Evaluation of co-culture of cellulolytic fungi for enhanced cellulase and xylanase activity and saccharification of untreated lignocellulosic material.

Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.

Production of a bacterial secretome highly efficient for the deconstruction of xylans.

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.

Cryptococcus laurentii: a wild yeast for xylanase production from agricultural by-products.

The development of technologies that allow the production of enzymes at a competitive cost is of great importance for several biotechnological applications, and the use of agro-industrial by-products is an excellent alternative to minimize costs and reduce environmental impacts. This study aimed to produce endo-xylanases using agro-industrial substrates rich in hemicellulose as sources of xylan in culture media. For this purpose, the yeast Cryptococcus laurentti and five lignocellulosic materials (defatted rice bran, rice husk, corn cob, oat husks, and soybean tegument), with and without pretreatment, were used as a source of xylan for enzyme production. To insert the by-products in the culture medium, they were dried and treated (if applicable) with 4% (w.v-1) NaOH and then added in a concentration of 2% (w.v-1). The cultures were agitated for 96 h, and the aliquots were removed to determine the enzymatic activities. Among the by-products studied, the maximum activity (8.7 U. mL-1 at pH 7.3) was obtained where rice bran was used. In contrast, corn cob was the by-product that resulted in lower enzyme production (1.6 U.mL-1). Thus, the defatted rice bran deserves special attention in front of the other by-products used since it provides the necessary substrate for producing endo-xylanases by yeast.

Exploring Bacillus species xylanases for industrial applications: screening via thermostability and reaction modelling.

CONTEXT: Xylanases derived from Bacillus species hold significant importance in various large-scale production sectors, with increasing demand driven by biofuel production. However, despite their potential, the extreme environmental conditions often encountered in production settings have led to their underutilisation. To address this issue and enhance their efficacy under adverse conditions, we conducted a theoretical investigation on a group of five Bacillus species xylanases belonging to the glycoside hydrolase GH11 family. Bacillus sp. NCL 87-6-10 (sp_NCL 87-6-10) emerged as a potent candidate among the selected biocatalysts; this Bacillus strain exhibited high thermal stability and achieved a transition state with minimal energy requirements, thereby accelerating the biocatalytic reaction process. Our approach aims to provide support for experimentalists in the industrial sector, encouraging them to employ structural-based reaction modelling scrutinisation to predict the ability of targeted xylanases. METHODS: Utilising crystal structure data available in the Carbohydrate-Active enzymes database, we aimed to analyse their structural capabilities in terms of thermal-stability and activity. Our investigation into identifying the most prominent Bacillus species xylanases unfolds with the help of the semi-empirical quantum mechanics MOPAC method integrated with the DRIVER program is used in calculations of reaction pathways to understand the activation energy. Additionally, we scrutinised the selected xylanases using various analyses, including constrained network analyses, intermolecular interactions of the enzyme-substrate complex and molecular orbital assessments calculated using the AM1 method with the MO-G model (MO-G AM1) to validate their reactivity.

Sugarcane bagasse derived xylooligosaccharides produced by an arabinofuranosidase/xylobiohydrolase from Bifidobacterium longum in synergism with xylanases.

Arabinoxylan is a major hemicellulose in the sugarcane plant cell wall with arabinose decorations that impose steric restrictions on the activity of xylanases against this substrate. Enzymatic removal of the decorations by arabinofuranosidases can allow a more efficient arabinoxylan degradation by xylanases. Here we produced and characterized a recombinant Bifidobacterium longum arabinofuranosidase from glycoside hydrolase family 43 (BlAbf43) and applied it, together with GH10 and GH11 xylanases, to produce xylooligosaccharides (XOS) from wheat arabinoxylan and alkali pretreated sugarcane bagasse. The enzyme synergistically enhanced XOS production by GH10 and GH11 xylanases, being particularly efficient in combination with the latter family of enzymes, with a degree of synergism of 1.7. We also demonstrated that the enzyme is capable of not only removing arabinose decorations from the arabinoxylan and from the non-reducing end of the oligomeric substrates, but also hydrolyzing the xylan backbone yielding mostly xylobiose and xylose in particular cases. Structural studies of BlAbf43 shed light on the molecular basis of the substrate recognition and allowed hypothesizing on the structural reasons of its multifunctionality.

Temperature-dependent structural changes in xylanase II from Trichoderma longibrachiatum.

Endo-ß-1,4-xylanases degrade heteroxylans that constitute the lignocellulosic plant cell wall. This enzyme is widely used in the food, paper, textile, and biorefinery industries. Temperature affects the optimum activity of xylanase and is an important factor in its application. Various structural analyses of xylanase have been performed, but its structural influence by temperature is not fully elucidated. To better understand the structural influence of xylanase due to temperature, the crystal structure of xylanase II from Trichoderma longibrachiatum (TloXynII) at room and cryogenic temperatures was determined at 2.1 and 1.9 Å resolution, respectively. The room-temperature structure of TloXynII (TloXynIIRT) showed a B-factor value 2.09 times higher than that of the cryogenic-temperature structure of TloXynII (TloXynIICryo). Subtle movement of the catalytic and substrate binding residues was observed between TloXynIIRT and TloXynIICryo. In TloXynIIRT, the thumb domain exhibited high flexibility, whereas in TloXynIICryo, the finger domain exhibited high flexibility. The substrate binding cleft of TloXynIIRT was narrower than that of TloXynIICryo, indicating a distinct finger domain conformation. Numerous water molecule networks were observed in the substrate binding cleft of TloXynIICryo, whereas only a few water molecules were observed in TloXynIIRT. These structural analyses expand our understanding of the temperature-dependent conformational changes in xylanase.

Deciphering heterogeneous enzymatic surface reactions on xylan using surface plasmon resonance spectroscopy.

Xylans' unique properties make it attractive for a variety of industries, including paper, food, and biochemical production. While for some applications the preservation of its natural structure is crucial, for others the degradation into monosaccharides is essential. For the complete breakdown, the use of several enzymes is required, due to its structural complexity. In fact, the specificity of enzymatically-catalyzed reactions is guided by the surface, limiting or regulating accessibility and serving structurally encoded input guiding the actions of the enzymes. Here, we investigate enzymes at surfaces rich in xylan using surface plasmon resonance spectroscopy. The influence of diffusion and changes in substrate morphology is studied via enzyme surface kinetics simulations, yielding reaction rates and constants. We propose kinetic models, which can be applied to the degradation of multilayer biopolymer films. The most advanced model was verified by its successful application to the degradation of a thin film of polyhydroxybutyrate treated with a polyhydroxybutyrate-depolymerase. The herein derived models can be employed to quantify the degradation kinetics of various enzymes on biopolymers in heterogeneous environments, often prevalent in industrial processes. The identification of key factors influencing reaction rates such as inhibition will contribute to the quantification of intricate dynamics in complex systems.

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library.

Xylanases are key biocatalysts in the degradation of the ß-1,4-glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-ß-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. KEY POINTS: • A GH10 endo-1,4-ß-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB's in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis.

Are phenolic compounds produced during the enzymatic production of prebiotic xylooligosaccharides (XOS) beneficial: a review.

Phenolics produced during xylooligosaccharide production might inhibit xylanases and enhance the antioxidant and antimicrobial activities of XOS. The effects of phenolic compounds on xylanases may depend on the type and concentration of the compound, the plant biomass used, and the enzyme used. Understanding the effects of phenolic compounds on xylanases and their impact on XOS is critical for developing viable bioconversion of lignocellulosic biomass to XOS. Understanding the complex relationship between phenolic compounds and xylanases can lead to the development of strategies that improve the efficiency and cost-effectiveness of XOS manufacturing processes and optimise enzyme performance.

Behavior of endo-xylanases on wheat milling products in relation with variable solid loading conditions.

To investigate the incubation conditions encountered by enzymes in cereal-based product transformation processes, this study aims to provide comprehensive information on the effect of low (18 %) to high (72 %) solid loading on the behavior of bacterial and fungal xylanases towards wheat grain fractions, i.e. white flour, ground whole grain and bran. Both enzymes are effective from 30 % water content. A water content of 50 % appears as the threshold for optimal arabinoxylan solubilisation. The specificity of enzymes was influenced by low hydration conditions, particularly in wheat bran, which contains arabinoxylan with diverse structures. Especially the bacterial xylanase became more tolerant to arabinose substitution as the water content decreased. Time Domain-NMR measurements revealed four water mobility domains in all the fractions. The water populations corresponding to 7.5 nm to 15 nm pores were found to be the most restrictive for enzyme activity. These results define the water content limits for the optimal xylanase action in cereal products.

Trends in the development and current perspective of thermostable bacterial hemicellulases with their industrial endeavors: A review.

Hemicellulases are enzymes that hydrolyze hemicelluloses, common polysaccharides in nature. Thermophilic hemicellulases, derived from microbial strains, are extensively studied as natural biofuel sources due to the complex structure of hemicelluloses. Recent research aims to elucidate the catalytic principles, mechanisms and specificity of hemicellulases through investigations into their high-temperature stability and structural features, which have applications in biotechnology and industry. This review article targets to serve as a comprehensive resource, highlighting the significant progress in the field and emphasizing the vital role of thermophilic hemicellulases in eco-friendly catalysis. The primary goal is to improve the reliability of hemicellulase enzymes obtained from thermophilic bacterial strains. Additionally, with their ability to break down lignocellulosic materials, hemicellulases hold immense potential for biofuel production. Despite their potential, the commercial viability is hindered by their high enzyme costs, necessitating the development of efficient bioprocesses involving waste pretreatment with microbial consortia to overcome this challenge.

The serine-threonine protein kinase Snf1 orchestrates the expression of plant cell wall-degrading enzymes and is required for full virulence of the maize pathogen Colletotrichum graminicola.

Colletotrichum graminicola, the causal agent of maize leaf anthracnose and stalk rot, differentiates a pressurized infection cell called an appressorium in order to invade the epidermal cell, and subsequently forms biotrophic and necrotrophic hyphae to colonize the host tissue. While the role of force in appressorial penetration is established (Bechinger et al., 1999), the involvement of cell wall-degrading enzymes (CWDEs) in this process and in tissue colonization is poorly understood, due to the enormous number and functional redundancy of these enzymes. The serine/threonine protein kinase gene SNF1 identified in Sucrose Non-Fermenting yeast mutants mediates de-repression of catabolite-repressed genes, including many genes encoding CWDEs. In this study, we identified and functionally characterized the SNF1 homolog of C. graminicola. Δsnf1 mutants showed reduced vegetative growth and asexual sporulation rates on media containing polymeric carbon sources. Microscopy revealed reduced efficacies in appressorial penetration of cuticle and epidermal cell wall, and formation of unusual medusa-like biotrophic hyphae by Δsnf1 mutants. Severe and moderate virulence reductions were observed on intact and wounded leaves, respectively. Employing RNA-sequencing we show for the first time that more than 2,500 genes are directly or indirectly controlled by Snf1 in necrotrophic hyphae of a plant pathogenic fungus, many of which encode xylan- and cellulose-degrading enzymes. The data presented show that Snf1 is a global regulator of gene expression and is required for full virulence.

Multi-point immobilization of GH 11 endo-ß-1,4-xylanase on magnetic MOF composites for higher yield of xylo-oligosaccharides.

GH 11 endo-ß-1,4-xylanase (Xy) was a crucial enzyme for xylooligosaccharides (XOS) production. The lower reusability and higher cost of purification has limited the industrial application of Xy. Addressing these challenges, our study utilized various immobilization techniques, different supports and forces for Xy immobilization. This study presents a new method in the development of Fe3O4@PDA@MOF-Xy which is immobilized via multi-point interaction forces, demonstrating a significant advancement in protein loading capacity (80.67 mg/g), and exhibiting remarkable tolerance to acidic and alkaline conditions. This method significantly improved Xy reusability and efficiency for industrial applications, maintaining 60 % activity over 10 cycles. Approximately 23 % XOS production was achieved by Fe3O4@PDA@MOF-Xy. Moreover, the yield of XOS from cobcorn xylan using this system was 1.15 times higher than that of the free enzyme system. These results provide a theoretical and applicative basis for enzyme immobilization and XOS industrial production.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

Discovery of novel alkaline-tolerant xylanases from fecal microbiota of dairy cows.

Xylo-oligosaccharides (XOS) are considered as a promising type of prebiotics that can be used in foods, feeds, and healthcare products. Xylanases play a key role in the production of XOS from xylan. In this study, we conducted a metagenomic analysis of the fecal microbiota from dairy cows fed with different types of fodders. Despite the diversity in their diets, the main phyla observed in all fecal microbiota were Firmicutes and Bacteroidetes. At the genus level, one group of dairy cows that were fed probiotic fermented herbal mixture-containing fodders displayed decreased abundance of Methanobrevibacter and increased growth of beneficial Akkermansia bacteria. Additionally, this group exhibited a high microbial richness and diversity. Through our analysis, we obtained a comprehensive dataset comprising over 280,000 carbohydrate-active enzyme genes. Among these, we identified a total of 163 potential xylanase genes and subsequently expressed 34 of them in Escherichia coli. Out of the 34 expressed genes, two alkaline xylanases with excellent temperature stability and pH tolerance were obtained. Notably, CDW-xyl-8 exhibited xylanase activity of 96.1 ± 7.5 U/mg protein, with an optimal working temperature of 55 â„ƒ and optimal pH of 8.0. CDW-xyl-16 displayed an activity of 427.3 ± 9.1 U/mg protein with an optimal pH of 8.5 and an optimal temperature at 40 â„ƒ. Bioinformatic analyses and structural modeling suggest that CDW-xyl-8 belongs to GH10 family xylanase, and CDW-xyl-16 is a GH11 family xylanase. Both enzymes have the ability to hydrolyze beechwood xylan and produce XOS. In conclusion, this metagenomic study provides valuable insights into the fecal microbiota composition of dairy cows fed different fodder types, revealing main microbial groups and demonstrating the abundance of xylanases. Furthermore, the characterization of two novel xylanases highlights their potential application in XOS production.

Arabinoxylan source and xylanase specificity influence the production of oligosaccharides with prebiotic potential.

Cereal arabinoxylans (AXs) are complex polysaccharides in terms of their pattern of arabinose and ferulic acid substitutions, which influence their properties in structural and nutritional applications. We have evaluated the influence of the molecular structure of three AXs from wheat and rye with distinct substitutions on the activity of ß-xylanases from different glycosyl hydrolase families (GH 5_34, 8, 10 and 11). The arabinose and ferulic acid substitutions influence the accessibility of the xylanases, resulting in specific profiles of arabinoxylan-oligosaccharides (AXOS). The GH10 xylanase from Aspergillus aculeatus (AcXyn10A) and GH11 from Thermomyces lanuginosus (TlXyn11) showed the highest activity, producing larger amounts of small oligosaccharides in shorter time. The GH8 xylanase from Bacillus sp. (BXyn8) produced linear xylooligosaccharides and was most restricted by arabinose substitution, whereas GH5_34 from Gonapodya prolifera (GpXyn5_34) required arabinose substitution and produced longer (A)XOS substituted on the reducing end. The complementary substrate specificity of BXyn8 and GpXyn5_34 revealed how arabinoses were distributed along the xylan backbones. This study demonstrates that AX source and xylanase specificity influence the production of oligosaccharides with specific structures, which in turn impacts the growth of specific bacteria (Bacteroides ovatus and Bifidobacterium adolescentis) and the production of beneficial metabolites (short-chain fatty acids).

Propeptide-Mediated Allosteric Regulation of Xylanase Xyl-1: An Integrated Experimental and Computational Analysis.

Most GH11 family endo-ß-1,4-xylanases contain a propeptide region linked to the N-terminal region. The mechanistic basis of this region harboring key regulation information for enzyme function, however, remains poorly understood. We reported an investigation on the allosteric regulation mechanism of the propeptide based on biochemical characterization, molecular dynamics simulations, and evolutionary analysis. We discovered that the mutant of truncated propeptide shows a remarkably increased thermal stability (melting temperature increased by 11.5 °C) and catalytic efficiency (1.7-fold kcat/Km value of wild type). Molecular dynamics simulations reveal that long-range fluctuations in the propeptide lead to a conformational perturbation in the catalytic pocket and the thumb region. The probability of sampling the active conformation during the glycosylation step is reduced (i.e., catalytic efficiency). In-depth sequence analysis indicates that the propeptide has a strong plasticity and degeneration trend, and propeptide truncation experiments of the homologous enzyme XynB validated the feasibility of the truncation strategy. This work reveals the role of GH11 family propeptides in functional regulation and provides a straightforward and practical method to increase the robustness of GH11 family xylanases.

An insight into microbial sources, classification, and industrial applications of xylanases: A rapid review.

Endo 1,4-ß-d-xylanases (EC3.2.1.8) are one of the key lignocellulose hydrolyzing enzymes. Xylan, which is present in copious amounts on earth, forms the primary substrate of endo-xylanases, which can unchain the constituent monosaccharides linked via ß-1,4-glycosidic bonds from the xylan backbone. Researchers have shown keen interest in the xylanases belonging to glycoside hydrolase families 10 and 11, whereas those placed in other glycoside hydrolase families are yet to be investigated. Various microbes such as bacteria and fungi harbor these enzymes for the metabolism of their lignocellulose fibers. These microbes can be used as miniature biofactories of xylanase enzymes for a plethora of environmentally benign applications in pulp and paper industry, biofuel production, and for improving the quality of food in bread baking and fruit juice industry. This review highlights the potential of microbes in production of xylanase for industrial biotechnology.

Endo-1,4-ß-xylanase-containing glycoside hydrolase families: characteristics, singularities and similarities.

Endo-1,4-ß-xylanases (EC 3.2.1.8) are O-glycoside hydrolases that cleave the internal ß-1,4-D-xylosidic linkages of the complex plant polysaccharide xylan. They are produced by a vast array of organisms where they play critical roles in xylan saccharification and plant cell wall hydrolysis. They are also important industrial biocatalysts with widespread application. A large and ever growing number of xylanases with wildly different properties and functionalites are known and a better understanding of these would enable a more effective use in various applications. The Carbohydrate-Active enZYmes database (CAZy), which classifies evolutionarily related proteins into a glycoside hydrolase family-subfamily organisational scheme has proven powerful in understanding these enzymes. Nevertheless, ambiguity currently exists as to the number of glycoside hydrolase families and subfamilies harbouring catalytic domains with true endoxylanase activity and as to the specific characteristics of each of these families/subfamilies. This review seeks to clarify this, identifying 9 glycoside hydrolase families containing enzymes with endo-1,4-ß-xylanase activity and discussing their properties, similarities, differences and biotechnological perspectives. In particular, substrate specificities and hydrolysis patterns and the structural determinants of these are detailed, with taxonomic aspects of source organisms being also presented. Shortcomings in current knowledge and research areas that require further clarification are highlighted and suggestions for future directions provided. This review seeks to motivate further research on these enzymes and especially of the lesser known endo-1,4-ß-xylanase containing families. A better understanding of these enzymes will serve as a foundation for the knowledge-based development of process-fitted endo-1,4-ß-xylanases and will accelerate their development for use with even the most recalcitrant of substrates in the biobased industries of the future.

BsEXLX of engineered Trichoderma reesei strain as dual-active expansin to boost cellulases secretion for synergistic enhancement of biomass enzymatic saccharification in corn and Miscanthus straws.

In this study, bacterial BsEXLE1 gene was overexpressed into T. reesei (Rut-C30) to generate a desirable engineered TrEXLX10 strain. While incubated with alkali-pretreated Miscanthus straw as carbon source, the TrEXLX10 secreted the ß-glucosidases, cellobiohydrolases and xylanses with activities raised by 34%, 82% and 159% compared to the Rut-C30. Supplying EXLX10-secreted crude enzymes and commercial mixed-cellulases for two-step lignocellulose hydrolyses of corn and Miscanthus straws after mild alkali pretreatments, this work measured consistently higher hexoses yields released by the EXLX10-secreted enzymes for synergistic enhancements of biomass saccharification in all parallel experiments examined. Meanwhile, this study detected that the expansin, purified from EXLX10-secreted solution, was of exceptionally high binding activities with wall polymers, and further determined its independent enhancement for cellulose hydrolysis. Therefore, this study raised a mechanism model to highlight EXLX/expansin dual-activation roles for both secretion of stable biomass-degradation enzymes at high activity and biomass enzymatic saccharification in bioenergy crops.