The paper industry faces two critical challenges: the scarcity of raw materials and the environmental impact of chemical waste pollution. Addressing the first challenge involves harnessing alternative, sustainable raw materials, while the second challenge can be mitigated through the adoption of bio-bleaching processes, which significantly reduce chemical consumption while enhancing paper brightness and quality. This study proposes a solution to both challenges by using non-woody Calotropis procera (Ankara) and a xylanase-producing microbial consortium for sustainable handmade paper production, a combination not extensively explored in prior research. To evaluate this approach, the process was divided into three stages. In stage I, Ankara fibre was pulped through open hot digestion. In stage II, the pulp was subjected to bio-bleaching in two experimental setups: Set I (without sucrose) and Set II (with sucrose) for 5 days. In stage III, chemical bleaching was used to improve the final brightness of the treated pulps. A novel comparison was made between the bio-bleaching efficiency of an individual isolate g5 (BI) and a bacterial consortium (BC). This research highlighted that bio-bleaching with the consortium effectively removed lignin (140±60 mg/l) and colour (1830±50 PCU), especially in the presence of sucrose, compared to using a single xylanase isolate. Pulp residue/filtrate collected at each stage was estimated based on parameters such as colour and lignin content. After stage III (chemical bleaching), the release of colour and lignin in pulp filtrate was higher in BI compared to BC, indicating the consortium\'s effectiveness during bio-bleaching, which leaves fewer degradable lignin structures for the chemical bleaching stage. Papers crafted from consortium-treated pulp also exhibited higher brightness than those treated with the isolate. This study reveals the synergistic effect of microbial consortia, leading to more efficient lignin degradation and enhanced bio-bleaching capabilities, supporting the development of greener industrial processes. Ultimately, this study demonstrates a unique and eco-friendly approach to papermaking, combining C. procera and enzymatic bio-bleaching to reduce dependency on hazardous chemicals and support sustainable industry practices.

Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (LeGH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7-5 and GH7-6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with β-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the LeGH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops.

Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.

The drive to enhance enzyme performance in industrial applications frequently clashes with the practical limitations of exhaustive experimental screening, underscoring the urgency for more refined and strategic methodologies in enzyme engineering. In this study, xylanase Xyl-1 was used as the model, coupling evolutionary insights with energy functions to obtain theoretical potential mutants, which were subsequently validated experimentally. We observed that mutations in the nonloop region primarily aimed at enhancing stability and also encountered selective pressure for activity. Notably, mutations in this region simultaneously boosted the Xyl-1 stability and activity, achieving a 65% success rate. Using a greedy strategy, mutant M4 was developed, achieving a 12 °C higher melting temperature and doubled activity. By integration of spectroscopy, crystallography, and quantum mechanics/molecular mechanics molecular dynamics, the mechanism behind the enhanced thermal stability of M4 was elucidated. It was determined that the activity differences between M4 and the wild type were primarily driven by dynamic factors influenced by distal mutations. In conclusion, the study emphasizes the pivotal role of evolution-based approaches in augmenting the stability and activity of the enzymes. It sheds light on the unique adaptive mechanisms employed by various structural regions of proteins and expands our understanding of the intricate relationship between distant mutations and enzyme dynamics.

Over the last decade, xylooligosaccharides (XOS) have attracted great attentions because of their unique chemical properties and excellent prebiotic effects. Among the current strategies for XOS production, enzymatic hydrolysis is preferred due to its green and safe process, simplicity in equipment, and high control of the degrees of polymerization. This paper comprehensively summarizes various lignocellulosic biomass and marine biomass employed in enzymatic production of XOS. The importance and advantages of enzyme immobilization in XOS production are also discussed. Many novel immobilization techniques for xylanase are presented. In addition, bioinformatics techniques for the mining and designing of new xylanase are also described. Moreover, XOS has exhibited great potential applications in the food industry as diverse roles, such as a sugar replacer, a fat replacer, and cryoprotectant. This review systematically summarizes the current research progress on the applications of XOS in food sectors, including beverages, bakery products, dairy products, meat products, aquatic products, food packaging film, wall materials, and others. It is anticipated that this paper will act as a reference for the further development and application of XOS in food sectors and other fields.

Agarwood oil is one of the costliest essential oils used in perfumery, medicine and aroma. Production of the oil traditionally involves a soaking/fermentation step. Studies have indicated a definite role of the diverse microorganisms growing during the open soaking step, and in the emergent aroma of the essential oil. However, the temporal nature of fermentation and a key functional aspect i.e., the enzymatic properties of the microbes from the fermentation basin have not been studied yet. A total of 20 bacteria and 14 fungi isolated from fermentation basins located in Assam, India, at different soaking periods classified as early (0-20 days), medium (20-40 days) and late (40-60 days) clearly pointed towards an early fungal domination followed by succession of bacteria. The physico-chemical transformations of the wood are controlled by enzymatic properties (cellulase, xylanase, amylase and lipase) of the isolates. The results indicated a strong lignocellulosic substrate modulation potential in the four isolates, viz- Purpureocillium lilacinum (0.354 mg/mL), Mucor circinelloides (0.331 mg/mL), Penicillium citrinum (0.324 mg/mL) and Bacillus megaterium (0.152 mg/mL). The highest culturable abundance (CFU/mL) was found in M. circinelloides (2 × 109) among fungi and B. megaterium (4.5 × 109) among bacteria. The highest cellulase activity was shown by P. lilacinum (0.354 mg/mL) while xylanase and lipase by M. circinelloides (0.873 and 0.128 mg/mL). An interesting revelation was that a substantial proportion of the isolates (70% bacteria and 78% fungi) were positive for lipase activity. This is the first report on the \"culturable microbiome\" of the agarwood fermentation basin from a temporal and functional bioactivity perspective.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s12088-024-01257-y.

Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into plant-pathogen interactions and facilitating plant cell wall biotechnological utilisation.

This research investigated and compared the prebiotic properties of a rice bran extract obtained through commercial xylanase extraction in comparison with water extraction. Prebiotic properties were evaluated by probiotic growth stimulation (Lacticaseibacillus casei and Lactiplantibacillus plantarum) and gastrointestinal pathogen inhibition (Bacillus cereus and Escherichia coli). The rice bran extract obtained with xylanase (RB1) displayed significantly higher total polysaccharide and total reducing sugar contents than those obtained with water (RB2; p<0.05). After extraction for 30 min, RB1 exhibited the highest total polysaccharide and total reducing sugar contents. HPLC (high performance liquid chromatography) analysis revealed that RB1 primarily contained xylose, while RB2 contained less glucose and lacked other sugar derivatives. RB1 proved effective in stimulating the growth of L. casei and L. plantarum, surpassing even inulin (a commercial prebiotic). Furthermore, it demonstrated a high potential for inhibiting the growth of pathogenic B. cereus and E. coli, comparable to inulin. In contrast, RB2 exhibited lower inhibitory capacity against B. cereus and E. coli.

Its high dietary fiber and protein contents and nutritional quality make defatted wheat germ (DWG) a valuable cereal by-product, yet its negative impact on food structure limits its use as a food ingredient. In this research, DWG underwent air classification, which identified two fractions with high fiber (HF) and low fiber/high protein (LF) contents, and a bioprocessing protocol, involving treatment with xylanase and fermentation with selected lactic acid bacterial strains. The degree of proteolysis was evaluated through electrophoretic and chromatographic techniques, revealing differences among fractions and bioprocessing options. Fermentation led to a significant increase in free amino acids (up to 6 g/kg), further enhanced by the combination with xylanase. When HF was used as an ingredient in bread making, the fiber content of the resulting bread exceeded 3.6 g/100 g, thus reaching the threshold required to make a \"source of fiber\" claim according to Regulation EC No.1924/2006. Meanwhile, all breads could be labeled a \"source of protein\" since up to 13% of the energy was provided by proteins. Overall, bioprocessed ingredients lowered the glycemic index (84 vs. 89) and increased protein digestibility (80 vs. 63%) compared to control breads. Technological and sensory analysis showed that the enzymatic treatment combined with fermentation also conferred a darker and more pleasant color to the bread crust, as well as better crumb porosity and elasticity.

Xylanases derived from fungi, including phytopathogenic and nonpathogenic fungi, are commonly known to trigger plant immune responses. However, there is limited research on the ability of bacterial-derived xylanases to trigger plant immunity. Here, a novel xylanase named CcXyn was identified from the myxobacterium Cystobacter sp. 0969, which displays broad-spectrum activity against both phytopathogenic fungi and bacteria. CcXyn belongs to the glycoside hydrolases (GH) 11 family and shares a sequence identity of approximately 32.0%-45.0% with fungal xylanases known to trigger plant immune responses. Treatment of Nicotiana benthamiana with purified CcXyn resulted in the induction of hypersensitive response (HR) and defence responses, such as the production of reactive oxygen species (ROS) and upregulation of defence gene expression, ultimately enhancing the resistance of N. benthamiana to Phytophthora nicotianae. These findings indicated that CcXyn functions as a microbe-associated molecular pattern (MAMP) elicitor for plant immune responses, independent of its enzymatic activity. Similar to fungal xylanases, CcXyn was recognized by the NbRXEGL1 receptor on the cell membrane of N. benthamiana. Downstream signalling was shown to be independent of the BAK1 and SOBIR1 co-receptors, indicating the involvement of other co-receptors in signal transduction following CcXyn recognition in N. benthamiana. Moreover, xylanases from other myxobacteria also demonstrated the capacity to trigger plant immune responses in N. benthamiana, indicating that xylanases in myxobacteria are ubiquitous in triggering plant immune functions. This study expands the understanding of xylanases with plant immune response-inducing properties and provides a theoretical basis for potential applications of myxobacteria in biocontrol strategies against phytopathogens.