Microbial enrichment and meta-omics analysis identify CAZymes from mangrove sediments with unique properties.

Although lignocellulose is the most abundant and renewable natural resource for biofuel production, its use remains under exploration because of its highly recalcitrant structure. Its deconstruction into sugar monomers is mainly driven by carbohydrate-active enzymes (CAZymes). To develop highly efficient and fast strategies to discover biomass-degrading enzymes for biorefinery applications, an enrichment process combined with integrative omics approaches was used to identify new CAZymes. The lignocellulolytic-enriched mangrove microbial community (LignoManG) established on sugarcane bagasse (SB) was enriched with lignocellulolytic bacteria and fungi such as Proteobacteria, Bacteroidetes, Basidiomycota, and Ascomycota. These microbial communities were able to degrade up to 55 % of the total SB, indicating the production of lignocellulolytic enzymes. Metagenomic analysis revealed that the LignoManG harbors 18.042 CAZyme sequences such as of cellulases, hemicellulases, carbohydrate esterases, and lytic polysaccharide monooxygenase. Similarly, our metaproteomic analysis depicted several enzymes from distinct families of different CAZy families. Based on the LignoManG data, a xylanase (coldXynZ) was selected, amplified, cloned, expressed, and biochemically characterized. The enzyme displayed psicrofilic properties, with the highest activity at 15 °C, retaining 77 % of its activity when incubated at 0 °C. Moreover, molecular modeling in silico indicated that coldXynZ is composed of a TIM barrel, which is a typical folding found in the GH10 family, and displayed similar structural features related to cold-adapted enzymes. Collectively, the data generated in this study represent a valuable resource for lignocellulolytic enzymes with potential biotechnological applications.

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.

A Novel Carbohydrate-binding Module from Sugar Cane Soil Metagenome Featuring Unique Structural and Carbohydrate Affinity Properties.

Carbohydrate-binding modules (CBMs) are appended to glycoside hydrolases and can contribute to the degradation of complex recalcitrant substrates such as the plant cell wall. For application in bioethanol production, novel enzymes with high catalytic activity against recalcitrant lignocellulosic material are being explored and developed. In this work, we report the functional and structural study of CBM_E1, which was discovered through a metagenomics approach and is the founding member of a novel CBM family, CBM81. CBM_E1, which is linked to an endoglucanase, displayed affinity for mixed linked ß1,3-ß1,4-glucans, xyloglucan, Avicel, and cellooligosaccharides. The crystal structure of CBM_E1 in complex with cellopentaose displayed a canonical ß-sandwich fold comprising two ß-sheets. The planar ligand binding site, observed in a parallel orientation with the ß-strands, is a typical feature of type A CBMs, although the expected affinity for bacterial crystalline cellulose was not detected. Conversely, the binding to soluble glucans was enthalpically driven, which is typical of type B modules. These unique properties of CBM_E1 are at the interface between type A and type B CBMs.

A Novel Member of GH16 Family Derived from Sugarcane Soil Metagenome.

Glycoside hydrolases (GHs) are enzymes found in all living kingdoms that are involved in multiple physiological functions. Due to their multiple enzymatic activities, GHs are broadly applied in bioethanol, food, and paper industry. In order to increase the productivity of these industrial processes, a constant search for novel and efficient enzymes has been proved to be necessary. In this context, metagenomics is a powerful approach to achieve this demand. In the current study, we describe the discovery and characterization of a novel member of GH16 family derived from the sugarcane soil metagenome. The enzyme, named SCLam, has 286 amino acid residues and displays sequence homology and activity properties that resemble known laminarases. SCLam is active against barley beta-glucan, laminarin, and lichenan (72, 33, and 10 U mg(-1), respectively). The optimal reaction conditions were identified as 40 °C and pH 6.5. The low-resolution structure was determined using the small-angle X-ray scattering technique, revealing that SCLam is a monomer in solution with a radius of gyration equal to 19.6 Å. To the best of our knowledge, SCLam is the first nonspecific (1,3/1,3:1,4)-ß-D-glucan endohydrolase (EC 3.2.1.6) recovered by metagenomic approach to be characterized.

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome.

In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Šresolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space group I23, with unit-cell parameters a = b = c = 88.07 Å.

Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-ß-D-mannanase.

The breakdown of ß-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of ß-mannanases. In this work, a two-domain ß-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/ß)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1.

Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20°C, and exclusively xylobiose at 90°C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.