Chang_2021_Extremophiles__

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 k(cat)/K(m) 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.