Fabio M. Squina

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%.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168.

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass.

Background Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the worlds second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant. Results Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source. Conclusion Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.

Ferulic acid and derivatives: molecules with potential application in the pharmaceutical field

Ferulic acid is a phenolic acid widely distributed in the plant kingdom. It presents a wide range of potential therapeutic effects useful in the treatments of cancer, diabetes, lung and cardiovascular diseases, as well as hepatic, neuro and photoprotective effects and antimicrobial and anti-inflammatory activities. Overall, the pharmaceutical potential of ferulic acid can be attributed to its ability to scavenge free radicals. However, recent studies have revealed that ferulic acid presents pharmacological properties beyond those related to its antioxidant activity, such as the ability to competitively inhibit HMG-CoA reductase and activate glucokinase, contributing to reduce hypercholesterolemia and hyperglycemia, respectively. The present review addresses ferulic acid dietary sources, the pharmacokinetic profile, antioxidant action mechanisms and therapeutic effects in the treatment and prevention of various diseases, in order to provide a basis for understanding its mechanisms of action as well as its pharmaceutical potential.

Functional characterization and synergic action of fungal xylanase and arabinofuranosidase for production of xylooligosaccharides

Plant cell wall degrading enzymes are key technological components in biomass bioconversion platforms for lignocellulosic materials transformation. Cost effective production of enzymes and identification of efficient degradation routes are two economic bottlenecks that currently limit the use of renewable feedstocks through an environmental friendly pathway. The present study describes the hypersecretion of an endo-xylanase (GH11) and an arabinofuranosidase (GH54) by a fungal expression system with potential biotechnological application, along with comprehensive characterization of both enzymes, including spectrometric analysis of thermal denaturation, biochemical characterization and mode of action description. The synergistic effect of these enzymes on natural substrates such as sugarcane bagasse, demonstrated the biotechnological potential of using GH11 and GH54 for production of probiotic xylooligosaccharides from plant biomass. Our findings shed light on enzymatic mechanisms for xylooligosaccharide production, as well as provide basis for further studies for the development of novel enzymatic routes for use in biomass-to-bioethanol applications.

High-yield secretion of multiple client proteins in Aspergillus

Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.

Genomics Review of Holocellulose Deconstruction by Aspergilli

SUMMARY Biomass is constructed of dense recalcitrant polymeric materials: proteins, lignin, and holocellulose, a fraction constituting fibrous cellulose wrapped in hemicellulose-pectin. Bacteria and fungi are abundant in soil and forest floors, actively recycling biomass mainly by extracting sugars from holocellulose degradation. Here we review the genome-wide contents of seven Aspergillus species and unravel hundreds of gene models encoding holocellulose-degrading enzymes. Numerous apparent gene duplications followed functional evolution, grouping similar genes into smaller coherent functional families according to specialized structural features, domain organization, biochemical activity, and genus genome distribution. Aspergilli contain about 37 cellulase gene models, clustered in two mechanistic categories: 27 hydrolyze and 10 oxidize glycosidic bonds. Within the oxidative enzymes, we found two cellobiose dehydrogenases that produce oxygen radicals utilized by eight lytic polysaccharide monooxygenases that oxidize glycosidic linkages, breaking crystalline cellulose chains and making them accessible to hydrolytic enzymes. Among the hydrolases, six cellobiohydrolases with a tunnel-like structural fold embrace single crystalline cellulose chains and cooperate at nonreducing or reducing end termini, splitting off cellobiose. Five endoglucanases group into four structural families and interact randomly and internally with cellulose through an open cleft catalytic domain, and finally, seven extracellular β-glucosidases cleave cellobiose and related oligomers into glucose. Aspergilli contain, on average, 30 hemicellulase and 7 accessory gene models, distributed among 9 distinct functional categories: the backbone-attacking enzymes xylanase, mannosidase, arabinase, and xyloglucanase, the short-side-chain-removing enzymes xylan α-1,2-glucuronidase, arabinofuranosidase, and xylosidase, and the accessory enzymes acetyl xylan and feruloyl esterases.

The Penicillium echinulatum Secretome on Sugar Cane Bagasse

Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases). Glycoside hydrolase (GH) family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.

Joint X‐ray crystallographic and molecular dynamics study of cellobiohydrolase I from Trichoderma harzianum: deciphering the structural features of cellobiohydrolase catalytic activity

Aiming to contribute toward the characterization of new, biotechnologically relevant cellulolytic enzymes, we report here the first crystal structure of the catalytic core domain of Cel7A (cellobiohydrolase I) from the filamentous fungus Trichoderma harzianum IOC 3844. Our structural studies and molecular dynamics simulations show that the flexibility of Tyr260, in comparison with Tyr247 from the homologous Trichoderma reesei Cel7A, is enhanced as a result of the short side‐chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. T. harzianum cellobiohydrolase I also has a shortened loop at the entrance of the cellulose‐binding tunnel, which has been described to interact with the substrate in T. reesei Cel7A. These structural features might explain why T. harzianum Cel7A displays higher kcat and Km values, and lower product inhibition on both glucoside and lactoside substrates, compared with T. reesei Cel7A.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-β-1,3-glucanase from Thermotoga petrophila

1,3-β-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal β-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.