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Deveer,* Ruud Dijkman,* Marijke Leuveling Tjeenk,: Hubertus M. Verheij,*,* Gerardus H. de Haas,* Edda Wenzig," and Fritz Gotzl Department of Biochemistry, State University of Utrecht, Padualaan 8, 3584 CH Utrecht. The Netherlands, Lehrstuhl fur Mikrobiologie. Technische Universitat Miinchen, Arcisstrasse 21, 0-8000 Miinchen 2, FRG, and Mikrobielle Genetik. Universitat Tiibingen, Auf der Morgenstelle 28, 0-7400 Tiibingen 1, FRG Received February 6, 1989; Revised Manuscript Received June 21, 1989 ABSTRACT: The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the prepsence of Sr2+or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from zyxwvu S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity. L i p a s e s (glycerol ester hydrolase, EC 3.1 .I .3) and bacterial lipases in particular are widespread in nature. These enzymes hydrolyze triglycerides to diglycerides, monoglycerides, gly- cerol, and fatty acids. Interest in bacterial lipases has increased markedly in the last two decades. A large number of lipases have been screened for applications in medicine (digestive enzymes) or as food additives (flavor-modifying enzymes), 'This zyxwvutsrqpo work has been supported by the Dutch Organization for Ad- vancement of Pure Science (NWO) and by the European Economical Community (BAP Contracts 0071-NL and 0196-D). TState University of Utrecht. $Present address: TNO Sectie Biochemie, Postbus 15, 6700 AA Wageningen, The Netherlands. 11 Technische Universitat Miinchen. Universitat Tubingen. industrial reagents (glyceride-hydrolyzing enzymes), and cleaners (detergent additives). Several lipases have been isolated and biochemically analyzed (Muruoka et al., 1982; Tyski et al., 1983; Rollof et al., 1987). These enzymes are characterized by a wide substrate specificity since they hy- drolyze tri-, di-, and monoglycerides as well as poly(oxy- ethylene) sorbitan fatty acyl esters (Tweens). The lipase gene of Staphylococcus hyicus has been cloned and expressed to high levels in Staphylococcus carnosus (Gotz et al., 1985; Lechner et al., 1988). A preprotein with 641 amino acids was predicted from the DNA sequence with a molecular mass of 7 1.4 kDa, although the apparent molecular mass from SDS-PAGE gels was about 86 kDa. In S. hyicus, but not in S. carnosus, the 86-kDa protein was rapidly transformed into a 46-kDa form. In this paper we describe 0006-2960/89/0428-9278$01.50/0 0 1989 American Chemical Society