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A novel cellulase gene encoding a thermostable endoglucanase from the thermophilic eubacterium Fervidobacterium nodosum Rt17-B1 was cloned and expressed, which is the first cellulase cloned from the organisms of genus Fervidobacterium and designated as FnCel5A for being a member of glycoside hydrolase family 5, and the enzymatic properties were characterized. The cellulase was overexpressed in Escherichia coli with a high protein content and good solubility in water, and could be easily purified. The purified recombinant cellulase shows high hydrolytic activities on carboxylmethyl cellulose, regenerated amorphous cellulose, β- d -glucan from barley and galactomannan, with the optimum temperature of 80–83 °C and the optimum pH of 5.0–5.5. Furthermore, this enzyme is highly thermostable and has a half-life of 48 h at 80 °C. With such a combination of thermostability and high activities, this cellulase is expected to be useful for hydrolysis of cellulosic and hemicellulosic substrates at high temperatures, and for industrial hydrolysis of plant cellulose during long-time processing at the elevated temperatures, particularly in converting biomass into biofuels.

Thermophilic enzymes have significant potential for industry applications due to their high inherent stability and high reaction rates at high temperatures. In this study a gene coding for a putative lipase composed of 302 amino acids from an anaerobic thermophilic bacterium Fervidobacterium nodosum Rt17-B1 was cloned and characterized. Phylogenetic analysis suggests that this novel lipase represents a new subfamily of family I of bacterial lipases, annotated as family I.8. The recombinant protein can catalyze the hydrolysis of p-nitrophenyl esters and shows substrate preference for p-nitrophenyl caprate with a kcat/Km of 22,500 s−1 μM−1. Most importantly, it can hydrolyze triacylglycerols with long acyl chains. In the test conditions applied here it has a maximum activity at 70 °C and pH 9.0 and displays extreme thermal stability. Interestingly, it was not only stable, but was also activated by treatment with polar organic solvents including propanol, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). Structural modeling showed that it is composed of an α/β-hydrolase fold and a lid domain comprised of four α-helices. A canonical catalytic triad consisting of Ser119, Asp206 and His282 was verified by site-directed mutagenesis.

The ring-opening polymerization of ɛ-caprolactone catalyzed by a novel thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time, reaction medium, and water activity on monomer conversion and product molecular weight were investigated. Poly(ɛ-caprolactone) was obtained in almost 100% of the monomer conversion, with a number-average molecular weight of 1400 in toluene at 80 °C for 72 h. Furthermore, the Michaelis–Menten kinetic analysis showed that the enzyme had the highest affinity for ɛ-caprolactone, with a Km value of 0.093 mol/l compared with other reported lipases. The possible structural and energetic effects of the enzyme on the Km value were investigated, using molecular docking studies.