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Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine, p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coil after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39[degrees]C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.