• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.111-116
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding $\beta$-xylosidase was cloned and sequenced previously. The transcriptional start site of the xylA gene cloned in E. coli was identified to be the guanine (G) by primer extension analysis. This supports that the expression of xylA gene is also directed in the E. coli cells by the previously determined transcription initiation signals, -10 sequence (CATAAT) and -35 sequence (TTGTTA) separated by 12 bp. To increase the expression of $\beta$-xylosidase, firstly the spacer region of xylA promoter was extended from 12 to 17 bp, and then the -10 and -35 elements were converted into their respective consensus sequences. The mutant promoters thus obtained were tested for their activities in both the E. coli and B. subtilis host cells. The change of the length of the spacer region from 12 to 17 bp resulted in a 1.6- and 2.5-fold increase in promoter strength in comparison with the wild type promoter in E. coli and B. subtilis cells, respectively. Also, strength of the promoter with the fourth T to A transversion on its -35 element increased in the transcription level by about 35 times compared with that of wild-type promoter. However, surprisingly the 5' end C-to-T transition of the -10 hexamer showed a 5- to 15-fold reduction in $\beta$-xylosidase activity in both E. coli and B. subtilis. Together, the present data demonstrated that the 5' end nucleotide C of the -10 sequence CATAAT and the fourth nucleotide A of the -35 hexamer are two most critical nucleotides for the promoter activity in the context of the xylA promoter.

• Toxicological Research
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• v.32 no.4
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• pp.337-343
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• 2016

We determined the toxicity of mixtures of ethyl acetate (EA), isopropyl alcohol (IPA), methyl ethyl ketone (MEK), toluene (TOL) and xylene (XYL) with half-maximal effective concentration ($EC_{50}$) values obtained using human hepatocytes cells. According to these data, quantitative property-activity relationships (QPAR) models were successfully proposed to predict the toxicity of mixtures by multiple linear regressions (MLR). The leave-one-out cross validation method was used to find the best subsets of descriptors in the learning methods. Significant differences in physico-chemical properties such as boiling point (BP), specific gravity (SG), Reid vapor pressure (rVP) and flash point (FP) were observed between the single substances and the mixtures. The $EC_{50}$ of the mixture of EA and IPA was significantly lower than that of contained TOL and XYL. The mixture toxicity was related to the mixing ratio of MEK, TOL and XYL (MLR equation $EC_{50}=3.3081-2.5018{\times}TOL-3.2595{\times}XYL-12.6596{\times}MEK{\times}XYL$), as well as to BP, SG, VP and FP (MLR equation $EC_{50}=1.3424+6.2250{\times}FP-7.1198{\times}SG{\times}FP-0.03013{\times}rVP{\times}FP$). These results suggest that QPAR-based models could accurately predict the toxicity of polar and nonpolar mixtures used in rotogravure printing industries.

• Proceedings of the Zoological Society Korea Conference
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• 2000.10a
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• pp.128.2-128
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• 2000

No Abstract, See Full Text

• Korean Journal of Microbiology
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• v.34 no.3
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• pp.115-119
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• 1998

We cloned the 4.8 kb BglII fragment containing genes downstream pHENX7 from Pseudomonas sp. strain DJ77. The restriction map of the resultant clone, recombinant plasmid pYCS500, was determined. Sequencing analysis of the 465 bp HindIII-ClaI fragment revealed an open reading frame of 282 bp that was then designated phnM. The deduced polypeptide is 93 amino acid residues long with a $M_r$ of 10,008. The PhnM has 37.3-53.9% identity with plant-type ferredoxin proteins such as NahT, XylT, DmpQ, AtdS, PhlG, PhhQ and TbuW and contains the motif similar to well-conserved functional domains of those proteins.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.331-335
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• 1996

The second $\beta$-Xylosidase gene (xylB) from Bacillus stearothermophilus was isolated from the genomic library, cloned into pBR322, and subsequently transferred into Escherichia coli HB101. Six out of 10, 000 transformants were selected from the selective LB medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf) and ampicillin ($50\mu g$/ml) based on their ability to form a yellow ring around the colony. One of the clones was found to harbor the recombinant plasmid with 5.0 kb foreign DNA, which was identical to the $\alpha$-L-arabinofuranosidase gene (arfI) previously cloned in this lab, while the other five had 3.5 kb of the foreign DNA. Southern blotting experiments confirmed that the 3.5 kb insert DNA was from B. stearothermophilus chromosomal DNA. A zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate revealed that the cloned gene product was one of the mutiple $\alpha$-L-arabinofuranosidases produced by B. stearothermophilus. Unlike the arfI gene product, the product of the gene on the insert DNA (xylB) showed an activity not only on pNPAf but also on oNPX suggesting that the cloned gene product could be a bifunctional enzyme having both $\alpha$-L-arabinofuranosidase and $\beta$-xylosidase activities.

• Korean Journal of Food Science and Technology
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• v.42 no.2
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• pp.223-232
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• 2010

Crude polysaccharide (WG-PL-CP) was fractionated from fermented ginseng with Phellinus linteus in solid culture to enhance the immunostimulation of ginseng. WG-PL-CP produce three active polysaccharide-rich fractions (WG-PL-CP-II, III, and IV) on DEAE-Sepharose CL-6B ($Cl^-$ form). WG-PL-CP-III displayed higher mitogenic activity (1.98-fold of the saline control at $100\;{\mu}g/mL$) than did WG-CP-III or PL-CP-III (1.60- or 1.65-fold, respectively), and potent intestinal immune system modulating activity through Peyer's patch was obtained by WG-PL-CP-IV only (1.56-fold). Meanwhile, WG-PL-CP-II and III significantly enhanced macrophage stimulating activity (2.01- and 1.94-fold) compared to WG-CP-II and III (1.73- and 1.66-fold) or PL-CP-II and III (1.79- and 1.72-fold). In addition, WG-PL-CP-III and IV mainly contained neutral sugar (73.5 and 67.3%) and uronic acid (23.2 and 24.6%). Component sugar analysis also showed that WG-PL-CP-III consisted mainly of uronic acid as well as the neutral sugars Glc, Ara, Gal, Rha and Xyl (molar ratio of 0.81:1.00:0.49:0.42:0.28:0.20), whereas WG-PL-CP-IV was mainly comprised of uronic acid, Ara, Rha, Gal, Xyl and Glc (1.00:0.75:0.69:0.63:0.42:0.34). Therefore, it is assumed that these active polysaccharides play an important role in enhancing the immunostimulation of fermented ginseng with P. linteus in solid culture.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.930-938
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• 2012

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA-335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Ni^{2+}$, $Fe^{2+}$, $Fe^{3+}$, $Zn^{2+}$, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of $60^{\circ}C$ and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of $70^{\circ}C{\sim}80^{\circ}C$), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.574-582
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• 1992

Exo-xylanase encoded by the xylA gene of Bacillus stearothermoPhillus was produced from Escherichia coli ]M109 carrying a recombinant plasmid pMGL Synthesis of the enzyme was observed to be cell-associated, and about 94% of the enzyme synthesized was located in the cytoplasmic region. The maximum production was attained when the E. coli strain was grown at $37^{\circ}C$ for 8 hours on the medium containing 0.5% fructose, 1.0% tryptone, 1.0% sodium chloride, and 0.5% yeast extract. The exo-xylanase was purified to homogeneity using a combination of salting out with ammonium sulfate, DEAE-Sepharose CL-6B ion exchange chromatography, Sephadex G-IOO gel filtration, and Sephadex G-150 gel filtration. The' purified enzyme was most active at pH 6.0 and $45^{\circ}C$. $Ca^{2+}$ and $Co^{2+}$ activated the exo-xylanase activity by about 20% while $Ag^{2+}$, $Fe^{2+}$, $Mg^{2+}$ and $Zn^{2+}$ inhibited the enzyme activity by up to 60%. The $K_m$, value on p-nitrophenyl-$\beta$-D-xylanopyranoside was 2.75 mM. The enzyme had a pI value of 4.7. The estimated molecular weight of the native protein was 200,000 daL SDS-polyacrylamide gel electrophoresis analysis suggested that the native enzyme was a trimer composed of three identical 66,000 da!. polypeptides. The purified enzyme efficiently converted all the xylo-oligosaccharides tested to xylose. It was also confirmed that the enzyme split xylans in an exo-manner even though the degree of hydrolysis was fairly low. The xylanolytic enzyme was, therefore, classified to be one of the few bacterial exo-xylanases lacking transferase activity.

• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.23-29
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• 1997

The nucleotide sequence of the estI gene encoding acetylxylan esterase I of Bacillus stearothermophilus was determined and analyzed. The estI gene was found to consist of a 810 base pair open reading frame coding for a polypeptide of 270 amino acids with a deduced molecular weight of 30 kDa. This was in well agreement with the molecular weight (29 kDa) estimated by SDS-PAGE of the purified esterase. The coding sequence was preceded by a putative ribo some binding site 10 bp upsteam of the ATG codon. Further 53 bp upstream, the transcription initiation signals were identified. The putative $_{-}$10 sequence (TCCAAT) and $_{-}$35 seqence (TTGAAT) corresponded closely to the respective consensus sequences for the Bacillus subtiis major RNA polymerase. The G+C content of the coding region of the estI was 51% whereas that of the third position of codone was 60.2%. The N-terminal amino acid sequence of the EstI deduced from the nucleotide sequence perfectly matched the corresponding region of the purified esterase described previously. Comparison with the amino acid sequence of other esterases and lipases reported so far allowed us to identify a sequence, GLSMG at positions 123 to 127 of the EstI which was reported to be the highly conserved active site sequence for those enzymes. The nucleotide sequence of the estI revealed 55.7% homology to that of the xylC coding for the acetylxylan esterase of Caldocellum saccharolyticum.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.606-612
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• 2018

The enzyme xylose isomerase (E.C. 5.3.1.5, XI) is responsible for the conversion of an aldose to ketose, especially xylose to xylulose. Owing to the ability of XI to isomerize glucose to fructose, this enzyme is used in the food industry to prepare high-fructose corn syrup. Therefore, we studied the characteristics of XI from Anoxybacillus kamchatkensis G10, a thermophilic bacterium. First, the gene coding for XI (xylA) was inserted into the pET-21a(+) expression vector and the construct was transformed into the Escherichia coli competent cell BL21 (DE3). The expression of recombinant XI was induced in the absence of isopropyl-thio-${\beta}$-galactopyranoside and purified using Ni-NTA affinity chromatography. The optimum temperature of recombinant XI was $80^{\circ}C$ and measurement of the heat stability indicated that 55% of residual activity was maintained after 2 h incubation at $60^{\circ}C$. The optimum pH was found to be 7.5 in sodium phosphate buffer. Magnesium, manganese, and cobalt ions were found to increase the enzyme activity; manganese was the most effective. Additionally, recombinant XI was resistant to the presence of $Ca^{2+}$ and $Zn^{2+}$ ions. The kinetic properties, $K_m$ and $V_{max}$, were calculated as 81.44 mM and $2.237{\mu}mol/min/mg$, respectively. Through redundancy analysis, XI of A. kamchatkensis G10 was classified into a family containing type II XIs produced by the genera Geobacillus, Bacillus, and Thermotoga. These results suggested that the thermostable nature of XI of A. kamchatkensis G10 may be advantageous in industrial applications and food processing.