Improved secretory expression and characterization of thermostable xylanase and ß-xylosidase from Pseudothermotoga thermarum and their application in synergistic degradation of lignocellulose.

Xylanase and ß-xylosidase are the key enzymes for hemicellulose hydrolysis. To further improve hydrolysis efficacy, high temperature hydrolysis with thermostable hemicellulases showed promise. In this study, thermostable xylanase (Xyn) and ß-xylosidase (XynB) genes from Pseudothermotoga thermarum were cloned and secretory expressed in Bacillu subtilis. Compared with Escherichia coli expression host, B. subtilis resulted in a 1.5 time increase of enzymatic activity for both recombinant enzymes. The optimal temperature and pH were 95°C and 6.5 for Xyn, and 95°C and 6.0 for XynB. Thermostability of both recombinant enzymes was observed between the temperature range of 75-85°C. Molecular docking analysis through AutoDock showed the involvement of Glu525, Asn526, Trp774 and Arg784 in Xyn-ligand interaction, and Val237, Lys238, Val761 and Asn76 in XynB-ligand interaction, respectively. The recombinant Xyn and XynB exhibited synergistic hydrolysis of beechwood xylan and pretreated lignocellulose, where Xyn and XynB pre-hydrolysis achieved a better improvement of pretreated lignocellulose hydrolysis by commercial cellulase. The observed stability of the enzymes at high temperature and the synergistic effect on lignocellulosic substrates suggested possible application of these enzymes in the field of saccharification process.

Advances in lytic polysaccharide monooxygenases with the cellulose-degrading auxiliary activity family 9 to facilitate cellulose degradation for biorefinery.

One crucial step in processing the recalcitrant lignocellulosic biomass is the fast hydrolysis of natural cellulose to fermentable sugars that can be subsequently converted to biofuels and bio-based chemicals. Recent studies have shown that lytic polysaccharide monooxygenase (LPMOs) with auxiliary activity family 9 (AA9) are capable of efficiently depolymerizing the crystalline cellulose via regioselective oxidation reaction. Intriguingly, the catalysis by AA9 LPMOs requires reductant to provide electrons, and lignin and its phenolic derivatives can be oxidized, releasing reductant to activate the reaction. The activity of AA9 LPMOs can be enhanced by in-situ generation of H2O2 in the presence of O2. Although scientific understanding of these enzymes remains somewhat unknown or controversial, structure modifications on AA9 LPMOs through protein engineering have emerged in recent years, which are prerequisite for their extensive applications in the development of cellulase-mediated lignocellulosic biorefinery processes. In this review, we critically comment on advances in studies for AA9 LPMOs, i.e., characteristic of AA9 LPMOs catalysis, external electron donors to AA9 LPMOs, especially the role of the oxidization of lignin and its derivatives, and AA9 LPMOs protein engineering as well as their extensive applications in the bioprocessing of lignocellulosic biomass. Perspectives are also highlighted for addressing the challenges.

Designer biocatalysts for direct incorporation of exogenous galactose into globotriose.

Galactose is ubiquitous. The synthesis of galactose-containing oligosaccharides using Leloir galactosyltransferase requires uridine diphosphate (UDP)-galactose as the precursor. Of all UDP-galactose synthesis pathways developed for in vitro synthesis, the salvage pathway represents the simplest route. In this study, for the first time, we designed and constructed an Escherichia coli strain to use salvage pathway for UDP-galactose synthesis, demonstrating effective and direct incorporation of exogenous galactose into globotriose (Gb3). Successful establishment of salvage pathway enabled a complete delineation of carbon and energy source. Consequently, the designed biocatalyst was able to achieve high yield synthesis from galactose (0.95 moles of Gb3/moles galactose consumed) and a high product titer (2 g/L) in shaker flask within 24 hr. Elimination of limitation in acceptor sugar via homologous overexpression of LacY, the transporter for lactose, further improved the synthesis, raising Gb3 titer to 6 g/L in 24 hr and 7.5 g/L in 48 hr. The design principles successfully demonstrated in this study could be broadly applied for synthesis of other galactose-containing oligosaccharides. This study also illustrates a valid strategy to overcome limitation in the transport of acceptor sugar. As lactose is one of the most important basal structures, the significant improvement in synthesis through its enhanced transport could be emulated in numerous other lactose-based oligosaccharides.

Synthesis, characterization and enzymatic surface roughing of cellulose/xylan composite films.

Upgrading renewable cellulose biopolymer to various high-value material/chemical is of great importance in building a sustainable bio-economy. This work assessed the technical feasibility of fabricating transparent cellulose/xylan composite films using facile solution-casting method. More importantly, this work also initially assessed the technical potential of xylanase treatment to selectively modify the surface of the obtained composite films with the goal of extending their applications. When bleached Kraft xylan addition was lower than 20 wt%, the composite films could still retain their original mechanical and structural advantages. Xylanase treatment specifically removed 26.0% and 32.3% xylan of the composite films with an enzyme loading of 2 and 5 mg g-1 cellulose, respectively. It was shown that xylan component was heterogeneously located in the surface of the composite films during film-casting process, which allowed the subsequent surface etching/roughing at nanoscale using facile xylanase treatment without compromising their structural advantages.

TCA cycle-powered synthesis of fucosylated oligosaccharides.

Microbial catalysis has recently emerged as one of the most promising approaches in oligosaccharide synthesis. However, despite significant progress, microbial synthesis still requires much improvement in efficiency and in reduction of process complexity. Additionally, given the stunning diversity and many varied applications of glycans, broadening the range of glycans accessible via microbial synthesis is of paramount importance. Major challenges in microbial synthesis include catabolite repression and high cellular energy requirement. Here we demonstrated a new approach to overcome these challenges by directly tapping into the cellular "power house," the TCA cycle, to provide the cellular energy for synthesis. This approach not only circumvents catabolite repression but also eliminates acidic glycolysis by-products. As such, the whole-cell biocatalysis can be carried out without sophisticated fed-batch feeding and pH control in the synthesis stage. The system could achieve several grams per liter (3-4 g/L) within a 24-h period in shaker flask cultivation for two targets, fucosyllactose and fucosyllactulose, demonstrating efficiency of the biocatalyst developed and its applicability to both natural and non-natural targets. To the best of our knowledge, this is the first use of TCA cycle intermediates as the energy source for oligosaccharide synthesis and the first description of successful synthesis of fucosyllactulose with titers in several grams per liter.

Thermostable xylanase-aided two-stage hydrolysis approach enhances sugar release of pretreated lignocellulosic biomass.

One of the challenges in biorefinery is the still too much enzyme involved in the saccharification of the cellulosic component. High-temperature hydrolysis with thermostable enzyme showed promise. In this study, a temperature-elevated two-stage hydrolysis, including xylan "coat" removal at high-temperature by thermostable xylanase (Xyn10A) from Thermotoga thermarum DSM 5069 followed with saccharification step by commercial cellulase, was introduced to improve biomass deconstruction. Results showed that high-temperature xylanase treatment considerably increased cellulose accessibility/hydrolyzability towards cellulases, with smoothed fiber surface morphology. Comparing with commercial xylanase (HTec) treatment at 50 °C, thermostable Xyn10A pre-hydrolysis at 85 °C was able to achieve a slightly better improvement of cellulose hydrolysis with much lower xylanase loading (about 5 times lower than HTec). It appeared that the increased temperature during thermostable xylanase treatment facilitated biomass slurry viscosity reduction, which exhibited more benefits during hydrolysis of various steam pretreated substrates at increased solid content (up to 10% w/w).

A xylanase-aided enzymatic pretreatment facilitates cellulose nanofibrillation.

Although biological pretreatment of cellulosic fiber based on endoglucanases has shown some promise to facilitate cellulose nanofibrillation, its efficacy is still limited. In this study, a xylanase-aided endoglucanase pretreatment was assessed on the bleached hardwood and softwood Kraft pulps to facilitate the downstream cellulose nanofibrillation. Four commercial xylanase preparations were compared and the changes of major fiber physicochemical characteristics such as cellulose/hemicellulose content, gross fiber properties, fiber morphologies, cellulose accessibility/degree of polymerization (DP)/crystallinity were systematically evaluated before and after enzymatic pretreatment. It showed that the synergistic cooperation between endoglucanase and certain xylanase (Biobrite) could efficiently "open up" the hardwood Kraft pulp with limited carbohydrates degradation (<7%), which greatly facilitated the downstream cellulose nanofibrillation during mild sonication process (90Wh) with more uniform disintegrated nanofibril products (50-150nm, as assessed by scanning electron microscopy and UV-vis spectroscopy).