• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.943-953
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• 2014

The XylH gene (1,167-bp) encoding a novel hemicellulase (41,584 Da) was identified from the genome of Microbacterium trichothecenolyticum HY-17, a gastrointestinal bacterium of Gryllotalpa orientalis. The enzyme consisted of a single catalytic domain, which is 74% identical to that of an endo-${\beta}$-1,4-xylanase (GH10) from Isoptericola variabilis 225. Unlike other endo-${\beta}$-1,4-xylanases from invertebrate-symbiotic bacteria, rXylH was an alkali-tolerant multifunctional enzyme possessing endo-${\beta}$-1,4-xylanase activity together with ${\beta}$-1,3/${\beta}$-1,4-glucanase activity, which exhibited its highest xylanolytic activity at pH 9.0 and 60oC, and was relatively stable within a broad pH range of 5.0-10.0. The susceptibilities of different xylosebased polysaccharides to the XylH were assessed to be as follows: oat spelts xylan > beechwood xylan > birchwood xylan > wheat arabinoxylan. rXylH was also able to readily cleave p-nitrophenyl (pNP) cellobioside and pNP-xylopyranoside, but did not hydrolyze other pNP-sugar derivatives, xylobiose, or hexose-based materials. Enzymatic hydrolysis of birchwood xylan resulted in the product composition of xylobiose (71.2%) and xylotriose (28.8%) as end products.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.28-33
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• 1998

A gene coding for ${\beta}$-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isloated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-${\beta}$-D xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is ${\beta}$-xylosidase gene. The ${\beta}$-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 ${\beta}$-xylosidase was highly homologous to the ${\beta}$-xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable ${\beta}$-xylosidase from Bacillus stearothermophilus.

• 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.4
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• pp.535-540
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• 2012

${\beta}$-D-Xylosidase (E.C. 3.2.1.37) from Bifidobacterium breve K-110, which hydrolyzes ginsenoside Ra1 to ginsenoside Rb2, was cloned and expressed in Escherichia coli. The ($His_6$)-tagged recombinant enzyme, designated as XlyBK-110, was efficiently purified using $Ni^{2+}$-affinity chromatography (109.9-fold, 84% yield). The molecular mass of XylBK-100 was found to be 55.7 kDa by SDS-PAGE. Its sequence revealed a 1,347 bp open reading frame (ORF) encoding a protein containing 448 amino acids, which showed 82% identity (DNA) to the previously reported glycosyl hydrolase family 30 of Bifidobacterium adolescentis ATCC 15703. The $K_m$ and $V_{max}$ values toward p-nitrophenyl-${\beta}$-D-xylopyranoside (pNPX) were 1.45mM and 10.75 ${\mu}mol/min/mg$, respectively. This enzyme had pH and temperature optima at 6.0 and $45^{\circ}C$, respectively. XylBK-110 acted to the greatest extent on xyloglucosyl kakkalide, followed by pNPX and ginsenoside Ra1, but did not act on p-nitrophenyl-${\alpha}$-L-arabinofuranoside, p-nitrophenyl-${\beta}$-D-glucopyranoside, or p-nitrophenyl-${\beta}$-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of ${\beta}$-D-xylosidase-hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.8-14
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• 1992

Nucleotide Sequence of phnE Gene Encoding Extradiol Dioxygenase fromPseudomonas sp. Strain DJ77Kim, Young-Chang'.", Myeong-Su Shin1, Kil-Sang Younl, Young-Soon Park1, andUg-Hyeon Kim'.' (Department of Microbiology, C'hungbuk National University.Cheongju 360-763, KOREA. and 'Research Center for Molecular Microbiology,Seoul National University)nal University)

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.3
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• pp.428-431
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• 1997

Chemical components of red algae (Gelidium amansii, Gracilaria verrucosa, Gigartina tenella, Carpopeltis cornea, Plocamium ovicornis, Callophyllis adnata, Lomentaria hakodatensis, Gymnogongrus flabelliformis, Chondrus pinnulatus, Adinotrichia fragilis, Gloipeltis tenax and Campylaephora hypnaeoides) were examined. The contents of carbohydrate, ash, crude protein and fat in dried red algae are $40.38\~69.88\%,\;8.57\~30.23\%,\;18.11\~33.90\%\;and\;0.11\~0.90\%$ on dry base, respectively. Yields of water-soluble compound in dried red algae and contents of sulfate in water-soluble compound extracted red algae were $4.32\~55.78\%\;and\;1.65\~19.48\%$, respectively. The principal sugars of water-soluble compound extracted from red algae were galactose, 3, 6-anhydrogalactose, glucose and xylose.

• Food Science and Preservation
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• v.15 no.4
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• pp.532-541
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• 2008

A central composite design was applied to investigate the effects of the independent variables of NaOH concentration(X1) and extraction time(X2) on dependent variables such as Yield(Y1), Xyl/Ara ratio(Y2), uronic acid(Y3), $\beta$-glucan(Y4) and total sugars(Y5) of hemicelluloses separated from rice bran. The Coefficients of determination(R2) in various models ranged from 0.8626 to 0.9319. Yield increased with increased NaOH concentration and extraction time. The optimum extraction conditions were NaOH concentration at 2.45M and extraction time of 24.2 h. Predicted values at the optimized conditions were acceptable, compared with experimental values. The structural characteristics of an optimum hemicellulose extract were explored. As a result, it showed that the surfaces of hemicellulose had a highly irregular reticulated structure. And also it was both small and large molecular particle in the hemicelluloses. Their average molecular weights were in the ranges $235{\sim}240$ kDa and $8.0{\sim}9.4kDa$, respectively.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.258-263
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• 1998

${\beta}$-Xylosidase was purified from the recombinant Escherichia coli carrying the Bacillus sp. KK-1 ${\beta}$-xylosidase gene (xylB). The molecular mass of the purified enzyme was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the apparent molecular mass of the ${\beta}$-xylosidase was 140 kDa, indicating that the native ${\beta}$-xylosidase has an oligomeric structure composed of two identical subunits. The isoelectric point was determined to be pH 5.5. The enzyme was highly active on p-nitrophenyl-$\beta$-D-xylopyranoside but it barely hydrolyzed xylan substrates, and did not exhibit activity towards carboxymethylcellulose and p-nitrophenyl-${\beta}$-D- glucopyranoside. The enzyme had a pH optimum for its activity at pH 6.5 and a temperature optimum at $40^{\circ}C$. The enzyme activity was completely inhibited by the presence of $Hg^{++}$, and also markedly inhibited by D-xylose and D-glucose.

• Journal of Microbiology
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• v.35 no.4
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• pp.300-308
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• 1997

The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.

• 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.