Screening and characterization of an esterase from a metagenomic library

A metagenomic library was constructed using a fosmid vector, and total genomic DNA was extracted directly from soil at Cisolok (hot spring area, Indonesia). This library was composed of 10,214 clones and screened for lipolytic enzyme on tributyrin agar plates. An esterase gene (estMa) was subcloned and sequenced from a positive lipolytic active clone. Esterase EstMa was encoded by a 954-bp open reading frame and showed low (11-33%) amino acid similarity to known esterases. The amino acid sequence analysis demonstrated that the enzyme is a new member of lipolytic enzyme family VI. The estMa gene encodes a preprotein of 317 amino acids with a predicted molecular mass of 34,799 Da. The purified enzyme exhibited optimal activity at 50°C and pH 6.5. The K_m and V_max values of EstMa for the hydrolysis of p-nitrophenyl valerate were 45.3 μM and 4.45 U/mg, respectively.