• 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.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.277-285
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• 2009

The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-$\beta$-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, $\alpha$-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.534-538
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• 2001

The xynA gene encoding an alikali-tolerant endo-1,4-${\beta}$-xylanase (XYN) was cloned from the alkalophilic Bacillus pumilus A-30. The nucleotide sequence of a 974-bp DNA fragment containing the xynA was determined. An ORF of 684 nucleotides that encoded a protein of 228 amino aicds was detected. Asparagine-71 of XYN from B. Pumilus A-30 showed to be highly conservative in alkaline xylanases of family G/11, upon comparing the amino acid sequences of 17 family G/11 xylanases. Site-directed mutation of N71D of the xynA gene resulted in a decrease of 12.4% in the specific acitivity and a significant decline in the enzyme activity in the alkaline pH range.

• 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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• 제12권6호
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• pp.890-894
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• 2002

A xylanase-producing Trichoderma strain was isolated from soil. Xylanase from Trichoderma strain SY was purified 21-fold to an apparent homogeneity, with a $17.4\%$ yield. The optimum pH and temperature were determined to be 5.5 and $50^{\circ}C$, respectively, and its molecular weight was 21-kDa by SDS-PAGE. The corresponding gene, named xyl, was cloned by RT-PCR. DNA blot analysis of xyl showed that this gene is present as a single copy. The amino acid sequence of the Xyl protein showed similarity to those of other xylanases derived from various fungi. mRNA of xyl was highly expressed when this fungus was grown on cellulose or xylan as a sole carbon source, but undetectable when grown on sucrose. Extracts of Escherichia coli cells expressing xyl were found to have xylanase activity. It was confirmed that xyl from this isolate encodes xylanase.

• 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.

• BMB Reports
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• 제44권10호
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• pp.653-658
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• 2011

Sclerotinia sclerotiorum fungus has three endoxylanases induced by wheat bran. In the first part, a partial xylanase sequence gene (90 bp) was isolated by PCR corresponding to catalytic domains (${\beta}5$ and ${\beta}6$ strands of this protein). The high homology of this sequence with xylanase of Botryotinia fuckeliana has permitted in the second part to amplify the XYN1 gene. Sequence analysis of DNA and cDNA revealed an ORF of 746 bp interrupted by a 65 bp intron, thus encoding a predicted protein of 226 amino acids. The mature enzyme (20.06 kDa), is coded by 188 amino acid (pI 9.26). XYN1 belongs to G/11 glycosyl hydrolases family with a conserved catalytic domain containing $E_{86}$ and $E_{178}$ residues. Bioinformatics analysis revealed that there was no Asn-X-Ser/Thr motif required for N-linked glycosylation in the deduced sequence however, five O-glycosylation sites could intervene in the different folding of xylanses isoforms and in their secretary pathway.

• 미생물학회지
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• 제36권3호
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• pp.196-202
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• 2000

Bacillus circulans ATCC21367의 Cellulolytic Xylanase 유전자를 pUC19을 vector 로 하여 대장균내에서 클로닝 하였다. 재조합 plasmid pXL180은 B. circulans 로부터 유래 된 0.5 kb와 1.3 kb Pst I 절편으로 이루어진 총 1.8 kb 삽입 절편을 포함 하고 있었다. 1.3 kb 절편의 상류영역에 있는 0.5 kb 절편은 클로닝된 유전자의 정상 발현뿐만아니라 과잉 발 현에 대해서도 필수 불가결함을 확인하였다. 형질전환체는 모균 B. circulans에 의해 생산된 것보다 135배 이상 많은 xylanase를 생산하였다. 클로닝된 효소의 최적 pH와 온도는 각각 pH 5.2와 $60^{\circ}C$ 였다. $55^{\circ}C$에서 1시간 동안의 사전 연처리에도 이 효소는 활성의 감소를 일 으키지 않았다. 이 효소는 xylan과 carboxymethyl cellulose (CMC), lichenan에 대하여 기질 특이성을 보였는데, 주요 산물로서 xylose(EH는 G1)와 xylobiose(또는 G2), xylotriose(또는 G3)를 생산하였다. 그러므로 우리는 클로닝된 유전자의 효소를 cellulolytic xylanase로 명명 하였다. 액체 배양된 Eschericxhia coli DH5$\alpha$(pXL180)의 전체 세포 추충물이나 배양 상등 액으로부터 조제된 시료들의 SDSPAGE와 zymogram을 통하여 이 효소의 분자량은 45kDa 이었으며, 대장균 내에서 주목할 만한 processing은 일어나지 않았다.

• 한국미생물·생명공학회지
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• 제48권2호
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• pp.158-166
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• 2020

Paenibacillus woosongensis의 xylanase 유전자를 클로닝하고 그 염기서열을 결정하였다. Xylanase 유전자는 xyn10A로 명명되었으며, 481 아미노잔기로 구성된 단백질을 코드하는 1,446개 뉴클레오티드로 구성되었다. 추론된 아미노산 배열에 따르면 Xyn10A는 glycosyl hydrolase family 10 xylanase와 상동성이 높은 활성영역과 카르복실 말단에 탄수화물을 결합하는 것으로 추정되는 영역이 포함된 다영역 효소로 확인되었다. DEAE-Sepharose와 Phenyl-Separose 컬럼 크로마토그래피 과정을 통해 P. woosongensis xyn10A 유전자를 함유한 재조합 대장균의 균체 파쇄상등액으로부터 Xyn10A를 정제하였다. 정제된 Xyn10A의 아미노 말단 배열이 GIANGSKF로 결정되었으며 이는 SignalP5.0 server로 예측된 signal peptide의 다음 아미노산 배열과 정확하게 일치하였다. 정제된 Xyn10A는 33 kDa 크기의 절단된 단백질이며 균체내 분해에 의해 카르복시 말단에서 CBM이 제거된 것으로 판단된다. 정제된 효소는 최적 pH와 온도가 6.0과 55-60℃이며 oat spelt xylan에 대한 반응 동력학적 계수 V_max와 K_m이 298.8 U/mg과 2.47 mg/ml로 각각 나타났다. 효소는 birchwood xylan이나 oat spelt xylan보다 arabinoxylan에 대한 활성이 높았으며 para-nitrophenyl-β-xylopyranoside에 대해 낮은 활성을 보였다. Xyn10A의 활성은 Cu²⁺, Mn²⁺과 SDS에 의해서 크게 저해되었으며 K⁺, Ni²⁺과 Ca²⁺에 의해는 상당하게 증진되었다. 또한 이 효소는 xylobiose 보다 중합도가 큰 자일로올리고당을 분해하였으며, 자일로올리고당의 최종 가수분해 산물은 xylose와 xylobiose로 확인되었다.

• 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.