• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.14-20
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• 1998

Syntheses of the B. stearothermophilus xylanolytic enzymes such as xylanases, ${\beta}$-xylosidases, ${\alpha}$-arabinofurano-sidases, and esterases, were observed to be regulated by the carbon source present in the culture media. Xylan induced synthesis of ${\beta}$-xylosidase at the highest level while xylose gave about 30% of the ${\beta}$-xylosidase activity induced by xylan. The lowest syntheses of the xylanolytic enzymes above mentioned were detected in the basal medium containing glucose as a sole carbon source. When a mixture of xylan and glucose was used as a carbon source, we could observe glucose repression of xylanase (about 70-fold) and ${\beta}$-xylosidase (about 40-fold) syntheses. Whereas, the level of the glucose repression of the expression of the xylA gene encoding the major ${\beta}$-xylosidase of B. stearothermophilus was assessed to be about l0-fold when the relative amounts of the xylA transcript were determined. From the sequence of the xylA gene, we could find two CRE-like sequences (CRE-l: nucleotides ＋124 to ＋136 and CRE-2:＋247 to ＋259) within the reading frame of the xylA gene, either or both of which were suspected to be involved in catabolite repression of the xylA gene.

• BMB Reports
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• 제38권1호
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• pp.17-23
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• 2005

During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.

• KSBB Journal
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• 제26권5호
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• pp.465-470
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• 2011

Twenty six microorganisms were isolated from soil and horse manure samples from in Iowa, U.S. Microorganisms were cultivated and screened by using plate count agar (PCA) at $35^{\circ}C$ containing 1% (w/v) oat spelt xylan instead of glucose. The xylanase activities of bacterial strains were analyzed by measuring the concentration of reducing sugar by DNS method. All isolated strains were characterized as the rod form and gram positive strains. Among the isolated strains, the HM6 strains gave the highest xylanase activity. This strain was identified as Bacillus pumilus HM6 by 16S rDNA sequence, morphological and biochemical analysis. Optimal culture temperature and initial medium pH for B. pumilus HM6 were $30-35^{\circ}C$ and pH 6-7, respectively. The maximum xylanase activity of 6879 IU/mL was obtained after growth of HM6 with 1% (w/v) oat spelt xylan at $35^{\circ}C$ for 6 days. Studies on enzymatic properties showed that the optimum conditions for the highest xylanase activity were $60^{\circ}C$ and pH 8.0. In addition, xylanase activity was stable over 2 hours at $50^{\circ}C$, whereas activity decreased after 30 min at $70^{\circ}C$.

• 한국미생물·생명공학회지
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• 제22권6호
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• pp.599-606
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• 1994

Bacillus stearothermomophilus, a strong xylan degrader, was confirmed to express multiple esterase activities in addition to the major xylanolytic enzymes. One of the genes encoding the esterases was isolated from the genomic library of B. stearothermophilus constructed with EcoRl restriction endonuclease and pBR322 plasmid. Three recombinant plasmids showing the tributyrin degrading activity were selected from approximately 7, 000 E. coli HB101 transformants, and were found to have the same insert of a 3.2 kb DNA fragment. Restriction mapping and hybridization studies revealed that the gene(estII) on the hybrid plasmid (pKMG7) had originated from the B. stearothermophilus chromosome, and was distinct from the estl, another esterase gene of B. stearothermophilus isolated in the previous work. The E. coli cells harboring pKMG7 produced an acetylxylan esterase that exibited similar substrate specificity to the esterase encoded by the estI gene.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제26권1호
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• pp.50-58
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• 2013

A facultative bacterium producing cellulolytic and hemicellulolytic enzymes was isolated from the rumen of a native Korean goat. The bacterium was identified as a Bacillus licheniformis on the basis of biochemical and morphological characteristics and 16S rDNA sequences, and has been designated Bacillus licheniformis JK7. Endoglucanase activities were higher than those of ${\beta}$-glucosidase and xylanase at all temperatures. Xylanase had the lowest activity among the three enzymes examined. The optimum temperature for the enzymes of Bacillus licheniformis JK7 was $70^{\circ}C$ for endoglucanase (0.75 U/ml) and $50^{\circ}C$ for ${\beta}$-glucosidase and xylanase (0.63 U/ml, 0.44 U/ml, respectively). All three enzymes were stable at a temperature range of 20 to $50^{\circ}C$. At $50^{\circ}C$, endoglucanse, ${\beta}$-glucosidase, and xylanase had 90.29, 94.80, and 88.69% residual activity, respectively. The optimal pH for the three enzymes was 5.0, at which their activity was 1.46, 1.10, and 1.08 U/ml, respectively. The activity of all three enzymes was stable in the pH range of 3.0 to 6.0. Endoglucanase activity was increased 113% by $K^+$, while $K^+$, $Zn^+$, and tween 20 enhanced ${\beta}$-glucosidase activity. Xylanase showed considerable activity even in presence of selected chemical additives, with the exception of $Mn^{2+}$ and $Cu^{2+}$. The broad range of optimum temperatures (20 to $40^{\circ}C$) and the stability under acidic pH (4 to 6) suggest that the cellulolytic enzymes of Bacillus licheniformis JK7 may be good candidates for use in the biofuel industry.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.584-590
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding ${\beta}-xylosidase$, a major xylanolytic enzyme, was previously cloned and sequenced by the present authors. Sequence analysis indicated that translation of the xylA gene was initiated from the noncanonical initiation codon UUG, confirmed by analyzing three different amber (UAG) mutants of the xylA gene. In the present study, the UUG initiation codon was mutated into AUG or GUG, and the effects of the mutations on the XylA synthesis were examined. The AUG initiation codon was found to direct the highest level of ${\beta}-xylosidase$ synthesis; three-fold and fourteen-fold more enzyme activity than the UUG codon in E. coli and B. subtilis cells, respectively. Surprisingly, contrary to other systems reported to date, the UUG start codon was found next to AUG in the relative order of translational efficiency in both organisms. In addition, a greater abundance of the xylA mRNA was detected with the AUG start codon in both of these host cells than with GUG or UUG. Northern blot and Toeprint assays revealed that this was due to enhanced stability of mRNA with the AUG initiation codon. As expected, the ${\beta}-xylosidase$ protein level in the bacterial cells containing mRNA with the AUC start codon was also much higher than the levels with the other two different mRNAs.

• 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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• 제24권7호
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• pp.943-953
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• 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.

• 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.