• Title/Summary/Keyword: Streptomyces thermocyaneoviolaceus

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Thermostable Xylanase Encoded by xynA of Streptomyces thermocyaneoviolaceus: Cloning, Purification, Characterization and Production of Xylooligosaccharides

  • CHOI JUN-HO;LEE OH-SEUK;SHIN JAE-HO;KWAK YUN-YOUNG;KIM YOUNG-MOG;RHEE IN-KOO
    • Journal of Microbiology and Biotechnology
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    • v.16 no.1
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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.

Production of Xylooligosaccharides with Thermostable Xylanases from the Streptomyces thermocyaneo-violaceus (내열성 방성균 Streptomyces thermocyaneoviloaceus 의 Xylanases를 이용한 자일로올리고당의 생산)

  • 이오석;최충식;최준호;주길재;이인구
    • Microbiology and Biotechnology Letters
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    • v.29 no.4
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    • pp.221-226
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    • 2001
  • Streptomyces themocyaneovio-laceus producing the thermostable xylanase was used for the production of xylooligosaccharides from xylan. The optimal conditions for the xylanase production were investigated in jar fermentor, which operated at 2 vvm aera-tion and 400 rpm agitation speed at $50^{\circ}C$ for 24 h. The optimal reaction condtion for the production of xylooli-gosaccharides with xylanases which were prepared by the percipitation with ammonium sulfate were obtained by the reaction at $60^{\circ}C$ for 12 h in the mixture composed of 10% birchwood xylan in 50 mM sodium phosphate buffer (pH 6.0)and 10 unit/ml of xylanase. In this optimal condition for the xylooligosaccharides production the mixture of xylooligosaccharides (58.8 g/I) which were composed of 20.1 g/I of xyobiose, 8.9 g/I of xylotriose 4.5 g/I of xylotetraose 16.2g/I of xylopentaose and 9.1 g/I xylohexaose and 5.0 g/I of xylose was produced from 100 g/I of birchwood xylan by the xylanases of S thermocyaneoviolaceus .

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Production Conditions of Xylanase from Streptomyces thermocyaneoviolaceus and Production of Xylooligosaccharides (Streptomyces thermocyaneoviolaceus의 Xylanase 생산조건 및 Xylooligo당의 생산)

  • Choi, Jun-Ho;Kwon, Dal-Ho;Lee, Oh-Seuk;Joo, Gil-Jae;Park, Heui-Dong;Rhee, In-Koo
    • Current Research on Agriculture and Life Sciences
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    • v.16
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    • pp.45-54
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    • 1998
  • A thermotolerant bacterium, Streptomyces thermocyaneoviolaceus which produced xylan-degrading enzymes, utilized excellently xylan of wheat bran by producing the enzymes in comparison with that of birchwood or oat spelts. Optimal enzyme production was achieved in WB medium containing 0.8% wheat bran, 0.06% yeast extract, 0.06% bactopeptone, 0.05% $MgSO_4{\cdot}7H_2O$, 0.05% $FeSO_4{\cdot}7H_2O$, 0.05% $KH_2PO_4$ and, 0.2% $K_2HPO_4$(pH 7.0) at $50^{\circ}C$ for 24 hrs. The optimal pH and temperature for the hydrolysis of xylan were pH 5.5 and $65^{\circ}C$, respectively. The enzyme activity was retained more than 80% at the range from pH 4.5 to pH 9.5 at $4^{\circ}C$ for 12 hrs and 94% on the heat-treatment at $65^{\circ}C$ for 1 hr. Xylobiose, xylotriose, xylose, and other xylooligosaccharides were produced as end products from hydrolysis of birchwood xylan by the xylanase of S. thermocyaneoviolceus.

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Construction of Xylose-Inducible Expression Vector Using xylA Promoter of Escherichia coli (대장균 xylA 프로모터를 이용한 xylose 유도성 발현벡터의 구축)

  • Kim, Hyun-Ho;So, Jai-Hyun;Rhee, In-Koo
    • Journal of Applied Biological Chemistry
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    • v.53 no.1
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    • pp.1-7
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    • 2010
  • xylA promoter is a major promoter in xylose operon of Escherichia coli. xylA promoter is sufficient as the promoter for the construction of new expression vector because this promoter was tightly controlled and induced by the addition of xylose. For the construction of xylose-inducible expression vector, 600 bp of xylA promoter was ligated between AatII and HindIII of pUC18, named pXA600. In order to investigate the effect of XylR protein encoded by xylR gene on the xylA promoter, 1,988 bp of xylR gene including its promoter was ligated into downstream of multiple cloning site to the opposite direction of xylA promoter in pXA600, named pXAR600. For the measurement of expression level, 3,048 bp of lacZ structural gene was fused into xylA promoter in both plasmids pXA600 and pXAR600 as a reporter gene, named pXA600-lacZ and pXAR600-lacZ, respectively. The $\beta$-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E. coli JM109 was determined to be 1,641 and 2,304 unit by the induction with xylose in LB medium, respectively. The $\beta$-galactosidase activity of pXAR600-lacZ/JM109 was about 1.4 times higher by the induction with xylose than that of pXA600-lacZ/JM109. The $\beta$-galactosidase activity of pXA600-lacZ and pXAR600-lacZ in E.coli JM109 showed 6,282 and 9,320 unit by the induction with xylose in DM minimal medium, respectively. A regulator, xylR protein works as an activator for the gene expression by the addition of xylose in the xylose-inducible vectors because the level of gene expression in pXA600 is increased by the insertion of xylR gene into the same vector. The xynA gene of Streptomyces thermocyaneoviolaceus cloned in pXA600 and pXAR600 was successfully expressed in E. coli BLR(DE3). As a result, plasmids pXA600 and pXAR600 using xylA promoter are sufficient as new expression system to produce a foreign protein in E. coli.