# Green Production and Biotechnological Applications of Cell Wall Lytic Enzymes
## 5 Bio-Applications of CWLEs
### Use of CWLEs for the Production of Biofuels from Agricultural Feedback  
Chemical or enzymatic treatments are employed to convert lignocellulosic biomass to fermentable sugars that, in turn, are used to produce biofuels. Chemical methods are harmful to the environment, clashing with the rationale of using lignocellulosicose as a clean fuel source. On the other hand, lignocellulosic treatment with microbial CWLEs is limited by the costs and low efficiency [132; 133]. Four major factors contribute to decreasing the enzymatic degradation efficiency of lignocellulosicose: (i) incomplete knowledge of the reactions and of the enzymes at the basis of such a process, (ii) the hydrophobic nature of lignin and cellulose that hinders the accessibility of glycoside hydrolases to the substrate, (iii) the broad heterogeneity of hemicellulose and pectin that requires many specialized CWLEs for their efficient depolymerization [34], and (iv) the property of lignin and of certain oligosaccharides to act as CWLE inhibitors. Enzymatic hydrolysis of lignocellulosic biomass can be improved by using selected mixtures of CWLEs and optimized reaction conditions. Under this perspective, the choice of proper enzymatic blend is a critical step, since the number of CWLEs is constantly increasing and part of them are characterized by novel and unknown functions. Nowadays, despite the many efforts related to the isolation and characterization of novel CWLEs, chemical and physical pretreatments of lignocellulosic biomass are required before proceeding with enzymatic treatment. According to this view, latest-generation enzyme-based products, such as those belonging to the Cellic CTech(r) series (Novozymes A/S, Bagsvaerd, Denmark), require chemically treated lignocellulosic (i.e., by diluted acid treatment) for efficient hydrolysis; such products are mainly composed of cellulases and LPMOs (application sheets of Cellic(r) CTec2, HTec2-Enzymes, and Cellic(r) CTec3 for hydrolysis of lignocellulosic materials), while ligninases, hemicellulases, and pectinases are present in lower amounts [27]. A promising application of CWLEs is in the sector of renewable fuels, namely second- and third-generation biofuels. Soluble sugars obtained from agricultural feedstock (e.g., bagasse, straw, corn cob, grass, bran) as well as the same residual biomass (insoluble) upon CWLE treatment can be used as feed for sustaining the fermentative process of yeasts in order to produce ethanol [134]. Under this perspective, cellulose is the most energetic substrate since it is exclusively composed of glucose, the monosaccharide with the highest yield in ethanol conversion. In recent years,  
Figure 4: Biotechnological applications of CWLEs. (**a**) Possible applications of plant-expressed CWLEs in agriculture, industrial, and medical fields; both an increased degradability and biomass productivity positively impact the release of fermentable sugars from plant biomass. (**b**) Use of plant-expressed CWLEs for the production of second- and third-generation biofuels; sugars released from waste materials upon CWLE-treatment are used as feed for yeasts, microalgae, and methanogenic bacteria. CO2 emitted from these metabolic processes may be converted into CWLEs and O2 by the plant biofactory. bioethanol production has also been extended to photosynthetic microorganisms such as cyanobacteria and microalgae [135]; however, although photo-fermentation is promising for bioethanol production, only little information is available from industrial applications. Enzymatically treated lignocellulosic can be used to produce other forms of biofuels, such as those obtained from oleaginous microalgae, namely, third-generation biofuels. Hydrolysates from lignocellulosic materials can be used as feed for sustaining the mixotrophic growth of oleaginous microalgae such as _Botryococcus braunii_, _Monoraphidium meglectum_, and those belonging to _Chlorella_ spp. [136; 137; 138].