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. 2013 Sep 11;3(1):56.
doi: 10.1186/2191-0855-3-56.

Biochemical and Kinetic Characterisation of a Novel Xylooligosaccharide-Upregulated GH43 β-D-Xylosidase/α-L-Arabinofuranosidase (BXA43) From the Probiotic Bifidobacterium Animalis Subsp. Lactis BB-12

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Biochemical and Kinetic Characterisation of a Novel Xylooligosaccharide-Upregulated GH43 β-D-Xylosidase/α-L-Arabinofuranosidase (BXA43) From the Probiotic Bifidobacterium Animalis Subsp. Lactis BB-12

Alexander Holm Viborg et al. AMB Express. .
Free PMC article

Abstract

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a β-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-β-d-xylopyranoside (pNPX), para-nitrophenyl-α-L-arabinofuranoside (pNPA), β-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2-4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I - V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40°C and at pH 4.0-8.0 and showed maximum activity at pH 5.5 and 50°C. Km and kcat for pNPX were 15.6 ± 4.2 mM and 60.6 ± 10.8 s-1, respectively, and substrate inhibition became apparent above 18 mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for β-d-xylosidase (XynB3) from Geobacillus stearothermophilus T‒6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn2+ and Ag+, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg505Tyr506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus.

Figures

Figure 1
Figure 1
Phylogenetic tree of GH43 enzymes annotated as characterised in the CAZy database (prepared by Dendroscope). Five distinct groups (I-V) are observed which represent different specificity clusters. BXA43 (in bold) is found in group IV. Enzyme activities have been abbreviated as X: β-d-xylosidase (EC 3.2.1.37). X13: β-1,3-xylosidase (EC 3.2.1.-). A: α-l-arabinofuranosidase (EC 3.2.1.55). eA: endo-arabinanase (EC 3.2.1.99), eX: endo-xylanase (EC 3.2.1.8). G: galactan 1,3-β-galactosidase (EC 3.2.1.145). The entries are numbered and further details like the full organism names and UniProt identifiers can be found in Additional file 1: Table S1.
Figure 2
Figure 2
Temperature and pH parameters of BXA43. (A) Stability () and activity (relative Vmax) () pH profiles of BXA43 using pNPX as substrate. (B) Stability () and activity (relative Vmax) () pH profiles of BXA43 using pNPX as substrate. (B) Stability (■) and activity (relative Vmax) (●) of BXA43 as a function of temperature using pNPX as substrate. (C) Arrhenius plot of the effect of temperature on the t½ for thermal inactivation of BXA43 in the temperature range 40–60°C (see Methods for details).
Figure 3
Figure 3
Michaelis-Menten kinetics of BXA43 toward pNPX. The fit is to uncompetitive substrate inhibition. All results are expressed as means ± standard error of the mean (SEM) n = 4 (see Methods for details).
Figure 4
Figure 4
Model of BXA43 (green) superimposed with the structure of the close homolog XynB3 (grey, 2EXH), showing the two catalytic residues D14 and E187 (in blue; BXA43 numbering). Xylotetraose (yellow) was manually fitted from the structure of the complex with BsAXH-m2,3 on Bacillus subtilis (grey, 3C7G) (Vandermarliere et al., 2009) occupying the +1, +2, +3, and +4 subsites and illustrating a predicted +3 subsite involvement of Y506 (red). Arg505 in BXA43 may play a role in the positioning of Tyr506.

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