Bacterial lipases for biotechnological applications

Lipase genes originating from the Gram-negative bacteria Serrutiu marcescens and Pseudomonus urruginosa were cloned. S. marcescens lipase was overexpressed in Escherichia coli yielding inclusion bodies which were purified and finally refolded to give enzymatically active lipase. The lipase operon of P. aeruginosa consisting of genes 1ipA and lip14 was cloned behind the T7 410 promoter and overexpressed in a lipase-negative P. aeruginosa strain carrying a chromosomal insertion of the gene encoding T7 RNA polymerase. A 3D structural model was built for P. aeruginosu lipase using the coordinates of the Burkholderiu cepacia lipase structure which has recently been solved in its open conformation by X-ray crystallography. Both lipases have been purified to homogeneity and were tested for their potential to catalyze biotechnologically important reactions. S. marcescens lipase stereoselectively hydrolyzed racemic isopropylideneglycerol acetate which is a basic building block in a variety of organic synthesis reactions. P. aeruginosa lipase was successfully used for kinetic resolution of chiral alcohols and amines giving enantiomeric excess values of 2 95% at reaction rates of 40-50%. Our results demonstrate that both lipases can be produced at levels of 100 mg/l for S. marcescens and 150 mg/l for P. arruginosa. The recombinant lipase proteins are promising candidates for biotechnological applications.