Structure-activity relationship analysis of the nitrilase form the cyanobacterium Synechocystis sp. PCC6803 through site-directed mutagenesis: improving catalytic properties for bioprocesses

• Antragstellerin: Dr. Maria Laura Magri
• Fachliche Zuordnung: Mikrobielle Ökologie und Angewandte Mikrobiologie
• Förderung: Förderung von 2007 bis 2008
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 36228800

A novel nitrilase has been previously detected by an in silico approach in the genome of Synechocystis sp. PCC6803 and recombinantly expressed and characterized. This nitrilase demonstrated a preference for aliphatic nitriles, a quite unusual property among these enzymes; therefore, it represents a unique opportunity to study structure-activity correlations for an “aliphatic” nitrilase. In the proposed project the enzyme will be rationally modified by site-directed mutagenesis and the enzymatic properties of the mutants analyzed. The results obtained will allow to identify the structural features which enable the enzyme to preferentially convert aliphatic substrates and this will finally result in a detailed model that relates certain amino acid residues with certain functions and abilities of the enzyme.Nitrilases that are active on alpha-substituted nitriles are of great interest for industrial biotransformations. The enantioselective hydrolysis of these nitriles produces chiral alpha-substituted carboxylic acids, valuable chemicals of commercial interest. The ability of the Synechocystis nitrilase to transform these alpha-substituted nitriles will be analyzed and genetically improved. It will be attempted to obtain point mutants with improved characteristics in terms of substrate specificity, enantioselectivity and/or specific activity towards these substrates. The mutated enzyme will be purified and immobilized for operational stability and the feasibility of applying this nitrilase to larger scale biotransformations will be analyzed in terms of yield, stability and costs.This project will contribute to structural knowledge of nitrilases and will also improve the industrial availability of these enzymes for a wide range of potential substrates.

• DFG-Verfahren: Forschungsstipendien
• Gastgeber: Professor Dr. Andreas Stolz