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)

Molecular cloning, expression, purification and structural characterization of endoglucanase from Trichoderma harzianum aiming the development of enzymatic blends for production of lignocellulosic ethanol

O aumento na demanda mundial por energia, a perspectiva de encolhimento dos recursos energéticos e a preocupação global com a questão ambiental, despertaram o interesse por fontes alternativas de energia. A biomassa lignocelulósica é abundante e de baixo custo, com potencial para complementar a produção em larga escala de combustíveis. A degradação das moléculas constituintes da parede celular à açúcares fermentescíveis e então à etanol, ocorre através da hidrólise enzimática da biomassa. Contudo, a utilização de enzimas para esse fim encontra-se em estágio exploratório e representa um gargalo na implementação de tecnologias de etanol 2G em escala industrial, desencadeando a busca de celulases bioquimicamente mais ativas, estáveis e economicamente viáveis. O presente trabalho visou a caracterização da endoglucanase I do fungo Trichoderma harzianum, e para isso foi realizada expressão, ensaios bioquímicos e biofísicos do domínio catalítico (ThCel7B-CCD) e da proteína inteira (ThCel7B-full). A enzima exibiu um perfil acidofílico, com atividade ótima em pH 3,0 a 55°C. A proteína também se mostrou capaz de hidrolisar uma variedade de substratos, sendo a maior atividade hidrolítica em β-glucano (75 U mg-1). Ao analisar a estabilidade térmica medida a 55°C em pH 5, a atividade residual manteve-se intacta por mais de 2 meses. Outra característica relevante foi o elevado grau de sinergismo entre ThCel7B e ThCel7A. Análises de microscopia eletrônica de flocos de aveia submetidas à hidrólise com ThCel7B evidenciaram os efeitos de degradação do substrato em relação às amostras controle. O conjunto desses resultados, além de importante para a compreensão do mecanismo molecular de ThCel7B e de outras endoglucanases da família GH7, também revelou uma enzima de interesse biotecnológicos uma vez que o comportamento ácido e sua estabilidade térmica são características relevantes para aplicações industriais sob condições extremamente ácidas.The increase in global energy demand, shrinkage perspective of energy resources and global concern about environmental issues, aroused interest in finding alternative sources of energy. Lignocellulosic biomass is plentiful and inexpensive, and has the potential to complement the large-scale production of fuel. The degradation of molecules that constitute the cell wall to fermentable sugars and then to ethanol, occurs via the enzymatic hydrolysis of biomass. However, the use of enzymes for this purpose is in exploratory stage and represents a bottleneck in the implementation of 2G ethanol technologies in industrial scale, supporting studies about biochemically most active, stable and economically viable cellulases. This work aimed the characterization of endoglucanase I from fungus Trichoderma harzianum and for that, was performed expression and biochemical and biophysical assays of the catalytic domain (ThCel7B-CCD) and full protein (ThCel7B-full). The enzyme exhibited a acidophilic profile, with optimal activity at pH 3.0 at 55°C. The protein was also able to hydrolyze a variety of substrates, with higher hydrolytic activity in β-glucano (75 U mg-1). Analyzing the thermal stability as pH 5, residual activity remained intact for over 2 months. Another important feature was the high degree of synergy between ThCel7B and ThCel7A (DS 3). Electron microscopy analysis of oat samples subjected to hydrolysis with ThCel7B show the substrate degradation effects in relation to the control samples. All these results, as well as important for understanding the molecular mechanism of ThCel7B and other endoglucanases of GH7 family, also disclose an enzyme of biotechnological interest because the acid behavior and thermal stability are promises of industrial applications under extremely acidic conditions

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