Detailed Kinetic Analysis of a Family 52 Glycoside Hydrolase:  A β-Xylosidase from Geobacillus stearothermophilus

Abstract

Geobacillus stearothermophilus T-6 encodes for a beta-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl beta-D-xylopyranoside substrates bearing different leaving groups. The Brønsted plot of log k(cat) versus the pK(a) value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases. Hydrolysis rates for substrates with poor leaving groups (pK(a) > 8) vary widely with the aglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, no such dependence is observed for more reactive substrates (pK(a) < 8), indicating that in this case hydrolysis of the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that the intermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, and bond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several gluco analogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl beta-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as 1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.

Strategy for Identification of Novel Fungal and Bacterial Glycosyl Hydrolase Hybrid Mixtures that can Efficiently Saccharify Pretreated Lignocellulosic Biomass

“…Recent work with a fungal endo-xylanase isolated from Trichoderma has shown that the Geobacillus enzyme has three-to fivefold higher activity on oat spelt xylan under identical conditions [28]. LβX was found to have high xylosidase activity but poor α-arabinofuranosidase activity, comparable to what has been reported earlier [32]. The bacterial LβG had significantly lower cellobiose activity than its fungal counterpart (βG).…”

“…Interestingly, the bacterial β-xylosidase has been reported earlier to retain about 40% activity at pH 5 vs. pH 6.5 [32]. We noticed only a 25% loss in activity based on the overall xylan conversions for AFEX corn stover (Fig.…”

“…This would suggest that in the presence of a suitable bacterial exo/endo-cellulase complex (that have an opti- mum activity at pH 6.5), it would be possible to further lower the total enzyme loading (and maximize glucan/xylan conversion). There have also been several reports on the activity of GH family 43 β-xylosidase, with very few publications on their GH 52 counterparts [32]. This study is one of the first that reports the activity of GH 52 β-xylosidases on pretreated lignocellulosic biomass.…”

“…One of the limitations of hydrolyzing the substrate at pH 5 is the relatively lower activity of these bacterial enzymes (40-50% of optimum at pH 6.5) reported by Bravman et al [32]. This would suggest that in the presence of a suitable bacterial exo/endo-cellulase complex (that have an opti- mum activity at pH 6.5), it would be possible to further lower the total enzyme loading (and maximize glucan/xylan conversion).…”

A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides inGeobacillus stearothermophilus

“…When grown in the presence of xylan, strain T-6 secretes a single extracellular endo 1,4-␤ xylanase (XynA) that hydrolyzes the polymer's main backbone, producing short modified oligoxylose units of two or more sugars in length. These modified xylosaccharides enter the cell by specialized ATP-binding cassette (ABC) sugar transporters (73), and they are further degraded to monomers by intracellular hydrolases, including a glycoside hydrolase family 10 (GH10) xylanase (77), two GH51 ␣-L-arabinofuranosidases (29), an ␣-glucuronidase (GH67) (23,68,89), three ␤-xylosidases (GH39, GH43, and GH52) (6,(8)(9)(10)(11), and two xylan acetyl esterases (CE4).…”