β-D-Xylosidase (Selenomonas ruminantium)

A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 3²-α-L-Araf-(1-4)-β-D-xylobiose (A³X), 2³-α-L-Araf-(1-4)-β-D-xylotriose (A²XX), 3³-α-L-Araf-(1-4)-β-D-xylotriose (A³XX), 2²-α-L-Araf-(1-4)-β-D-xylotriose (XA²X), 3²-α-L-Araf (1-4)-β-D-xylotriose (XA³X), 2³-α-L-Araf-(1-4)-β-D-xylotetraose (XA²XX), 3³-α-L-Araf-(1-4)-β-D-xylotetraose (XA³XX), 2³ ,3³-di-α-L-Araf-(1-4)-β-D-xylotriose (A²⁺³XX), 2³,3³-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA²⁺³XX), 2⁴,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA²⁺³XXX) and 3³,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA³A³XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A^2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

Arabinoxylans, β-glucans, and dextrins influence the brewing industry's filtration process and product quality. Despite their relevance, only a maximum concentration of β-glucans is recommended. Nevertheless, filtration problems are still present, indicating that although the chemical concentration is essential, other parameters should be investigated. Molar mass and conformation are important polymer physical characteristics often neglected in this industry. Therefore, this research proposes an approach to physically characterize enzymatically isolated beer polysaccharides by asymmetrical flow field-flow fractionation coupled to multi-angle light scattering and differential refractive index detector. Based on the obtained molar masses, root-mean-square radius (rrms from MALS), and hydrodynamic radius (rhyd), conformational properties such as apparent density (ρapp) and rrms/rhyd can be calculated based on their molar mass and size. Consequently, the ρapp and rrms/rhyd behavior hints at the different structures within each polysaccharide. The rrms/rhyd 1.2 and high ρapp values on low molar mass dextrins (1-2·10⁵ g/mol) indicate branches, while aggregated structures at high molar masses on arabinoxylans and β-glucans (2·10⁵ -6·10⁶ g/mol) are due to an increase of ρapp and a rrms/rhyd (0.6-1). This methodology provides a new perspective to analyze starch and non-starch polysaccharides in cereal-based beverages since different physical characteristics could influence beer's filtration and sensory characteristics.

Background: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L^− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Nonionic surfactant Tween 80 is effective in alleviating the adsorption of lignocellulose-degrading enzymes onto lignin. However, the impacts of lignin structural characteristics on this mitigation effect of Tween 80 were still not clear. Herein, effects of Tween 80 on cellulase and xylanase adsorption on different enzymatic residual lignins (ERLs) and poplar samples were investigated. Besides, effects of Tween 80 on desorption of the two enzyme preparations from different ERLs and poplar samples were also evaluated. By adding Tween 80, the inhibition of ERLs to cellulase hydrolysis was offset and the positive role of Tween 80 in Avicel hydrolysis with presence of ERLs was more effective. Binding strength of cellulase with ERLs was alleviated from 236.0-410.9 to 43.9-116.6 mL/g after adding 0.5 mg/mL Tween 80. When ERLs was more hydrophobic and had more condensed phenolic OH, the alleviation effect of Tween 80 on cellulase adsorption on ERLs was intensified but its positive effect on enhancing cellulase desorption was weakened. Tween 80 was effective in increasing xylanase activity and preventing xylanase deactivation. With the increase of lignin content and phenolic OH in pretreated poplar, the effect of Tween 80 in alleviating xylanase adsorption on poplar samples was intensified but the effect in enhancing xylanase desorption from poplar samples was weakened. The results provided distinct findings to understand the interaction between Tween 80 and cellulase/xylanase in enzymatic saccharification of lignocelluloses.

Since xylose is the second most abundant sugar in lignocellulose, using microorganisms able to metabolize it into bio-based chemicals like lactic acid is an attractive approach. In this study, Lactobacillus pentosus CECT4023T was evolved to improve its xylose fermentation capacity even at acid pH by adaptive laboratory evolution in repeated anaerobic batch cultures at increasing xylose concentration. The resulting strain (named MAX2) presented between 1.5 and 2-fold more xylose consumption and lactic acid production than the parental strain in 20 g L⁻¹ xylose defined media independently of the initial pH value. When the pH was controlled in bioreactor, lactic acid productivity at 16 h increased 1.4-fold when MAX2 was grown both in xylose defined media and in wheat straw hydrolysate. These results demonstrated the potential of this new strain to produce lactic acid from hemicellulosic substrates at low pH, reducing the need of using neutralizing agents in the process.

Xylan, the most abundant hemicellulose in lignocellulosic biomass, requires a consortium of xylanolytic enzymes to achieve its complete de-polymerisation. As global interest in using xylan-containing lignocellulosic feedstocks for biofuel production increases, an accompanying knowledge on how to efficiently depolymerise these feedstocks into fermentable sugars is required. Since it has been observed that the same enzyme [i.e. an enzyme with the same EC (Enzyme Commission) classification] from different GH families can display different substrate specificities and properties, we evaluated GH10 (XT6) and 11 (Xyn2A) xylanase performance alone, and in combination, during xylan depolymerisation. Synergistic enhancement with respect to reducing sugar release was observed when Xyn2A at 75% loading was supplemented with 25% loading of XT6 for both beechwood glucuronoxylan (1.14-fold improvement) and wheat arabinoxylan (1.1-fold improvement) degradation. Following this, the optimised xylanase mixture was dosed with an oligosaccharide reducing-end xylanase (Rex8A) from either Bifidobacterium adolescentis or Bacillus halodurans for further synergistic enhancement. Dosing 75% of the xylanase mixture (Xyn2A:XT6 at 75:25%) with 25% loading of Rex8A led to an enhancement of reducing sugar (up to an 1.1-fold improvement) and xylose release (up to an 1.5-fold improvement); however, this effect was both xylan and Rex8A specific. Using thin layer chromatography, synergism appeared to be a result of the GH10 and 11 xylanases liberating xylo-oligomers that are preferred substrates of the processive Rex8As. Rex8As then hydrolysed xylo-oligomers to xylose - and xylobiose which was the preferred substrate for xylosidase, SXA. This likely explains why there was a significant improvement in xylose release in the presence of Rex8As. Here, it was shown that Rex8As are key enzymes in the efficient saccharification of hetero-xylan into xylose, a major component of lignocellulosic substrates.

In this study, two selected hardwoods were subjected to sodium chlorite delignification and steam explosion, and the impact of pre-treatments on synergistic enzymatic saccharification evaluated. A cellulolytic core-set, CelMix, and a xylanolytic core-set, XynMix, optimised for glucose and xylose release, respectively, were used to formulate HoloMix cocktail for optimal saccharification of various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of 75%: 25%, while for untreated and steam exploded biomass the HoloMix consisted of 93.75%: 6.25% protein dosage, CelMix: XynMix, respectively. Saccharification by HoloMix (27.5 mg protein/g biomass) for 24 h achieved 70-100% sugar yields. Pre-treatment of the hardwoods, especially those with a higher proportion of lignin, with a laccase improved saccharification by HoloMix. This study provided insights into enzymatic hydrolysis of various pre-treated hardwood substrates and showed the same lignocellulolytic cocktail comparable to/if not better than commercial enzyme preparations can be used to efficiently hydrolyse different hardwood species.

In this work, the roles of hemicellulases, endoxylanase and β-xylosidase, in improving the hydrolysis of corn stover pretreated by aqueous ammonia (CS–AA) and dilute acid (CS–DA) were evaluated. Synergistic actions of endoxylanase and β-xylosidase were observed in the release of xylose in the hydrolysis of both isolated xylan and xylan in pretreated corn stover. Endoxylanase significantly reduced the negative effect of xylan on the action of cellulases, especially on cellobiohydrolase I. The addition of β-xylosidase from Selenomonas ruminantium increased the xylose yields from 9.6% and 13.0% to 31.7% and 47.6% in the hydrolysis of CS–AA by cellulases and xylanase at 40°C and 50°C, respectively. Furthermore, the addition of thermostable β-xylosidase from Entamoeba coli increased glucose yields from 40.3% and 20.7% to 44.0% and 26.6% in the hydrolysis of CS–AA and CS–DA by cellulases and xylanase at 50°C, respectively. β-xylosidase significantly reduced xylo-oligosaccharides inhibition on cellobiohydrolase I by converting most of xylo-oligosaccharides (93.6%) to the less inhibitory xylose, showing the importance and potential benefits of β-xylosidase in efficient and complete hydrolysis of lignocelluloses.

Background: Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Results: Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. Conclusion: The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.

Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30°C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.