• Journal of Microbiology and Biotechnology
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• v.6 no.3
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• pp.179-183
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• 1996

Synergism among endo-xylanase, $\beta$-xylosidase, and $\alpha$-L-arabinofuranosidase from Bacillus stearothermophilus upon xylan hydrolysis was investigated by using birchwood, oat spelt, and arabinoxylan as substrates. Endo-xylanase and $\beta$-xylosidase showed the cooperative action on all three substrates tested, revealing the fact that $\beta$-xylosidase assists endo-xylanase action in xylan hydrolysis by relieving the endproduct inhibition upon endo-xylanase conferred by xylooligomers. $\alpha$-L-Arabinofuranosidase also exhibited synergic effects with endo-xylanase and $\beta$-xylosidase on oat spelt and arabinoxylan, which contained significant amounts of arabinose side chains, whereas no synergism was detected on birchwood xylan which had only trace amounts of the side chain. Thus, the hydrolysis of xylan containing arabinose side chains required $\alpha$-L-arabinofuranosidase as well as endo-xylanase and $\beta$-xylosidase for the better hydrolysis of the substrates, and these enzymes work cooperatively in order to maximize the extent and rate of xylan hydrolysis.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.647-654
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• 1992

In order to study the physiological properties of the intestinal bacteria, we isolated the intestinal bacteria of Koreans and tested the enzymatic patterns. Isolated Bifidobacterium sp. Int-57 had the higher activity of $\alpha$-glucosidase, $\beta$-glucosidase, $\alpha$-galactosidase, $\beta$-galactosidase. $\beta$-xylosidase and $\alpha$-arabinofuranosidase than other intestinal microorganisms. The effect of the carbon sources on the production of each enzymes of Bijidobacterium sp. Int-57 was investigated. The most suitable carbon source for the production of $\beta$-glucosidase was maltose, for a-glucosidase cellobiose, for $\alpha$-galactosidase raffinose, for $\beta$-galactosidase lactose, and for $\beta$-xylosidase and $\alpha$-arabinofuranosidase xylose, respectively. In addition, we investigated the optimal conditions and pH stability of each crude enzymes. The optimal condition of a-glucosidase was pH 6.0 and $40^{\circ}C$. that of Jj-glucosidase pH 7.0 and 50oe, that of $\beta$-galactosidase pH 7.0 and $50^{\circ}C$, that of $\beta$-xylosidase pH 6.0 and $40^{\circ}C$ , and that of $\alpha$-arabinofuranosidase pH 5.0 and $50^{\circ}C$. respectively. a-Glucosidase was stable at pH 4.0-9.0. Jj-glucosidase at pH 4.0-7.0. $\beta$-galactosidase at pH 4.0-9.0, $\beta$-xylosidase at pH 4.0-6.0, and /3-arabinofuranosidase at pH 7.0-9.0, respectively.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1711-1716
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• 2010

A gene encoding the ${\beta}$-xylosidase/${\alpha}$-arabinofuranosidase (XylC) of Paenibacillus woosongensis was cloned into Escherichia coli. This xylC gene consisted of 1,425 nucleotides, encoding a polypeptide of 474 amino acid residues. The deduced amino acid sequence exhibited an 80% similarity with those of both Clostridium stercorarium ${\beta}$-xylosidase/${\alpha}$-N-arabinosidase and Bacillus cellulosilyticus ${\alpha}$-arabinofuranosidase, belonging to the glycosyl hydrolase family 43. The structural gene was subcloned with a C-terminal His-tag into a pET23a(+) expression vector. The His-tagged XylC, purified from a cell-free extract of a recombinant E. coli BL21(DE3) Codon Plus carrying a xylC gene by affinity chromatography, was active on para-nitrophenyl-${\alpha}$-arabinofuranoside (pNPA) as well as para-nitrophenyl-${\beta}$-xylopyranoside (pNPX). However, the enzymatic activities for the substrates were somewhat incongruously influenced by reaction pHs and temperatures. The enzyme was also affected by various chemicals at different levels. SDS (5 mM) inhibited the enzymatic activity for pNPX, while enhancing the enzymatic activity for pNPA. Enzyme activity was also found to be inhibited by addition of pentose or hexose. The Michaelis constant and maximum velocity of the purified enzyme were determined for hydrolysis of pNPX and pNPA, respectively.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.297-302
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• 1998

$\beta$-Xylosidase B was produced by Escherichia coli HB101/pKMG12 carrying the xylB gene of Bacillus stearothermophilus No.236 on its recombinant plasmid. The $\beta$-xylosidase B produced was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Sephacryl S-200 and Superdex 200 HR gel filtration. The purified enzyme showed the highest activity at pH 6.5 and 5$0^{\circ}C$. But, the enzyme was observed to be very sensitive to the pH and temperature of the reaction mixture. The enzyme was activated about 35% of its original activity in the presence of 1 mM of $Mn^{2+}$ but it was completely inhibited by $Ag^{+}$, $Cu^{2+}$and $Hg^{2+}$ions. In contrast with the $\beta$-xylosidase A, the B enzyme was found to have $\alpha$-arabinofuranosidase activity though the activity was fairly low compared with the $\alpha$-arabinofuranosidase produced from the arfI gene of the same Bacillus stearothermophilus. Therefore, $\beta$-xylosidase B is considered to be more suitable than $\beta$-xylosidase A at least for the biodegradation of arabinoxylan. The $K_{m}$ and V$_{max}$ values of the $\beta$-xylosidase B for o-nitrophenyl-$\alpha$-D-xylopyranoside were 6.43 mM and 1.45 $\mu$mole/min, respectively. Molecular mass of the enzyme was determind to be about 54 kDa by SDS-PAGE and 160 kDa by Superdex 200HR gel filtration, indicating that the functional $\beta$-xylosidase B was composed of three identical subunits.s.

• 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.

• Microbiology and Biotechnology Letters
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• v.24 no.2
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• pp.197-205
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• 1996

The $\beta$-xylosidase was purified 99- fold from the culture supernatant of Pseudo onas sp. CB-33 by ammonium sulfate precipitation, PEI precipita- tion, DEAE-Sephadex column chromatography, Sephadex G-75 gel filtration chromatography and preparative disc gel electrophoresis. Molecular weight of the enzyme was estimated to be 44,000 by SDS polyacrylamide gel electrophoresis. The enzyme has a pH optimum for activity at 7.0 and is stable over pH 6.5-9.0. The optimal temperature of the enzyme was 45$\circ$C, and its enzymatic activity was completely inactivated at 55$\circ$C for 30 min. Km value of the enzyme for p-nitrophenyl-$\beta$-D-xylopyranoside was calculated to be 4.6 mM. The effect of various reagents on the $\beta$-xylosidase activity was investigated. The enzyme activity was completely inhibited by Hg$^{2+}$, Cu$^{2+}$ and Zn$^{2+}$. The $\beta$-xylosidase was inactivated by tryptophan-specific reagent, N-bromosuccinimide and tyrosine-specific reagent, iodine. The enzyme could degrade xylo-oligosaccharides to xylose and the enzyme was competitively inhibited by xylose. The $\beta$-xylosidase and endoxylanase from Psedomonas sp. CB-33 hydrolized xylan synergically. The purified enzyme also showed $\alpha$-L-arabinofuranosidase activity.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.474-482
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• 2004

The $\alpha$-L-arabinofuranosidase (Arfase) gene of Bacillus stearothermophilus No. 236 was cloned and sequenced. The ORF of the gene, designated arfA, encoded a 507 -residue polypeptide with calculated molecular mass of 57 kDa. The Arfase produced by a recombinant Escherichia coli strain containing the arfA gene was purified to apparent homogeneity and characterized. The molecular mass of the Arfase determined by SDS-PAGE was 60 kDa. However, according to gel filtration, it was estimated to be approximately 190 kDa. These results indicated that the functional form of the Arfase is trimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and $55^{\circ}C$, respectively. The half-life of the enzyme at $60^{\circ}C$ was about 6 h. Kinetic experiments at $45^{\circ}C$ with pNPM (p-nitrophenyl $\alpha$-L-arabinofuranoside) as a substrate gave the $K_m and V_{max}$ values of 1.19 mM and 26.1 U/ mg, respectively. When the enzyme was combined with Bacillus stearothermophilus No. 236 endoxylanase and $\beta$-xylosidase, it hydrolyzed arabinoxylan into L-arabinose and xylose more efficiently than Arfase alone. This synergistic effect suggested that the complete hydrolysis of xylan with large amounts of arabinose side chains required Arfase as well as endoxylanase and $\beta$-xylosidase.

• Korean Journal of Food Science and Technology
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• v.25 no.2
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• pp.191-195
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• 1993

The intestinal microflora of humans is an extraordinarily complex mixture of microorganisms, the majority of which are anaerobic bacteria. Amongst them, most prevalent bacteria are Bacteroides, Eubacterium, Peptococcus, Bifidobacteria. We isolated a Bacteroides fragilis strain from a Korean adult and examined various glycosidase activities of this strain. The activities of $N-acetyl-{\beta}-glucosaminidase,\;{\alpha}-fucosidase$, ${\beta}-glucuronidase$, chitobiase and PNPCase were stronger in Bacteroides fragilis Roid 8 than in other intestinal anaerobic bacteria. $N-acetyl-{\beta}-glucosaminidase$ was strongest, followed by ${\alpha}-fucosidase$, ${\beta}-glucuronidase$ and PNPCase. The activities of ${\beta}-galactosidase$, ${\beta}-xylosidase,\;{\alpha}-arabinofuranosidase$ were not present or very low. The activities of ${\alpha}-glucosidase$, ${\beta}-glucosidase$ and ${\alpha}-galactosidase$ were present but at a lower level than in Bifidobacterium. The effect of the carbon sources on the production of $N-acetyl-{\beta}-glucosaminidase$, ${\alpha}-fucosidase$, ${\beta}-glucuronidase$ and PNPCase of Bacteroides fragilis Roid 8 was investigated. :.actose and glucose lowered the production of the varous glycosidase enzymes studied in this work. In addition, we investigated the optimum temperature and pH of each glycosidase from Bacteroides fragilis Roid-8 using crude enzyme preparations.

• Journal of Microbiology and Biotechnology
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• v.21 no.3
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• pp.284-292
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• 2011

An endocellulase-free multienzyme complex was produced by a thermophilic anaerobic bacterium, Thermoanaerobacterium thermosaccharolyticum strain NOI-1, when grown on xylan. The temperature and pH optima for growth were $60^{\circ}C$ and 6.0, respectively. The bacterial cells were found to adhere to insoluble xylan and Avicel. A scanning electron microscopy analysis showed the adhesion of xylan to the cells. An endocellulase-free multienzyme complex was isolated from the crude enzyme of strain NOI-1 by affinity purification on cellulose and Sephacryl S-300 gel filtration. The molecular mass of the multienzyme complex was estimated to be about 1,200 kDa. The multienzyme complex showed one protein on native PAGE, one xylanase on a native zymogram, 21 proteins on SDS-PAGE, and 5 xylanases on a SDS zymogram. The multienzyme complex consisted of xylanase, ${\beta}$-xylosidase, ${\alpha}$-L-arabinofuranosidase, ${\beta}$-glucosidase, and cellobiohydrolase. The multienzyme complex was effective in hydrolyzing xylan and corn hulls. This is the first report of an endocellulase-free multienzyme complex produced by a thermophilic anaerobic bacterium, T. thermosaccharolyticum strain NOI-1.