PSEUDOMONAS-GLUMAE LIPASE - INCREASED PROTEOLYTIC STABILITY BY PROTEIN ENGINEERING

The feasibility of stabilizing proteins towards proteolytic degradatio n was explored by engineering the primary proteolytic cleavage site(s) . This novel approach does not require information on the 3-D structur e of the native enzyme. As a model system, the extracellular lipase of Pseudomonas glumae was chosen, which is sensitive towards degradation by subtilisin-type proteases. The primary proteolytic cleavage in the lipase appeared to be located between amino acids serine 153 and hist idine 154. Since subtilisins are known to show a preference towards am ino acid residues surrounding the scissile bond, non-preferred amino a cids were introduced in this area. Two concepts were tested: the intro duction of arginine or glutamate residues (charge concept) and the int roduction of proline residues (proline concept). Although the mutant l ipases produced according to either of these concepts were still cleav ed in the same area, they showed a considerably increased stability to wards proteolytic degradation.