Douglas A. A. Paixão

Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase

Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-L-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

Addition of feruloyl esterase and xylanase produced on-site improves sugarcane bagasse hydrolysis

Accessory enzymes that assist biomass degradation could be used to improve the recovery of fermentable sugar for use in biorefineries. In this study, different fungal strains isolated from the Amazon rainforest were evaluated in terms of their ability to produce feruloyl esterase (FAE) and xylanase enzymes, and an assessment was made of the contributions of the enzymes in the hydrolysis of pretreated sugarcane bagasse. In the selection step, screening using plate assays was followed by shake flask submerged cultivations. After carbon source selection and cultivation in a stirred-tank bioreactor, Aspergillusoryzae P21C3 proved to be a promising strain for production of the enzymes. Supplementation of a commercial enzyme preparation with 30% (v/v) crude enzymatic complex from A. oryzae P21C3 increased the conversion of cellulose derived from pretreated sugarcane bagasse by 36%. Supplementation with FAE and xylanase enzymes produced on-site can therefore be used to improve the hydrolysis of sugarcane bagasse.

Structure and function of a novel cellulase 5 from sugarcane soil metagenome.

Cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars. However, their low performance on complex substrates and reduced stability under industrial conditions remain the main obstacle for the large-scale production of cellulose-derived products and biofuels. Thus, in this study a novel cellulase with unusual catalytic properties from sugarcane soil metagenome (CelE1) was isolated and characterized. The polypeptide deduced from the celE1 gene encodes a unique glycoside hydrolase domain belonging to GH5 family. The recombinant enzyme was active on both carboxymethyl cellulose and β-glucan with an endo-acting mode according to capillary electrophoretic analysis of cleavage products. CelE1 showed optimum hydrolytic activity at pH 7.0 and 50 °C with remarkable activity at alkaline conditions that is attractive for industrial applications in which conventional acidic cellulases are not suitable. Moreover, its three-dimensional structure was determined at 1.8 Å resolution that allowed the identification of an insertion of eight residues in the β8-α8 loop of the catalytic domain of CelE1, which is not conserved in its psychrophilic orthologs. This 8-residue-long segment is a prominent and distinguishing feature of thermotolerant cellulases 5 suggesting that it might be involved with thermal stability. Based on its unconventional characteristics, CelE1 could be potentially employed in biotechnological processes that require thermotolerant and alkaline cellulases.

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

Understanding the function of conserved variations in the catalytic loops of fungal glycoside hydrolase family 12

Enzymes that cleave the xyloglucan backbone at unbranched glucose residues have been identified in GH families 5, 7, 12, 16, 44, and 74. Fungi produce enzymes that populate 20 of 22 families that are considered critical for plant biomass deconstruction. We searched for GH12‐encoding genes in 27 Eurotiomycetes genomes. After analyzing 50 GH12‐related sequences, the conserved variations of the amino acid sequences were examined. Compared to the endoglucanases, the endo‐xyloglucanase‐associated YSG deletion at the negative subsites of the catalytic cleft with a SST insertion at the reducing end of the substrate‐binding crevice is highly conserved. In addition, a highly conserved alanine residue was identified in all xyloglucan‐specific enzymes, and this residue is substituted by arginine in more promiscuous glucanases. To understand the basis for the xyloglucan specificity displayed by certain GH12 enzymes, two fungal GH12 endoglucanases were chosen for mutagenesis and functional studies: an endo‐xyloglucanase from Aspergillus clavatus (AclaXegA) and an endoglucanase from A. terreus (AtEglD). Comprehensive molecular docking studies and biochemical analyses were performed, revealing that mutations at the entrance of the catalytic cleft in AtEglD result in a wider binding cleft and the alteration of the substrate‐cleavage pattern, implying that a trio of residues coordinates the interactions and binding to linear glycans. The loop insertion at the crevice‐reducing end of AclaXegA is critical for catalytic efficiency to hydrolyze xyloglucan. The understanding of the structural elements governing endo‐xyloglucanase activity on linear and branched glucans will facilitate future enzyme modifications with potential applications in industrial biotechnology. Biotechnol. Bioeng. 2014;111: 1494–1505.

Biochemical and biophysical properties of a metagenome-derived GH5 endoglucanase displaying an unconventional domain architecture

Endoglucanases are key enzymes in the degradation of cellulose, the most abundant polymer on Earth. The aim of this work was to perform the biochemical and biophysical characterization of CelE2, a soil metagenome derived endoglucanase. CelE2 harbors a conserved domain from glycoside hydrolase family 5 (GH5) and a C-terminal domain with identity to Calx-beta domains. The recombinant CelE2 displayed preference for hydrolysis of oat beta-glucan, followed by lichenan and carboxymethyl cellulose. Optimum values of enzymatic activity were observed at 45°C and pH 5.3, and CelE2 exhibited considerable thermal stability at 40°C for up to 360min. Regarding the cleavage pattern on polysaccharides, the release of oligosaccharides with a wide degree of polymerization indicated a characteristic of endoglucanase activity. Furthermore, the analysis of products generated from the cleavage of cellooligosaccharides suggested that CelE2 exhibited transglycosylation activity. Interestingly, the presence of CaCl2 positively affect CelE2, including in the presence of surfactants. SAXS experiments provided key information on the effect of CaCl2 on the stability of CelE2 and dummy atom and rigid-body models were generated. To the best of our knowledge this is the first biochemical and biophysical characterization of an endoglucanase from family GH5 displaying this unconventional modular organization.

Food Storage by the Savanna Termite Cornitermes cumulans (Syntermitinae): a Strategy to Improve Hemicellulose Digestibility?

It has been suggested that food storage inside the nest may offer termites with a nutritional provision during low resource availability. Additionally, feces employed as construction material provide an excellent environment for colonization by microorganisms and, together with the storage of plant material inside the nest, could thus provide some advantage to the termites in terms of lignocellulose decomposition. Here, we conducted for the first time a comprehensive study of the microbial communities associated to a termite exhibiting food storage behavior using Illumina sequencing of the 16S and (ITS2) regions of rRNA genes, together with enzymatic assays and data collected in the field. Cornitermes cumulans (Syntermitinae) stored grass litter in nodules made from feces and saliva located in the nest core. The amount of nodules increased with nest size and isolation, and interestingly, the soluble fraction of extracts from nodules showed a higher activity against hemicellulosic substrates compared to termite guts. Actinobacteria and Sordariales dominated microbial communities of food nodules and nest walls, whereas Spirochetes and Pleosporales dominated gut samples of C. cumulans. Within Syntermitinae, however, gut bacterial assemblages were dissimilar. On the other hand, there is a remarkable convergence of the bacterial community structure of Termitidae nests. Our results suggest that the role of nodules could be related to food storage; however, the higher xylanolytic activity in the nodules and their associated microbiota could also provide C. cumulans with an external source of predigested polysaccharides, which might be advantageous in comparison with litter-feeding termites that do not display food storage behavior.

Draft Genome Sequence of the Thermophile Thermus filiformis ATCC 43280, Producer of Carotenoid-(Di)glucoside-Branched Fatty Acid (Di)esters and Source of Hyperthermostable Enzymes of Biotechnological Interest.

ABSTRACT Here, we present the draft genome sequence of Thermus filiformis strain ATCC 43280, a thermophile bacterium capable of producing glycosylated carotenoids acylated with branched fatty acids and enzymes of biotechnological potential.

Microbial Communities of the Gut and Nest of the Humus- and Litter-Feeding Termite Procornitermes araujoi (Syntermitinae)

The evolution of the symbiotic association with microbes allowed termites to decompose ingested lignocellulose from plant-derived substrates, including herbivore dung and soil humus. Representatives of the Syntermitinae (Termitidae) range in their feeding habits from wood and litter-feeding to humus-feeding species. However, only limited information is available about their feeding ecology and associated microbial communities. Here we conducted a study of the microbial communities associated to the termite Procornitermes araujoi using Illumina sequencing of the 16S and ITS rRNA genes. This species has been previously included in different feeding guilds. However, most aspects of its feeding ecology are unknown, especially those associated to its symbiotic microbiota. Our results showed that the microbial communities of termite guts and nest substrates of P. araujoi differed significantly for bacteria and fungi. Firmicutes dominated the bacterial gut community of both workers and soldiers, whereas Actinobacteria was found in higher prevalence in the nest walls. Sordariomycetes was the most abundant fungal class in both gut and nest samples and distinguish P. araujoi from the grass/litter feeding Cornitermes cumulans. Our results also showed that diversity of gut bacteria were higher in P. araujoi and Silvestritermes euamignathus than in the grass/litter feeders (C. cumulans and Syntermes dirus), that could indicate an adaptation of the microbial community of polyphagous termites to the higher complexity of their diets.