A novel thermophilic lipase from Fervidobacterium nodosum Rt17-B1 representing a new subfamily of bacterial lipases

Abstract

Abstract 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.