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http://dx.doi.org/10.4014/mbl.2002.02013

Gene Cloning, Purification and Characterization of Xylanase 10A from Paenibacillus woosongensis in Escherichia coli  

Yoon, Ki-Hong (Food Science and Biotechnology Major, Woosong University)
Publication Information
Microbiology and Biotechnology Letters / v.48, no.2, 2020 , pp. 158-166 More about this Journal
Abstract
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 Vmax and Km 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.
Keywords
Paenibacillus woosongensis; xylanase 10A; purification; characterization;
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