• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.254-264
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• 2010

To obtain an efficient natural lignocellulolytic complex enzyme, we screened an efficient lignocellulose-degrading composite microbial system (XDC-2) from composted agricultural and animal wastes amended soil following a long-term directed acclimation. Not only could the XDC-2 degrade natural lignocelluloses, but it could also secrete extracellular xylanase efficiently in liquid culture under static conditions at room temperature. The XDC-2 degraded rice straw by 60.3% after fermentation for 15 days. Hemicelluloses were decomposed effectively, whereas the extracellular xylanase activity was dominant with an activity of 8.357 U/ml on day 6 of the fermentation period. The extracellular crude enzyme noticeably hydrolyzed natural lignocelluloses. The optimum temperature and pH for the xylanase activity were $40^{\circ}C$ and 6.0. However, the xylanase was activated in a wide pH range of 3.0-10.0, and retained more than 80% of its activity at $25-35^{\circ}C$ and pH 5.0-8.0 after three days of incubation in liquid culture under static conditions. PCR-DGGE analysis of successive subcultures indicated that the XDC-2 was structurally stable over long-term restricted and directed cultivation. Analysis of the 168 rRNA gene clone library showed that the XDC-2 was mainly composed of mesophilic bacteria related to the genera Clostridium, Bacteroides, Alcaligenes, Pseudomonas, etc. Our results offer a new approach to exploring efficient lignocellulolytic enzymes by constructing a high-performance composite microbial system with synergistic complex enzymes.

• 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.

• 한국버섯학회지
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• 제13권4호
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• pp.305-309
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• 2015

Cellulase와 xylanase 분비능이 우수한 부숙촉진 세균을 분리하기 위하여 진주시 집현면 소재의 느타리버섯 재배농장으로부터 느타리버섯 수확후배지를 수집하였다. 느타리버섯 수확후배지로부터 19종의 균주를 분리하였으며 이 중 cellulase와 xylanase을 동시에 분비하는 균주를 최종 선발하여 CA105로 명명하였다. Bacillus ID kit와 VITEK 2 system를 이용하여 분리균 CA105의 생화학적 특성을 조사한 결과에서도 분리균 CA105은 B. subtilis와 유사한 특징을 나타내었으며 16S rRNA 염기서열 분석 결과에서는 B. subtilis와 98.9%의 상동성을 나타내었다. 이와 같은 결과를 종합하여 분리균 CA105은 Bacillus subtilis CA105로 동정되었다. 분리균이 분비하는 cellulase와 xylanase 활성은 분리균이 증식함에 따라 대수증식기 중반부터 급격히 증가하였고 정지기에 진입하면 효소활성이 더 이상 증가하지 않는 것으로 나타났다.

• 한국미생물·생명공학회지
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• 제20권5호
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• pp.574-582
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• 1992

Bacillus stearothermophilus exo-xylanase 유전자 DNA가 삽입된 재조합 plasmid pMG1을 가지고 있는 E.coli JM109 exo-xylanase 생산 최적 배양 조건, 생산 효소의 정제 및 정제 효소의 특성 등을 조사 연구하였다. 상기 재조합 E.coli 균주는 0.5 fructose, 0.5 yeast extract, 1.0 tryptone 및 1.0 sodium chloride가 함유된 배지에서 약 10시간 배양했을 때 최대량의 효소를 생산하였으며 생산효소의 94는 세포내에 존재하는 것으로 분석되었다. 생산 효소는 ammonium sulfate 분획, ion exchange chromatography 및 gel filtration 등의 과정을 거쳐 단일 단백질로 정제하였으며 정제 효소는 pH 6.0과 $45^{\circ}C$에서 가장 높은 효소 활성을 나타내었다.또한 1mM $Ca^{2+}$과 $Co^{2+}$ 이온의 첨가는 각각 약 25% 정도의 활성화 효과를 나타내는 반면, 본 효소의 pNPX에 대한 $K_{m}$은 2.75mM, pl값을 4.7, 그리고 분자량은 gel-filtration 법으로는 약 200,000dal., SDS-polyacrylamide gel 전기영동법으로는 약 66,000dal 으로 측정되어 세 개의 동일한 subunit로 구성된 효소 단백질인 것으로 추정되었다. 본 정제 효소는 xylobiose, xylotrioxe 및 xylotetraose 등의 xylo-oligosaccharide를 효과적으로 분해함은 물론이고, 분해율은 낮으나 birchwood xylan, larchwood xylan 및 oatspelt xylan 등의 xyland에도 작용, xylose 생산을 확인함으로써 본 효소는 그 예가 극히 드문 bacterial exo-xylanase인 것으로 분류되었다.

• Journal of Microbiology and Biotechnology
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• 제16권11호
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• pp.1753-1759
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• 2006

Paenibacillus sp. HY-8 isolated from the digestive tracts of the longicorn beetle, Moechotypa diphysis, produced an extracellular endoxylanase with a molecular weight of 20 kDa estimated by SDS-PAGE. The xylanase was purified to near electrophoretic homogeneity from the culture supernatant after ammonium sulfate precipitation, gel filtration, and ionexchange chromatography. The purified xylanase exhibited the highest activities at pH 6.0 and $50^{\circ}C$. The $K_m\;and\;V_{max}$ values were 7.2 mg/ml and 16.3 U/mg, respectively, for birchwood xylan as the substrate. Nucleotide sequence of the PCR-cloned gene was determined to have the open reading frame encoding a polypeptide of 212 amino acids. The N-terminal amino acid sequence and the nucleotide sequence analyses predicted that the precursor xylanase contained a signal peptide composed of 28 amino acids and a catalytically active 19.9-kDa peptide fragment. The deduced amino acid sequence shared extensive similarity with those of the glycoside hydrolase family 11 of xylanases from other bacteria. The predicted amino acid sequence contained two glutamate residues, previously identified as essential and conserved for active sites in other xylanases of the glycoside hydrolase family 11.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.873-880
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• 2009

A novel xylanase gene, Kxyn, was cloned from Kocuria sp. Mn22, a bacteria isolated from the deep sea of the east Pacific. Kxyn consists of 1,170 bp and encodes a protein of 390 amino acids that shows the highest identity (63%) with a xylanase from Thermohifida fusca YX. The mature protein with a molecular mass of approximately 40 kDa was expressed in Escherichia coli BL21 (DE3). The recombinant Kxyn displayed its maximum activity at $55^{\circ}C$ and at pH 8.5. The $K_m,\;V_{max}$, and $k_{cat}$ values of Kxyn for birchwood xylan were 5.4 mg/ml, $272{\mu}mol/min{\cdot}mg$, and 185.1/s, respectively. Kxyn hydrolyzed birchwood xylan to produce xylobiose and xylotriose as the predominant products. The activity of Kxyn was not affected by $Ca^{2+},\;Mg^{2+},\;Na^+,\;K^+$, ${\beta}$-mercaptoethanol, DTT, or SDS, but was strongly inhibited by $Hg^{2+},\;Cu^{2+},Zn^{2+}$, and $Pb^{2+}$. It was stable over a wide pH range, retaining more than 80% activity after overnight incubation at pH 7.5-12. Kxyn is a cellulase-free xylanase. Therefore, these properties make it a candidate for various industrial applications.

• Journal of Microbiology and Biotechnology
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• 제22권8호
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• pp.1133-1140
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• 2012

Ribonucleic acid interference (RNAi) inhibits the expression of target genes in a sequence-specific manner, and shows potential for gene knockdown in filamentous fungi, in which the locus-specific gene knockout occurs in low frequency. In this study, the function of the repressor of cellulase expression I (ACEI) was verified in Trichoderma koningii (T. koningii) YC01 through RNAi, and ace1-silenced strains with improved cellulase productivity were obtained. An expression cassette that transcribed the interfering double-stranded RNA (dsRNA) of ace1 was constructed and transformed into T. koningii, and the transformants, in which the expression of ace1 was successfully silenced, were selected. As a result of the ace1 gene silencing, the expression levels of the main cellulase and xylanase genes were elevated, and the enhanced production of total proteins, cellulase, and xylanase was observed in the cultivation. In addition, the down-regulation of ace1 resulted in an increasing expression of xyr1, but no clear variation in the expression of cre1, which suggested that ACEI acted as a repressor of the xyr1 transcription, but was not involved in the regulation of the cre1 expression. The results of this work indicate that ace1 is a valid target gene for enhancing enzyme production in T. koningii, and RNAi is an appropriate tool for improving the properties of industrial fungi.

• 한국미생물·생명공학회지
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• 제45권1호
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• pp.43-50
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• 2017

본 연구로부터 WJ-1 균주는 제주도에서 수집된 토양샘플로부터 동정되었는데, 형태분화관찰 및 16S rRNA 유전자 염기서열분석과 DNA-DNA hybridization 분석을 통하여 S. glaucescens의 신아종으로 분류되었다. 균주 WJ-1의 주요 cellular fatty acid와 게놈내 G+C 농도는 각각 $C_{15:0}$ anteiso (42.99%)와 74.73 mol%였다. 이 균은 배양액으로부터 준비된 조효소액의 xylanase 활성은 중성 pH 조건 및 $55^{\circ}C$에서 활성이 가장 높았다. S. glaucescens의 조효소액을 이용하여 xylan으로부터 xylotriose 및 xylotetraose를 포함하는 xylooligosaccharide를 제조할 수 있다. 본 연구는 S. glaucescens의 아종에 관한 최초의 보고이며, 관련 종에서 xylanase 활성에 관한 최초의 보고이다. 본 연구 결과로부터, WJ-1 균주는 lignocellulosic biomass의 이용 및 기능성 xylooligosacchade 생산에 유용하게 활용될 수 있을 것으로 기대된다.

• 한국미생물·생명공학회지
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• 제40권4호
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• pp.419-423
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• 2012

A xylanolytic bacterial strain, MX47, was isolated from rotting plant matter in soil. The strain was aerobic and gram positive, and grew between pH 6.0 and 11.0. Cells were susceptible to thiostrepton and chloramphenicol. The major fatty acids (>3%) comprised 64.55% of iso-$C_{15:0}$, 22.76% of anteiso-$C_{15:0}$, and 3.92% of iso-$C_{17:0}$. The G/C content of the DNA was 44.15 mol%. The predominant respiratory quinone was menaquinone 7 (MK-7). Searches for 16S rRNA gene sequence similarity as well as phylogenetic analyses strongly suggested that the strain should be classified to the genus Bacillus. However, its biochemical characteristics, including acid production and enzyme activities, are different from those of other Bacillus strains in the same clade, and therefore, we propose the name Bacillus sp. MX47.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1077-1084
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• 2009

A genomic library was constructed to clone a xylanase gene (Mxyn10) from Demequina sp. JK4 isolated from a deep sea. Mxyn10 encoded a 471 residue protein with a calculated molecular mass of 49 kDa. This protein showed the highest sequence identity (70%) with the xylanase from Streptomyces lividans. Mxyn10 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10) and a carbohydrate-binding module (CBM) belonging to family 2. The optimum pH and temperature for enzymatic activity were pH 5.5 and $55^{\circ}C$, respectively. Mxyn10 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 3.5 to 10.0. The protein was not significantly affected by a variety of chemical reagents, including some compounds that usually inhibit the activity of other related enzymes. In addition, Mxyn10 showed activity on cellulose. These properties mark Mxyn10 as a potential enzyme for industrial application and saccharification processes essential for bioethanol production.