Novel thermostable enzymes for industrial biotechnology (THERMOGENE)

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

There is an increasing demand for new thermostable enzymes with enhanced performance and/or novel functionalities that could provide savings in time, money and energy for industrial processes in the areas of high value chemical production and many other "white" biotechnology applications.
Enzyme chemistry can make reactions feasible that are currently unavailable using conventional chemical methods. Use of enzymes for chemical processes is a route to lower energy consumption and reduced waste generation. In addition the selectivity of enzymatic processes reduces raw material costs and the safety issues surrounding the production of wasteful bi-products. Optimised enzyme production will lead to economically viable and cost effective, sustainable production.
THERMOGENE will focus on the discovery of selected transferase enzymes with known and potential commercial applications. These include enzymes able to transfer 2-carbon units, transketolases; able to transfer amine groups, transaminases; able to transfer isoprenyl or prenyl groups, prenyltransferases and able to transfer methyl and hydroxymethyl groups, methyl and hydroxymethyl transferases.
The use of enzymes in green chemistry and biotechnology is increasingly important. This is partially driven by the pharmaceutical sector in the development of new therapeutic agents that are required to be optically pure compounds. The number of new drug intermediates produced by enzymes is expected to rise significantly in the next few years.
One disadvantage of using enzymes is that proteins are often not stable during the reaction conditions required for the industrial process. This is still a major bottleneck in the commercial use of enzyme catalysts.
This proposal will concentrate on discovery of novel enzymes from marine and terrestrial environments. This will use newly sequenced genomes of thermophilic bacteria and archaea and metagenomes isolated from thermophilic environments. The latter will allow us to sample the DNA from organisms not able to be cultured in the laboratory and also the genetic material from viruses. The enzymes isolated from organisms able to grow at high temperatures will have higher stability in water based and solvent based conditions which are required for the industrial processes. It will also seek to discover enzymes with new and novel activities which are often found in these organisms that have evolved to have different fats in their cell membrane and different pathways in their metabolism.
At the end of the project we will identify many new biological catalysts that can be used to replace and work together with traditional chemical processes. This will lead to a greener and more sustainable environment for future generations.

Technical Summary

The application of enzymes in 'white biotechnology' for the synthesis of industrially important chiral compounds is becoming increasingly important for the pharmaceutical industry. Many companies who were traditionally not incorporating biocatalysis in their drug production programmes are now very keen to develop the technology. The wealth of genome data now available makes searching for enzymes using both advanced bioinformatic and substrate screening approaches an area for development. Also more of the classified enzyme groups are being investigated for their application in industrial biocatalytic processes which are often used with a traditional chemistry synthetic step.
This project will concentrate on the transferase class of enzymes. These include the transaminase enzymes that are already accepted for commercial production of chiral amines and amino alcohols. These are key building blocks in many important drugs. The transketolase enzymes that have also been demonstrated for use in large scale biocatalysis for the production of unusual sugars (Lilly et al., 1996). In addition other transfer enzymes including the soluble prenyl transferases (some unique to Archaea; Ohnuma et al.,2000) include trans-polyprenyl diphosphate synthases, cis-polyprenyl diphosphate synthases and ABBA aromatic prenyltransferases. The aromatic prenyltransferases found in bacteria allow a promiscous prenylation of different aromatic substrates which is advantageous for their chemoenzymatic synthesis of bioactive compounds (Heide, 2009). The methyl and hydroxymethyl transferases have potential for development as commercial biocatalyts (Vidal et al., 2005, Koehl, 2005).

Planned Impact

The THERMOGENE project will discover and develop enzymes that can transfer a variety of chemical groups from one molecule to another. The proposal will develop more industrially stable counterparts of two already studied transfer enzymes, that can transfer two carbon groups, transketolases and amine groups, transaminases. It has already been shown that these enzymes can work together in tandem to product high value chemical compounds. In particular the less studied class IV and class VI will be studied. In addition other transferase enzymes studied in any detail as regard for their application for commercial biocatalysis namely soluble prenyl transferases and methyl and hydroxymethyl transferases. This combination of different transferase enzymes will ensure a wealth of new thermally stable enzymes will be discovered with industrially relevant applications.
The enzyme discovery in WP2 will develop new bioinformatic approaches to search for different transferase enzymes in recently sequenced thermophilic genomes which have not been investigated to date. This will produce an impact into evolution of different transferase enzymes and their mechanism. It will also have an impact on the identification of new stable enzymes that can be used to drive the bio-economy in Europe.
The cloning, expression and enzyme production in WP 3 will develop new expression hosts and vectors that can be used to produce industrial scale quantities of soluble robust biocatalysts. This will have an impact on the over-expression of other thermophilic enzymes of industrial interest. The screening in this WP will have an impact on the development of new screening techniques where the technology can be transferred to screening of other biocatalysts.
The overall information gained will have an impact on the supply of enzyme preparations by partner 4 and other major pharmaceutical companies.
The biochemical and structural studies in WP4 will have an impact academically on our understanding of the structures of the different transferase enzymes and how this can help in our understanding of 'natural' methods of protein stabilization. It will also impact on our understanding of enzyme mechanism and substrate specificity.
Overall the results from the THERMOGENE project will make a major advance in enzyme discovery and will bring a variety of new transferase biocatalysts to the European industrial community in the area of 'white biotechnology'. The project will therefore be of the upmost importance to help European companies maintain their expertise and leading role in this area.
This project will provide cutting-edge tools and resources to the European and international scientific/biotechnological community, and also nucleate a European network of laboratories/industry aiming to exploit these tools and resources for biotechnological innovation.
The project will train early career scientists and graduate students in this multidiscplinary area and promote exchange between countries within Europe. It is anticipated for each partner to host at least one meeting in their own country and to encourage personnel employed and associated with the project to visit each the different laboratories in order to exchange expertise as appropriate at different stages in the research programme.

Publications

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Antranikian G (2017) Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island. in Extremophiles : life under extreme conditions

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Cutlan R (2020) Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases. in Biochimica et biophysica acta. Proteins and proteomics

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Ferrandi EE (2018) New Thermophilic a/ß Class Epoxide Hydrolases Found in Metagenomes From Hot Environments. in Frontiers in bioengineering and biotechnology

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Lebedev AA (2014) Space-group and origin ambiguity in macromolecular structures with pseudo-symmetry and its treatment with the program Zanuda. in Acta crystallographica. Section D, Biological crystallography

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Littlechild JA (2015) Archaeal Enzymes and Applications in Industrial Biocatalysts. in Archaea (Vancouver, B.C.)

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Littlechild JA (2015) Enzymes from Extreme Environments and Their Industrial Applications. in Frontiers in bioengineering and biotechnology

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Littlechild JA (2017) Improving the 'tool box' for robust industrial enzymes. in Journal of industrial microbiology & biotechnology

 
Description For enzymes to be used commercially they need to be stable to industrial conditions such as temperature and solvents. This project is developing stable new transfer enzymes, which can add different chemical functionalities to molecules, that have currently not been studied in detail for their biocatalytic applications. It aims to make them available to others so that new processes can be developed for industry to carry out chemical synthesis in an environmentally friendly and sustainable manner. This will result in new drug intermediates and other applications for food and domestic products.
The use of enzymes to carry out synthetic reactions contributes to a green and sustainable environment. It also reduces the use of petroleum based products and contributes to a circular economy where waste materials are used to produce new products. The enzymes developed on this project are both novel in their properties and are more stable and therefore better suited to industrial processes where they can be reused for several cycles of the reaction process.
Exploitation Route Specific enzyme production can be scaled up to larger production for marketing to interested companies
Other papers resulting from this grant will be submitted within the next 6 months
These include:
A 'Split-gene' transketolase from the hyper-thermophilic bacterium Carboxydothermus hydrogenoformans: structure and biochemical characterisation
Paul James, Michail N. Isupov, Simone Antonio De Rose, Christopher Sayer, Isobel Cole and Jennifer Littlechild (To be submitted 9th March to Frontiers in Microbiology)
New thermostable novel sugar transaminases- biochemical and structural characterisation
Paul James, Antonio Gonzalez, Simone De Rose, Christopher Sayer, Michail N. Isupov, Nils Bikeland and Jennifer A. Littlechild (manuscript in preparation)
New thermostable hydroxymethyl transferase enzymes -biochemical and structural characterisation,
Antonio Gonzalez, Christopher Sayer, Michail Isupov, Nils Birkeland and Jennifer A Littlechild (manuscript in preparation)
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.ex.ac.uk/biosciences/biocatalysis
 
Description The novel enzymes that have arisen from this project have received interest from Sigma Aldrich /Merck in collaboration with Prof Roland Wohlgemuth. Interest has been expressed to scale-up production of some enzymes with Biocatalysts, UK and to market them. Need to establish some follow-on funding to achieve these goals and further objectives.
First Year Of Impact 2018
Sector Chemicals,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description ERA-!B
Amount € 1,155,000 (EUR)
Funding ID ERA-IB-16-049 Subcontractor since BBSRC did not contribute to this call 
Organisation Science and Technology Norway 
Sector Academic/University
Country Norway
Start 02/2018 
End 01/2020
 
Description ERA-CoBiotech
Amount £412,012 (GBP)
Funding ID BB/R02166X1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Title Protein database 
Description Two entries for new protein structures generated from this project have been submitted to the protein data base Branched Chain transaminases 5MQZ and 5MR0 Other entries to follow for hydroxymethyl transferase and sugar transaminases 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Understanding of protein structure in respect of protein stability, substrate specificity and potential applications in industrial biotechnology 
 
Description Collaborations with Prof Elizaveta Bonch-Osmolovskaya and Prof Vladimir Popov, Russia, Prof Nils Birkeland, Norway, Prof Peter Schoheit, Germany 
Organisation Russian Academy of Sciences
Country Russian Federation 
Sector Public 
PI Contribution My group co-ordinated the project. Carried out the bioinformatics to search for new transfer enzymes in thermophilic genomes, cloned,over-expressed and structural characterised these enzymes. Main enzymes studied at Exeter, sugar transaminases, branched chain transaminases, split archaeal transketolases. Also carried out structural studies for other members of the consortium hydroxymethyl transfers, full length transketolases and collaborated on prenyl transferases with Russian group.
Collaborator Contribution Norway team carried out bioinformatics searches from their own marine metagenomes and other novel organisms from Norweigen oil wells. Carried out cloning and over-expression of selected proteins and their biochemical characterisation. Russian team composed of National Academy of Sciences for microbiology who isolated and sequenced new thermophiic genomes and biochemistry who carried out cloning, over-expression and biochemical and structural studies on prenyl transferases and branched chain transaminases. German team, metabolic links to thermophilic bacterial and archaeal metabolic pathways to aid cloning and over-expressed some transferase enzymes and carried out detailed kinetic studies with Exeter branched chain transaminases and biochemical characterisation of other split archaeal transketolase enzymes. Dr Roland Wohlegemuth from Sigma Aldrich was involved with the scientific advisory body
Impact 2 joint papers and another 1 ready for submission and 2 in preparation. Collaboration is multidisciplinary. Bioinformatics from thermophilic genomes and metagenomes, gene cloning and over-expression, enzyme biochemical characterisation, enzyme crystallisation and structural determination, enzyme mechanism, applications in industrial biocatalysis with industrial advisory partner, Prof Roland Wohlgemuth, Sigma/Aldrich/Merck.
Start Year 2013
 
Description Collaborations with Prof Elizaveta Bonch-Osmolovskaya and Prof Vladimir Popov, Russia, Prof Nils Birkeland, Norway, Prof Peter Schoheit, Germany 
Organisation University of Bergen
Country Norway 
Sector Academic/University 
PI Contribution My group co-ordinated the project. Carried out the bioinformatics to search for new transfer enzymes in thermophilic genomes, cloned,over-expressed and structural characterised these enzymes. Main enzymes studied at Exeter, sugar transaminases, branched chain transaminases, split archaeal transketolases. Also carried out structural studies for other members of the consortium hydroxymethyl transfers, full length transketolases and collaborated on prenyl transferases with Russian group.
Collaborator Contribution Norway team carried out bioinformatics searches from their own marine metagenomes and other novel organisms from Norweigen oil wells. Carried out cloning and over-expression of selected proteins and their biochemical characterisation. Russian team composed of National Academy of Sciences for microbiology who isolated and sequenced new thermophiic genomes and biochemistry who carried out cloning, over-expression and biochemical and structural studies on prenyl transferases and branched chain transaminases. German team, metabolic links to thermophilic bacterial and archaeal metabolic pathways to aid cloning and over-expressed some transferase enzymes and carried out detailed kinetic studies with Exeter branched chain transaminases and biochemical characterisation of other split archaeal transketolase enzymes. Dr Roland Wohlegemuth from Sigma Aldrich was involved with the scientific advisory body
Impact 2 joint papers and another 1 ready for submission and 2 in preparation. Collaboration is multidisciplinary. Bioinformatics from thermophilic genomes and metagenomes, gene cloning and over-expression, enzyme biochemical characterisation, enzyme crystallisation and structural determination, enzyme mechanism, applications in industrial biocatalysis with industrial advisory partner, Prof Roland Wohlgemuth, Sigma/Aldrich/Merck.
Start Year 2013
 
Description Collaborations with Prof Elizaveta Bonch-Osmolovskaya and Prof Vladimir Popov, Russia, Prof Nils Birkeland, Norway, Prof Peter Schoheit, Germany 
Organisation University of Kiel
Country Germany 
Sector Academic/University 
PI Contribution My group co-ordinated the project. Carried out the bioinformatics to search for new transfer enzymes in thermophilic genomes, cloned,over-expressed and structural characterised these enzymes. Main enzymes studied at Exeter, sugar transaminases, branched chain transaminases, split archaeal transketolases. Also carried out structural studies for other members of the consortium hydroxymethyl transfers, full length transketolases and collaborated on prenyl transferases with Russian group.
Collaborator Contribution Norway team carried out bioinformatics searches from their own marine metagenomes and other novel organisms from Norweigen oil wells. Carried out cloning and over-expression of selected proteins and their biochemical characterisation. Russian team composed of National Academy of Sciences for microbiology who isolated and sequenced new thermophiic genomes and biochemistry who carried out cloning, over-expression and biochemical and structural studies on prenyl transferases and branched chain transaminases. German team, metabolic links to thermophilic bacterial and archaeal metabolic pathways to aid cloning and over-expressed some transferase enzymes and carried out detailed kinetic studies with Exeter branched chain transaminases and biochemical characterisation of other split archaeal transketolase enzymes. Dr Roland Wohlegemuth from Sigma Aldrich was involved with the scientific advisory body
Impact 2 joint papers and another 1 ready for submission and 2 in preparation. Collaboration is multidisciplinary. Bioinformatics from thermophilic genomes and metagenomes, gene cloning and over-expression, enzyme biochemical characterisation, enzyme crystallisation and structural determination, enzyme mechanism, applications in industrial biocatalysis with industrial advisory partner, Prof Roland Wohlgemuth, Sigma/Aldrich/Merck.
Start Year 2013
 
Description ERA-IB Partner in Germany, Norway and Russia 
Organisation University of Bergen
Country Norway 
Sector Academic/University 
PI Contribution Partner in Exeter, UK Involved in cloning, biochemical and structural characterisation of thermophilic transketolaes,transaminases,prenyl transferases and their industrial applications in biocatalysis
Collaborator Contribution ERA-IB Partners on project THERMOGENE Academic and industrial Funding going into project from partners local funding Prof Peter Schoenheit, University of Kiel, Germany Enzyme cloning and biochemical characterisation Funding 362,000euro Bundesministerium fur Bildung und Forschung Prof Nils-Kare Birkeland, University of Bergen Funding 296,602 euro Research Council of Norway Moltech Ltd and Russian Academy of Sciences, Moscow 306,378euro
Impact Mid term report and presentation sent to ERA-IB and TSB Meeting in Brussels to disseminate work presented by Prof Jenny Littlechild Final Reporting Meeting in Berlin, Feb 2016 to disseminate work presented by Prof Jenny Littlechild Wrote article on THERMOGENE for ERA-net projects funded in same round for dissemination. Multidisciplinary project to discover new thermophilic transferase enzymes with industrial applications encompassing sampling of biological diversity, identification of new archaeal and bacterial thermophilic species, genome and metagenome sequencing, bioinformatic identification of enzymes of interest, gene cloning and over-expression, biochemical and structural characterisation of enzymes of interest, industrial applications in biocatalysis and other applications. Collaboration continuing and further papers in preparation
Start Year 2013
 
Description ERA-IB Partner in Germany, Norway and Russia 
Organisation University of Kiel
Country Germany 
Sector Academic/University 
PI Contribution Partner in Exeter, UK Involved in cloning, biochemical and structural characterisation of thermophilic transketolaes,transaminases,prenyl transferases and their industrial applications in biocatalysis
Collaborator Contribution ERA-IB Partners on project THERMOGENE Academic and industrial Funding going into project from partners local funding Prof Peter Schoenheit, University of Kiel, Germany Enzyme cloning and biochemical characterisation Funding 362,000euro Bundesministerium fur Bildung und Forschung Prof Nils-Kare Birkeland, University of Bergen Funding 296,602 euro Research Council of Norway Moltech Ltd and Russian Academy of Sciences, Moscow 306,378euro
Impact Mid term report and presentation sent to ERA-IB and TSB Meeting in Brussels to disseminate work presented by Prof Jenny Littlechild Final Reporting Meeting in Berlin, Feb 2016 to disseminate work presented by Prof Jenny Littlechild Wrote article on THERMOGENE for ERA-net projects funded in same round for dissemination. Multidisciplinary project to discover new thermophilic transferase enzymes with industrial applications encompassing sampling of biological diversity, identification of new archaeal and bacterial thermophilic species, genome and metagenome sequencing, bioinformatic identification of enzymes of interest, gene cloning and over-expression, biochemical and structural characterisation of enzymes of interest, industrial applications in biocatalysis and other applications. Collaboration continuing and further papers in preparation
Start Year 2013
 
Description 12th International Conference on Renewable Resources and Biorefineries, RRB12 , Ghent, Belgium 30th May - 1st June 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Dissemination of THERMOGENE project and results obtained to academic, business and policy makers
Year(s) Of Engagement Activity 2016
 
Description BBSRC BIOCATNET meeting, London, 2014 Oral presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Study participants or study members
Results and Impact Novel Thermostable Transfer Enzymes for Industrial Biocatalysis.
Year(s) Of Engagement Activity 2014
 
Description Biotrans Meeting 2015, Vienna, Austria Oral Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Thermogene- Novel Thermostable Transfer Enzymes for Biocatalysis, Littlechild
Year(s) Of Engagement Activity 2015
 
Description COST Action in Systems Biocatalysis 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Part of COST Action on Systems Biocatalysis
Support to attend associated meetings and funds to exchange postgraduate students on project involved with transaminase enzymes
Year(s) Of Engagement Activity 2014,2015,2016
 
Description International Extremophile Meeting, 2014 St Petersburg, Moscow Oral Presentation. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact A novel Archaeal 'Split Transketolase' Enzyme: reconstitution, structural and evolutionary perspectives Littlechild oral
Poster THERMOGENE, All partners
Year(s) Of Engagement Activity 2014
 
Description International Thermophile Meeting, 2013 Regensburg, Germany Oral Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Thermophilic Enzymes for Industrial Biocatalysis. Littlechild.
Poster THERMOGENE, All partners
Year(s) Of Engagement Activity 2013
 
Description International Thermophile Meeting, 2015 Chile Oral and Poster presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Novel thermophilic enzymes for industrial biocatalysis. oral presentation Littlechild
Structural studies, substrate identification and activity of sugar aminotransferase enzymes from thermophilic (meta)genomes Karki, García-Moyano, Sayer, Isupov, Littlechild and Birkeland
Academic and Industrial Interest
Year(s) Of Engagement Activity 2015
 
Description Invited Lecture, Russian Biochemical Society Meeting, Sochi, Russia, Oct.4th -9th 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dissemination of results of THERMOGENE to International audience
Year(s) Of Engagement Activity 2016
 
Description Keynote Lecture International Enzymology and Biotechnology Meeting , London, 5th-6th March 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited lecture to International audience industry and academics
Year(s) Of Engagement Activity 2018
 
Description Keynote Lecture International Protein Stabilisation Meeting, ProStab, Vilnius, Lithuania, May 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Keynote Lecture International Protein Stabilisation Meeting, ProStab, Vilnius, Lithuania, May 2018
Talked about THERMOGENE grant and the application of Thermophilic enzymes to an academic and industrial International audience
Year(s) Of Engagement Activity 2018
 
Description Keynote Lecture at International Thermophile Meeting, South Africa, 24th August to 1st Sep, 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Keynote Lecture at International Thermophile Meeting
Year(s) Of Engagement Activity 2017
 
Description Plenary Lecture, European Congress on Biotechnology, ECB16 Krakow, Poland, 3rd to 6th July, 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dissemination of results of THERMOGENE to academics, industry and policy makers
Year(s) Of Engagement Activity 2016
 
Description ProStab Meeting 2014 Stressa, Italy Oral Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Thermostable Enzymes for Industrial Biocatalysis. Sayer and Littlechild
Year(s) Of Engagement Activity 2014
 
Description SIMB Meeting, Philadelphia, USA, 2015, USA Oral Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Thermophilic Enzymes and Applications as Industrial Biocatalysts, Littlechild
Year(s) Of Engagement Activity 2015
 
Description TRANSAM Meeting Greitswald, Germany 2015. Oral Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Structural Studies on Transaminase Enzymes and Applications in Biocatalysis, Littlechild. Poster Sayer, James, Isupov and Littlechild
Year(s) Of Engagement Activity 2015