The main objective of this proposal is to generate and improve techniques for forced evolution of desired enzymatic activities.
Two target enzymes 1. Savinase (subtilisin 309) and 2. Lipolase (H. lanuainosa Lipase) have been chosen for attempts to 1. change substrate specificity (charged residue in P4) and 2. optimize performance in a non-natural environment (improved substrate binding in presence of detergent). The sub-objectives are:
1) To develop in vitro selection principles based on catalytic activity.
2) To develop techniques for highly efficient regional in vivo mutagenesis.
3) To develop techniques for efficient in vivo recombination between libraries.
4) To extend on structure-function knowledge of serine proteases and lipases by X-ray crystallographic structure determination of a large number of variants, individually as well as complexed with inhibitors.
5) To further develop techniques for semiautomatic electron-density fitting and model building.
Libraries of the enzymes mono-valently displayed as M13 GIII fusion proteins, and variegated in regions selected on basis of structure-function relations, will be generated. Selection of improved variants will be performed by reaction with suicide inhibitor-labels or covalent transistion state analogs, allowing subsequent panning of labelled phages for amplification. After several selection/amplification cycles, possibly exerted at gradually increased selection pressure (set by concentration of inhibitor and reaction time), the resulting population is characterised by DNA sequencing and activity assays. Selected variants will be recloned in adequate expression organisms and purified in sufficient quantities for the subsequent biochemical characterization and 3-D structure determination by X-ray crystallography. In order to extend the space of diversity obtainable through exsting techniques (appr. 10 mill.), we will attempt to establish a regional plasmid specific and highly error prone replication system, for the generation of regional diversity in vivo. Further, we will attempt the construction of forced in vivo recombination systems, allowing efficient in vivo recombination among different libraries. Construction of similar in vivo mutagenesis and recombination systems in B. subtilis will facilitate generation and screening of enzyme variants in this production relevant organism.
Overall this project aims to improve the techniques available for the development of enzymes with novel properties, and at the same time provide detailed structure -function relationships which can be used in rational design.
Funding SchemeCSC - Cost-sharing contracts
100 44 Stockholm
YO1 5DD York