Soliman_2007_Process.Biochem_42_1090

Genes encoding an esterase (EstA) and lipase (LipA) from Geobacillus thermoleovorans YN, a strain isolated from Egyptian desert soil, were cloned and the respective proteins were expressed in Escherichia coli and characterized. Whereas LipA was cloned directly by PCR amplification from genomic DNA, a genomic library composed of 3000 clones was screened on tributyrin agar plates to find EstA. An open reading frame of 744bps encoding a polypeptide of 247 amino acid residues was identified as esterase due to its conserved GXSXG motif and its high similarity toward other carboxyl esterases. LipA (416 aa residues) is encoded by an ORF of 1251bps and constitutes a pre-protein with a calculated molecular mass of 46kDa including a signal sequence of 28 aa resulting in a mature lipase of 43kDa. Both, LipA and EstA were sub-cloned and expressed under control of the temperature-inducible ?-promoter and purified by IMAC and gel filtration. The molecular mass of the purified EstA was 29kDa. Both enzymes were most active at pH ~9.5 and remarkably stable at pH 5 and 10.5. Temperature optima and stabilities (up to 70C) of both enzymes as well as their reaction kinetics and substrate spectra were determined.