Two Novel Esterases from Deep-Sea Sediment Reveal Key Residues for Thermostability

Abstract Thermostability is one of the major concerns in the industrial application of enzymes. In this study, two novel family IV esterases, Est2 and Est4, were identified from a deep-sea sediment metagenomic library. The two enzymes had high amino acid sequence identity (96%) with only twelve different residues. Characteristic analysis indicated that both enzymes shared most of the enzymatic properties, including optimum p-nitrophenyl substrates (p-nitrophenyl butyrate and hexanoate), temperature (40°C) and pH (7.0–8.0). Interestingly, Est2 showed higher thermostability at 50°C than Est4. Mutagenesis analysis of Est2 identified two out of twelve differential amino acids, Asp18 and Lys289, that were crucial for the thermostability of the enzymes. Asp18 determined both the thermostability and catalytic activity of Est2. Structural analysis showed that Asp18 was located at the cap domain of Est2 and might be involved in the mobility of the cap domain. Lys289, located at the surface of Est2, determined the discrepancy in the surface potential between the two enzymes. Our results provide inspiration for research on the thermostability of esterases and improve the application potential of deep sea-derived esterases in industrial production.