A gene (estA', 804 bp) from Streptomyces lividans TK24 was artificially synthesized and successfully overexpressed as a 6His-tagged fusion protein in Escherichia coli. It encoded a carboxylesterase (EstA) that composed of 267 amino acids with a predicted molecular weight of 28.56 kDa. Multiple sequence alignment indicated that EstA has typical characteristics of esterases, including a catalytic triad (Ser93-Asp194-His224) and a conserved pentapeptide motif (Gly91-Leu92-Ser93-Met94-Gly95). Simultaneously, phylogenetic analysis indicated that EstA belongs to family VI. Biochemical characterization displayed its optimum enzyme activity was at 55 ℃ and pH 8.5. Additionally, EstA exhibited higher activity towards short carbon substrates and showed the outstanding catalytic efficiency for pNPA2 with kcat/Km of 2296.14 ± 10.35 s-1 mM-1. Notably, EstA has hyper-thermostability and good alkali stability. The activity of EstA did not change obviously when incubated at 50 and 100 ℃ for 337 and 1 h, independently. Besides, by incubating at 100 ℃ for 6 h, EstA remained about half of its initial activity. Moreover, EstA showed stability at pH ranging from 8.0 to 11.0, and about 90% residual enzyme activity was reserved by being treated at pH 8.0 or 9.0 for 80 h, especially. Such multiple features prepare EstA for a potential candidate in the field of biological catalysis of some industrial applications under harsh conditions.
Keywords: Esterase; Kinetics; Structural modeling; Thermostability.