• 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.

• 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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• 제14권1호
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• pp.171-177
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• 2004

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 into the downstream of the yeast ADH1 promoters, resulting in the pAEDX-1 (7.63 kb) and pAEX (8.47 kb) plasmids, respectively. When the yeast transformants, S. cerevisiae SEY2102 harboring pAEDX-1 or pAEX, were grown on YPD medium, the total activities of the enzymes were approximately 9.8 unit/ml for endoxylanase and 2.9 unit/m1 for $\beta$-xylosidase. When the three kinds of xylan from oat spelts, birch wood, and corncob were hydrolyzed by treating with recombinant endoxylanase and $\beta$-xylosidase, it was found that xylose, xylobiose, and xylotriose were produced. To efficiently hydrolyze xylan, various reaction conditions such as amount of enzymes, substrate type, substrate concentration, temperature, and reaction time were examined. The optimized conditions for the hydrolysis of xylan were as follows: amount of endoxylanase, 10 units; amount of $\beta$-xylosidase, 10 units; temperature, $50^\circ{C}$; substrate type, oat spelts xylan; substrate concentration, 6%; reaction time, 1 h. Under the optimal condition, xylose was mainly produced from oat spelts xylan by cooperative action of endoxylanase and $\beta$-xylosidase.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.136-142
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• 1992

토양 분리균인 B.stearothermophilus chromosome의 유전자 은행으로부터 E.coli HB101 균주에 $\beta$-D-xylosidase 생산능력을 갖게하는 5.4Kb와 6.4Kb의 두 DNA 절편을 분리, pBR322에 크로닝하여 각각 pMG01과 pMG02의 재조합 플라스미드를 얻었다. 상기 두 B.stearothermophilus DNA 절편의 restriction map을 작성하고 이것을 기초로 하여 $\beta$-D-xylosidase 유전자인자의 위치를 확인함과 동시에 pUC18에 subcloning하여 각각 2.2kb와 1.0kb의 DNA 단편이 삽입된 $\beta$-D-xylosidase 양성의 pMG1와 pMG2를 분리하였다.

• 한국미생물·생명공학회지
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• 제20권3호
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• pp.271-279
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• 1992

내알카리성 및 내열성 xylanase를 생산하는 토양분리균인 Bacillus stearothermophilus의 chromosomal DNA와 pBR322 plasmid DNA의 HindIII 절단 DNA 단편을 ligation 시켜 E.coli HB101들 형질전환, 약 5천개의 형질전환체를 얻었으며 이들 중에서 세개의 xylanase 양성 형질전환체를 분리하였다. 상기 세 xylanase 양성 형질전환체들로부터 분리한 제조합 plasmid(pMG11, pMG12 및 pMG13)는 다 같이 xylanase 활성과 관계되는 B.stearothermophilus 유래의 동일 4kb 외래 DNA를 가지고 있었으나, pMG13은 외래 DNA의 삽입 방향만이 다름을 확인하였다.

• 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.

• 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.

• Animal Bioscience
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• 제34권3_spc호
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• pp.371-384
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• 2021

Objective: Wheat bran (WB) was co-fermented with Aspergillus oryzae and phytase (Phy) to determine whether co-fermentation improve WB phosphorus and fiber utilization in Isa-brown layers. Methods: A total of 112 Isa brown layer were randomly divided into 7 treatments with 8 replicates per a treatment and 2 hens per a replicate. The treatments included basal diet (control), basal diet supplemented with 250 unit/kg Phy (control+Phy), diet with 10% WB (10% WB), diet with 5% WB and 250 unit/kg Phy (5% WB+Phy) diet with 10% WB and 250 unit/kg Phy (10% WB+Phy), diet with 5% fermented WB supplemented with molasses and phy (PCFWH) and 125 unit/kg Phy (5% PCFWH), and diet with 10% PCFWH (10% PCFWH). The intestinal microbial population, intestinal morphology, serum antioxidant enzyme activities, and excreta phosphorus content were assessed. Results: In PCFWH, spore counts, protease activity, xylanase activity, and ferulic acid were 8.50 log/g dry matter (DM), 190 unit/g DM, 120 unit/g DM, and 127 ㎍/g, respectively. Xylobiose and xylotriose were released in PCFWH, while they were not detectable in WB. Antioxidant capacity was also enhanced in PCFWH compared to WB. The 10% WB+Phy and 10% PCFWH groups produced higher egg mass, but hens fed 5% WB+Phy had the lowest amount of feed intake. Eggs from 10% PCFWH had better eggshell weight, eggshell strength, and eggshell thickness. Birds fed with 10% PCFWH also had higher serum superoxide dismutase and catalase activities. Compare to control, 10% PCFWH significantly reduced excreta phosphorus content. Conclusion: Diet inclusion of 10% PCFWH improved egg quality, antioxidant status, and excreta phosphorus content of laying hens.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.861-868
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• 2011

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at $55^{\circ}C$ and was thermostable at $50^{\circ}C$ and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent $K_m$ and $V_{max}$ values were 1.18 mg/ml and 1,961 ${\mu}mol$/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.