Pseudomonas glumae lipase: increased proteolytic stabifity by protein engineering

The feasibility of stabilizing proteins towards proteolyticdegradation was explored by engineering the primary proteolyticcleavage site(s). This novel approach does not require informationon the 3-D structure of the native enzyme. As a model system,the extracellular lipase of Pseudomonas glumae was chosen, whichis sensitive towards degradation by subtilisin-tvpe proteases.The primary proteolytic cleavage in the lipase appeared to belocated between amino acids serine 153 and histidine 154. SincesubtUisins are known to show a preference towards amino acidresidues surrounding the scissile bond, non-preferred aminoadds were introduced in this area. Two concepts were tested:the introduction of arginine or glutainate residues (chargeconcept) and the introduction of proline residues (proUne concept).Although the mutant Upases produced according to either of theseconcepts were still cleaved in the same area, they showed aconsiderably increased stability towards proteolytic degradation.