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Graphical abstract Schematic representation of TPC2 role, investigated through genetic ablation and drug blocker, in the inhibition of Coronaviruses release into the cells.

Additional file 1. Schematic representation of the TeTrCBH I expression construct in S. cerevisiae.

Additional file 2. High performance size exclusion chromatography UV and RI chromatograms.

Additional file 1: Figure S1. SDS-PAGE analyses of T. atroviride culture filtrate and purified Trichoderma spp. Cel7A enzymes. Figure S2. Substrate dependence plots and Hanes-Wolff plots from enzyme kinetics experiments with TatCel7A, ThaCel7A and TreCel7A, using pNP-Lac as substrate and cellobiose as inhibitor. Additional information regarding the mathematical model for quasi-steady state kinetics of processive cellulose hydrolysis by GH7 cellobiohydrolases and the derivation of kinetic parameters by non-linear regression fitting to real-time progress curves of the initial stage of cellulose hydrolysis. Figure S3. A) Real-time progress curves. B) Derivative of the progress curves in A). Figure S4. A) Simplified reaction scheme for a processive cellulase. B) Illustration of the molecular steps involved in the reaction scheme. Figure S5. Non-linear regression fit to real-time progress curves. Figure S6. Bar diagram of kinetic parameters derived from initial hydrolysis of BMCC. Additional information regarding correlation of kinetic parameters derived by non-linear regression fit to initial hydrolysis data. Table S1. Parameter correlation matrix for TreCel7A. Figure S7. Kinetic parameter fit to simulated data with 2.5% random noise added, and to experimental data recorded for TreCel7A during initial hydrolysis of BMCC. Table S2. Comparison of kinetic parameters from the fit to simulated data with 2.5% random noise, and to experimental data recorded for TreCel7A during initial hydrolysis of BMCC. Figure S8. Sequence alignment of the GH7 CBH catalytic domains used for RCA analysis. Figure S9. Phylogenetic tree of GH7 catalytic domain protein sequences from Trichoderma spp. and Fusarium spp. Table S3. S scores from RCA analysis for residues of interest for TatCel7A, ThaCel7A and TreCel7A. Additional MD simulation results Figure S10. RMSD as a function of time for each 100-ns, ligand-bound MD simulation of TatCel7A, ThaCel7A and TreCel7A catalytic domains.

Cellulases, hemicellulases (xylanases) and accessory enzymes (e.g.arabinofuranosidases, pectinases, mannanases) are needed for lignocellulose conversion into fermentable sugars for second generation bioethanol production. The high production costs of the above enzymes limit the bioethanol competitiveness,pushing towards the development of new biocatalysts, with improved catalytic properties, and new processes, whith lower costs. In this work, industrial waste based compost and natural habitats were investigated as a source of new (hemi)cellulolytic bacteria and fungi for discovery of new (hemi)cellulases. Tomato processing waste was shown a suitable substrate for the production of a new alpha-L-arabinofuranosidase (PoAbf) by solid state fermentation (SSF) with the basidiomycete Pleurotus ostreatus. Cellulolyitc activities of Bacillus amyloliquefaciens B31C and Streptomyces sp. G12 isolated from industrial waste based compost were characterized. An endoglucanase (CelB31C) from B.amyloliquefaciens B31C was identified, purified and characterized. The enzyme follows a Michaelis-Menten kinetics towards carboxymethyl-cellulose. It shows a retention of 90% of its activity for at least 6 days of incubation at 40 °C and a range of optimal temperatures from 50 to 70 °C. Investigating the enzymes responsible for cellulase activity produced by Streptomyces sp. G12, proteomic analyses led to the identification of three enzymes acting on cellulose. Gene coding for one of them, named CelStrep, was cloned and sequenced. Recombinant expression of CelStrep was carried out in Escherichia coli and the recombinant enzyme (rCelStrep) was characterized. rCelStrep follows a Michaelis-Menten kinetics towards carboxymethyl-cellulose and exhibits a half life of 24 h and 96 h at 60°C and 50°C, respectively and a retention of around 80% of activity after 96 h at 40°C. 93 microorganisms were isolated from 50 samples collected in the Western Ghat region (India, Kerala). Their screening for cellulases and xylanases production led to the selection of 7 xylanolytic and 14 cellulolytic microorganisms, identified by 16S rRNA sequencing as belonging to the species Bacillus, Streptomyces, Lysinobacillus and Paenibacillus. A further screening in liquid medium revealed the strain Lysinobacillus xylanolyticus XR84 able to produce both cellulase and xylanase activities. Due to the interest in thermo-resistant cellulases and to the ability of filamentous fungi to grow on agro-industrial wastes by SSF, 150 thermophilic filamentous fungi isolated from a "maasra" were screened for cellulase activity production at 45°C, leading to the selection of four Aspergillus strains, a Malbranchea strain and a Myceliophthora thermophila strain, all representing a potential reservoir of thermostable enzymes to be further identified and characterized. Proteomic analyses led to the identification of an alpha-L-arabinofuranosidase (PoAbf) responsible for the xylanase activity produced by P.ostreatus SSF on tomato waste. cDNA coding for PoAbf was synthesized and sequenced. Recombinant expression systems were set up in Kluyveromyces lactis and Pichia pastoris, the latter being the best host for enzyme production (180 mg L-1). Recombinant PoAbf (rPoAbf) follows Michaelis-Menten kinetics. It has a durable activity in a broad range of pH, particularly at pH 5 (t1/2= 51 days), which is also its optimal pH. It shows high stability (t1/2= 7 days) at 30°C and 40°C, the latter being its optimal temperature. It was shown to be a versatile enzyme, able to hydrolyze arabinooligosaccharides, arabinan and arabinoxylan. Glycosylation in position S160 positively affects the enzyme stability as demonstrated by site-directed mutagenesis experiments.