Enzymes are amazing catalysts which effortlessly, and with extreme precision, regulate reactions of biosynthetic pathways in a biological system. We are taking enzymes from different sources, those that offer the optimal characteristics for our chemistry, and recreate artificial cascades ex-vivo using a flow reactor. Enzymes we produce, are directly and covalently immobilised onto solid supports and packed into column reactors. Each enzymatic biotransformation can be individually optimised so that the starting material is fully converted into the product by the time the flow takes it out of the column. Two, three more columns containing different enzymes can be connected sequencially, and several steps can be performed with high efficiency and fast flow.  Continuous flow biocatalysis is the ultimate evolution of continuous processing and has brought enzymes to a whole new dimension. Have a look at the enzymes we work with below.

We have a special interest in enzymes from extremophilic microbes which have adapted to life in  high salt environments (Dead Sea, salterns, etc). The mechanism developed by the organisms to  withstand molar concentrations of NaCl is either by accumulation of high amounts of KCl in the cytoplasm, or by efficient production of soluble organic molecules. In both cases the enzymes expressed by these bacteria or achaea have evolved structurally to be able to perform in a water depleted system and therfore are excellent candidates for synthetic applications which normally require organic solvents.

Transaminases are a family of enzymes with high potential in biotechnological applications. They can be very useful for the enantioselective production of a series of compounds with high value such as chiral amines and enantiopure amino alcohols which find use in many chemical fields; above all, for the synthesis of biologically active compounds. The synthesis of enantiopure amines by enzymatically catalyzed reactions presents several advantages as an alternative to traditional approaches such as mild reaction conditions, high stereoselectivity, fewer synthetic steps, potential total substrate conversion and no environmental issues unlike in the case of transition metal catalysts. 

The reaction is reversible and transaminases can also be used for the mild oxidation of amines into aldehydes and ketones.

We have isolated and investigated a transaminase from Halomonas elongata (HEWT) which has shown high enantioselectivity, large substrate spectra and stability in organic solvents, HEWT a highly suitable enzyme for biotechnological applications in the production of chiral amines. For these reasons, we are currently developing different projects regarding its application, including continuous flow biotransformation and enzyme evolution to improve its catalytic performance against non natural substrates.

Redox enzymes encompass a number of different biocatalysts of great industrial interest. We have a collection of enzymes from both salt adapted and mesophilic sources such as alcohol dehydrogenases (ADHs) and ketoreductases (KR) which we have used to synthesise chiral alcohols (in the reductive direction) and in the dynamic kinetic resolution of profenic aldehydes. These enzymes pose an additional challenge as they are cofactor dependent (NAD(P)H). The cofactor cannot be used stoichiometrically as it is prohibitively expensive. We have therefore developed simple recycling strategies which are easily adaptable to flow biocatalysis.

Hydrolytic enzymes are another large family of very useful biocatalysts. We have recently expanded our research into reactions which include ester and amide hydrolysis (and eventually also synthesis) as part of an artificial enzymatic cascade. A selection of esterases and amidases are now part of our library. In a separate project six ß-glycosyl hydrolyses, all classified as GH1, have been selected to investigate how environmental adaptation affects biocatalytic properties, as well as stability at different temperatures, pHs and in the presence of solvents. Furthermore, we analysed whether the subtle differences in the active site of the proteins may indicate a pattern for a preferential substrate recognition. GH1 from Thermobaculum terrenum (Tte) and Thermus Nonproteolyticus (Tno) have been chosen as thermophilic examples, Halothermothrix orenii (Hor) and Halobacillus halophilus (Hha) as two halophilic proteins (with Hor being also thermophilic), and finally Colwellia psychrerythraea (Cps) and Marinomonas profundimaris (Mpr) as psychrophilic ones. 

Metals are part of biological molecules and cover different roles. It is fascinating how inorganic elements are pivotal in many cases for biological activity. In collaboration with Prof. Albrecht in Switzerland we have started looking at the amino acid residues that hold in place a catalytic metal in the active site of a number of enzymes. Specifically we are investigating the role of histidine as a ligand in copper proteins such as azurin. Azurin is a bacterial blue copper protein that acts as electron shuttle in bacteria denitrification process, where its metal center undergoes oxidation-reduction between Cu(I) and Cu (II) during the electron transfer. We have taken azurin as a model to evaluate the hypothesis that carbon-metal bonding in proteins (other than nitrogen-metal bonding) could play an important role since the interconversion of histidine between the weakly π-acidic imine (N-bound form) and the strongly σ-donating C-bound carbene tautomer2 will have substantial implications on the activity and oxidation-reduction chemistry of the coordinated metal center.

Under this same heading it is worth mentioning that our ADH proteins contain a catalytic zinc in the active site which coordinates to the substrate and facilitates its biotransformation. We are interested in investigating better the role of the zinc and its role in substrate selectivity. What happens if we change the metal for a different one? Can substrates other than alcohols/carbonyls be accommodated and transformed?

Peptidomimetics are widely used in medicinal chemistry as therapeutic agents to act as agonists or antagonists on receptor or enzyme targets. They are also valuable tools for investigating the relationship between peptide structure and function as they elucidate key residues that are required to achieve a desired biological response. The incorporation of cyclic structures into peptidomimetics was shown to decrease their conformational flexibility resulting in increased selectivity and affinity with their target and improved enzymatic stability and bioavailability.

While trying to synthesise the side chain of an non-natural amino acid, we serendipitously discovered a side product of our reaction which looked more interestingly than the product we wanted to make. In 2013 we published the synthesis of the non-natural δ-amino acid ACCA (cis-3-(aminomethyl)cyclobutanecarboxylic acid) (O’Reilly et al. 2013, Amino acids, 511-518). ACCA contains a cyclobutane ring that gives the molecule conformational rigidity and locks the amino and carboxylic acid group in a cis conformation.

ACCA shows structural similarity to glutamate, making it an interesting building block for the synthesis of glutamate analogues. A set of dipeptides containing ACCA and a natural amino acid (glycine, valine, phenylalanine, cysteine) have been synthesized and are evaluated for their application as glutamate analogues selectively targeting the cystine-glutamate exchanger (X_c^–).

Angiotensin-(1-7) is a heptapeptide hormone of the renin-angiotensin system (RAS). We are particularly interested in the potential use of Angiotensin-(1-7) in anti-cancer therapy. Its anti-angiogenic and anti-proliferative properties have been investigated Prof. Gallagher at Wake Forest University (NC, USA). A phase II clinical trial recently completed by the Wake Forest School of Medicine successfully showed its therapeutic potential as a second or third line treatment of patients with unresectable or metastatic sarcomas.

Ang-(1-7) mediates its biological responses by interacting with the G protein-coupled mas. It is readily cleaved by the dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) resulting in short-half life. We have synthesised a series of Ang-(1-7) analogues incorporating ACCA to decrease enzymatic metabolism thereby overcoming the problem of the short half-life of the endogenous heptapeptide and improving its properties as a therapeutic agent. This work has led to a patent application (Gallagher et al. U.S. Provisional Application No. 62/266,410, 2015).