Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass.

Abstract

Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to β-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60°C and is thermo-resistance at 65°C. XynA maintains 66% of the enzymatic activity at high temperatures (90°C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50μmolmL-1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78μmolmL-1) and increasing the KCat/KM ratio (1.63 U s-1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.