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.