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

Abstract

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 kcat/KM for xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s−1 at 37°C) and X4 (770 s−1), and kcat for hydrolysis of X2 (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 X2 and X3 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 X3 may be present.

Production of prebiotic xylooligosaccharide from aqueous ammonia‐pretreated rice straw by β‐xylosidase of Weissella cibaria

“…Values with different superscripts in the same column are significantly different (P < 0.05). The main hydrolysis product xylose, X1, a product of the reaction catalysed by X2 and X3, is the same as previously reported (Falck et al 2016;L opez-Hern andez et al 2018). W. confuse L9 exert the b-xylosidase which degraded xylobiose and xylotriose to xylose as a final product (L opez-Hern andez et al 2018).…”

“…The main hydrolysis product xylose, X1, a product of the reaction catalysed by X2 and X3, is the same as previously reported (Falck et al . ; López‐Hernández et al . ).…”

“…The β‐xylosidase activity assay was performed according to Falck et al . () to measure the amount of reducing sugars released from p ‐nitrophenyl‐β‐ d ‐xylopyranoside. The reaction mixture (1·5 ml) contained 0·15 ml p ‐nitrophenyl‐β‐ d ‐xylopyranoside and 0·15 ml of suitably diluted enzyme solutions in a 50 mmol l −1 phosphate buffer (pH 7), and the reaction was maintained at 37°C for 1 min.…”

“…Using the Weissella sp. strain 92, a novel gene sequence encoding a putative GH43 b-xylosidase was identified and was then expressed in Escherichia coli and characterized for XOS utilization Falck et al (2016). To date, XOS production from rice straw is limited compared to that from corncobs and other agriculture residues.…”

Structural Considerations on the Use of Endo-Xylanases for the Production of prebiotic Xylooligosaccharides from Biomass

“…XOS are not digestible by human enzymes, but stimulate selectively the growth of beneficial gastrointestinal flora, such as lactic acid bacteria and bifidobacteria. In vitro assays have shown that X 2 and X 3 promote the growth of Bifidobacterium adolescentis , B. infantis , B. longum , B. bifidum (reviewed by Vázquez et al [ 123 ]) and lactic acid bacteria such as Lactobacillus brevis [ 37 ] including new potential probiotic strains of Weissella [ 124 , 125 ] while short AXOS are more selective for Bifidobacterium strains [ 14 , 31 ]. Other biological effects tested in vitro are antimicrobial effects of acidic xylooligosaccharides, such as aldotetrauronic and aldopentauronic acids [ 58 ], and antioxidant activities of XOS from sugarcane bagasse [ 70 ].…”