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.

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.

Engineering lower inhibitor affinities in beta-D-xylosidase.

Beta-D-Xylosidase catalyzes hydrolysis of xylooligosaccharides to D-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by D-xylose and D-glucose (K (i) values of approximately 10(-2) M). Higher K (i)'s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1 degrees screen selects for activity (active/inactive screen) and the 2 degrees screen selects for a higher K (i(D-xylose)) and its subsequent use in screening approximately 5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, K (i(D-xylose)) is threefold and K (i(D-glucose)) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite -1 or subsite +1 indicate that the changed affinity stems from changed affinity for D-xylose in subsite +1 and not in subsite -1 of the two-subsite active site. Trp145 is 6 A from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite -1.

Biochemical characterization of a novel dual-function arabinofuranosidase/xylosidase isolated from a compost starter mixture.

The gene encoding a glycoside hydrolase family 43 enzyme termed deAX was isolated and subcloned from a culture seeded with a compost starter mixed bacterium population, expressed with a C-terminal His(6)-tag, and purified to apparent homogeneity. deAX was monomeric in solution and had a broad pH maximum between pH 5.5 and pH 7. A twofold greater k (cat)/K (m) for the p-nitrophenyl derivative of alpha-L: -arabinofuranose versus that for the isomeric substrate beta-D-xylopyranose was due to an appreciably lower K (m) for the arabinofuranosyl substrate. Substrate inhibition was observed for both 4-methylumbelliferryl arabinofuranoside and the xylopyranoside cogener. While no loss of activity was observed over 4 h at 40 degrees C, the observed t (1/2) value rapidly decreased from 630 min at 49 degrees C to 47 min at 53 degrees C. The enzyme exhibited end-product inhibition, with a K (i) for xylose of 145 mM, 18.5 mM for arabinose, and 750 mM for glucose. Regarding natural substrate specificity, deAX had arabinofuranosidase activity on sugar beet arabinan, 1,5-alpha-L-arabinobiose, and 1,5-alpha-L-arabinotriose, and wheat and rye arabinoxylan, while xylosidase activity was detected for the substrates xylobiose, xylotriose, xylotetraose, and arabinoxylan from beech and birch. Thus, deAX can be classified as a dual-function xylosidase/arabinofuranosidase with respect to both artificial and natural substrate specificity.

Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08.

The gene encoding a glycoside hydrolase family 43 beta-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-beta-D: -xylopyranose (4NPX) and p-nitrophenyl-alpha-L: -arabinofuranose (4NPA), and it was found that the ratio k (cat)/K (m) 4NPA/k (cat)/K (m) 4NPX was approximately 7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k (cat)/K (m) muA/k (cat)/K (m) muX was approximately 5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K (i) of 6.8 +/- 0.62 mM and xylose K (i) of 76 +/- 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 degrees C, with a t (1/2) of 35 min at 57.5 degrees C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.