Cloning, nucleotide sequence and primary biochemical characterization of esterase EstA from Ralstonia eutropha CH34

A genomic library of Ralstonia eutropha CH34 was screened in Escherichia coli S17-1 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 1,711 bp DNA fragment was subcloned from an esterase-positive clone and sequenced. Esterase EstA was encoded by a 825-bp open reading frame and exhibited significant amino acid similarities with the enzymes involved in the meta-cleavage pathway. EstA is composed of 275 amino aicds with a predicted molecular mass of 30785 Da. The optimal pH for EstA was 7.0, and the enzyme retained more than 65% activity when incubated in buffers with pH 3.8–9.2 for 2 h. EstA was active at temperatures up to 80 °C and retained more than 77% activity after exposure to temperatures below 60 °C for 2 h.