Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92

In this work, we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold β-propeller catalytic domain and a C-terminal β-sandwich domain of unknown function. WXyn43 hydrolyzed short (1–4)-β-d-xylooligosaccharides, with similar k(cat)/K(M) for xylobiose (X(2)) and xylotriose (X(3)) and clearly lower efficiency in xylotetraose (X(4)) conversion. WXyn43 displays the highest reported k(cat) for conversion of X(3) (900 s(−1) at 37°C) and X(4) (770 s(−1)), and k(cat) for hydrolysis of X(2) (907 s(−1)) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer–monomer hydrogen bonds, while the bonds creating dimer–dimer interactions only involved residues from the N-terminal domain. Docking of X(2) and X(3) in the active site shows interactions corresponding to subsites −1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X(3) may be present.