OVEREXPRESSION, IMMOBILIZATION AND BIOTECHNOLOGICAL APPLICATION OF PSEUDOMONAS LIPASES

Pseudomonas lipases play an important role in biotechnology both as hy drolases for detergent additives and as synthases catalyzing the kinet ic resolution of racemic compounds. Large-scale production of Pseudomo nas lipases requires correct folding and secretion through the bacteri al membranes. Controllable expression of the gene lipH encoding a lipa se-specific foldase proves to be important for overexpression in the h omologous host Escherichia coli. Construction of appropriate His-tagge d fusion proteins permitted overexpression, secretion and one-step pur ification of lipase from culture supernatants of the homologous host P seudomonas aeruginosa. The immobilization of lipases in hydrophobic so l-gel materials derived from alkylsilane precursors of the type RSi(OC H3)(3) or mixtures of RSi(OCH3)(3) and Si(OCH3)(4) provides highly act ive chemically and thermally stable heterogeneous biocatalysts. The en trapped lipases are excellent catalysts in a variety of synthetic orga nic transformations. Using directed evolution based on error prone PCR , the enantioselectivity of the hydrolysis of a chiral ester, catalyze d by the lipase from P. aeruginosa, can be increased from ee 2 to ee 8 1% in just four mutagenesis cycles. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.