• The Korean Journal of Food And Nutrition
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• v.14 no.1
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• pp.65-68
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• 2001

${\alpha}$-L-Arabinofuranosidase was purified from the culture supernatant of Bacillus sp. DSNC 101. The enzyme had a molecular weight of 56 kDa. Optimum temperature and pH for ${\alpha}$-L-arabinofuranosidase activity were 55$^{\circ}C$ and 7.0 respectively. The Michaelis constant(Km) and maximal reaction velo-city(Vmax) for p-nitrophenyl-${\alpha}$-L-arabinofuranoside were 1.0 mM and 113.6 U/mg protein, respe-ctively. ${\alpha}$-L-Arabinofuranosidase was completely inhibited by HgCl$_2$ and CuSO$_4$. The enzyme was spe-cific for the ${\alpha}$-linked arabinoside in the furanoside configuration. The enzyme was produced during growth on agricultural residue such as rice straw, but not during growth on spelt xylan, glucose or cellobiose.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.607-613
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• 1994

The Bacillus stearothermophilus arfI gene encoding a-arabinofuranosidase was isolated from the genomic library, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101. The recombinant E. coli was selected from approximately 10,000 transformants screened by making use of its ability to produce a yellow pigment around the colony on the selective medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf), a chromogenic substrate. The functional clone was found to harbor a recombinant plasmid, pKMG11 with an insertion of about 5 kb derived from the B. stearothermophilus chromosomal DNA. Identity of the arfI gene on the insert DNA was confirmed by a zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate. The $\alpha$-arabinofuranosidase from the recombinant E. coli strain showed very high substrate specificity; the enzyme displayed high activity only with pNPAf among many other p- or $o$-nitrophenyl derivatives of several sugars, and acted only on arabinoxylan among various natural arabinose containing polysaccharides tested.

• 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.23 no.4
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• pp.446-453
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• 1995

$\alpha $-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observed when E. coli HB101 cells were grown at 37$\circ$C for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The $\ALPHA $-arabinofuranosidase produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 6B-100 gel filtration. The purified enzyme was most active at 55$\circ$C and pH 6.5. The K$_{m}$ and V$_{max}$ values of the enzyme on $\rho $-nitrophenyl-$\alpha $-arabinofuranoside was determined to be 2.99 mM and 0.43 $\mu $mole/min (319.74 $\mu $mole/min/mg), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel clectrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the a-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg( \ulcorner )-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.483-488
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• 2013

Ginsenoside Rd was produced from ginsenoside Rc using a thermostable recombinant ${\alpha}-{\small{L}}$-arabinofuranosidase from Caldicellulosiruptor saccharolyticus. The optimal reaction conditions for the production of ginsenoside Rd from Rc were pH 5.5, $80^{\circ}C$, 227 U enzyme/ml, and 8.0 g/l ginsenoside Rc in the presence of 30% (v/v) n-hexane. Under these conditions, the enzyme produced 7.0 g/l ginsenoside Rd after 30 min, with a molar yield of 100% and a productivity of 14 g $l^{-1}\;h^{-1}$. The conversion yield and productivity of ginsenoside Rd are the highest reported thus far among enzymatic transformations.

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.37-43
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• 2019

The gene encoding an ${\alpha}-{\text\tiny{L}}-arabinofuranosidase$ (BvAF) GH51 from Bacillus velezensis FZB42 was cloned and expressed in Escherichia coli. The corresponding open reading frame consists of 1,491 nucleotides which encode 496 amino acids with the molecular mass of 56.9 kDa. BvAF showed the highest activity against sugar beet (branched) arabinan in 50 mM sodium acetate buffer (pH 6.0) at $45^{\circ}C$. However, it could hardly hydrolyze debranched arabinan and arabinoxylans. The time-course hydrolyses of branched arabinan and arabinooligosaccharides (AOS) revealed that BvAF is a unique exo-hydrolase producing exclusively ${\text\tiny{L}}-arabinose$. BvAF could cleave ${\alpha}-(1,2)-$ and/or ${\alpha}-(1,3)-{\text\tiny{L}}-arabinofuranosidic$ linkages of the branched substrates to produce the debranched forms of arabinan and AOS. Although the excessive amount of BvAF could liberate ${\text\tiny{L}}-arabinose$ from linear AOS, it was extremely lower than that on branched AOS. In conclusion, BvAF is the arabinan-specific exo-acting ${\alpha}-{\text\tiny{L}}-arabinofuranosidase$ possessing high debranching activity towards ${\alpha}-(1,2)-$ and/or ${\alpha}-(1,3)-linked$ branches of arabinan, which can facilitate the successive degradation of arabinan by $endo-{\alpha}-(1,5)-{\text\tiny{L}}-arabinanase$.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.236-244
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• 2014

The gene encoding ${\alpha}-\small{L}$-arabinofuranosidase (AFase) from Paenibacillus sp. DG-22 was cloned, sequenced, and expressed in Escherichia coli. The AFase gene (abfA) comprises a 1,509 bp open reading frame encoding 502 amino acids with a molecular mass of 56,520 daltons. The deduced amino acid sequence of the gene shows that AbfA is an enzyme consisting of only a catalytic domain, and that the enzyme has significant similarity to AFases classified into the family 51 of the glycosyl hydrolases. abfA was subcloned into the pQE60 expression vector to fuse it with a six-histidine tag and the recombinant AFase (rAbfA) was purified to homogeneity. The specific activity of the recombinant enzyme was 96.7 U/mg protein. Determination of the apparent molecular mass by gel-filtration chromatography indicated that AbfA has a tetrameric structure. The optimal pH and temperature of the enzyme were 6.0 and $60^{\circ}C$, respectively. The enzyme activity was completely inhibited by 1 mM $HgCl_2$. rAbfA was active only towards p-nitrophephenyl ${\alpha}-\small{L}$-arabinofuranoside and exhibited $K_m$ and $V_{max}$ values of 3.5 mM and 306.1 U/mg, respectively. rAbfA showed a synergistic effect in combination with endoxylanase on the degradation of oat spelt xylan and wheat arabinoxylan.

• 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.

• 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.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.273-277
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• 2004

$\alpha$-L-Arabinofuranosidase ($\alpha$-L-AFase, EC 3.2.1.55) was isolated from hyperthermophilic microorganism, Thermotoga maritima. The open reading frame (ORF) of $\alpha$-L-AFase gene is 1,455 bp long and encodes 484 amino acid residues with a molecular weight of 55,265 Da. The ORF of $\alpha$-L-AFase gene was introduced into the E. coli expression vector, $_p/RSET-B, and overexpressed in E. coli BL21. The purified recombinant $\alpha$-L-AFase showed the highest activity at 10$0^{\circ}C$ and pH 5.5. The purified enzyme appeared to have no metal cofactor requirement. The Km and specific activity values of the recombinant enzyme were 0.99 mM and 1,200 U/mg on p-nitrophenyl-$\alpha$-L-arabinofuranoside. It released only L-arabinose from sugar beet arabinan, sugar beet debranched arabinan and oat spelts arabinoxylan but had no activity onarabinogalactan and gum arabic. This result suggests that L-arabinose could be produced from natural polysaccharides using this enzyme. Mutant enzymes which Glu26, Glu172 and Glu281 residues were replaced to alanine, aspartic acid or glutamine caused Kcat to decrease by a factor of between 10$^3$ and 10$^4$. Glu172 and Glu281 residues of $\alpha$-L-AFase are seemed to be the acid/base and nucleophile in catalytic reaction, respectively, and Glu26 is supposed to playa key role in substrate binding.ng.