title: Cloning and characterization of a multidomain GH10 xylanase from Paenibacillus sp. DG-22.
abstract: The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419 bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and 65 °C and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM Cu(2+) and Hg(2+), whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.
authors.affiliation: Department of Biotechnology, Dongguk University, Gyeongju 780-714, Republic of Korea.
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Yong-Eok
authors.initials: YE
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases