dr. Linda Otten

T: 015 - 27 89969
E: L.G.Otten@remove-this.tudelft.nl

Room C2.
Van der Maasweg 9
2629 HZ Delft
The Netherlands

 

 

Read more about Linda here.

Molecular enzyme evolution

Many enzymes are superior catalysts with high regio-, chemo- and stereoselectivity and are able to catalyse a great variety of chemical transformations. Unfortunately, enzymes are optimised for natural processes and conditions during evolution and not for specific, non-natural biotransformations in a harsh industrial environment.

In order to tune enzymes towards specific reactions we use knowledge from nature on different levels. First of all we use the Directed Evolution technique in which we mimic natural evolution processes in a test tube by introducing mutations in a random or site directed way and recombining different mutations afterwards. This evolution process can be directed towards the desired enzyme by applying selective pressure (survival of the fittest) or by screening all mutant enzymes in order to find the desired properties. Setting-up robust and cost-effective screening methods is one of the research lines in this area. A fully equipped screening lab including pipetting and picking robots is available.

Since it is impossible to probe all potential mutations in an enzyme we use bio-informatics tools to visualise and calculate the most promising mutations for an envisaged reaction.  These tools use existing enzyme structures and known mutations to compute new activities, which can be tested in the lab. This in silico pre-selection reduces the screening effort and improves the chance of finding the best biocatalyst. Finding the right mix of calculation and randomness in preparing a library is crucial to a successful outcome.

Generic enzyme assays

Enzymes are superior catalysts of chemical reactions. In order to measure the performance of an enzyme, usually, changes in substrate or product concentrations are measured. This means that, in principle, for every enzyme and every substrate/product a different assay has to be developed. This is currently a bottleneck in high-throughput approaches to systematically engineer industrially useful biocatalysts.

Calorimetry offers a way to bypass the need to measure substrate/product levels, but instead measure the heat that is being developed (or consumed) during a chemical reaction. Current state-of-the-art microcalorimeters are able to accurately measure the small heat changes during enzyme catalysed reactions. However, the proper analysis to obtain accurate kinetic parameters (Vmax, Km, KI) for any enzyme still requires development.

 

© 2017 TU Delft

Metamenu