Cellulases are a heterogeneous group of enzymes that synergistically catalyze the hydrolysis of cellulose, the major component of plant biomass. Such reaction has biotechnological applications in a broad spectrum of industries, where they can provide a more sustainable model of production. As a prerequisite for their implementation, these enzymes need to be able to operate in the conditions the industrial process requires. Thus, cellulases retrieved from extremophiles, and more specifically those of thermophiles, are likely to be more appropriate for industrial needs in which high temperatures are involved. Metagenomics, the study of genes and gene products from the whole community genomic DNA present in an environmental sample, is a powerful tool for bioprospecting in search of novel enzymes. In this review, we describe the cellulolytic systems, we summarize their biotechnological applications, and we discuss the strategies adopted in the field of metagenomics for the discovery of new cellulases, focusing on those of thermophilic microorganisms.

The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had only been used for thermalism activities. One of such hot springs, Muiño da Veiga, has now been explored using metagenomics to study the microbial community that inhabits these high-temperature circumneutral continental waters. Sequencing of the metagenome allowed the characterization of its composition, diversity, metabolic connections and potential as a source for thermozymes, as well as its ability to assemble MAGs. A diverse microbial community dominated by Bacteria domain members was revealed, particularly from the early-branching Aquificales group. The most abundant genus was Sulfurihydrogenibium, known for its implication in sulfur cycling and for forming mats that enable novel niches. The variety of primary producers with autotrophic pathways (and specifically the sulfur oxidizing pathway) expands the range of available nutrients, and the increase in biomass forms thicker mats, resulting in more available niches and broader microbial diversity. Nonetheless, certain metabolic pathways were attributed to less abundant members of the microbial community, reinforcing the idea that the rare biosphere plays important roles in the network of interactions present in an ecosystem and acts as genetic reservoirs. In addition, three of the assembled MAGs represent novel microbial diversity found in this hot spring. Moreover, the presence of enzymes and microorganisms with possible biotechnological applications was confirmed, including proteases, lipases and cell-wall degrading enzymes, pointing to the potential for the hot spring as a source for thermozymes.

Functional screenings were conducted on two metagenomic libraries from hot springs in order to find novel thermozymes with potential biotechnological applications. These included enzymes acting on plant cell walls such as endoglucanases and exoglucanases, β-glucosidases, xylanases, and β-xylosidases, and broad application enzymes such as proteases and lipolytic hydrolases. Of all the enzymes found by this bioprospection, we selected a novel lipolytic enzyme for further characterization. The protein was found to belong to the SGNH/GDSL family of hydrolases. It was purified and its biochemical parameters determined. We found that the enzyme was most active at 60 °C and pH 9 using pNP-laurate as substrate and was highly thermostable. It also showed preference for short-chained substrates and activation with temperature and with certain detergents such as Tween 80. Proteins of this family of hydrolases are relevant for their broad substrate specificity, that coupled with this protein’s high temperature optima, broad pH range, and thermostability further highlights its biotechnological potential.

Β-galactosidases (EC.3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in the food industry: the production of low-lactose milk and dairy goods for lactose intolerant people and the generation of galacto-oligosaccharides (GOS) by transgalactosylation reactions. Owing to their thermostability, β-galactosidases derived from thermophilic microorganisms are highly attractive for industrial applications, as elevated temperatures can enhance initial enzymatic productivity, improve substrate solubility, and reduce the risk of microbial contamination. In this study, a novel thermostable β-galactosidase (BWbg1) was obtained through functional screening of a metagenomic library from As Burgas hot spring (Ourense, Spain). The analysis of the protein amino acid sequence reveals that the enzyme belongs to GH35 family. The purified enzyme shows maximum activity at 80 °C and pH 7 and retains more than 72% of its activity after incubation at 55 °C for 6 h. BWbg1 can produce up to 48% (w/w) of GOS from a solution of 40% (w/v) of lactose at 70 °C following a 4 h reaction. Furthermore, remarkable heat activation, detected after 2 h of incubation at 65 °C, enhances its ability to hydrolyze lactose and produce GOS after HTST and VAT pasteurization of commercial milk. The results of this study reveal that BWbg1 is a versatile enzyme, capable of hydrolyzing lactose from commercial milk and producing GOS even at 8 °C, making it a strong candidate for industrial application. Key points: • Novel thermostable GH35 β-galactosidase (BWbg1) mined from a hot spring metagenome. • BWbg1 high activity at 80 °C enables efficient GOS synthesis from lactose and milk. • BWbg1 retains activity after pasteurization and remains active at 8 °C.