1. Introduction
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 cost1. 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, their low specific activity, their susceptibility to inactivation and the difficulty to recycle them2.
A group of naturally occurring cellulases are reported from heterotrophic microorganisms including bacteria and fungi3. These organisms secrete cellulases to utilize cellulose as a carbon source. Bioconversion processes involve the hydrolysis of cellulose to produce reducing sugars; further fermentation of the sugars to ethanol and other bioproducts 4. Cellulases hydrolyze the β-1,4 glycosidic 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 cellobiose3. Finally, the cellobiose molecules produced are converted to glucose by intra- and extracellular β -glucosidases (EC 3.2.1.21), celludextrinases (EC 3.2.1.4), and cellodextrin phosphorylases (EC 2.4.1.49), depending upon the characteristic of each cellulolytic species 5. Other than heterotrophs, cellulases belonging to glucoside hydrolase family (GH9) are also described from higher plants6. However, it has been reported that plant cellulases participate in the biosynthesis and/or remodeling of cellulose rather than in its degradation 6,7.
Algae are phototrophs, ubiquitous with versatile metabolic pathways, which have been well exploited to obtained multiple products through algal refinery 8,9. However, the presence of cellulases and cellulolytic activity was poorly described in algae. In 1966, Dvořáková-Hladká et al. reported the presence of β-glucosidase activity in S. obliquus , which allows it to grow using cellobiose as a substrate 10. In 1970 Burczyk and col. reported the presence of extracellular cellulases in Scenedesmus obliquusbecause cell walls accumulated in the medium as a result of mother cells autospore release were deprived of the cellulose layer present in daughter cells. 11. In 2012, Blifernez-Klassen and col. observed that the photoheterotrophic microalgae Chlamydomonas reinhardtii , was also capable of degrading and assimilating exogenous cellulose 3. This interesting finding led us to investigate the presence of cellulases in S. quadricauda . This organism is a freshwater, non-mobile green algae which usually forms colonies of four cells. It belongs to the same class of green algae (Chlorophyceae) as the genus Chlamydomonas . S. quadricaudahas gained great importance due to its high capacity for effluent treatment, CO2 capture and biofuel production as we showed in a previous work 9. The S. quadricaudaLWG002611 genome was sequenced and functional genes of different metabolic pathways were identified, such as those involved in the synthesis of triacyl glycerol (TAG) 9. However, no evidence on cellulase secretion and cellulose utilization in this alga was found or reported, as well as genes that encode proteins with glycoside hydrolase activity have not yet been described.
In this work, we have identified different genes in the genome sequence of S. quadricauda LWG002611, belonging to GH1, GH5, GH9 and GH10 families. Furthermore, a comparative bioinformatic analysis was conducted in several available Scenedesmaceae algae genome (Scenedesmus obliquus EN0004 v1.0, Scenedesmus obliquusUTEX B 3031, Scenedesmus obliquus var. DOE0013 v1.0,Scenedesmus sp. NREL 46B-D3 v1.0, and S. quadricaudaLWG002611) to identify multiple homologs of endoglucanase, β-glucosidase and exocellulase genes. Additionally, a phylogenetic analysis and a 3D protein modeling were achieved. Our results showed that the 3D structures of all the modeled domains obtained and the main catalytic amino acid residues implicated in cellulolytic activity are well conserved in the Scenedesmus analyzed enzymes.
These new findings open the opportunity to identify new cellulases from algae, as well as carry out their functional characterization to be used in biotechnological applications.