ABSTRACT
A xylanase gene (thxyn11A) from the Thermobifida halotolerans strain YIM 90462(T) was cloned and expressed in Escherichia coli. The open reading frame (ORF) of thxyn11A has 1,008 bp encoding a mature xylanase with a high degree of similarity (80 %) to the xylanase from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111. This enzyme (Thxyn11A) also possesses a glycosyl hydrolases family 11 (GH11) domain and a high isoelectric point (pI = 9.1). However, Thxyn11A varies from most GH11 xylanases, due to its large molecular mass (34 kDa). Recombinant Thxyn11A demonstrated a strong pH and temperature tolerance with a maximum activity at pH 9.0 and 70 °C. Xylotriose, the end-product of xylan hydrolysis by Thxyn11A, serves as a catalyst for hemicellulose pretreatment in industrial applications and can also function as a food source or supplement for enterobacteria. Due to its attractive biochemical properties, Thxyn11A may have potential value in many commercial applications.
Subject(s)
Actinomycetales/enzymology , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/metabolism , Actinomycetales/classification , Actinomycetales/genetics , Base Sequence , Biotechnology , Cloning, Molecular , Endo-1,4-beta Xylanases/genetics , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Hydrolysis/drug effects , Isoelectric Point , Kinetics , Phylogeny , Substrate Specificity , Temperature , Trisaccharides/metabolism , Xylans/metabolismABSTRACT
The endoglucanase gene, thcel9A, from Thermobifida halotolerans YIM 90462(T) was cloned and expressed in Escherichia coli BL 21(DE). The 2895-bp full-length gene encodes a 964-residue polypeptide (Thcel9A) containing a catalytic domain belonging to glycosyl hydrolases (GH) family 9. Phylogenetic analysis indicated that Thcel9A is closely related to Cel9A of Thermobifidafusca YX. Thcel9A was purified from the culture supernatant by Ni(2+)-affinity chromatography and the purified enzyme exhibited optimal activity at 55°C and pH 8.0. Substrate specificity assays showed that it not only had CMCase activity, but also hydrolase activity on microcrystalline cellulose and filter paper. These properties suggested that Thcel9A is a classical GH9 group A endoglucanase.
