• Journal of Microbiology and Biotechnology
• /
• 제23권3호
• /
• pp.397-404
• /
• 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.

• 한국응용곤충학회지
• /
• 제46권2호
• /
• pp.303-311
• /
• 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
• /
• 제43권4호
• /
• pp.330-335
• /
• 2015

Bacillus agaradhaerens 균주의 효소를 정제하여 그 특성을 확인하였다. 균주가 생산하는 xylanase를 70% ammonium sulfate로 침전하고 CM-sepharose ion exchange chromatography와 ultrafiltration(PM-10)을 행하여 16.7%의 수율과 6.7배의 정제도를 지니는 효소를 얻을 수 있었으며 SDS-PAGE를 통하여 23 kDa의 분자량을 갖는 효소임을 확인하였다. 효소의 최적 pH는 6.0으로서 sodium phosphate buffer에서 24시간까지 안정하였으며, 최적반응온도는 $60^{\circ}C$, EDTA의 첨가 시 효소활성이 증가되었고 $40^{\circ}C$에서 24시간까지 안정하였다. 효소는 xylan만을 기질로 이용할 수 있었으며 Birchwood xylan에 대하여 최대 활성을 나타내었고 $V_{max}$는 $49,724{\mu}M/min$, $K_m$은 6.08 mg/ml로 확인되었다.

• 한국미생물·생명공학회지
• /
• 제27권5호
• /
• pp.370-377
• /
• 1999

Thermo-tolerant bacterium producing the xylanase was isolated from soil and identified as Bacillus pumilus. This strain, named Bacillus pumilus TX703, was able to grow ad produce xylanase at the culture temperature of 5$0^{\circ}C$. The maximum xylanase production was obtained when 1%(w/v) birchwood xylan and 1% (w/v) soytone were used as carbon source and nitrogen source, respectively. The biosynthesis of xylanase was under the catabolite repression induced by glucose in the culture medium, and it was completely inhibited in the presence of 0.2% (w/v) glucose. The maximum activity of xylanase was observed from pH8.0 to 9.0 and from 50 to 6$0^{\circ}C$ and the enzyme was highly heat-stable, whose activity remained was over 50% at 8$0^{\circ}C$, and was quite stable from pH5.0 to 10.0.

• Journal of Microbiology and Biotechnology
• /
• 제12권1호
• /
• pp.153-156
• /
• 2002

The characterization of a partially purified, cellulase-free, thermostable alkaline xylanase from thermoalkalophilic Bacillus sp. JB-99 was investigated. The xylanase production was the highest when birchwood xylan was added to a medium containing finely powdered rice bran, showing 4,826 IU$ml^-1$ of activity for 15 h of incubation. The partially purified xylanase exhibited an optimum temperature and pH at $70^C{\circ}$ and 10, respectively. The enzyme was stable at pH 5-11 at $50^C{\circ}$. The xylanase activity was strongly inhibited by $Hg^2+$, while dithiothreitol, cysteine, and ${\beta}$-mercaptoethanol enhanced the activity.

• 한국미생물·생명공학회지
• /
• 제45권2호
• /
• pp.155-161
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• 제24권7호
• /
• pp.943-953
• /
• 2014

The XylH gene (1,167-bp) encoding a novel hemicellulase (41,584 Da) was identified from the genome of Microbacterium trichothecenolyticum HY-17, a gastrointestinal bacterium of Gryllotalpa orientalis. The enzyme consisted of a single catalytic domain, which is 74% identical to that of an endo-${\beta}$-1,4-xylanase (GH10) from Isoptericola variabilis 225. Unlike other endo-${\beta}$-1,4-xylanases from invertebrate-symbiotic bacteria, rXylH was an alkali-tolerant multifunctional enzyme possessing endo-${\beta}$-1,4-xylanase activity together with ${\beta}$-1,3/${\beta}$-1,4-glucanase activity, which exhibited its highest xylanolytic activity at pH 9.0 and 60oC, and was relatively stable within a broad pH range of 5.0-10.0. The susceptibilities of different xylosebased polysaccharides to the XylH were assessed to be as follows: oat spelts xylan > beechwood xylan > birchwood xylan > wheat arabinoxylan. rXylH was also able to readily cleave p-nitrophenyl (pNP) cellobioside and pNP-xylopyranoside, but did not hydrolyze other pNP-sugar derivatives, xylobiose, or hexose-based materials. Enzymatic hydrolysis of birchwood xylan resulted in the product composition of xylobiose (71.2%) and xylotriose (28.8%) as end products.

• Current Research on Agriculture and Life Sciences
• /
• 제14권
• /
• pp.101-110
• /
• 1996

Jar fermentor 배양에서 S. chibaensis J-59로 부터 포도당 이성화효소의 최적 생산조건을 확립하기 위하여 반응표면분석을 이용하였다. 실험계획은 중심계획합성법을 이용하였고, 3가지 실험요인으로는 공기주입량(vvm), 교반속도(rpm), 포도당의 농도(%)로 선별하였다. 요인실험의 결과로 회귀계수를 산출한 후 반응표면 회귀식을 구하였다. 효소 생산량의 변화에 대한 회귀식의 결정계수(R2)는 0.9776이였고, 분산분석의 F-ratio가 12.487이었으며, 이때 유의성(Prob>F)은 5% 이내로 인정되었다. 능선분석 결과, 효소생산 최대값은 공기주입량 2.227vvm, 교반속도 587.5rpm, glucose 농도 0.586%로 나타났으며 이때 반응표면 회귀식에 의해 추정하는 예상 효소활성은 7.67 단위로 나타났다. Jar fermentor 배양에서의 최적화 회귀식에 따라 1% birchwood xylan, 1.5% CSL, 0.1% $MgSO_4{\cdot}7H_2O$, 0.012% $CoCl_2{\cdot}6H_2O$, (pH 7.0)로 구성된 배지에 초기 pH를 7.0으로 조정하여 배양 종료까지 7.0을 유지하면서 공기주입량을 2.2vvm, 교반속도를 580rpm, glucose 농도를 0.586%로 하여 $30^{\circ}C$에서 42 시간 배양하였을때, 실제 효소생산량은 7.43 단위로 상기 분석법에서 예상한 효소 생산량의 97%에 달하였고, 플라스크배양의 효소생산량 (2.78 GIU/ml)보다 약 2.7배 가량 효소생산이 증가되었다.

• Journal of Microbiology and Biotechnology
• /
• 제6권2호
• /
• pp.79-85
• /
• 1996

By using a T7 expression system, a large amount of Bacillus stearothermophilus endo-xylanase A (XynA) could be produced in Escherichia coli cells. The overproduced enzyme formed inclusion bodies, and so the protein could be more easily purified to homogeneity. The molecular weight of the purified enzyme was estimated to be 22 kDa by SDS-polyacrylamide gel electrophoresis and 43 kDa by Sephacryl S-200 gel filtration, suggesting that the native enzyme was a homodimer. The pI value was determined to be 8.4. The Michaelis constants for birchwood xylan and oat spelts xylan were calculated to be 3.83 mg/ml and 5.03 mg/ml, respectively, and the $V_{max}$ max/ values for both xylans were 2.86 $\mu mole$/min. The purified enzyme was most active at $55^{\circ}C$ and pH 8.0, and stable up to $60^{\circ}C$ and in the near neutral pH range. From the zymogram, Bacillus stearothermophilus was found to have at least three xylanases and the purified one was the smallest among them.

• BMB Reports
• /
• 제28권4호
• /
• pp.348-352
• /
• 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).