Project Details
Projekt
Lipase function and folding analyzed by in vivo high throughput screening of combinatorial enzyme libraries
Subject Area
Biochemistry
Term
from 2004 to 2011
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 5427318
Directed evolution constitutes the method of choice to create biocatalysts with desired properties like improved activity or stability, unique substrate specificity or high enantioselectivity. Many different and powerful methods have been developed to generate large mutant libraries consisting of hundreds of thousands up to billions of variant proteins. However, it is still very difficult and often impossible to identify among all these variant proteins the biocatalyst having the desired property. We present here a novel concept for ultra-high throughput screening of biocatalysts in vivo by using a combination of cell-surface display and fluorescence-activated cell sorting. The proposed project aims at demonstrating that a FACS-based ultra-high throughput screening technology enables the rapid and efficient identification of enzymes with predefined and novel substrate specificities. Lipases and esterases form the most important group of biocatalysts for biotechnological applications. Bacteria belonging to the genus Pseudomonas produce several different lipases and esterases which have been shown to catalyze the hydrolysis and synthesis of a wide range of different synthetic substrates. Several Pseudomonas lipases and esterases are already being used for different biotransformations by the biotechnology industries. Therefore, an esterase from Pseudomonas aeruginosa will be used as a model protein to optimize by directed evolution its substrate specificity and enantioselectivity. Furthermore, the best-performing newly evolved enzyme variants will be subjected to a detailed biochemical and structural characterization.
DFG Programme
Priority Programmes
