• Journal of Industrial Technology
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• v.29 no.A
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• pp.161-167
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• 2009

The bifunctional Xylanase-Cellulase hybrid protein was constructed by gene fusion. Two genes corresponding to endoxylanase gene (xylS) and endocellulase gene (celA) were amplified by PCR from Bacillus licleniformis NBL420. It was then linked through splicing by overlap extension (SOE) by PCR method. The two resulting fused hybrids, xyl/cel and cel/xyl, which differ by its orientation, were confirmed by its nucleotide sequencings. One of two fusion genes, xyl/cel was successfully expressed into pET22b(+) vector (pxyl/cel) with bifunctional xylanase-cellulase activity. On the contrary, the other cel/xyl fusion protein showed only cellulase activity with much decreased xylanase activity. Enzymatic properties of Xyl/Cel fusion protein were investigated regarding optimum pH, optimum temp, thermostability, and pH stability. It was revealed that Xyl/Cel fusion protein retained the bifunctional xylanase-cellulase activities eventhough two enzymes were connected with each other directly. These informations could be useful for construction of other hybrid proteins as well as increased range of substrate utilization.

• 한국신재생에너지학회:학술대회논문집
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• 2011.05a
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• pp.171-171
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• 2011

리그노셀룰로오스 바이오매스를 원료로 하여 발효당을 생산하고자 할 때 최종적인 당수율은 적용하는 전처리 기술의 종류와 전처리 조건에 따라 크게 달라질 수 있다는 사실은 널리 알려져 있다. 또한 전처리물의 효소당화에서 셀룰로오스의 당전환율은 효소의 종류와 사용량에 의존적이므로 부족한 전처리 효과를 일부 보완하기도 한다. 본 연구에서는 바이오매스의 효소당화에 흔히 사용되고 있는 특정효소와 최근 공급되기 시작한 새로운 효소를 시료로 하여 해바라기 줄기의 열수전처리에 이은 효소당화에서 효소의 종류가 최종 당수율, 효소당화 시간 및 전처리효과에 미치는 영향을 측정하였다. 해바라기 줄기 분말을 $180^{\circ}C$에서 30분 동안 열수전처리하였을 때 헤미셀룰로오스의 수율이 최대가 되었으나 이 조건에서는 후속 효소당화에 의한 포도당 수율이 높지 않았다. Celluclast 1.5L 혹은 Celluclast conc BG는 전처리 물의 당화속도가 상대적으로 빠른 편이었고, 이 효소에 의한 당수율은 해바라기 줄기 셀룰로오스 함량의 80% 내외였으며, 바이오매스 1g당 효소첨가량이 3ml까지 증가함에 따라 당수율도 꾸준히 증가하는 경향을 보였다. 반면에 노보자임 스코리아로부터 분양받은 효소로서 NS22074와 NS50010의 혼합물은 Celluclast보다 약 10% 더 높은 당수율을 보여주었으나 당화는 상대적으로 느리게 진행되어 72시간 이상이 소요되었다. Endoxylanase 혹은 hemicellulase 등 다른 효소를 NS22074와 NS50010의 혼합물에 가하여도 당수율의 증대 효과는 미미하거나 거의 없었다. 시험에 사용한 효소제제에는 포도당, 소르비톨 등 여러 가지 당들이 보존제 혹은 안정화제로 함유되어 있는 것으로 나타나서 사용에 주의할 필요가 이었다.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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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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• v.17 no.11
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• pp.1782-1788
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• 2007

Cellulosomes in Clostridium cellulovorans are assembled by the interaction between the repeated cohesin domains of a scaffolding protein (CbpA) and the dockerin domain of enzyme components. In this study, we determined the synergistic effects on cellulosic and hemicellulosic substrates by three different recombinant mini-cellulosomes containing either endoglucanase EngB or endoxylanase XynA bound to mini-CbpA with one cohesin domain (mini-CbpAl), two cohesins (mini-CbpA12), or four cohesins (mini-CbpAl234). The assembly of EngB or XynA with mini-CbpA increased the activity against carboxymethyl cellulose, acid-swollen cellulose, Avicel, xylan, and com fiber 1.1-1.8-fold compared with that for the corresponding enzyme alone. A most distinct improvement was shown with com fiber, a natural substrate containing xylan, arabinan, and cellulose. However, there was little difference in activity between the three different mini-cellulosomes when the cellulosomal enzyme concentration was held constant regardless of the copy number of cohesins in the cellulosome. A synergistic effect was observed when the enzyme concentration was increased to be proportional to the number of cohesins in the mini-cellulosome. The highest degree of synergy was observed with mini-CbpAl234 (1.8-fold) and then mini-CbpAl2 (1.3-fold), and the lowest synergy was observed with mini-CbpAl (1.2-fold) when Avicel was used as the substrate. As the copy number of cohesin was increased, there was more synergy. These results indicate that the clustering effect (physical enzyme proximity) of the enzyme within the mini-cellulosome is one of the important factors for efficient degradation of plant cell walls.

• Korean Chemical Engineering Research
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• v.54 no.6
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• pp.822-829
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• 2016

Our aim was to optimize the production of cellulase-free thermoactive xylanase by Aureobasidium pullulans CBS 135684 with statistical methodology based on experimental designs. Among eleven variables, the nutrient sources that had significant effect on xylanase production were corncob, $(NH_4)_2SO_4$, xylose, $KH_2PO_4$ and tween 80, identified by the initial screening method of Plackett-Burman. The optimum concentrations of these five components were subsequently investigated using response surface methodology. The optimal concentrations ($g{\cdot}l^{-1}$) for maximum production of xylanase were corncob, 39.0; $(NH_4)_2SO_4$, 3.0; xylose, 1.8; $KH_2PO_4$ 1.4; and tween 80, 1.4, respectively. An improved xylanase yield of $8.74{\pm}0.84U{\cdot}ml^{-1}$ was obtained with optimized medium which is 2.1-fold higher production than previously obtained results ($4.10{\pm}0.10U{\cdot}ml^{-1}$) after 48 h of cultivation. In addition, the xylanase production under optimal condition reached $10.09{\pm}0.27U{\cdot}ml^{-1}$ after 72 h of cultivation.

• The Korean Journal of Mycology
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• v.18 no.4
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• pp.209-217
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• 1990

The influence of cellulosic and hemicellulosic substrates on the production of cellulase and xylanase complexes in Aspergillus niger was investigated. The culture conditions with different substrates exhibited profound effects on the level of endoglucanase (CMCase), ${\beta}-glucosidase$, endoxylanase and ${\beta}-xylosidase$, and on their isozyme patterns. However, intracellular and extracellular isozyme patterns of cellulase and xylanase complexes were qualitatively identical and appeared to be simultaneous in the early growth phase. Prolonged incubation led to the increase in the concentrations of isozymes with a little changes in the relative proportions of those isozymes. These results suggest that the biosynthesis of cellulase and xylanase complexes in A. niger is coordinately regulated at the level of induction. Moreover, multiple forms of extracellular cellulase and xylanase complexes seem to be the outcome of specific gene expression and should not be considered solely as the consequence of post-secretional modification of synthesized enzymes.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.9-19
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• 2016

Xylanases sourced from different bacteria have significantly different enzymatic properties. Therefore, studying xylanases from different bacteria is important to their applications in different fields. A potential xylanase degradation gene in Massilia was recently discovered through genomic sequencing. However, its xylanase activity remains unexplored. This paper is the first to report a xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) from the genus Massilia. The gene encodes a 383-residue polypeptide (XynRBM26) with the highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, which could maintain more than 90% activity between pH 5.0 and 8.0. Moreover, XynRBM26 is stable at 37℃ and could maintain at least 96% activity after being placed at 37℃ for 1 h. This paper is the first to report that GH10 xylanase in an animal gastrointestinal tract (GIT) has salt tolerance, which could maintain 86% activity in 5 M NaCl. Under the optimum conditions, K_m, V_max, and k_cat of XynRBM26 to beechwood xylan are 9.49 mg/ml, 65.79 μmol/min/mg, and 47.34 /sec, respectively. Considering that XynRBM26 comes from an animal GIT, this xylanase has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and seafood processing, as well as other high-salt environmental biotechnological fields, because of its high catalytic activity in high-concentration NaCl.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.794-801
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• 2013

This paper reports the production and characterization of crude xylanase from the newly isolated Humicola sp. Ly01. The highest (41.8 U/ml) production of the crude xylanase was obtained under the optimized conditions (w/v): 0.5% wheat bran, 0.2% $KH_2PO_4$, and 0.5% peptone; initial pH 7.0; incubation time 72 h; $30^{\circ}C$; and 150 rpm. A considerable amount of the crude xylanase was induced using hulless barley bran or soybean meal as the carbon source, but a small amount of the enzyme was produced when supplementary urea was used as the nitrogen source to wheat bran. The crude xylanase showed apparent optimal cellulase-free xylanase activity at $60^{\circ}C$ and pH 6.0, more than 71.8% of the maximum xylanase activity in 3.0-30.0% (v/v) ethanol and more than 82.3% of the initial xylanase activity after incubation in 3.0-30.0% (v/v) ethanol at $30^{\circ}C$ for 2 h. The crude xylanase was moderately resistant to both acid and neutral protease digestion, and released 7.9 and 10.9 ${\mu}mol/ml$ reducing sugar from xylan in the simulated gastric and intestinal fluids, respectively. The xylooligosaccharides were the main products of the hydrolysis of xylan by the crude xylanase. These properties suggested the potential of the crude enzyme for being applied in the animal feed industry, xylooligosaccharides production, and high-alcohol conditions such as ethanol production and brewing.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.489-496
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• 2014

High levels of extracellular xylanase activity (211.79 IU/mg) produced by Paenibacillus sp. NF1 were detected when it was submerged-cultured. After three consecutive purification steps using Octyl-Sepharose, Sephadex G75, and Q-Sepharose columns, a thermostable xylanase (XynNF) was purified to homogeneity and showed a molecular mass of 37 kDa according to SDS-PAGE. The specific activity of the purified XynNF was up to 3,081.05 IU/mg with a 14.55-fold purification. The activity of XynNF was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Fe^{2+}$, $Zn^{2+}$, $Fe^{2+}$, $Cu^{2+}$, SDS, and EDTA. The purified XynNF displayed a greater affinity for oat spelt xylan with the maximal enzymatic activity at $60^{\circ}C$ and pH 6.0. XynNF, which was shown to be cellulose-free, with high stability at high temperature ($70^{\circ}C-80^{\circ}C$) and low pH range (pH 4.0-7.0), is potentially valuable for various industrial applications. The enzyme hydrolyzed oat spelt xylan to yield mainly xylooligosaccharides (95.8%) of 2-4 degree of polymerization (DP2-4). Moreover, the majority of the xylooligosacharides (DP2-4) products was xylobiose (61.5%). The thermostable xylanase (XynNF) thus seems potentially usefull in the production of xylooligosaccharides.