*5.2. Inhibition by* l*-arabinose*

l-arabinose is one of the monosaccharides produced during enzymatic saccharification of cellulosic biomass [7,8,28]. It is liberated by the action of α-l-arabinofuranosidases, and, during the saccharification process, its concentration may sufficiently increase to inhibit the activity of hemicellulolytic enzymes [115]. Indeed, l-arabinose has been identified as an inhibitor of various β-xylosidases (Table 4). For instance, 50 mM l-arabinose reduces the activity of the aryl β-xylosidase from *Caldocellum saccharolyticum* Tp8T6.3.3.1 by 15% [120], the β-xylosidase from *B. pumilus* 12 by 21% [113], and the bifunctionalβ-xylosidase/α-l-arabinofuranosidase from *P. chrysosporium* BKM-F-1767 by ~70% [43]. This is not surprising, since the stereochemistry of l-arabinose near the glycosidic bond is similar to that of d-xylose [61], explaining its binding in the active site of a β-xylosidase.



#: GH family is not assigned in the CAZy database.

In general, l-arabinose is a weaker inhibitor of β-xylosidase activity than d-xylose. For example, the β-xylosidase from the fungus *Trichoderma reesei* RUT C30 is strongly inhibited by d-xylose with a *K*<sup>i</sup> of 2.4 mM, but it is not inhibited by l-arabinose, even at a concentration of 500 mM [24]. Several other β-xylosidases can also withstand high concentrations of l-arabinose [95,122,141]. Finally, the β-xylosidase from *Enterobacter* sp. is competitively inhibited by l-arabinose, but with a quite high *K*<sup>i</sup> value of 102 mM [72], indicating that the enzyme has only low affinity for l-arabinose.

Intriguingly, at low concentration (~5 mM) l-arabinose stimulates rather than inhibits the β-xylosidase activity of the bifunctional β-xylosidase/α-l-arabinofuranosidase from *P. chrysosporium* BKM-F-1767, as also noticed for d-glucose [43]. Activation by l-arabinose has also been observed for a furan aldehyde-tolerant β-xylosidase/α-l-arabinofuranosidase procured from a metagenomic sample, which showed 65% higher β-xylosidase activity compared with the control without l-arabinose [109]. Although there are many data describing the effects of l-arabinose on inhibition/activation of β-xylosidase activity, the molecular basis of the effects on activity still needs further investigation.

#### *5.3. Inhibition by Other Monosaccharides*

Apart from d-xylose and l-arabinose, d-glucose is another monosaccharide that has been frequently reported to affect β-xylosidase activity. d-glucose inhibits the β-xylosidase activity of the β-xylosidases from *S. ruminantium* GA192 (*K*<sup>i</sup> 44 mM) [27], *B. pumilus* 12 (9% inhibition at 50 mM) [113], *T. harzianum* (3% inhibition at 5 mM) [83], and the bifunctional β-glucosidase/β-xylosidases RuBG3A and RuBG3B from the metagenome of yak rumen microorganisms (97.5% and 45.6% inhibition, respectively, at 5 mM) [111]. On the other hand, the sugar did not inhibit β-xylosidases from *Thermomonospora fusca* [159], *A. niger* 90196 [134], *A. oryzae* [138], and *N. crassa* ST A [147] at concentrations of up to 90, 20, 20, and 10 mM, respectively. The Mg2<sup>+</sup>-activated β-xylosidase RS223-BX could even withstand much higher d-glucose concentrations displaying a *Ki* value of 1270 mM on the substrate *p*-nitrophenyl-α-l-arabinofuranoside (*p*NPA) [19]. Apparently, inhibition by d-glucose varies considerably among β-xylosidases.

Finally, besides d-xylose, l-arabinose, and d-glucose, also other monosaccharides have been reported to inhibit β-xylosidases, including d-arabinose [26,113], d-erythrose [26], d-fructose [111,138], d-galactose [26,113,120], d-ribose, and l-xylose [26,113]. Again, the molecular details of the interactions of these sugars with the enzymes are not known.

#### *5.4. Inhibition Kinetics*

The inhibition of β-xylosidases by monosaccharides follows competitive, non-competitive, or un-competitive inhibition kinetics. For most β-xylosidases, d-xylose acts as a competitive inhibitor when using *p*-nitrophenyl-β-d-xyloside (*p*NPX) as substrate [113,114,120,134]. However, the β-xylosidase from *N. crassa* ST A showed non-competitive inhibition by this sugar [147]. Furthermore, d-xylose inhibition of the β-xylosidase from *S. ruminantium* GA192, which also displays α-l-arabinofuranosidase activity, followed non-competitive kinetics for its β-xylosidase activity on *p*NPX as substrate, but competitive kinetics for its α-l-arabinofuranosidase activity on *p*NPA as substrate [26]. This differs slightly from *A. carbonarius* KLU-93 β-xylosidase, for which d-xylose was a competitive inhibitor for the conversion of both substrates [130]. Similarly, l-arabinose acts as a competitive inhibitor for the hydrolysis of *p*NPA by the RS223-BX β-xylosidase [19], but it is a non-competitive inhibitor of *S. ruminantium* GA192 β-xylosidase hydrolyzing *p*NPX or *p*NPA [26]. With respect to these substrates, d-arabinose, d-glucose, and d-ribose are competitive inhibitors of *S. ruminantium* GA192 β-xylosidase, whereas d-erythrose and l-xylose are non-competitive [26]. As also observed for *S. ruminantium* GA192 β-xylosidase, d-glucose inhibition of RS223-BX was competitive when using *p*NPA as substrate [19]. Uniquely, un-competitive inhibition was displayed by d-fructose for the activity of *A. oryzae* β-xylosidase on *p*NPX [138]. Thus, commonly, competitive inhibition by d-xylose is observed. Other monosaccharides can display both competitive and non-competitive inhibition, and in one case, d-fructose, un-competitive inhibition takes place. The exact mechanism and the structural details of non-competitive and un-competitive inhibition remain unknown.

#### *5.5. Structural Details of Inhibitor Binding in the Active Site of* β*-Xylosidases*

As discussed above, the active site pockets of β-xylosidases contain two substrate-binding subsites, subsites –1 and +1, on either side of the scissile bond. In the active site of *S. ruminantium* GA192 β-xylosidase, the monosaccharides d-arabinose, l-arabinose, d-erythrose, and d-ribose can bind in both subsites –1 and +1, but d-xylose and l-xylose bind only in subsite –1 [26]. Similarly, d-glucose binds in one subsite only. Its binding position was speculated to be in subsite –1, with partial occupancy of subsite +1, because glucose is too large to fit in subsite –1 only. Alternatively, it could bind in such a way that it excludes binding of a second sugar [26].

In contrast, *G. thermoleovorans* IT-08 β-xylosidase shows rather dissimilar binding properties for l-arabinose and d-xylose compared to *S. ruminantium* GA192 β-xylosidase. Crystal structures of *G. thermoleovorans* IT-08 β-xylosidase revealed that l-arabinose binds exclusively in subsite –1, while d-xylose prefers subsite +1 [70]. Thus, depending on the enzyme, the –1 and +1 subsites differ in preference for different monosaccharides, which could also contribute to the differences in inhibition kinetics observed for the different enzymes.

### *5.6. Engineering to Reduce* β*-Xylosidase Inhibition by Monosaccharides*

During saccharification of cellulosic biomass, monosaccharides such as d-xylose, l-arabinose, and d-glucose, may reach concentrations that are high enough to inhibit β-xylosidase activity [26,27]. Therefore, β-xylosidases that are not affected by high monosaccharide concentrations are highly desirable for the efficiency of the saccharification process. To develop such β-xylosidase variants, the W145G mutation was introduced into *S. ruminantium* GA192 β-xylosidase, resulting in a variant with a 3-fold lower affinity for d-xylose and a 2-fold lower affinity for d-glucose [121]. Subjecting this variant to saturation mutagenesis of residue 145 yielded variants with even lower affinity for monosaccharides and higher catalytic activity than wild-type enzyme [27]. Mutation of Trp-145 alters the affinity of subsite +1 for d-xylose, but not that of subsite -1, where catalysis occurs, suggesting a strategy for reducing inhibition by monosaccharides by mutating residues of subsite +1 [27]. In the structure of *G. thermoleovorans* IT-08 β-xylosidase, d-xylose binds in subsite +1 interacting, among others, with Asp-198, which is not present in most other β-xylosidases [70]. Therefore, this residue may be a good target for mutation to obtain *G. thermoleovorans* IT-08 β-xylosidase variants with lower affinity for d-xylose.
