• Current Research on Agriculture and Life Sciences
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• v.11
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• pp.81-89
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• 1993

In order to isolate a mutant which was constitutively expressed in xylA gene, Pxyl-cat-xylA fusion gene was constructed by the insertion of cat gene between xylA promoter and xylA structural gene in pEX13 contained xylA gene. The expression of cat and xylA gene from transformants of xylA mutant DH77 with plasmid pEXC131 containing Pxyl-cat-xylA fusion gene was induced by the addition of 0.4% xylose to media. This results indicated that cat and xylA gene were expressed under control of xylA promoter the presence of xylR gene. We have also isolated constitutive mutant plasmid pEXC131-39 from pEXC131 by trementment with N-methyl-N'-nitro-N-nitrosoguanidine(NTG). cat and xylA gene from pEXC131-39 were constitutively expressed without induction of xylose regardless of xylR gene. Transformants of xylR mutant DH60 with pEXC131-39 also expressed chloramphenicol resistances and xylose isomerase without induction of xylose. This result shows that mutation in region of xylA promoter might make it possible to be constitutively expressed.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.21-27
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• 1998

The xylA gene of Bacillus stearothermophilus encoding the major ${\beta}$-xylosidase was previously cloned and sequenced. In the present study we examined the regulation of the cloned xylA gene expression in Bauillus subtilis MW15 carrying the xylA::aprA fusion plasmids. The induction of the fused xylA gene expression remained uninfluenced by any of the carbon sources tested but the gene expression was repressed about 2-3 fold in the presence of glucose. Two CRE-like sequences (CRE-1: nucleotides ＋ 124 to ＋136 and CRE-2: ＋247 to ＋259) were recognized within the reading frame region of the xylA gene. The deletion experiments showed that the CRE-2 sequence had a role in catabolite repression (CR) as a true CRE of the xylA gene, but the CRE-1 had no effect on CR of the xylA gene expression. Surprisingly, the deletion of the CRE- 1 sequence reduced about 2~3 fold of the expression of the xylA fused gene. The repression ratios of the xylA gene expression were estimated to be about 0.4 from the assay of subtilisin activity, and about 0.3 at the level of transcription by determining the amounts of xylA transcripts in B. subtilis. While, the level of CR of the xylA gene was assessed to be about l0-fold in previous work when the relative amounts of the xylA transcripts were measured in B. stearothermophilus.

• Current Research on Agriculture and Life Sciences
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• v.10
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• pp.125-135
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• 1992

Nine strains of xyl mutants that could not utilize xylose as a carbon source were isolated from E. coli JM109 by the treatment of NTG in order to investigate the regulation of xylose operon and to use recipient cells for the cloning of xylR gene. For the characterization of all isolated mutants, colony colors of all mutants on MacConkey-xylose and MacConkey-xylulose agar plate were observed for the utilization of xylose and xylulose, and the growth level and the activity of xylose isomerase and xylulokinase were determined in need. The isolated xylR/T mutants formed the white colony on MacConkey-xy-lose and MacConkey-xylulose agar plate. They did not detect the activity of xylose isomerase, and the activity of xylose isomerase was not restored in transformants of xylR/T mutant with pEX13 which contained xylA gene. xylR and xylT mutants were classified from xylR/T mutants depending upon the growth level in minimal medium. xylT mutants; DH13, DH121 and DH125 could grow a little in that medium, but xylR mutants; DH10, DH53, and DH60 could not grow that medium.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.946-949
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• 2010

The effects of two different sugars (glucose and xylose) on the expression levels and patterns of the xylose reductase (xyl1), xylitol dehydrogenase (xyl2), and xylulokinase (xyl3) genes were analyzed using Pichia stipitis. A significant increase in mRNA levels of xyl1 was observed after 6 h growth in culture conditions using xylose as a sole carbon source, but expressions of the three genes were not influenced by normal culture media with glucose. In addition, expressions of xyl2 and xyl3 were not observed during the entire culture period during which xylose was added. It also was found that the expression level of xyl1 increased as a function of the xylose concentration (40, 60, and 80 g/l) used in this study, indicating that xyl1 expression sensitively responded to xylose in the culture media. Although the induced level of xyl2 increased slightly after 48 h in the xylose-supplemented culture conditions, the expression of xyl2 was not observed in the xylitol-supplemented culture conditions. Finally, considering the expression of each gene in response to glucose or xylose, the absolute expression levels of the three genes indicate that xyl1 is induced primarily by exposure to xylose.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.837-844
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• 2008

Glucose (xylose) isomerases from Streptomyces sp. have been used for the production of high fructose corn syrup for industrial purposes. An 11-kb DNA fragment containing the xyl gene cluster was isolated from Streptomyces lividans TK24 and its nucleotide sequences were analyzed. It was found that the xyl gene cluster contained a putative transcriptional repressor (xylR), xylulokinase (xylB), and xylose isomerase (xylA) genes. The transcriptional directions of the xylB and xylA genes were divergent, which is consistent to those found in other streptomycetes. A gene encoding XylR was located downstream of the xylB gene in the same direction, and its mutant strain produced xylose isomerase regardless of xylose in the media. The enzyme expression level in the mutant was 4.6 times higher than that in the parent strain under xylose-induced condition. Even in the absence of xylose, the mutant strain produce over 60% of enzyme compared with the xylose-induced condition. Gel mobility shift assay showed that XylR was able to bind to the putative xyl promoter, and its binding was inhibited by the addition of xylose in vitro. This result suggested that XylR acts as a repressor in the S. lividans xylose operon.

• Journal of Life Science
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• v.27 no.12
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• pp.1403-1409
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• 2017

In this study, the xylitol dehydrogenase (XYL2) gene was expressed in Saccharomyces cerevisiae as a host cell for ease of use in the degradation of lignocellulosic biomass (xylose). To select suitable expression systems for the S.XYL2 gene from S. cerevisiae and the P.XYL2 gene from Pichia stipitis, $pGMF{\alpha}-S.XYL2$, $pGMF{\alpha}-P.XYL2$, $pAMF{\alpha}-S.XYL2$ and $pAMF{\alpha}-P.XYL2$ plasmids with the GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter to allow secretion. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$ strain and the xylitol dehydrogenase activity was investigated. The GAL10 promoter proved more suitable than the ADH1 promoter for expression of the XYL2 gene, and the xylitol dehydrogenase activity from P. stipitis was twice that from S. cerevisiae. The xylitol dehydrogenase showed $NAD^+$-dependent activity and about 77% of the recombinant xylitol dehydrogenase was secreted into the periplasmic space of the $SEY2102{\Delta}trp1/pGMF{\alpha}-P.XYL2$ strain. The xylitol dehydrogenase activity was increased by up to 41% when a glucose/xylose mixture was supplied as a carbon source, rather than glucose alone. The expression system and culture conditions optimized in this study resulted in large amounts of xylitol dehydrogenase using S. cerevisiae as the host strain, indicating the potential of this expression system for use in bioethanol production and industrial applications.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.443-448
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• 1996

In order to investigate the function of xylA promoter(Pxyl) as regulatory region Pxyl-lacZ fusion gene was constructed by the insertion of xylA promoter to the multiple cloning site of upstream of lacZ gene in a multicopy numbered plasmid pMC1403 containing promoterless lac operon, which was designated pMCX191, and Pxyl-lacZ fragment from pMCX191 was inserted to low copy numbered plasmid pLG339, designated pLGX191. The expressions of ${\beta}-galactosidase$ in these recombinant plasmids containing Pxyl-lacZ fusion gene were induced strongly by the addition of xylose, repressed by the addition of 0.2% glucose in the presence of xylose. The catabolite repressions were derepressed by the addition of 1 mM cAMP as same as native xylA gene. The fragment of xylA promoter was partially deleted from the upstream of xylA promoter by exonuclease III to investigate the regulation site of xylA promoter and the degrees of deletion derivatives of xylA promoter were analyzed by the DNA base sequencing. By the investigations of the induction by xylose, repression by glucose and derepression by cAMP on xylose isomerase production, the regulation site of xylA promoter may be located in segment between -165 and -59 bp upstream from the initiation site of xylA translation.

• Animal Bioscience
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• v.36 no.12
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• pp.1869-1879
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• 2023

Objective: This study was to evaluate the effects of individual or combinational use of phytase, protease, and xylanase on total tract digestibility of corn, soybean meal, and distillers dried grains with soluble (DDGS) fed to pigs. Methods: Each experiment had four 4×4 Latin squares using 16 barrows. Each period had 5-d adaptation and 3-d collection. All experiments had: CON (no enzyme); Phy (CON+phytase); Xyl (CON+xylanase); Pro (CON+protease); Phy+Xyl; Phy+Pro, Xyl+Pro, Phy+Xyl+Pro. Each Latin square had 'CON, Phy, Xyl, and Phy+Xyl'; 'CON, Phy, Pro, and Phy+Pro'; 'CON, Pro, Xyl, and Xyl+Pro'; and 'Phy+Xyl, Phy+Pro, Xyl+Pro, Phy+Xyl+Pro'. Results: The digestible energy (DE), metabolizable energy (ME), and nitrogen retention (NR) of corn were not affected by enzymes but the apparent total tract digestibility (ATTD) of phosphorus (P) was improved (p<0.01) by Phy. The DE and ATTD dry matter (DM) in soybean meal were increased (p<0.05) by Phy+Pro and the ATTD P was improved (p<0.01) by Phy, Phy+Pro, and Phy+Xyl. The DE, ME, and ATTD DM in DDGS were improved (p<0.05) by Phy+Xyl and the ATTD P was improved (p<0.01) by Phy, Phy+Pro, and Phy+Xyl. Conclusion: Phytase individually or in combination with xylanase and protease improved the Ca and P digestibility of corn, soybean meal, and DDGS, from the hydrolysis of phytic acid. The supplementation of protease was more effective when combined with phytase and xylanase in the soybean meal and DDGS possibly due to a higher protein content in these feedstuffs. Xylanase was more effective in DDGS diets due to the elevated levels of non-starch polysaccharides in these feedstuffs. However, when xylanase was combined with phytase, it demonstrated a higher efficacy improving the nutrient digestibility of pigs. Overall, combinational uses of feed enzymes can be more efficient for nutrient utilization in soybean meal and DDGS than single enzymes.

• 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.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1002-1010
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• 2001

The D-xylose operon in Escherichia coli is known to be regulated by a transcriptional activator protein, XyIR, which is responsible for the expression of both xylAB and xylFGH gene clusters. The XyIR was purified to homogeneity by using the maltose binding protein fusion expression and purification systems involving two chromatography steps. The purified XyIR protein was composed of two subunits of 45 kDa, which was determined by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The purified XyIR was specifically bounded to the xylA promoter, regardless of adding xylose to the reaction mixture, but binding of XylR was specifically bounded to the xylA promoter, regardless of adding xylose to the reaction mixture, but binding of XylR to the xylA promoter was enhanced by adding xylose. The enhanced binding ability of XyIR in the presence of xylose was not diminished by adding glucose. The presumed XyIR binding site is located between 120 bp to 100 bp upstream the xylA initiation codon.