1,4-xylanase임을 확인하였다. 본 연구로부터 동정된 Paenibacillus sp. HX-1은 다양한 산업에 응용이 가능한 새로운 자일라네이즈를 제공할 수 있는 중요한 균으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.29-36
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• 2007

The xynA gene encoding the xylanase A of Paenibacillus sp. DG-22 was isolated with a DNA probe obtained by PCR amplification, using degenerated primers deduced from the amino acid residues of the known N-terminal region of the purified enzyme and the conserved region in the family 11 xylanases. The positive clones were screened on the LB agar plates supplemented with xylan, by the Congo-red staining method. The xynA gene consists of a 630-bp open reading frame encoding a protein of 210 amino acids, and the XynA preprotein contains a 28-residues signal peptide whose cleavage yields a l82-residues mature protein of a calculated molecular weight of 20,000Da and pI value of 8.77. The cloned DNA fragment also has another ORF of 873 nucleotides that showed 76% identity to the putative transcriptional activator of Bacillus halodurans C-125. Most of the xylanase activity was found in the periplasmic space of E. coli. The xynA gene was subcloned into pQE60 expression vector to fuse with six histidine-tag. The recombinant xylanase A was purified by heating and immobilized metal affinity chromatography. The optimum pH and temperature of the purified enzyme were 6.0 and $60^{\circ}C$, respectively. This histidine-tagged xylanase A was less thermostable than the native enzyme.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1525-1535
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• 2014

The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and $65^{\circ}C$ and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM $Cu^{2+}$ and $Hg^{2+}$, whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1014-1021
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• 2004

Two thermostable xylanases, designated XynA and XynB, were purified to homogeneity from the culture supernatant of Paenibacillus sp. DG-22 by ion-exchange and gel-filtration chromatography. The molecular masses of xylanases A and B were 20 and 30 kDa, respectively, as determined by SDS-PAGE, and their isoelectric points were 9.1 and 8.9, respectively. Both enzymes had similar pH and temperature optima (pH 5.0-6.5 and $70^{\circ}C$), but their stability at various temperatures differed. Xylanase B was comparatively more stable than xylanase A at higher temperatures. Xylanases A and B differed in their $K_m$ and $V_{max}$ values. XynA had a $K_m$ of 2.0 mg/ml and a $V_{max}$ of 2,553 U/mg, whereas XynB had a K_m$ of 1.2 mg/ml and a $V_{max}$, of 754 U/mg. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on birchwood xylan, but showed different modes of action. Xylotriose was the major product of XynA activity, whereas XynB produced mainly xylobiose. These enzymes utilized small oligosaccharides such as xylotriose and xylotetraose as substrates, but did not hydrolyzed xylobiose. The amino terminal sequences of XynA and XynB were determined. Xylanase A showed high similarity with low molecular mass xylanases of family 11.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2003년도 제40회 국제학술심포지움
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• pp.78-78
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• 2003

• 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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• 제22권7호
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• pp.930-938
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• 2012

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA-335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Ni^{2+}$, $Fe^{2+}$, $Fe^{3+}$, $Zn^{2+}$, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of $60^{\circ}C$ and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of $70^{\circ}C{\sim}80^{\circ}C$), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.