THE (PHOSPHO)LIPASE FROM STAPHYLOCOCCUS-HYICUS - EXPRESSION IN ESCHERICHIA-COLI, LARGE-SCALE PURIFICATION AND APPLICATION IN ESTERIFICATIONREACTIONS

The gene coding for the mature part of the (phospho)lipase from Staphy lococcus hyicus has been cloned and overexpressed in Escherichia coli. The recombinant enzyme accumulated in the cytoplasm and was purified using a combination of cation- and anion-exchange chromatography and r efolding from guanidine/HCl. The purification protocol yielded 0.46 g of pure S. hyicus (phospho)lipase from 100 gram wet E. coli cells. Alt hough the recombinant enzyme has an N-terminal extension of 35 amino a cids, the activity towards lipids and phospholipids of different chain -length is identical to that of the wild-type S. hyicus (phospho)lipas e. Application of the (phospho)lipase in esterification reactions demo nstrated that the enzyme is inactive in microemulsions of charged dete rgents, but active in the presence of the neutral detergent Triton X-1 00 and in the absence of a detergent. These results were compared to t he performance of a series of other lipases from microbial origin. The results are discussed in view of the possible application of the S. h yicus (phospho)lipase in the modification of phospholipids.