# Recombinant Clostridium acetobutylicum Endoxylanase for Xylooligosaccharide Production from Pretreated Lignocellulosic Biomass

## TL;DR

This paper explores using a recombinant enzyme from Clostridium acetobutylicum to produce prebiotic xylooligosaccharides from plant biomass.

## Contribution

The study introduces a recombinant endo-β-1,4-xylanase (XynA) from Clostridium acetobutylicum for efficient XOS production from lignocellulosic biomass.

## Key findings

- The recombinant XynA enzyme efficiently hydrolyzed xylan substrates to produce xylobiose.
- XynA successfully catalyzed the conversion of xylotriose into xylobiose and xylose.
- The enzyme effectively hydrolyzed pretreated coconut husk biomass to release XOS.

## Abstract

Xylooligosaccharides (XOS) are functional oligosaccharides with recognized prebiotic properties and growing industrial relevance, typically obtained through enzymatic depolymerization of xylan-rich lignocellulosic substrates. In this study, a recombinant endo-β-1,4-xylanase (XynA) from Clostridium acetobutylicum was employed for XOS production. The xynA gene was cloned into the expression vector pET-21a(+) and heterologously expressed in Escherichia coli BL21(DE3) under induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein, with an estimated molecular mass of 37.5 kDa, was verified by SDS-PAGE and Western blot analysis. Functional characterization via thin-layer chromatography revealed that XynA efficiently hydrolyzed beechwood xylan and rye arabinoxylan, predominantly yielding xylobiose. Additionally, the enzyme catalyzed the conversion of xylotriose into xylobiose and trace amounts of xylose. Notably, XynA demonstrated hydrolytic activity against autohydrolysed and alkali-pretreated coconut husk biomass, facilitating the release of XOS. These results underscore the potential of C. acetobutylicum XynA as a biocatalyst for the valorization of lignocellulosic residues into high-value oligosaccharides.