THE LIPASE FROM STAPHYLOCOCCUS-AUREUS - EXPRESSION IN ESCHERICHIA-COLI, LARGE-SCALE PURIFICATION AND COMPARISON OF SUBSTRATE-SPECIFICITY TOSTAPHYLOCOCCUS-HYICUS LIPASE

The genes coding for the mature part of the lipases from Staphylococcu s aureus NCTC8530 and Staphylococcus hyicus have been cloned and overe xpressed in Escherichia coli as fusion proteins with an N-terminal hex a-histidine tag. The enzymes accumulated in the cytoplasm and were pur ified using sequential precipitation with protamine sulphate and ammon ium sulphate, followed by metal-affinity and hydroxyapatite chromatogr aphy. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus li pase and 13 mg/g for S. hyicus lipase. The purified enzymes need calci um for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with f ull retention of activity of S. aureus lipase. Whereas S. hyicus lipas e is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are p oor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain l ength. The results are discussed in view of the large sequence similar ity between both lipases.