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Rice husk is an abundant agricultural waste generated from rice production, but its application is limited. Considering its complex components, the rice husk was hydrolyzed by different enzymes to enhance its saccharification. In this study, saccharification of the rice husk by cellulase, xylosidase, and xylanase was first investigated. The synergistic effect of LPMO on the above hydrolases and different enzyme combinations in the saccharification process was then explored. Thereafter, the formulation of the enzyme cocktail and the degradation conditions were optimized to obtain the highest saccharification efficiency. The results showed that the optimum enzyme cocktail consists of Celluclast 1.5 L (83.3 mg/g substrate), the key enzymes in the saccharification process, worked with Bp Xyl (20 mg/g substrate), Bp Xyn11 (24 mg/g substrate), and R17L/N25G (4 mg/g substrate). The highest reducing sugar concentration (1.19 mg/mL) was obtained at pH 6.0 and 60 ℃ for 24 h. Fourier transform infrared spectroscopy and scanning electron microscopy were employed to characterize the structural changes in the rice husk after degradation. The results showed that the key chemical bonds in cellulose and hemicellulose were broken. This study illuminated the concept of saccharifying lignocellulose from rice husk using LPMO synergistically assisted combined-hydrolase including cellulase, xylosidase, and xylanase, and provided a theoretical basis for lignocellulose biodegradation. • Synergistic effect of xylanase and xylosidase with cellulase is identified to saccharify rice husk. • LPMO can synergize with the above three hydrolases to saccharify rice husk. • A combination of all four enzymes provided 1.19 mg/mL of reducing sugar production. • The conditions of the degradation process are optimized. [ABSTRACT FROM AUTHOR]

The generation of a complex microbial consortium is a promising approach for efficient biomass decomposition. An anaerobic thermophilic alkaliphilic microbial consortium with efficient degradation ability was screened from bovine manure compost using non-pretreated milling corn stover (CS) and rice straw (RS). A stable microbial consortium ISHI-3 with high degradation ability for CS and RS was isolated by the roll tube technique. ISHI-3 comprised Herbivorax saccincola and bacteria belonging to the classes Pelotomaculum, Tepidanaerobacter , and Tepidimicrobium , as determined by DGGE of the PCR-generated 16S rRNA genes. Furthermore, metagenomics analysis using a 16S rRNA library was carried out to determine the bacterial distribution during degradation of CS and RS. H. saccincola and bacteria belonging to Pelotomaculum were relatively abundant in the beginning to middle periods of culture with CS and RS whereas bacteria belonging to Tepidanaerobacter and Tepidimicrobium gradually increased in the population during the later stages. To understand the role of non-cellulolytic bacteria in the consortium, novel strains ET1 and GL4, which were most closely related to Tepidimicrobium ferriphilum and Tepidanaerobacter acetatoxydans , were isolated from ISHI-3. Based on their carbon source usage, morphology, and phylogenetic analysis, we propose that strains ET1 and GL4 should be classified as a novel genus or species. Bacteria ET1 and GL4 can utilize different organic compounds as carbon and energy sources such as organic acids, alcohols, sugars, and amino acids, showing a preference for organic acids and alcohols rather than sugars such as glucose and cellobiose. These results indicated that ET1 and GL4 help to accelerate efficient lignocellulose degradation of H. saccincola . [ABSTRACT FROM AUTHOR]

Oil palm trunk (OPT) is one of the most promising lignocellulosic bioresources. To develop effective biodegradation, thermophilic, anaerobic microorganisms were screened from bovine manure compost using fibrillated OPT (f-OPT) pretreated by wet disk milling as the substrate. One thermophilic, anaerobic bacterium, strain CL-2, whose 16S rDNA gene has 98.6% sequence identity with that of Caldicoprobacter faecale DSM 20678 T , exhibited high degradation activity (32.7% reduction in total dry solids of f-OPT). Strain CL-2 did not use cellulose as a carbon source, but used hemicelluloses such as xylan, arabinoxylan, starch and pectin at 70 °C. Phylogenetic and morphologic analyses and the polysaccharide use suggest that CL-2 may be classified as a novel species of Caldicoprobacter , named Caldicoprobacter sp. CL-2. To characterize enzymatic activities of CL-2, extracellular enzymes were prepared from culture broth using beechwood xylan as the carbon source. The extracellular enzymes showed high xylanase activity, but low cellulase activity, suggesting that f-OPT degradation may depend on xylanase activity. To understand the xylanase system of CL-2, a major xylanase was cloned and characterized. The xylanase ( Cal Xyn11A) had a modular structure consisting of a glycoside hydrolase (GH) family-11 domain and a family 36 carbohydrate-binding module. Cal Xyn11A did not show f-OPT degradation activity, but a strong synergistic effect was observed when Cal Xyn11A was added to the extracellular enzyme preparation. These results indicate that, rather than working alone, Cal Xyn11A has an important role in enhancing total lignocellulose degradation activity by cooperation with other GHs. [ABSTRACT FROM AUTHOR]

Cellulosic ethanol production relies on the biochemical (enzymatic) conversion of lignocellulose to fermentable sugars and ultimately to bioethanol. However, the cost of lignocellulolytic enzymes is a limiting factor in the commercialisation of this technology. This therefore necessitates the optimisation of lignocellulolytic enzyme cocktails through the elucidation of synergistic interactions between enzymes so as to improve lignocellulose hydrolysis and also lower protein loadings in these reactions. However, many factors affect the synergism that occurs between these lignocellulolytic enzymes, such as enzyme ratios, substrate characteristics, substrate loadings, enzyme loadings and time. This review examines the effect of time on the synergistic dynamics between lignocellulolytic enzymes during the hydrolysis of both complex (true) lignocellulosic substrates and model substrates. The effect of sequential and simultaneous application of the lignocellulolytic enzymes on the synergistic dynamics during the hydrolysis of these substrates is also explored in this review. Finally, approaches are further proposed for efficient and synergistic hydrolysis of both complex lignocellulosic substrates and model substrates. With respect to the synergistic enzymatic hydrolysis of lignocellulosic biomass, this review exposed knowledge gaps that should be covered in future work in order to fully understand how enzyme synergism works: e.g. elucidating protein to protein interactions that exist between these enzymes in establishing synergy; and the effect of lignocellulose degradation products of one enzyme on the behaviour of the other enzyme and ultimately their synergistic relationship. [ABSTRACT FROM AUTHOR]

A newly isolated endo -β-1,4-xylanase (Xyn10E) from Paenibacillus curdlanolyticus B-6 has a modular structure consisting of a family 22 carbohydrate-binding module (CBM), a glycoside hydrolase (GH) family 10 catalytic domain, two fibronectin type III (Fn3) domains, and a family 3 CBM at the C-terminus. Intact Xyn10E (rXyn10E), CBM22-deleted Xyn10E (X-CBM3), CBM3-deleted Xyn10E (X-CBM22), and GH10 catalytic domain only (X-GH10) were expressed in Escherichia coli . rXyn10E showed bifunctional degradation activity toward xylan and β-glucan and also degraded microcrystalline cellulose. Although X-CBM3 and X-GH10 had drastically reduced xylanase and β-glucanase activities, X-CBM22 mostly retained these activities. Similar K m values were obtained for rXyn10E and X-CBM3, but k cat and k cat / K m values for X-CBM3 and X-GH10 were lower than those for rXyn10E, suggesting that CBM22 of Xyn10E may contribute to catalytic efficiency. In binding assays, X-CBM3 was still able to bind to β-glucan, soluble xylan, insoluble xylan, and cellulose through GH10 and CBM3. These results indicate that CBM22 has an important role not only in binding to xylan and β-glucan but also in feeding both polysaccharides into the neighboring GH10 catalytic domain. rXyn10E showed remarkable synergism with rXyn11A, a major xylanase subunit of P. curdlanolyticus B-6, in the degradation of untreated corn stover and sugarcane bagasse; however, the combination of X-CBM3 and rXyn11A was not synergistic. These results indicate that Xyn10E and Xyn11A act synergistically on lignocellulosic biomass, and CBM22 is essential for efficient degradation of lignocellulosic materials. [ABSTRACT FROM AUTHOR]

The bacterial expansin, BsEXLX1, has been studied as a model to understand the synergistic effect of expansins on lignocellulose degradation and the structure-function relationships of expansins. However, the specific mechanism is still poorly understood. In this study, the binding, synergistic and structural characteristics of BsEXLX1 for loosening the main components of lignocellulose: lignin, xylan, and cellulose, were further characterized. Results showed that BsEXLX1 preferentially binds to xylan over lignin or cellulose under various conditions. The binding of BsEXLX1 to all substrates increased linearly with the initial concentration of BsEXLX1. But the changing rate of binding (i.e., slope of the line; k value) varied with the incubation temperature. Interestingly, the binding of BsEXLX1 to substrates did not saturate. Mutating residue-125, 126 or 171 indicated their importance for binding, but they were less important for binding to xylan. Through binding assays and homologous modeling, it was concluded that residue-125 and 171 play more important roles in the structural maintenance of BsEXLX1. By comparing the synergistic activity of BsEXLX1 or its mutants, it was found that synergistic activity is not correlated with specific binding. All these results can lead deeper understand about the mechanism of wall loosening by expansins, and further promote the application of expansins in lignocellulose degradation. [ABSTRACT FROM AUTHOR]

Highlights: [•] 44 white-rot lignocellulytic fungal strains are screened. [•] Ganoderma lucidum can grow on alkali lignin and expresses high laccase activity. [•] The G. lucidum laccase extract enhances cellulase-catalyzed lignocellulose degradation. [ABSTRACT FROM AUTHOR]