title: Molecular characterization of a cold-active recombinant xylanase from Flavobacterium johnsoniae and its applicability in xylan hydrolysis.
abstract: A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30 °C, but Xyn10A retained 50% activity at 4 °C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-β-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose.
authors.affiliation: Dept of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. shicheng@msu.edu
authors.forename: Shicheng
authors.initials: S
authors.lastname: Chen
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kaufman
authors.forename: Kerri L
authors.initials: KL
authors.lastname: Miazgowicz
authors.forename: Michael
authors.initials: M
authors.lastname: Bagdasarian
authors.forename: Edward D
authors.initials: ED
authors.lastname: Walker
mesh_terms: D000595:Amino Acid Sequence; D003080:Cold Temperature; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D041965:Flavobacteriaceae / Q000145:classification / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry*; D013045:Species Specificity; D014990:Xylans / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D014990:Xylans; D043364:Endo-1,4-beta Xylanases