• Korean Journal of Microbiology
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• v.46 no.4
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• pp.397-400
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• 2010

The activities of mannanase, ${\beta}$-mannosidase, and ${\alpha}$-galactosidase were detected in culture filtrate of Paenibacillus woosongensis showing mannanolytic activity for locust bean gum. Optimal conditions occurred at pH 5.5 and $60^{\circ}C$ for mannanase toward locust bean gum, pH 6.5 and $50^{\circ}C$ for ${\beta}$-mannosidase toward para-nitrophenyl-${\beta}$-D-mannopyranoside, and pH 6.0-6.5 and $50^{\circ}C$ for ${\alpha}$-galactosidase toward para-nitrophenyl-${\alpha}$-D-galactopyranoside in the culture filtrate, respectively. The mannanolytic enzyme of culture filtrate hydrolyzed mannobiose as well as manno-oligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. It could also hydrolyze ${\alpha}$-1,6 linked galacto-oligosaccharides such as melibiose, raffinose and stachyose to liberate galactose residue. From these results, it is assumed that P. woosongensis produces three enzymes required for the complete decomposition of galactomannan.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.141-146
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• 2012

A gene encoding the xylanase (XynA) predicted from partial genomic sequence of Paenibacillus woosongensis was cloned into Escherichia coli by PCR. This xynA gene consisted of 633 nucleotides, encoding a polypeptide of 211 amino acid residues. The deduced amino acid sequence exhibited 85-89% identity with those of several Paenibacillus xylanases, belonging to the glycosyl hydrolase family 11. As a results of expression of the structural gene by T7 promoter of a pET23a(+) expression vector, xylanase activity was higher in cell-free extract than culture filtrate of a recombinant Escherichia coli BL21(DE3) CodonPlus. However, the expression level of xylanase was not sufficient be detected by SDS-PAGE. The cell-free extract showed maximal xylanase activity at $60^{\circ}C$ and pH 5.5. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylbiose.

• 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.48 no.2
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• pp.158-166
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• 2020

A gene coding for the xylanase was cloned from Paenibacillus woosongensis, followed by determination of its complete nucleotide sequence. This xylanase gene, designated as xyn10A, consists of 1,446 nucleotides encoding a polypeptide of 481 amino acid residues. Based on the deduced amino acid sequence, Xyn10A was identified to be a modular enzyme composed of a catalytic domain highly homologous to the glycosyl hydrolase family 10 xylanase and a putative carbohydrate-binding module (CBM) in the C-terminus. By using DEAE-sepharose and phenyl-sepharose column chromatography, Xyn10A was purified from the cellfree extract of recombinant Escherichia coli carrying a P. woosongensis xyn10A gene. The N-terminal amino acid sequence of the purified Xyn10A was identified to exactly match the sequence immediately following the signal peptide predicted by the Signal5.0 server. The purified Xyn10A was a truncated protein of 33 kDa, suggesting the deletion of CBM in the C-terminus by intracellular hydrolysis. The purified enzyme had an optimum pH and temperature of 6.0 and 55-60℃, respectively, with the kinetic parameters V_max and K_m of 298.8 U/mg and 2.47 mg/ml, respectively, for oat spelt xylan. The enzyme was more active on arabinoxylan than on oat spelt xylan and birchood xylan with low activity for p-nitrophenyl-β-xylopyranoside. Xylanase activity was significantly inhibited by 5 mM Cu²⁺, Mn²⁺, and SDS, and was noticeably enhanced by K⁺, Ni²⁺, and Ca²⁺. The enzyme could hydrolyze xylooligosaccharides larger than xylobiose. The predominant products resulting from xylooligosaccharide hydrolysis were xylobiose and xylose.

• Microbiology and Biotechnology Letters
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• v.45 no.2
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• pp.155-161
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• 2017

A gene coding for the xylanase predicted from the partial genomic sequence of Paenibacillus woosongensis was cloned by PCR amplification and sequenced completely. This xylanase gene, designated xyn11B, consisted of 1,071 nucleotides encoding a polypeptide of 356 amino acid residues. Based on the deduced amino acid sequence, Xyn11B was identified to be a modular enzyme, including a single carbohydrate-binding module besides the catalytic domain, and was highly homologous to xylanases belonging to glycosyl hydrolase family 11. The SignalP4.1 server predicted a stretch of 26 residues in the N-terminus to be the signal peptide. Using DEAE-Sepharose and Phenyl-Sepharose column chromatography, Xyn11B was partially purified from the cell-free extract of recombinant Escherichia coli carrying a copy of the P. woosongensis xyn11B gene. The partially purified Xyn11B protein showed maximal activity at $50^{\circ}C$ and pH 6.5. The enzyme was more active on arabinoxylan than on oat spelt xylan and birchwood xylan, whereas it did not exhibit activity towards carboxymethylcellulose, mannan, and para-nitrophenyl-${\beta}$-xylopyranoside. The activity of Xyn11B was slightly increased by $Ca^{2+}$ and $Mg^{2+}$, but was significantly inhibited by $Cu^{2+}$, $Ni^{2+}$, $Fe^{3+}$, and $Mn^{2+}$, and completely inhibited by SDS.

• Journal of Life Science
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• v.27 no.9
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• pp.1003-1010
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• 2017

An open reading frame coding for mannanase predicted from the partial genomic sequence of Paenibacillus woosongensis was cloned into Escherichia coli by polymerase chain reaction amplification, and completely sequenced. This mannanase gene, designated man26AT, consisted of 3,162 nucleotides encoding a polypeptide of 1,053 amino acid residues. Based on the deduced amino acid sequence, Man26AT was identified as a modular enzyme, which included a catalytic domain belonging to the glycosyl hydrolase family 26 and two carbohydrate-binding modules, CBM27 and CBM11. The amino acid sequence of Man26AT was homologous to that of several putative mannanases, with identity of 81% for P. ihumii and identity of less than 57% for other strains of Paenibacillus. A cell-free extract of recombinant E. coli carrying the man26AT gene showed maximal mannanase activity at $55^{\circ}C$ and pH 5.5. The enzyme retained above 80% of maximal activity after preincubation for 1 h at $50^{\circ}C$. Man26AT was comparably active on locust bean gum (LBG), galactomanan, and kojac glucomannan, whereas it did not exhibit activity on carboxymethylcellulose, xylan, or para-nitrophenyl-${\beta}$-mannopyranoside. The common end products liberated from mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, or LBG by Man26AT were mannose, mannobiose, and mannotriose. Mannooligosacchrides larger than mannotriose were found in enzymatic hydrolyzates of LBG and guar gum, respectively. However, Man26AT was unable to hydrolyze mannobiose. Man26AT was intracellularly degraded into at least three active proteins with different molecular masses by zymogram.