Directed evolution of a stable transaminase – a Pilot study

Sammanfattning

Biocatalysis is an up-and-coming field in the search for sustainable alternatives to chemical catalysis. Biocatalysts are biodegradable, non-toxic and made from renewable sources. With their great stereo- and enantioselectivity they are especially attractive for the manufacture of pharmaceuticals and fine chemicals. Still, biocatalysis faces challenges in industrial applications, the most pressing being the fragility of enzymes. As they have evolved to function optimally in their natural conditions, in the cell, they are easily destabilized in non native conditions. A promising application of biocatalysis is the use of transaminases to catalyze the substitution of a keto- or aldehyde-group with an amino-group to form amines. This paper sets out to survey the current methodologies of enzyme engineering through directed evolution, with the aim to propose a solution to improve the operational stability of a native transaminase. The transaminase in question is the class III aminotransferase SpATA from the bacteria Silicibacter pomeroyi which was first discovered in 2012. The result is a research-based protocol describing a low-cost and high throughput method for improvement of operational stability of transaminases, in organic solvent, through directed evolution. The protocol proposes the procedure of generating a mutant libraryvia error prone PCR. The desired mutants are then selected through colony-based screening where o-xylylenediamine dihydrochloride, in a solution of DMSO, is used as the amine donor. The following reaction would yield a visibly colored product, making screening simple and efficient. The genetic material of the selected colony would then be retrieved and amplified before repeating the cycle of directed evolution.