• BMB Reports
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• 제44권1호
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• pp.52-57
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• 2011

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and $37^{\circ}C$, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.57-63
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• 2006

We have cloned a xylanase gene (xynA) from Streptomyces thermocyaneoviolaceus. The deduced amino acid sequences of the XynA, including the active site sequences of glycosyl hydrolase family 10, showed high sequence homology with several xylanases assigned in this category. The XynA was overexpressed under an IPTG inducible T7 promoter control in E. coli BLR(DE3). The overproduced enzymes were excreted into culture supernatants and periplasmic space. The purified XynA had an apparent molecular mass of near 54 kDa, which corresponds to the molecular mass calculated from its gene. The optimum pH and temperature of the purified XynA were determined to be 5.0 and $65^{\circ}C$, respectively. The XynA retained over $90\%$ its activity after the heat treatment at $65^{\circ}C$ for 30 min. The XynA was highly efficient in producing xylose (X1), xylobiose (X2), xylotriose (X3), and xylotetraose (X4) from xylan.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.662-671
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• 2015

To enrich the genetic resource of microbial xylanases with high activity and stability under alkaline conditions, a xylanase gene (xynSL4) was cloned from Planococcus sp. SL4, an alkaline xylanase-producing strain isolated from the sediment of soda lake Dabusu. Deduced XynSL4 consists of a putative signal peptide of 29 residues and a catalytic domain (30-380 residues) of glycosyl hydrolase family 10, and shares the highest identity of 77% with a hypothetical protein from Planomicrobium glaciei CHR43. Phylogenetic analysis indicated that deduced XynSL4 is closely related with thermophilic and alkaline xylanases from Geobacillus and Bacillus species. The gene xynSL4 was expressed heterologously in Escherichia coli and the recombinant enzyme showed some superior properties. Purified recombinant XynSL4 (rXynSL4) was highly active and stable over the neutral and alkaline pH range from 6 to 11, with maximum activity at pH 7 and more than 60% activity at pH 11. It had an apparent temperature optimum of 70℃ and retained stable at this temperature in the presence of substrate. rXynSL4 was highly halotolerant, retaining more than 55% activity with 0.25-3.0 M NaCl and was stable at the concentration of NaCl up to 4M. The enzyme activity was significantly enhanced by β-mercaptoethanol and Ca²⁺ but strongly inhibited by heavy-metal ions and SDS. This thermophilic and alkaline- and salt-tolerant enzyme has great potential for basic research and industrial applications.

• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.77-83
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• 1999

Molecular biological techniques that recently developed, have made it possible to realize some of new attempts in the research field of rumen microbiology. Those are 1) cloning of genes from rumen microorganisms mainly in E. coli, 2) transformation of rumen bacteria and 3) ecological analysis with nonculturing methods. Most of the cloned genes are for polysaccharidase enzymes such as endoglucanase, xylanase, amylase, chitinase and others, and the cloning rendered gene structural analyses by sequencing and also characterization of the translated products through easier purification. Electrotransformation of Butyrivibrio fibrisolvens and Prevotella ruminicola have been made toward the direction for obtaining more fibrolytic, acid-tolerant, depoisoning or essential amino acids-producing rumen bacterium. These primarily required stable and efficient gene transfer systems. Some vectors, constructed from native plasmids of rumen bacteria, are now available for successful gene introduction and expression in those rumen bacterial species. Probing and PCR-based methodologies have also been developed for detecting specific bacterial species and even strains. These are much due to accumulation of rRNA gene sequences of rumen microbes in databases. Although optimized analytical conditions are essential to reliable and reproducible estimation of the targeted microbes, the methods permit long term storage of frozen samples, providing us ease in analytical work as compared with a traditional method based on culturing. Moreover, the methods seem to be promissing for obtaining taxonomic and evolutionary information on all the rumen microbes, whether they are culturable or not.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1999년도 한국생물과학협회 학술발표대회
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• pp.273.1-273
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• 1999

No Abstract, See Full Text

• 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.

• 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.

• 생명과학회지
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• 제25권6호
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• pp.680-685
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• 2015

이전에 토양으로부터 cellulase와 xylanase 생산 균주로 단리하였다. 단리한 균주 유래의 16S rRNA 유전자 및 API 50 kit를 분석한 결과 Bacillus subtilis와 약 99.5%의 높은 상동성을 보였기에 본 균주를 B. subtilis NC1으로 명명하였다. Bacillus subtilis NC1 균주 유래 cellulase와 xylanase 유전자를 cloning 하여 유전자 배열을 규명하였다. 또한, 두 효소의 아미노산 배열을 이용하여 상동성을 검토한 결과 cellulase는 Glycoside hydrolase family (GH) 5 그리고 xylanase는 GH30에 속하는 효소임을 밝혔다. 본 연구에서는 B. subtilis NC1 의 cellulase 생산을 위한 배지성분의 최적 농도를 결정하기 위해 중심합성계획법(central composite design, CCD)을 기반으로 한 반응표면 분석법(Response Surface Methodology) 을 수행하였다. 세가지 독립변수로는 tryptone, yeast extract, 그리고 NaCl이 조사되었다. 반응값에 대하여 분산분석을 실시한 결과 결정계수(R²)는 0.96이었으며 전체 모델에 대한 유의확률이 0.0001로 매우 높은 유의성을 지님을 확인하였다. 반응표면분석법을 통하여 얻어진 B. subtilis NC1의 cellulase 활성을 위한 최적화 배지의 각 변수 농도는 tryptone 2.5%, yeast extract 0.5%, 그리고 NaCl 1.0%로 예측 되었다. 최적화 배지에서의 B. subtilis NC1의 cellulase 활성을 검증한 최적화를 실시하기 이전인 대조구의 cellulase 활성 0.5U/ml와 비교하면 24% 활성이 향상된 0.62U/ml의 높은 활성을 보였다.

• Journal of Applied Biological Chemistry
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• 제53권1호
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• pp.1-7
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• 2010

xylA 프로모터는 대장균의 xylose 대사에 관여하는 xylose 오페론 상의 중요한 프로모터이다. 이 프로모터는 xylose에 의해 강하게 조절을 받는다고 알려져 있다. 이러한 특징은 새로운 발현 백터를 구축하는데 충분한 조건을 갖추고 있다고 생각된다. 본 연구에서는 이러한 xylose에 의해 유도 되는 발현벡터를 구축하기 위하여 600 bp의 xylA 프로모터를 증폭하여 pUC18의 AatII와 HindIII 사이에 삽입하여 pXA600을 구축하였다. 또한 조절단백질인 XylR의 영향을 조사하기 위하여 xylR 유전자를 삽입하여 pXAR600을 구축하였다. 발현의 강도를 측정하기 위하여 3,048 bp의 lacZ유전자를 xylA 프로모터의 하류에 연결하여 pXA600-lacZ와 pXAR600-lacZ를 구축하고 대장균 JM109에 형질전환시켰다. 구축된 pXA600-lacZ와 pXAR600-lacZ는 LB 배지에서 배양하였을 때 xylose 유도하에서 각각 1,641 unit와 2,304 unit의 $\beta$-galactosidase 활성을 보였으며, DM 배지상에서 배양했을 때 xylose 유도 시 각각 6,282 unit와 9,320 unit의 $\beta$-galactosidase 활성을 보였다. 또한 왜래 유전자의 발현 가능성을 확인하기 위하여 S. thermocyaneoviolaceus의 내열성 xylanase를 코딩하는 xynA 유전자를 실제로 구축된 pXA600과 pXAR600에서 발현을 확인하여 pXA600 및 pXAR600이 새로운 xylose 유도성 발현벡터로서의 사용 가능성을 확인하였다.