CONICET | Buscador de Institutos y Recursos Humanos

Generation of renewable energy resources and waste management are the major concern in twenty first century. Lignocellulosic agricultural and forest wastes are the promising feedstock for production of biofuel and value-added products due its high availability and low cost. Nevertheless, no commercial process has still been reported for the enzymatic hydrolysis of cellulose. The main reason is the high cost of the required enzymes, low specific activity, susceptible to inactivation and difficult to recycle.A group of naturally occurring cellulases has been reported from heterotrophic microorganisms including bacteria and fungi. They secrete cellulases to utilize cellulose as sole carbon source. Bioconversion process involves the hydrolysis of cellulose to produce reducing sugars, further fermentation of the sugars to ethanol and other bioproducts. Cellulases hydrolyze the β-1,4 glucosidic bonds of the glucose polymer by two different ways, endoglucanases cut random positions along the cellulose chain, and exoglucanases progressively act on the terminal ends of the polymer, releasing either glucose molecules, or cellobiose. Finally, the cellobiose molecules produced are converted to glucose by intra- and extracellular beta-glucosidases (EC 3.2.1.21), celludextrinasas (EC 3.2.1.4), and celudextrin phosphorylases (EC 2.4.1.49), depending upon the characteristic of each cellulolytic species. Other than heterotrophs, cellulases belonging to glucoside hydrolase family (GH9) are also described from higher plants. However, it has been reported that plant cellulases participates in biosynthesis of cellulose rather than degradation.Algae are phototrophs, ubiquitous with versatile metabolic pathways. Which have been well exploited to obtain multiple products through algal refinery. However, presence of cellulases and cellulolytic ability is poorly reported form algae. In 1970 Burczyk et al. reported the presence of extracellular cellulases in Scenedesmus obliquus by comparing the composition of their cell wall with that of the stem cell spore walls accumulated in the culture medium. In 1965, Dvořáková-Hladká et al. reported the presence of beta-glucosidase activity in Scenedesmus obliquus, which allows it to grow using cellobiose as a substrate. In 2012, Blifernez-Klassen et al. observed that the photoheterotrophic microalgae Chlamydomonas reinhardtii is also capable of degrading and assimilating exogenous cellulose. This interesting finding led us to search for cellulases in Scenedesmus quadricauda. It is freshwater, not mobile green algae usually forming colonies of four cells. It belongs to the same class of green algae (Chlorophyceae) as the genus Chlamydomonas. S. quadricauda has gained great importance due to high capacity for effluent treatment, CO2 capture and biofuel production as evident by our previous study also.In the present work, we identified cellulases (GH1, GH5 and GH9) gene in the genome sequence of S. quadricauda LWG002611. Furthermore, we have carried out a comparative bioinformatic analysis in several available Scenedesmaceae algae genome (Scenedesmus obliquus EN0004 v1.0, Scenedesmus obliquus UTEX B 3031, Scenedesmus obliquus var. DOE0013 v1, Scenedesmus sp. NREL 46B-D3 v1.0, Tetradesmus deserticola SNI-2 v1.0 with and S. quadricauda LWG002611 to identify multiple homologs of endoglucanase, β-glucosidase and exocellulase genes. We were able to identify 57 endoglucanase sequences, 28 cellobiase sequences, and 12 exocellulase sequences. For each of them we identified the catalytic and carbohydrate-binding domains, analyzed by phylogeny their evolutionary relationship, and obtained 3D models of the most important catalytic modules and domains. The sequences were taken from phytozome or NCBI. Conserved domains, Signal peptide, and GH-family assignment were identified with Prosite patterns SignalP and predAlgo. For the phylogenetic analysis the sequences were aligned with Clustal Omega and the alignment submitted to MEGA 6 software. The 3D homology models were generated with RaptorX server. The regions implicated in substrate binding and activity were manually annotated using the pattern sequences or 3D structure of cellulase templates available in Prosite and pdb database. Our new finding would open the opportunity for future applications of algal cellulases for biotechnological applications.

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