STAPHYLOCOCCALLIPASES AND PHOSPHOLIPASES

Abstract

For many years it has been known that certain Staphylococci produce and release lipolytic enzymesinto the culture medium. In order to obtain more information about the genetic and biochemical properties, the lipase genes of Staphylococcus hyicus and Staphylococcus aureus were cloned and sequenced. The S. hyicus lipase gene was cloned and expressed in Staphylococcus carnosus. From this organism, the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kD. This protein was purified and the N-terminal sequence revealed that the primary gene product was processed at the proposed signal peptide cleavagesite. In S. hyicus, the DNA-donorstrain, the lipase was further processed by an extracellular protease to a 46 kD protein, which exhibited a 3-fold higher specific activity as compared to the 86 kD protein. The 46 kD protein waspurified to homogeneity. The lipolytic activity was dependent upon the presence of Ca2*. The purified lipase revealed an extremely broad specificity. The enzyme hydrolysed not only triglycerides, but also naturally occurring phosphatidylcholines and lysophospholipidsto free fatty acids and water-soluble products. The 641 aminoacid residue S. hyicus lipase is organized as a pre-pro-lipase, consisting of a 38 aminoacid signal peptide, a 207 aminoacid pro-peptide, and a 396 amino acid mature lipase. Secondary structure predictions revealed that the pro-peptideis characterized by hydrophilic protein domains, while in the mature lipase, hydrophobic protein domains are predominate. It was demonstrated by gene fusionstudies that the pro-peptide is necessary for efficient lipase secretion.