Biochemical characterization and low-resolution SAXS shape of a novel GH11 exo-1,4-β-xylanase identified in a microbial consortium

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic β-1,4- and β-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 β-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small angle Xray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 oC. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 oC. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis)

Targeted metatranscriptomics of compost derived consortia reveals a GH11 exerting an unusual exo-1,4-β-xylanase activity

Background: Using globally abundant crop residues as a carbon source for energy generation and renewable chemicals production stands out as a promising solution to reduce current dependency on fossil fuels. In nature, such as in compost habitats, microbial communities efficiently degrade the available plant biomass using a diverse set of synergistic enzymes. However, deconstruction of lignocellulose remains a challenge for industry due to recalcitrant nature of the substrate and the inefficiency of the enzyme systems available, making the economic production of lignocellulosic biofuels difficult. Metatranscriptomic studies of microbial communities can unveil the metabolic functions employed by lignocellulolytic consortia and identify new biocatalysts that could improve industrial lignocellulose conversion. Results: In this study, a microbial community from compost was grown in minimal medium with sugarcane bagasse sugarcane bagasse as the sole carbon source. Solid-state nuclear magnetic resonance was used to monitor lignocellulose degradation; analysis of metatranscriptomic data led to the selection and functional characterization of several target genes, revealing the first glycoside hydrolase from Carbohydrate Active Enzyme family 11 with exo-1,4-β-xylanase activity. The xylanase crystal structure was resolved at 1.76 Å revealing the structural basis of exo-xylanase activity. Supplementation of a commercial cellulolytic enzyme cocktail with the xylanase showed improvement in Avicel hydrolysis in the presence of inhibitory xylooligomers. Conclusions: This study demonstrated that composting microbiomes continue to be an excellent source of biotechnologically important enzymes by unveiling the diversity of enzymes involved in in situ lignocellulose degradation

Sugarcane bagasse pretreatment optimization strategies for the production of second generation ethanol via enzymatic hydrolysis

Atualmente, o aumento da preocupação com a sustentabilidade ambiental, alinhado às perspectivas de esgotamento das reservas de petróleo, tem direcionado às buscas por fontes renováveis de energia. O emprego de resíduos agroindustriais, principalmente de usinas sucroalcooleiras destaca-se como sendo uma alternativa para a produção de etanol de segunda geração. Dentre as metodologias aplicadas para disponibilização dos açúcares fermentescíveis está a hidrólise enzimática. Ainda, para facilitar esta etapa e torná-la mais acessível, submete-se, previamente, o material lignocelulósico a um pré-tratamento, com o objetivo de contribuir com a susceptibilidade da celulose a ataques enzimáticos. No entanto, devido à complexidade das estruturas lignocelulósicas, os processos de hidrólise e pré-tratamento precisam se tornar mais eficientes e economicamente viáveis. Desta forma, o objetivo desse trabalho foi avaliar e caracterizar os pré-tratamentos hidrotérmico e organossolve (etanol 50%), isoladamente, e estes combinados em diferentes condições, assim como a influência destes procedimentos na estrutura e composição da biomassa, bem como na hidrólise enzimática. Os resultados demonstraram que os pré-tratamentos hidrotérmicos a 160 ºC nas condições analisadas foram pouco efetivos na melhora do acesso enzimático durante a etapa de hidrólise, pois atuaram de maneira branda na parede celular, pouco solubilizando a hemicelulose e lignina, conforme as análises físicas comprovaram. Os tratamentos combinados hidrotérmico 30 min e 60 min a 160 ºC seguidos pelo organossolve por 150 min apresentaram semelhança morfológica e alta solublização da lignina e hemicelulose, justificando os valores de hidrólise. Nossos resultados abrem perspectivas de novos estudos que visam a otimização dos pré-tratamentos hidrotérmicos e organossolve, além da compreensão das alterações composicionais e morfológicas que levam à melhoria da hidrólise enzimática na biomassa lignocelulósica.The concerns with environmental sustainability and perspectives of petroleum reserves depletion motivated exploration of new and sustainable energy sources. In this context, renewable energies start to receive significant attention in the world´s energy matrix, with biofuels playing a special role. The use of agro-industrial residues, mainly from the sugarcane industry, stands out as a viable alternative for the production of second-generation ethanol. The enzymatic hydrolysis of the biomass has a number of advantages for polysaccharides depolimerization, such as high substrate specificity, low environmental impact and lack of corrosion issues. To further facilitate this procedure and to make biomass more accessible, the lignocellulosic material has to be previously submitted to a pretreatment in order to increase the cellulose accessibility and susceptibility to the enzymatic action. This process aims at the disorganization of the chemical structure of the lignocellulosic matter, facilitating the further steps of hydrolysis and fermentation. Due to the complexity of the lignocelluloses structures, their pretreatment and hydrolysis processes have to become more efficient and economically viable to be efficiently applied at an industrial scale. Therefore, the objective of this work is to evaluate the hydrothermal and organosolv (50% ethanol) pretreatments, separately and combined in different conditions, and the influence of these procedures on the structure and composition of the biomass and on the efficiency of enzymatic hydrolyses. Our results demonstrated that the hydrothermal pretreatments at 160ºC within the analyzed reaction conditions had minor effects on improving the enzymatic efficiency, being not harsh enough to introduce significant modifications of the cell wall composition and structure, as demonstrated by our physical and chemical analyses. The combined hydrothermal treatments lasting 30 min and 60 min at 160ºC followed by the organosolv step for 150 min resulted in significant morphological changes and high lignin and hemicelluloses solubilization, resulting in an efficient enzymatic hydrolysis. Our results open perspectives of further studies aimed at optimization of hydrothermal and organosolv pretreatments and comprehension of compositional and morphological changes which lead to improved enzymatic hydrolysis of the lignocelulosic biomass

Physical-chemical characterization of sugarcane bagasse after several pretreatments aiming to improve enzymatic hydrolysis yield

No Brasil o emprego de resíduos sucroalcooleiros, como o bagaço da cana-de-açúcar, destaca-se como uma alternativa para ampliar a produção de etanol e reduzir as dependências com combustíveis fósseis. Os principais constituintes da parede celular desta biomassa são carboidratos que podem ser clivados em açúcares fermentescíveis via hidrólise enzimática. Para facilitar esta etapa e tornar o substrato acessível às enzimas, o material lignocelulósico deve ser submetido a um pré-tratamento, o qual irá influenciar as etapas subsequentes. No entanto é preciso investigar o efeito do pré-tratamento para melhor compreensão de como o mesmo irá afetar as etapas subsequentes. A utilização de diferentes técnicas físico-químicas auxilia no estudo de como a composição e estrutura da fibra está relacionada com a sacarificação enzimática. Assim, com o objetivo de contribuir neste estudo, foram empregados diferentes pré-tratamentos em diversas condições, sendo eles ácidos, hidrotérmico e explosão a vapor com e sem catalisadores externos, a fim de se compreender a sua atuação e influência no contexto de conversão glicosídica. Foi realizado a caracterização química por cromatografia líquida de alta eficiência e por ressonância magnética nuclear, além da hidrólise com diferentes cargas enzimáticas, utilizando Accelerase® suplementado com β-glicosidase, Cellic CTec e xilanase. Também foi realizado análise morfológica da biomassa, através de microscopia eletrônica de varredura e microscopia confocal. As análises físico-químicas revelaram a correlação entre o tempo de reação do pré-tratamento como proporcional ao efeito de desprendimento das fibras que constituem o bagaço, bem como a formação de aglomerados de lignina na superfície do mesmo. Também foi possível confirmar que há forte correlação entre a estrutura da lignina e a conversão enzimática, sendo que a mesma também é afetada pela presença de alto teor de derivados da hemicelulose, inibindo a ação de celulases. Por fim, por meio deste trabalho foi verificado que o fator de severidade não é suficientemente robusto para relacioná-lo com a eficiência do pré-tratamento aplicado.In Brazil, the use of sugar and alcohol residues, such as sugarcane bagasse, stands out as an alternative to expand ethanol production and reduce dependencies on fossil fuels. The main constituents of the cell wall of this biomass are carbohydrates that can be cleaved into fermentable sugars via enzymatic hydrolysis. To facilitate this step and make the substrate accessible to enzymes, the lignocellulosic material must be subjected to a pretreatment, which will influence the subsequent steps. However, it is necessary to investigate the effect of the pre- treatment to better understand how it will affect the subsequent steps. The use of different physical-chemical techniques contributes to the study of how the composition and structure of the fiber is related to enzymatic saccharification. Thus, in order to contribute to this study, different pre-treatments were used in different conditions, acids, hydrothermal and steam explosion with and without external catalysts, in order to understand their performance and influence in the context of glycosidic conversion. Chemical characterization was performed by high performance liquid chromatography and nuclear magnetic resonance, in addition to hydrolysis with different enzyme charges, using Accelerase® supplemented with β-glycosidase, Cellic CTec and xylanase. Morphological analysis of biomass was also performed, using scanning electron microscopy and confocal microscopy. The physical-chemical analyzes revealed the correlation between the reaction time of the pretreatment as proportional to the effect of detachment of the fibers that make up the bagasse, as well as the formation of lignin clusters on its surface. It was also possible to confirm that there is a strong correlation between the structure of lignin and enzymatic conversion, and it is also affected by the presence of high content of hemicellulose derivatives, inhibiting the action of cellulases. Finally, through this work it was verified that the severity factor is not robust enough to relate it to the efficiency of the applied pretreatment

Evaluating the Potential of Culms from Sugarcane and Energy Cane Varieties Grown in Argentina for Second-Generation Ethanol Production

The efficient transformation of lignocellulosic biomass into fermentable sugars is essential for building bioeconomies. Sugarcane is an important agricultural crop in a number of Latin American countries, including Brazil and Argentina. Herein culms from two different sugarcane (SC384 and SC724) and two energy cane varieties (EC3116 and EC3118) bred in Argentina were evaluated for sustainable production of second-generation biofuels and green chemicals. Changes in the biomass crystallinity, structure, and morphology introduced by pretreatments were investigated using X-ray diffraction (DRX), confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) techniques. Enzymatic hydrolysis yields of untreated and pretreated sugarcane and energy cane culms were determined and correlated with physical analyses and chemical composition characterizations. Overall, after combined acid and alkali pretreatment, enzymatic convertibility was highly efficient for all studied sugarcane and energy cane varieties, reaching over 97% of theoretical conversion yields. High crystallinity indices and crystallite sizes of pretreated culms and SEM results and CLSM were consistent with the removal of lignin, solubilization of hemicellulose, and amorphous parts of lignocellulose imprinted by the pretreatments. High potential of culms from sugarcane and energy cane varieties cultivated in Argentina for sustainable production of renewable lignocellulosic sugars and their transformation into green chemicals and fuels was demonstrated. Graphic Abstract: [Figure not available: see fulltext.]Fil: Kane, Aissata Ousmane. Universidade de Sao Paulo; BrasilFil: Pellergini, Vanessa O. Arnoldi. Universidade de Sao Paulo; BrasilFil: Espirito Santo, Melissa C.. Universidade de Sao Paulo; BrasilFil: Ngom, Balla Diop. Universite Cheikh Anta Diop; SenegalFil: Garcia, Jose Maria. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Tucuman-Santiago del Estero. Estación Experimental Agropecuaria Famaillá; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; ArgentinaFil: Acevedo, Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Erazzú, Luis Ernesto. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Polikarpov, Igor. Universidade de Sao Paulo; Brasi

Leaves from four different sugarcane varieties as potential renewable feedstocks for second-generation ethanol production: Pretreatments, chemical composition, physical structure, and enzymatic hydrolysis yields

Development of Bioeconomy is impossible without establishing efficient technologies of lignocellulosic biomass valorization via transformation into fermentable sugars. Sugarcane is a valuable agricultural crop in several Asian, African, and Latin American countries. Here, leaves from two sugarcane and two energy cane varieties were evaluated for sustainable production of second-generation sugars, and their enzymatic hydrolysis yields were compared. Structural, morphological, and chemical composition changes in sugar and energy cane leaves submitted to acid, and acid-alkaline pretreatments were analyzed using X-ray diffraction, scanning electron microscopy, confocal laser microscopy, high-performance liquid chromatography, and low-field solid-state NMR techniques. Enzymatic hydrolysis assays were conducted to evaluate saccharification yields of untreated and pretreated leaves. Jointly, our results revealed the significant potential of leaves from two commercial sugar cane cultivars currently bred in Argentina as possible lignocellulose substrates for the 2G ethanol industry.EEA FamailláFil: Espirito Santo, Melissa. University of Sao Paulo. Sao Carlos Institute of Physics; BrasilFil: Ousmane Kane, Aissata. University of Sao Paulo. Sao Carlos Institute of Physics; BrasilFil: Arnoldi Pellergini, Vanessa. University of Sao Paulo. Sao Carlos Institute of Physics; BrasilFil: Thema, Force Tefo. Botswana University of Agriculture and Natural Resources; BotswanaFil: García, José María. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Famaillá; Argentina.Fil: García, José María. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Acevedo, Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; ArgentinaFil: Erazzú, Luis E. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Famaillá; Argentina.Fil: Guimaraes, Francisco E.G. University of Sao Paulo. Sao Carlos Institute of Physics; BrasilFil: Azevedo, Eduardo R. de. University of Sao Paulo. Sao Carlos Institute of Physics; BrasilFil: Polikarpov, Igor. University of Sao Paulo. Sao Carlos Institute of Physics; Brasi

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Additional file 1. Additional figures and tables

Enzymatic production of xylooligosaccharides from corn cobs: Assessment of two different pretreatment strategies

Corn cobs (CCs) are abundant xylan-rich agricultural wastes. Here, we compared CCs XOS yields obtained via two different pretreatment routs, alkali and hydrothermal, using a set of recombinant endo- and exo-acting enzymes from GH10 and GH11 families, which have different restrictions for xylan substitutions. Furthermore, impacts of the pretreatments on chemical composition and physical structure of the CCs samples were evaluated. We demonstrated that alkali pretreatment route rendered 59 mg of XOS per gram of initial biomass, while an overall XOS yield of 115 mg/g was achieved via hydrothermal pretreatment using a combination of GH10 and GH11 enzymes. These results hold a promise of ecologically sustainable enzymatic valorization of CCs via “green” and sustainable XOS production.Fil: de Mello Capetti, Caio Cesar. Universidade de Sao Paulo; BrasilFil: Oliveira Arnoldi Pellegrini, Vanessa. Universidade de Sao Paulo; BrasilFil: Espirito Santo, Melissa Cristina. Universidade de Sao Paulo; BrasilFil: Abreu Cortez, Anelyse. Universidade de Sao Paulo; BrasilFil: Falvo, Maurício. Universidade de Sao Paulo; BrasilFil: Aprigio da Silva Curvelo, Antonio. Universidade de Sao Paulo; BrasilFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Gonçalves Filgueiras, Jefferson. Universidade Federal Fluminense; Brasil. Universidade Federal do Rio de Janeiro; BrasilFil: Gontijo Guimaraes, Francisco Eduardo. Universidade de Sao Paulo; BrasilFil: Ribeiro de Azevedo, Eduardo. Universidade de Sao Paulo; BrasilFil: Polikarpov, Igor. Universidade de Sao Paulo; Brasi