Display of active subtilisin 309 on phage: Analysis of parameters influencing the selection of subtilisin variants with changed substrate specificityfrom libraries using phosphonylating inhibitors

Many attempts have been made to endow enzymes with new catalytic activities . One general strategy involves the creation of random combinatorial Librar ies of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypep tides with desired properties by establishing a close Link between the poly peptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus len tils on the surface of filamentous fd phage and to develop selection scheme s that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenz yme that mature in active enzyme autocatalytically. They have a broad subst rate specificity but exhibit a significant preference for hydrophobic resid ues and very Limited reactivity toward charged residues at the P4 site in t he substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is rele ased from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by rea ction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-L-Ala-L-Al a-L-Pro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 10 4 and 107 forming part of the S4 pocket have been randomised, with (biotin- N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-L-Lys-L-Ala-L-Pro-Phe(P )-diphenylester ter allowed us to select enzymes with increased specific ac tivity for a substrate containing a lysine in P4. Parameters influencing th e selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated. (C) 2000 Academic Press.