RESUMO
Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Evolução Molecular Direcionada/métodos , Inibidores Enzimáticos/farmacologia , Biblioteca de Peptídeos , Fosfatos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/química , Hidrólise , Modelos Moleculares , Estrutura Molecular , Mutação , Fosfatos/química , Conformação Proteica , Estrutura Terciária de Proteína , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Bacteriófago M13/enzimologia , Bacteriófago M13/genética , Lipase/metabolismo , Fagos Bacilares/enzimologia , Fagos Bacilares/genética , Bacillus subtilis/metabolismo , Células Cultivadas , Clonagem Molecular , Inibidores Enzimáticos/química , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Isomerismo , Lipase/química , Lipase/genética , Organofosfonatos/química , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
The esterase-catalyzed enantioselective hydrolysis of the fluoroacetate of pantolactone leads to fluoroacetic acid, a toxic compound which inhibits the growth of esterase-producing yeast; this forms the basis of an ee-assay.