Dinitrosalicylic Acid Hydrazide [HRP]

A novel xylanase of family 11 (Xyn11-1) was obtained from the metagenomic DNA of alkaline soil by touchdown-PCR and thermal asymmetric interlaced (TAIL) PCR methods. Xyn11-1 is composed of 645 nucleotides, which encodes a signal peptide of 19 amino acids and a mature protein of 196 amino acids. It is a novel GH11 xylanase sharing the highest identity (79 %) with the reported GH11 xylanase (XP_008721536) in GenBank database. In order to detect its biological activity, the recombinant plasmid xyn11-1-pET28a(+) was constructed and recombinant protein was successfully expressed in heterologous hosts Escherichia coli BL21 (DE3) induced by isopropy-beta-D-thiogalactopyranoside (IPTG). Using the 3,5-dinitrosalicylic acid (DNS) method, the xylanase activity of crude intracellular protein is 11.32 U/mL. The optimal inducing IPTG concentration and inducing temperature was examined at 0.2 mM and 15 degrees C, respectively.

A new insoluble dye substrate for xylan degrading enzymes was prepared. Xylan was dyed with Cibacron blue 3GA and cross-linked by 1,4-butanedioldiglycidylether. The substrate was stable at 25 degrees C for 60 h. Xylanase assay by the substrate was more sensitive than conventional dinitrosalicylic acid method and was not interfered with by a reducing sugar. The substrate could be used for simple and sensitive measurement of xylanase activities and preparation of agar plates for screening of xylanase producing microorganisms. (C) 1997 Elsevier Science Ireland Ltd.