Penicillium camemberti galacturonate reductase: C-1 oxidation/reduction of uronic acids and substrate inhibition mitigation by aldonic acids.

The enzyme galacturonate oxidoreductase PcGOR from Penicillium camemberti reduces the C-1 carbon of D-glucuronate and C-4 epimer D-galacturonate to their corresponding aldonic acids, important reactions in both pectin catabolism and ascorbate biosynthesis. PcGOR was active on both glucuronic acid and galacturonic acid, with similar substrate specificities (kcat/Km) using the preferred co-substrate NADPH. Substrate acceptance extended to lactone congeners, and D-glucurono-3,6-lactone was converted to L-gulono-1,4-lactone, an immediate precursor of ascorbate. Reaction with glucuronate showed only minor substrate inhibition, and the product L-gulonate and L-gulono-1,4-lactone were both found to be competitive inhibitors with Ki in the low mM range. In contrast, reaction with C-4 epimer galacturonate displayed marked substrate inhibition. Moreover, the product L-galactonate and L-galactono-1,4-lactone were observed to mitigate substrate inhibition by galacturonate, with the lactone having a greater effect than the acid.

Biochemical characterization of Caulobacter crescentus xylose dehydrogenase.

d-Xylose sugar is a common component of hemicellulose, the second largest fraction of biomass. Many groups have developed biological conversions of d-xylose to value-added products by recombinant expression of the xylose dehydrogenase enzyme from Caulobacter crescentus. This enzyme uses NAD+ as a cofactor to oxidize d-xylose to d-xylono-1,4-lactone. A detailed understanding of the mechanism of this enzyme could be useful in engineering more efficient versions. Therefore, we have conducted kinetic studies including both the forward and reverse physiological reactions of this enzyme. We demonstrate that the enzyme's substrate binding mode follows a sequential steady state ordered mechanism with NAD+ or NADH binding first. Furthermore, the kcat of the reaction in the direction of NAD+ reduction is 10-fold higher than that of the reverse reaction. From rapid reaction studies, we demonstrate the binding of NAD+ and NADH to the free enzyme and that hydride transfer occurs in a fast step followed by a much slower steady state. We calculate that the dissociations of the sugar products from the enzyme complexes are the major rate limiting steps in both directions.

Absence or presence of metal ion activation in two structurally similar GH43 ß-xylosidases.

Two GH43 ß-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus, that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% loss of activity for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases. The high activity of the BoXA mutants compared to that of the analogous RS223-BX mutants further undermines the possibility that BoXA maintains a tightly bound metal cofactor resistant to EDTA extraction. The results strengthen our conclusion that the very similar proteins differ in one being metal ion dependent and one not.

Expression and Characterization of Hyperthermostable Exopolygalacturonase RmGH28 from Rhodothermus marinus.

The gene RmGH28 from the organism Rhodothermus marinus, a putative glycosyl hydrolase family 28 polygalacturonase, was expressed in Escherichia coli and biochemically characterized. The gene was found to encode an exopolygalacturonase termed RmGH28, with galacturonic acid monomer and the polymer substrate (n-1) as the products released when acting on de-esterified polygalacturonic acid from citrus pectin. The enzyme at 25 °C had k cat ∼6 s-1 when acting on polygalacturonic acid, with K m ∼0.7 µM and a substrate inhibition constant K si ∼70 µM. The enzyme was hyperthermophilic, with one half initial enzyme activity remaining after 1-h incubation at 93.9 °C. Since the enzyme can function at high temperatures where reaction rates are increased and the risk of bacterial contamination is decreased, this indicates that RmGH28 can be useful in industry for generating galacturonic acid from pectin. The amino acid sequence of RmGH28 is highly homologous to the known hyperthermophilic exopolygalacturonases TtGH28 and Tm0437, which together can serve as starting points for structure-function studies and molecular breeding enzyme engineering approaches.

A general correction to catalytic rates determined for nonprocessive exo-depolymerases acting on both substrate and product in the initial-rate measurement.

We recently reported on the kinetics of the polygalacturonase TtGH28 acting on trimer and dimer substrates. When the starting substrate for hydrolysis is the trimer, the product dimer is also subject to hydrolysis, resulting in discrepancies when either the concentration of dimer or monomer product is used for analysis of trimer hydrolysis. Here, we derive a method for determining catalytic rates of exo-hydrolases acting on trimer (and higher order) substrates when products may also be substrates for hydrolysis and show how this correction may be applied for TtGH28.

Biochemical Characterization of a GH43 ß-Xylosidase from Bacteroides ovatus.

Divalent metal-activated glycoside hydrolase family 43 (GH43) ß-xylosidases have been found to have high k cat/K m for xylooligosaccharides and may demonstrate high efficacy in industrial reactors digesting hemicellulose. By searching an amino acid database, we found a Bacteroides ovatus GH43 ß-xylosidase termed BoXA that is 81% identical in overall amino acid sequence to a GH43, divalent metal-activated ß-xylosidase with high k cat/K m, and also it has 19 of 20 residues in the active site conserved. However, unlike its metal-activated homolog, the B. ovatus enzyme does not lose activity after extensive EDTA treatment nor does it gain activity by addition of divalent metal ions. Thus, either it cannot be activated by divalent metal or it maintains a tightly bound, non-exchangeable metal ion. At 25 °C and pH 6.0, the k cat is 69 s-1 for xylobiose and k cat/K m is 210 s-1 mM-1 for xylotriose, with the latter being 0.7 that of the highest known value. The determined K i for D-glucose is 4.9 M, which is the highest known for a ß-xylosidase. The enzyme has potential utility operating in bioreactors digesting plant biomass.

Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus.

D-galacturonic acid is a potential platform chemical comprising the principal component of pectin in the citrus processing waste stream. Several enzyme activities are required for the enzymatic production of galacturonic acid from pectin, including exo- and endo-polygalacturonases. The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in Escherichia coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and details of their catalytic mechanism have been elucidated shows that three catalytic Asp residues and several other key active site residues are strictly conserved. Purified TtGH28 was dimeric and hyperthermostable, with K t (0.5)  = 86.3 °C. Kinetic parameters for activity on digalacturonic acid, trigalacturonic acid, and polygalacturonic acid were obtained. No substrate inhibition was observed for polygalacturonate, while the K si values for the oligogalacturonides were in the low mM range, and K i for product galacturonic acid was in the low µM range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional.

Isolation and divalent-metal activation of a ß-xylosidase, RUM630-BX.

The gene encoding RUM630-BX, a ß-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (kcat) by divalent metals Mg(2+), Mn(2+) and Co(2+) but not by Ca(2+), Ni(2+), and Zn(2+). Activation of RUM630-BX by Mg(2+) (t0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (kcat/Km) of 299 s(-1) mM(-1) on xylotetraose being the highest reported.

Rate-limiting steps of a stereochemistry retaining ß-d-xylosidase from Geobacillus stearothermophilus acting on four substrates.

Kinetic experiments of GSXynB2, a GH52 retaining ß-xylosidase, acting on 2-nitrophenyl-ß-d-xylopyranoside (2NPX), 4-nitrophenyl-ß-d-xylopyranoside (4NPX), 4-methylumbelliferyl-ß-d-xylopyranoside (MuX) and xylobiose (X2) were conducted at pH 7.0 and 25 °C. Catalysis proceeds in two steps (xylodidation followed by dexylosidation): E + substrate TO E-xylose + leaving group TO E + xylose. kcat falls into two groups: 4NPX (1.95 s(-1)) and 2NPX, MuX and X2 (15.8 s(-1), 12.6 s(-1), 12.8 s(-1), respectively). Dexylosylation (E-xylose to E + xylose), the common step for the enzymatic hydrolysis of the four substrates, must exceed 15.8 s(-1). kcat of 4NPX would seem mainly limited by xylosylation (step 1) and the other three substrates would seem mainly limited by dexylosylation (step 2) - a conclusion that critically lacks chemical justification (compare 4NPX and 2NPX). Presteady-state rates indicate rapid xylosidation rates for all substrates so a later step (not dexylosidation) is rate-limiting for 4NPX. That 2NPX is an onlier and 4NPX is an outlier (both leaving group pKa of 7.2) of the Brønsted plot pattern (logkcat vs pKa of phenol leaving group) is thus possibly explained by 4NP release. The pH dependency of kcat 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7.

X-ray Crystal Structure of Divalent Metal-Activated ß-xylosidase, RS223BX.

We report the X-ray crystal structure of a glycoside hydrolase family 43 ß-xylosidase, RS223BX, which is strongly activated by the addition of divalent metal cations. The 2.69 Å structure reveals that the Ca(2+) cation is located at the back of the active-site pocket. The Ca(2+) is held in the active site by the carboxylate of D85, an "extra" acid residue in comparison to other GH43 active sites. The Ca(2+) is in close contact with a histidine imidazole, which in turn is in contact with the catalytic base (D15) thus providing a mechanism for stabilizing the carboxylate anion of the base and achieve metal activation. The active-site pocket is mirrored by an "inactive-site" pocket of unknown function that resides on the opposite side of the monomer.

Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans.

Enzyme catalysts will be vital in the development of synthetic biology approaches for converting pectinic monosaccharides from citrus and beet processing waste streams to value-added materials. We describe here the biophysical and mechanistic characterization of uronate dehydrogenases from a wide variety of bacterial sources that convert galacturonic acid, the predominate building block of pectin from these plant sources, and glucuronic acid to their corresponding dicarboxylic acids galactarate and glucarate, the latter being a DOE top value biochemical from biomass. The enzymes from Pseudomonas syringae and Polaromonas naphthalenivorans were found to have the highest reported kcat(glucuronic acid) values, on the order of 220-270 s(-1). The thermal stability of this enzyme type is described for the first time here, where it was found that the Kt((0.5)) value range was >20 °C, and the enzyme from Chromohalobacter was moderately thermostable with Kt((0.5))=62.2 °C. The binding mechanism for these bi-substrate enzymes was also investigated in initial rate experiments, where a predominately steady-state ordered binding pattern was indicated.

Directed evolution of GH43 ß-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis.

Directed evolution of ß-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K(t)°·5 by -1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K(t)°·5 by ~3 °C; none of the individual amino acid changes measurably affect K(t)°·5. Saturation mutagenesis of L186 identified the variant L186K as having the most improved K(t)°·5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K(t)°·5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k cat of xylobiose and 4-nitrophenyl-ß-D-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K(m), K(ss) (substrate inhibition), and K(i) (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.

Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases.

We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to ß-xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 ß-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the ß-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 ß-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose. kcat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-ß-d-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for kcat(4NPX).

Activation of a GH43 ß-xylosidase by divalent metal cations: slow binding of divalent metal and high substrate specificity.

RS223-BX of glycoside hydrolase family 43 is a ß-d-xylosidase that is strongly activated (k(cat)/K(m) as much as 116-fold) by the addition of divalent metal cations, Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+). Slow activation by Mg(2+) was demonstrated (k(on) 0.013 s(-1) mM(-1), k(off) 0.008 s(-1)) at pH 7.0 and 25 °C. k(off) and k(on) values are independent of Mg(2+) concentration, but k(off) and k(on) are slower in the presence of increasing levels of substrate 4-nitrophenyl-ß-D-xylopyranoside. The kinetics strongly suggest that M(2+) binds to the enzyme rapidly, forming E M(2+), followed by slow isomerization to the activated enzyme, E* M(2+). Moderately high values of kcat (7-30 s(-1)) were found for M(2+)-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25 °C. Certain M(2+)-activated RS223-BX exhibit the highest reported values of k(cat)/K(m) of any ß-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn(2+), 190 s(-1) mM(-1)), xylotriose (Ca(2+), 150 s(-1) mM(-1)) and xylotetraose (Ca(2+), 260 s(-1) mM(-1)). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations.

Divalent metal activation of a GH43 ß-xylosidase.

Depolymerization of xylan, a major fraction of lignocellulosic biomass, releases xylose which can be converted into transportation fuels and chemical feedstocks. A requisite enzyme for the breakdown of xylan is ß-xylosidase. A gene encoding the 324-amino acid ß-xylosidase, RS223-BX, was cloned from an anaerobic mixed microbial culture. This glycoside hydrolase belongs to family 43. Unlike other GH43 enzymes, RS223-BX can be strongly activated by exogenously supplied Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+) (e.g., 28-fold by Mg(2+)) and it is inhibited by Cu(2+) or Zn(2+). Sedimentation equilibrium centrifugation experiments indicated that the divalent metal cations mediate multimerization of the enzyme from a dimeric to a tetrameric state, which have equal catalytic activity on an active-site basis. Compared to the determined active sites of other GH43 ß-xylosidases, the predicted active site of RS223-BX contains two additional amino acids with carboxylated side chains that provide potential sites for divalent metal cations to reside. Thus, the divalent metal cations likely occupy the active site and participate in the catalytic mechanism. RS223-BX accepts as substrate xylobiose, arabinobiose, 4-nitrophenyl-ß-D-xylopyranoside, and 4-nitrophenyl-α-L-arabinofuranoside. Additionally, the enzyme has good pH and temperature stabilities and a large K(i) for D-glucose (1.3 M), favorable properties for performance in saccharification reactors.

Highly active ß-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates.

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. ß-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active ß-xylosidases, some of which could have practical applications. The biochemical details of four GH43 ß-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 ß-xylosidases. The K i values for D-glucose (83.3-357 mM) and D-xylose (15.6-70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t (0.5) ∼ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s(-1), 138 s(-1) mM(-1)), xylotriose (235 s(-1), 80.8 s(-1) mM(-1)), and xylotetraose (146 s(-1), 32.6 s(-1) mM(-1)).

Plant cell walls to ethanol.

Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s-1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional ß-D-xylosidase/α-L-arabinofuranosidase.

Conformational inversion occurs 7-8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-ß-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional ß-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite -1 residues decrease the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise k(cat)(X2/4NPX) and alanine mutations of subsite +1 and interface residues lower k(cat)(X2/4NPX). We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite -1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues.

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural.

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH↔HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).

Engineering lower inhibitor affinities in ß-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145.

ß-D-Xylosidase/α-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-ß-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (K (i) ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K(i)(D-glucose) and K(i)(D-xylose) twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k(cat)(D-xylobiose) and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K (i) values that are 1.5-2.7-fold (D-glucose) and 1.9-4.6-fold (D-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k(cat)(D-xylobiose) than that of the wild type. At 0.1 M D-xylobiose and 0.1 M D-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.