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Engineering promiscuous activities in N-N bond forming enzymes

Project description

We are interested in exploiting the promiscuous activities of N-N bond forming enzymes (NNzymes) to access a wide variety of N-N bond containing functional groups to manufacture pharmaceuticals and precursors thereof.

NNzymes from natural product biosynthetic gene clusters catalyze unique chemical reactions, but are tremendously underexplored for their potential to be applied as sustainable biocatalysts in the pharmaceutical industry. Versatile enzyme platforms that connect nitrogen-nitrogen (N-N) bonds therefore represent currently one of the largest gaps in biocatalysis. NNzymes have very recently been investigated in the context of their native reactions, but structural knowledge on how these powerful enzymes perform the challenging formation of an N-N bond is still very limited. This is the reason that their reactivity toward non-natural substrates and transformations has not yet been systematically explored.

Thus, the proposed project aims to engineer a NNzyme from Kutzneria sp. (called KtzT) towards intermolecular N-N bond formations. The natural activity of KtzT involves the intramolecular formation of the N-N bond in piperazic acid, but yet no knowledge is available whether this enzyme can also perform intermolecular N-N bond formations on aromatic substrates, which would provide access to important drugs (e.g. benserazide).

Since no crystal structure of KtzT is available yet, the project will pursue research along three lines: 1) improve existing expression and purification protocols for solving the crystal structure of KtzT; 2) perform directed evolution by random mutagenesis based on epPCR of the whole enzyme, and 3) develop a high-throughput assay to screen resulting libraries for novel activities in 96-well format.

Finally, the biocatalytic properties of identified ‘hits’ will be studied and the improved variants will be applied for the synthesis of model products.


The master research project will be conducted in the Synthetic Biotechnology group of Dr. Sandy Schmidt. Our laboratory is well equipped for molecular biology, enzymology, protein engineering, and biocatalysis work. We offer a stimulating research environment for a student interested in these topics. There are also good contacts with the pharmaceutical industry.


We are looking for highly motivated students interested in performing state-of-the-art protein engineering work.


For further information, please contact Dr. Sandy Schmidt.
Chemical and Pharmaceutical Biology, GRIP
Building 3215, room 9.29
Antonius Deusinglaan 1
9713 AV Groningen

Laatst gewijzigd:13 oktober 2022 13:17