Paradisi Rese@rch

Discovery Evolution Application
Francesca Paradisi
Prof. Francesca Paradisi

Associate Professor in Biocatalysis and Enzyme Engineering

About me...

I completed my MSc in Organic Chemistry from the University of Bologna in 1998 under the supervision of Prof. Cainelli. In 2002 I completed my PhD at the same institution with a thesis on the synthesis of non-natural amino acids via diketopiperazine scaffolds. During my PhD I spent a summer in Dublin as a visiting student in Trinity College working with Prof. Thorri Gunnlaugsson and it was a fantastic experience. So after my PhD I had no doubt I wanted to go abroad and I joined the group of Prof. Paul Engel at University College Dublin as Post-Doctoral Fellow where I discovered the wonders of biocatalysis. I remained in Paul’s group until 2005 developing several projects mainly focused on amino acid dehydrogenases and their applications in the synthesis of non-natural amino acids. I owe Paul all I know about enzymes and their reaction mechanisms!

I spent then a few months in Enzolve Technologies in 2005, a spin-off company of UCD where I worked on the use of mutant dehydrogeanse enzymes for neonatal screening of metabolic disorders. In 2006 I won the lotto and was appointed College Lecturer in Chemical Biology at the UCD School of Chemistry. I was promoted in 2014 to Senior Lecturer. I was fortunate to be always surrounded by a team of excellent students that made my research always interesting, certainly challenging, and thankfully rewarding.

A had the great opportunity to spend the summer of 2015 in UC Davis in California as a visiting academic and I joined the group of Dr. Justin Siegel who gave me the possibility of expanding my research to a different class of enzymes (glycosyl hydrolyses) and getting my hands dirty in the lab again was awesome as they say.  While I was in Davis and I thought things couldn’t get any better, I was offered the position of Associate Professor in Biocatalysis and Enzyme Engineering in the School of Chemistry at the Univeristy of Nottingham. I have started this new adventure since February 2016.

Outside my work life, I have a husband and two children, Oliver and Martina, and a cat. I love skiing and swimming and travelling too.

Research Highlights

Green Chemistry Paper : “Continuous flow biocatalysis: production and in-line purification of amines by immobilised transaminase from Halomonas elongata”

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OBC Paper : “Stereoelectronic effects in the reaction of aromatic substrates catalysed by Halomonas elongata transaminase and its mutants”

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Latest Post

May 2017: Well done Larah!

Larah Bruen recently received a prize for "Best Talk" at the Third Year Talks Programme in University College Dublin. This annual event brings together third year PhD students from UCD, Trinity College and Dublin Institute of Technology to present their research to...
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Research

Our Projects :
Alcohol dehydrogenases

Enzymes have evolved to perform highly regioselective and stereoselective reactions and can, under mild conditions, selectively resolve or create chiral centres without the need for deprotection chemistry. Alcohol dehydrogenase (ADHs) reduce pro-chiral ketones to optically active secondary alcohols, highly desirable chiral intermediates for the pharmaceutical industry. Halophilic enzymes are adapted to function in areas with low water activity; they posses a high number of acidic residues, have a low lysine content, and an increased number of small hydrophobic residues. The mechanisms governing haloadaptation of protein from haloarchaeal microorganisms could be transferable to an organic solvent environment.

We explored several ADH’s including HsADH2 from Halobacterium salinarium, HvADH1 fromHaloferax volcanii and HmADH12 from Haloarcula marismortui, His-HLADH-EE from Equus Caballus). Within our lab, we fully characterised ADH2 from Haloferax volcanii as the most stable halophilic enzyme, with the broadest substrate specificity and the highest optimal working temperature. The aim of this project was to generate a novel catalyst with broader substrate specificity and higher tolerance to organic solvents.

Homology modeling and docking studies, in conjuncion with site-directed mutagenesis, have been appied to unravel the substrate specificity of ADH2 from Haloferax volcanii. Solid and liquid colorimetric screening are currently being optimized for the halophilic system to identify hits from mutant libraries. We have expanded our searches to cold-adapted ADH’s which function at lower temperatures compared to their halophilic homologs. Our current research also looks at the behaviour of halophilic ADH’s in the presence of ionic liquids.

Amino transferases

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.

We recently identified, cloned, and expressed a putative amine transaminase (HEWT) from the genome of the bacterium Halomonas elongata; this is a moderate halophile which evolved an organic-osmolyte strategy to overcome the high osmotic pressure of its natural environment. H. elongata does not exhibit the extensive adaptation of the intracellular macromolecules present in archaea halophiles, which preferentially evolved to maintain their proper osmotic pressure by the accumulation of high cytoplasmic concentration of potassium chloride. On the contrary, H. elongata preserves an appropriate cytoplasm osmotic pressure by accumulation and/or biosynthesis of organic solute.

With its 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, such as continuous flow biotransformation and enzyme evolution to improve the catalytic feature against non natural substrates.

Glycosyl hydrolyses

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.

Results show a very interesting behaviour specially in the presence of miscible organic solvents; thermophilic proteins markedly favor DMSO vs CH3CN, while the opposite is true for the cold-adapted enzymes. Halophilic proteins have a more uniform behaviour with all tested solvents including alcohols (which are well tolerated also by thermo- and psychrophilic GH1). As expected, thermal stability and activity of the different enzymes reflects the environmental adaptation of the original organisms with thermophilic proteins being significantly more stable (as well as active) at 50-60 degrees than the cold adapted.

Kinetic parameters, measured with four different sugar substrates (PNP-Glu, PNP-Gal, PNP-Fuc, PNP-Xyl) also highlight significant differences among the GH1s specially for what concerns the affinity for the various substrates. A superimposed model of the active sites of all the enzymes reveals only very minor amino acidic alteration (thought the overall sequence identity is low) which appear not to rationalize the differences observed experimentally, however once again a pattern is apparent among cold adapted vs salt adapted vs thermophilic GH1s indicating that environmental adaptation may be a relevant factor to consider for biocatalytic activity.

Metallo enzymes

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 the 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 coordinated to the substrate and facilitate the 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?

Peptidomimeticts

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 (Xc).

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).

Group Members

Dr. Jennifer Cassidy

I am currently a Post-Doctoral researcher with Dr Paradisi. During my PhD in this group, my research focussed on the discovery and characterisation of novel enzymes from halophilic organisms.

Research area

My research interests include protein expression, purification, mutagenesis, homology modelling and in silico docking. At present, I am studying the effects of mutations on the substrate scope of alcohol dehydrogenases and how ionic liquids can be applied as green solvents.

Larah Bruen

I am a 3rd year PhD student focused on the area of biocatalysis, following a B.Sc. Chemistry degree in National University of Ireland, Galway.

Research area

My research is focused on an alcohol dehydrogenase from a halophilic archaea, and currently I am working to expand the substrate scope of this enzyme through in-silico docking and site-directed mutagenesis. In parallel, I am currently working on a novel method of screening mutant libraries of alcohol dehydrogenases.

Eimear Hegarty

I have recently joined the Paradisi Research Group as a 1st year PhD student having just completed my B.Sc. in Industrial Biochemistry at the University of Limerick (Ireland).

Research area

With a keen interest in biocatalysis and enzyme engineering my research will focus on exploiting the hyper-salinity requirements of Haloferax volcanii to the advantage of biotechnology by developing it as a new expression system for proteins of industrial relevance.

Dr. Martina Contente

I am a post-doctoral researcher currently working in Professor Francesca Paradisi’s research group at the University of Nottingham.

Research area

I am very interested in Biocatalysis research in particular in using enzymes or engineered whole cells as catalysts for the preparation of chiral pharmaceutical intermediates. I am working on immobilized transaminase from halophilic microorganism and its different mutants for the transformation of various substrates, interesting building blocks for the synthesis of pharmaceuticals. I am also exploring new synthetic techniques for flow chemistry reactors.

Benedetta Guidi

I am a PhD student in chemistry at University of Milan (Italy) currently visiting Dr. Paradisi’s research group

Research area

I am currently attempting to express the halophilic enzymes from the marine bacterium Virgibacillus pantothenticus in the archaea host Haloferax volcanii. Specifically, my PhD project is focused on the marine environment potential as a source of new and unusual biocatalysts. The idea concerns in exploiting the ability of these novel enzymes to catalyse enantioselective conversion of pharmaceutical chiral intermediates.

Matteo Planchestainer

I am a 3rd year PhD student in Biocatalysis, in the context of enzymology and protein engineering mostly of enzyme from extremophiles.

Research area

I am interested in redesigning enzymes by engineering novel properties and understanding their structural behaviour. I am working on the evolution of a halophilic amino transaminase to accept non-natural substrates. I am carrying out a comparative study on glycosyl ydrolases from different extreme organisms to investigate how environmental adaptation affects enzymatic properties. Finally, I am also investigating metallo-enzymes in attempt to understand the role of the residues involved metal coordination by developing semi-synthetic biocatalysts.

David Roura Padrosa

I am a 1st year PhD student focused on the area of flow chemistry combined with biocatalysis. I studied a Bachelor degree in Biology and an MSc in Molecular Biology and Biomedicine at the University of Girona.

Research area

I joined Dr. Paradisi group to widen my professional horizons by gaining new  knowledge and experience in the study and application of enzymes. My main interests are protein expression and characterization, directed mutagenesis and in silico approaches for the optimizati on and better understanding of enzyme functions.

Alessandro Orlandini

I joined Dr. Paradisi group to widen my professional horizons by gaining new knowledge and experience in the study and application of enzymes. My main interests are protein expression and characterization, directed mutagenesis and in silico approaches for the optimization and better understanding of enzyme functions.

Research area

I am collaborating with the Synthetic Biology Research Centre on the assembly of biosynthetic pathways for the production of 3-hydroxypropionic acid (3-HPO) and its derivatives. Within this project my efforts will focus on trying to evolve a specific aminomutase capable of a biotransformation that doesn’t occur in nature. The evolution of this enzyme will significantly impact the overall process.

Dr. Jennifer Cassidy

I am currently a Post-doctoral researcher with Dr Paradisi. During my PhD in this group, my research focussed on the discovery and characterisation of novel enzymes from halophilic organisms.

Research area

My research interests include protein expression, purification, mutagenesis, homology modelling and in silico docking. At present, I am studying the effects of mutations on the substrate scope of alcohol dehydrogenases and how ionic liquids can be applied as green solvents.

Matteo Planchestainer

I am a 3rd year PhD student in Biocatalysis, in the context of enzymology and protein engineering mostly of enzyme from extremophiles.

Research area

I am interested in redesigning enzymes by engineering novel properties and understanding their structural behaviour. I am working on the evolution of a halophilic amino transaminase to accept non-natural substrates. I am carrying out a comparative study on glycosyl ydrolases from different extreme organisms to investigate how environmental adaptation affects enzymatic properties. Finally, I am also investigating metallo-enzymes in attempt to understand the role of the residues involved metal coordination by developing semi-synthetic biocatalysts.

Larah Bruen

I am a 3rd year PhD student focused on the area of biocatalysis, following a B.Sc. Chemistry degree in National University of Ireland, Galway.

Research area

My research is focused on an alcohol dehydrogenase from a halophilic archaea, and currently I am working to expand the substrate scope of this enzyme through in-silico docking and site-directed mutagenesis. In parallel, I am currently working on a novel method of screening mutant libraries of alcohol dehydrogenases.

Dr. Martina Contente

I am a post-doctoral researcher currently working in Professor Francesca Paradisi’s research group at the University of Nottingham.

Research area

I am very interested in Biocatalysis research in particular in using enzymes or engineered whole cells as catalysts for the preparation of chiral pharmaceutical intermediates. I am working on immobilized transaminase from halophilic microorganism and its different mutants for the transformation of various substrates, interesting building blocks for the synthesis of pharmaceuticals. I am also exploring new synthetic techniques for flow chemistry reactors.

Benedetta guidi

I am a PhD student in chemistry at University of Milan (Italy) currently visiting Dr. Paradisi’s research group.

Research area

I am currently attempting to express the halophilic enzymes from the marine bacterium Virgibacillus pantothenticus in the archaea host Haloferax volcanii. Specifically, my PhD project is focused on the marine environment potential as a source of new and unusual biocatalysts. The idea concerns in exploiting the ability of these novel enzymes to catalyse enantioselective conversion of pharmaceutical chiral intermediates.

Eimear Hegarty

I have recently joined the Paradisi Research Group as a 1st year PhD student having just completed my B.Sc. in Industrial Biochemistry at the University of Limerick (Ireland). 

Research area

With a keen interest in biocatalysis and enzyme engineering my research will focus on exploiting the hyper-salinity requirements of Haloferax volcanii to the advantage of biotechnology by developing it as a new expression system for proteins of industrial relevance.

David Roura Padrosa

I am a 1st year PhD student focused on the area of flow chemistry combined with biocatalysis. I studied a Bachelor degree in Biology and an MSc in Molecular Biology and Biomedicine at the University of Girona.

Research area

I joined Dr. Paradisi group to widen my professional horizons by gaining new knowledge and experience in the study and application of enzymes. My main interests are protein expression and characterization, directed mutagenesis and in silico approaches for the optimization and better understanding of enzyme functions.

Alessandro Orlandini

I am a 1st year PhD student working in Paradisi Research Lab at the University of Nottingham. I completed both my Bachelor in Biotechnology and my MSc in Industrial and Molecular Biotechnology at the University of Bologna (Italy).

Research area

I am collaborating with the Synthetic Biology Research Centre on the assembly of biosynthetic pathways for the production of 3-hydroxypropionic acid (3-HPO) and its derivatives. Within this project my efforts will focus on trying to evolve a specific aminomutase capable of a biotransformation that doesn’t occur in nature. The evolution of this enzyme will significantly impact the overall process.

Those that shared the journey

Dr. Kevin Devine, Senior Lecturer, London Metropolitan University, UK
Dr. Daniele Balducci
Dr. Elena Lestini, Post Doc, Dublin City University, IE
Dr. Ann-Kathrin Liliensiek, Researcher, Federal Institute of Hydrology, Essen, DE
Dr. Maeve O’Neill, Team Leader, Biocatalysts Ltd, Cardiff, UKK

Dr. Elaine O’Reilly (2010), Assistant Professor, University of Nottingham, UK
Dr. Gabriele Gucciardo (2010), Team Leader, Almac Group, Northern Ireland, UK
Dr. Sabrina Devereux (2010), Project Manager, Horizon Pharma Ireland, IE
Dr. Leanne Timpson (2011), Novozymes,  Nottingham, UK
Dr. Daniela Quaglia (2012), Post Doctoral Researcher, Université de Montréal, Canada
Dr. Philip Conway (2012), Technical Development Chemist, GSK, Cork, IE
Dr. Lara Pes (2013), Post Doctoral Researcher, Weill Cornell Medical College, NY, USA
Dr. Keith Robertson (2013), Technical Development Chemist, GSK, Cork, IE
Dr. Diya Alsafadi (2013), Researcher, Royal Scientific Society, Jordan
Dr. Jennifer Cassidy (2015), Post Doctoral Researcher, University College Dublin, IE
Dr. Anita Wester (2016).

Cillin Mac Donnchadha (2009), LEO Pharma, Dublin, IE

Outputs

Journal Articles

Reviews & Book

Patents

August 2016: Big changes ahead!
The group will shortly officially move to Nottingham, and we will have a number of new researchers joining us, so we
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October 2016: Well done Anita!
Massive congratulations to Anita who successfully defended her thesis on the 4th of October 2016. Wishing you all the very
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March 2017: Lab B11 road trip
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October 2016: Nott’s crew… Check!
Here we are! The group is finally complete and ready to kick off this new adventure!!        
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September 2016: Leaving UCD
Emotional moment during Francesca’s leaving lecture in UCD few weeks ago. Many students, colleagues, and friends attended her fascinating summary of ten
Read more.
June 2016: The Tromsø Experience
Read more.
May 2017: Well done Larah!
Read more.
December 2016: Jenni’s poster astonishes at CSCB symposium
Read more.
October 2016: Shiny New Lab
Here we are! After a lot of unpacking and patience too, our lab is ready to run!      
Read more.
October 2016: Ben Feringa in Groningen
Pleasant surprise for Francesca e Matteo during the Novel Enzyme conference in Groningen (The Netherland) last week.   Bernard Lucas
Read more.

Collaborations

“A knotty puzzle may hold a scientist up for a century, when it may be that a colleague has

the solution already and is not even aware of the puzzle that it might solve.”

(Isaac Asimov, The Robots of Dawn)

Dr. Justin Siegel and Dr. Marc Facciotti, UC Davis, California, USA

Dr. Justin Siegel and Dr. Marc Facciotti, UC Davis, California, USA

Prof. Peg Gallagher and Dr. Ann Tallant

Prof. Peg Gallagher and Dr. Ann Tallant, Wake Forest University, North Carolina, USA

Dr. Cormac Murphy

Dr. Cormac Murphy, University College Dublin, Ireland

Prof. Prem Puri

Prof. Prem Puri, National Children’s Research Centre, Dublin, Ireland

Dr. Thorsten Allers

Dr. Thorsten Allers, University of Nottingham, UK

Dr. Adele Williamson

Dr. Adele Williamson, Tromso University, Norway

Prof. Martin Albrecht

Prof. Martin Albrecht, University of Bern, Switzerland

Prof. Francesco Molinari

Prof. Francesco Molinari, University of Milan, Italy

Dr. Alessandra Tolomelli

Dr. Alessandra Tolomelli, University of Bologna, Italy

Prof. Francesca Paradisi

Associate Professor in Biocatalysis and Enzyme Engineering

School of Chemistry |University of Nottingham| University Park Nottingham | NG7 2RD| UK

Phone: +44(0)115 74 86267

Email: francesca.paradisi@nottingham.ac.uk

School of Chemistry

University Park, Nottingham NG7 2RD, Regno Unito

Prof. Francesca Paradisi

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THANKS FOR VISITING US

Francesca Paradisi Research © 2016-2017  - School of Chemistry - University of Nottingham

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