Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-L-arabinofuranosidase and β-xylosidase.
McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Carbohydrate Research, 407, 79-96.
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 A2,3XX, 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.
Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases.
Badhan, A., Wang, Y., Gruninger, R., Patton, D., Powlowski, J., Tsang, A. & McAllister, T. (2014). BMC Biotechnology, 14(1), 31.
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.
New glycosidase substrates for droplet-based microfluidic screening.
Najah, M., Mayot, E., Mahendra-Wijaya, I. P., Griffiths, A. D., Ladame, S. & Drevelle, A. (2013). Analytical Chemistry, 85(20), 9807-9814.
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.
Role of hemicellulases in production of fermentable sugars from corn stover.
Xin, D., Sun, Z., Viikari, L. & Zhang, J. (2015). Industrial Crops and Products, 74, 209-217.
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.
Formulation of an optimized synergistic enzyme cocktail, HoloMix, for effective degradation of various pre-treated hardwoods.
Malgas, S., Chandra, R., Van Dyk, J. S., Saddler, J. N. & Pletschke, B. I. (2017). Bioresource Technology, In Press.
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.