Autodisplay of an endo-1,4-β-xylanase from Clostridium cellulovorans in Escherichia coli for xylans degradation

Abstract

The goal of this work was the autodisplay of the endo β-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and β-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants V_max and K_m were 149 U/g_DCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu²⁺, Zn²⁺ and Hg²⁺ as well as EDTA, detergents, and organic acids, and improved by Ca²⁺, Co²⁺ and Mn²⁺ ions. Ca²⁺ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl₂ were added. Also, Ca²⁺ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.

Keywords: AIDA; Autodisplay; Hemicellulose; Whole cell biocatalysts; Xylanase.