Abstract
The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 β-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)–beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize β-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications.
Highlights
The use of lignocellulosic residues as a renewable source to synthesize high-value products plays a crucial role in the establishment of a circular economy [1]
The mature XynSOS protein is composed of 389 amino acids and contains a catalytic domain that shows high identity with other GH10 enzymes (HMMER dbCAN2, E-value 3.6 × 10−98) and a C-terminal Carbohydrate-Binding Module (CBM) assigned to family 1 (HMMER dbCAN2, E-value 8.4 × 10−17) and connected by a Ser/Thr rich linker region (Figure S2), as previously described in other GH10 endoxylanases [30]
The xynSOS gene without the three introns and lacking the signal peptide was expressed in P. pastoris GS115, since several glycosyl hydrolases (GH) from this fungus have been successfully expressed in this model yeast to increase their production and facilitate their purification [26]
Summary
Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds. Ana Pozo-Rodríguez 1 , Juan A. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
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