Pseudomonas glumae lipase: increased proteolytic stabifity by protein engineering

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Abstract
The feasibility of stabilizing proteins towards proteolytic degradation was explored by engineering the primary proteolytic cleavage site(s). This novel approach does not require information on the 3-D structure of the native enzyme. As a model system, the extracellular lipase of Pseudomonas glumae was chosen, which is sensitive towards degradation by subtilisin-tvpe proteases. The primary proteolytic cleavage in the lipase appeared to be located between amino acids serine 153 and histidine 154. Since subtUisins are known to show a preference towards amino acid residues surrounding the scissile bond, non-preferred amino adds were introduced in this area. Two concepts were tested: the introduction of arginine or glutainate residues (charge concept) and the introduction of proline residues (proUne concept). Although the mutant Upases produced according to either of these concepts were still cleaved in the same area, they showed a considerably increased stability towards proteolytic degradation.

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