We have a special interest in enzymes with inherited stability. We believe they offer a better starting point to advance the implementation of biocatalysis on large scale. While extremophilic microbes which have adapted to life in  peculiar environments offer an excellent source of biocatalysts, we are also looking at mesophilic organisms which, for less clear reasons, also offer particularly stable enzymes. We identify new enzymes by a combination of genomic database scanning and in-silico modelling to handpick possible good hits exploiting our understanding of structural features which induce stability. Mutagenesis is of course also a very powerful tool to increase stability (quaternary structure stabilisation is key) and of course enzyme immobilisation. We have nowadays extensive experience in a range of immobilisation strategies which can be extremely efficient in lengthening the lifespan of a biocatalyst. For a review on this topic click here.

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 number of transaminases, one of our best his has been the one 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. This enzyme has been successfully incorporated in many flow-based project and continues to be our go to biocatalyst for amination reactions. However, we have expanded our very own transaminases kit to include an R-selective enzyme from Thermomyces stellatus which is highly stable and can be produced in excellent amounts. 

Acyl-transferases are another very nice class of enzymes which is normally used to make esters. We have developed several projects on an acyl-transferase from Mycobacterium smegmatis which we have used for the scale up of the synthesis of melatonine (an amide) and flavour esters.  

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. NADH oxidases (NOXs) can also be combined for the recycling of the cofactor when the primary reaction is the oxidation of alcohols.

Amino acid dehydrogenases are also redox enzymes which we use often in combination with other biocatalysts (transaminases for example, but also imine reductases, acyl-transferases etc) and have proven very useful in hydrogen-borrowing systems for the production of valuable chemicals.

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 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. We have looked at this enzymes also in the context of food chemistry applications, such as wine production and hydrolysis of valuable molecules from their glycol-conjugate form.

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 enzymatic cascades, leading to a variety of different molecules, ex-vivo, using a flow reactor. When compared with a microbial machinery, optimised multi-step enzymatic syntheses in flow avoid all unnecessary pathways and energy draining process which are typical of a cell, as well as the physical barrier to substrates and products which is represented by the cellular wall.  

The enzymes we produce, are immobilised onto a variety of 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 or 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. Flow-chemistry increases the sustainability of a process, specially when the “waste” can be also recycled. 

However, flow biocatalysis can be integrated with other technologies. We are looking at projects where we can generate starting materials from engineered microbial strain (growing on cheap carbon sources) and feed the substrate-rich broth in the flow system for further modifications which are more efficiently carried out extra-cellularly. Exciting times ahead!

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 here in Bern 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 tautomer is plausible and may have substantial implications on the activity and oxidation-reduction chemistry of the coordinated metal center. We have expanded this research to catalytically active enzyme (NiR) and we are interested in exploring the possible role of carbene incorporation in heme-containing biocatalysts. For recent articles on this topic click here and here.

Peptidic scaffolds. We have in the past worked on natural peptidic scaffolds to incorporate non-natural amino acids for applications in medicinal chemistry . 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. By stabilising such structure with a structurally rigid amino acid we labelled ACCA, the activity in vitro of the peptide was significantly enhanced.  

However, peptidic scaffolds can be used also to create artificial ligands in metal based catalysis and our research is now exploring the integration of synthetic ligands with enzymes to broaden the in-situ reactivity we can induce.

The overarching goal of our research is SUSTAINABILITY and we want to demonstrate that enzyme based catalysis can be integrated with more traditional synthetic methodologies. Exceptional progress has been madero date, and we are determined to add our contribution. Flow-chemistry, with its ability of compartmentalising reactions in separate reactors, offers an exceptional tool to advance in this filed. We are working on a range of new cascades to combine enzymatic biotransformaitons with synthetic steps. The compatibility of the reaction environments is important but no longer a must.  The versatility of our modular systems allows for each individual reaction step to be optimised and integrated in a cascade.