Fernanda Mandelli

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.

The characterization of a thermostable and cambialistic superoxide dismutase from Thermus filiformis

The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS‐PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0·41, 0·56 and 13·73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3‐dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis.

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.

The Coptotermes gestroi aldo-keto reductase: a multipurpose enzyme for biorefinery applications.

BackgroundIn nature, termites can be considered as a model biological system for biofuel research based on their remarkable efficiency for lignocellulosic biomass conversion. Redox enzymes are of interest in second-generation ethanol production because they promote synergic enzymatic activity with classical hydrolases for lignocellulose saccharification and inactivate fermentation inhibitory compounds produced after lignocellulose pretreatment steps.ResultsIn the present study, the biochemical and structural characteristics of the Coptotermes gestroi aldo–keto reductase (CgAKR-1) were comprehensively investigated. CgAKR-1 displayed major structural differences compared with others AKRs, including the differences in the amino acid composition of the substrate-binding site, providing basis for classification as a founding member of a new AKR subfamily (family AKR1 I). Immunolocalization assays with anti-CgAKR-1 antibodies resulted in strong fluorescence in the salivary gland, proventriculus, and foregut. CgAKR-1 supplementation caused a 32% reduction in phenolic aldehydes, such as furfural, which act as fermentation inhibitors of hemicellulosic hydrolysates, and improved ethanol fermentation by the xylose-fermenting yeast Scheffersomyces stipitis by 45%. We observed synergistic enzymatic interactions between CgAKR-1 and commercial cellulosic cocktail for sugarcane bagasse saccharification, with a maximum synergism degree of 2.17 for sugar release. Our data indicated that additive enzymatic activity could be mediated by reactive oxygen species because CgAKR-1 could produce hydrogen peroxide.ConclusionIn summary, we identified the founding member of an AKRI subfamily with a potential role in the termite digestome. CgAKR-1 was found to be a multipurpose enzyme with potential biotechnological applications. The present work provided a basis for the development and application of integrative and multipurpose enzymes in the bioethanol production chain.

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.