• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.536-539
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• 2003

A strong constitutive $P_{JH}$ promoter from Bacillus sp. was applied to overexpress the endoxylanase gene in Bacillus subtilis. The expression plasmid, pJHKJ4, was designed to contain the $P_{JH}$ promoter and open reading frame of endoxylanase including its own promoter. The plasmid was introduced into B. subtilis DB431 and the resulting transformant was grown on LB glucose medium. The endoxylanase activity in the culture supernatant reached about 140 unit/ml. The enzyme in the supernatant was efficiently concentrated to 70% by two-step treatments of ammonium sulfate saturation and ultrafiltration. The stabilization of concentrated enzyme solution at different storage temperatures was examined with various stabilizers such as NaCl, $CaCl_2$, sucrose, sorbitol, polyethylene glycol, and Tween-80.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.467-470
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• 2003

The endoxylanase (642 bp; 213 amino acids) and ${\beta}-xylosidase$ (1,602 bp; 533 amino acids) genes from Bacillus sp. were amplified by PCR and separately inserted downstream of the yeast ADH1 promoters, resulting in the pAEDX-1 and pAEX plasmid. When the yeast transformants, S. cerevisiae SEY2102 harboring pAEDX-1 or pAEX, were grown on YPD medium, the total activities of the enzymes reached about 9.8 unit/mL for endoxylanase and 2.9 unit/mL for ${\beta}-xylosidase$. When the three kinds of xylan from oat spelts, birch wood, and corncob were hydrolyzed by treatment of recombinant endoxylanase and ${\beta}-xylosidase$, it was found that xylose, xylobiose and xylotriose were produced and xylose was the major product after 12 h reaction. In addition, with the higher amount of enzymes, the more amount of xylose was produced.

• 한국미생물·생명공학회지
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• 제32권4호
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• pp.322-327
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• 2004

구성적 promoter인 $P_{JH}$ promoter 하류에 연결된 Bacillus sp. 유래의 endoxylanase를 B. subtilis에서 과발현 하였다. 발현된 plasmid, pJHKJ4는 자체 promoter($P_{B}$)와 Bacillus의 강력한 promoter($P_{JH}$)가 tandem으로 위치하여 transcription되었다. B.subtilis DB431에 형질전환된 균주를 glucose가 함유된 LB 배지에서 배양 후 총활성 및 분비효율은 140 U/ml 으로 나타났다. 배양상등액은 ultrafiltration(MW cut off 10 kDa and 30 kDa)과 염석법으로 농축하였다. 효소의 농축에서 ultrafiltration이 70% ammonium sulfate precipitation 보다 효과적이었다. $50^{\circ}C$ 에서 농축된 효소액의 안정화는 NaCl, glycerol, sorbitol, and $CaCl_{2}$ 등의 다양한 안정제를 농도별로 첨가하여 안정제의 효과를 검토한 결과 가장 효과적인 안정제는 NaCl과 $CaCl_{2}$ 이었다.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1476-1484
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• 2015

Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40℃. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0℃. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80℃ and 65℃, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

• 한국균학회지
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• 제17권2호
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• pp.57-65
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• 1989

Cellulose와 hemicellulose의 단일 유도기질과 그 혼합물을 이용하여 Aspergillus nidulans의 섬유질 분해효소계의 유도 특이성을 조사하였다. 섬유질 분해효소계의 생합성에 있어서 최적의 유도기질이 endoglucanase의 경우엔 carboxymethylcellulose, ${\beta}-glucosidase$는 cellobiose, 그리고 endoxylanase와 ${\beta}-xylosidase$는 xylan으로 알려져 왔으나 이들 단일기질보다 기질들의 혼합물 특히 CMC-xylan과 CMC-xylan-laminarin of cellulase와 xylanase complexes의 생합성을 증가시키는데 매우 효과적인 것으로 나타났다. 이것은 각각의 유도기질에 따른 endoglucanase와 ${\beta}-glucosidase$ 그리고 endoxylanase의 components 양상 및 비교 활성도 변화에 기인하는 것으로 polyacrylamide gel 전기영동과 활성염색의 결과에서도 나타났다. 섬유소 분해효소계 생합성을 위한 유도물질의 이와 같은 공조효과는 Aspergillus nidulans에서 Cellulose와 xylanase systems의 생합성 조절이 유도물질에 의한 효소의 유도 수준에서 상호 관련되고 있음을 시사한다.

• 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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• 제7권2호
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• pp.114-120
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• 1997

Microorganisms producing xylanase were screened for the enzymatic production of xylo-oligo saccharides from xylan. One of the bacteria isolated from compost produced an endoxylanase extracellularly. The bacterium was identified as Bacillus sp. according to its taxonomic characteristics examined. Xylanase production reached upto 5 U/ml after 22 h of culture in LB medium at $30^{\circ}C$. The xylanase was purified by ammonium sulfate precipitation and gel filtration. The molecular weight of the xylanase was estimated to be 20,400 by SDS-PAGE. Optimal temperature and pH for the xylanase activity was $60^{\circ}C$ and 6.5, respectively. The enzyme was stable at temperatures upto $40^{\circ}C$ and pH values from 4 to 10. The xylanase was completely inhibited by the addition of 2 mM mercury ion. Apparent $K_m$ and $V_max$ values for oat spelt xylan were 9.2 mg/ml and 1954 U/mg protein, respectively. For birchwood xylan, the values were 6.3 mg/ml and 1009 U/mg protein. The predominant products of the xylan hydrolysis were xylobiose, xylotriose and xylotetraose, indicating that the enzyme is an endoxylanase. Upto $85{\%}$ of the initially added enzyme (2 U/ml) was bound to 50 mg/ml of the insoluble fraction of oat spelt xylan after incubation at $30^{\circ}C$ for 30 min.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.474-482
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• 2004

The $\alpha$-L-arabinofuranosidase (Arfase) gene of Bacillus stearothermophilus No. 236 was cloned and sequenced. The ORF of the gene, designated arfA, encoded a 507 -residue polypeptide with calculated molecular mass of 57 kDa. The Arfase produced by a recombinant Escherichia coli strain containing the arfA gene was purified to apparent homogeneity and characterized. The molecular mass of the Arfase determined by SDS-PAGE was 60 kDa. However, according to gel filtration, it was estimated to be approximately 190 kDa. These results indicated that the functional form of the Arfase is trimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and $55^{\circ}C$, respectively. The half-life of the enzyme at $60^{\circ}C$ was about 6 h. Kinetic experiments at $45^{\circ}C$ with pNPM (p-nitrophenyl $\alpha$-L-arabinofuranoside) as a substrate gave the $K_m and V_{max}$ values of 1.19 mM and 26.1 U/ mg, respectively. When the enzyme was combined with Bacillus stearothermophilus No. 236 endoxylanase and $\beta$-xylosidase, it hydrolyzed arabinoxylan into L-arabinose and xylose more efficiently than Arfase alone. This synergistic effect suggested that the complete hydrolysis of xylan with large amounts of arabinose side chains required Arfase as well as endoxylanase and $\beta$-xylosidase.

• 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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.707-710
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• 2003

A strong constitutive $P_{JH}$ promoter from Bacillus was applied to overexpress the end oxylanase gene in Bacillus subtilis. The expression plasmid, pJHKJ4, was designed to contain the $P_{JH}$ promoter and endoxylanase promoter $(P_B)$, and introduced into Bacillus subtilis DB431 The total activities of the enzymes reached about 140 unit/ml by cultivation of B. subtilis DB431 harboring pJHKJ4 in LB glucose medium. Ultrafilteration is effective its yield is 70%.