Research
Current Research Projects
Enzyme Micro Reactors
The research involves the use of micro reactor systems for the immobilisation of enzymes and the use of developed systems for screening of enzymes for activity against a range of substrates. The work was done in collaboration with a group from the University of Hull and funded by the EPSRC. The research to date has focused on the immobilisation and activity investigation of an L-aminoacylase from Thermococcus litoralis and a γ-lactamase from Sulfolobus solfataricus in micro reactors designed and made in the Department of Chemistry, University of Hull. Two types of micro reactors were used, micro reactor chips (Figure 1, left) and capillary column reactors (Figure 1, right). Free enzyme was cross-linked to a monolith structure in the channels of the micro reactor chips and cross-linked enzymes were packed into silica-fritted capillary column reactors.
L-Aminoacylase
The L-aminoacylase from Thermococcus litoralis catalyses the removal of N-acyl protecting groups from L-amino acids, leaving D-amino acids untouched.
Scheme 1: L-Aminoacylase transformation
L-Aminoacylase was cross-linked to the monolith structure in micro reactor channels and investigated for activity on a range of substrates and also investigated for stability under operational conditions.
γ-Lactamase
The γ-lactamase from Sulfolobus solfataricus catalyses the formation of amino acids from amides.
Scheme 2: γ-Lactamase transformation
This enzyme fits into the amidase class and is capable of generating carboxylic acids from amides.
Scheme 3: Amidase transformation
γ-Lactamase was used as a cross-linked enzyme and trapped in capillary column reactors using a silica frit and investigated for activity on a range of substrates and also investigated for stability under operational conditions.
Current and future work
Ongoing and future research will focus on immobilisation of co-factor dependent enzymes, investigating immobilisation of enzyme only, co-factor only and enzyme and co-factor together. Of particular interest is the latter, since the reduction in the cost of enzyme and co-factor, brought about by immobilisation, could significantly enhance the attractiveness of co-factor dependent enzyme use for biocatalysis in an industrial setting.
Commercial Exploitation of Marine Enzymes
Project Outline
An existing collection of marine archaea and eubacteria will be screened for a range of enzyme activities, database searches will also be carried out to identify putative enzymes from thermophilic organisms. The search will focus on halogenating and dehalogenase enzymes involved with the production of novel halogenated bioactive natural products and active pharmaceuticals. Marine organisms have evolved to cope with and metabolise halogenated molecules and as such contain an untapped resource of novel enzymatic pathways representing a source of high value chemistry by a natural route. Any enzymes found can be exploited for anti-cancer and anti-infective healthcare products, drug intermediates and speciality chemicals.
Key Findings
Assays have been developed to screen for dehalogenase, bromoperoxidase, lipase, esterase and ADH activity. Using these assays over 500 bacterial strains have been screend for novel enzyme activities. A number of hits have been identified and some of these are being followed up. The enzymes responsible for the observed activity are being isolated using a range of techniques including cloning from genomic DNA, construction of expression libarys and purifying the enzyme from native cells by following the activity through a range of steps. Once pure enzyme is obtained its potential for commercialisation can be investigated.
Design of Effective and Substainable Control Strategies for Liver Fluke in Europe
DELiver is a € 3.54 million funded (Priority 5 of the 6th FP) EU project to investigate new methods for controlling liver fluke disease in livestock to minimise the use of anthelmintic drugs. The research includes investigation of vaccines, drug targets and drug resistance in addition to the development of reliable diagnostic tests and predictive models to assist in livestock management. Further details of all aspects of the project can be found on the project website.
Liver fluke disease (fasciolosis) causes annual losses over 3 $US billion to livestock production and the food industry worldwide. The prevalence of fasciolosis is dramatically increasing – in recent years for example increases of up to 12 fold have been recorded in EU member states. In the UK the prevalence of infection in cattle ranges from 45% to 84% and in Ireland alone annual losses have been estimated at €60 million. Fasciolosis is also an emerging human disease in many INCO countries (an estimated 17 million people are infected). Most of these human infections arise around areas where water cress is grown, where the infective life stage of the parasite is deposited from it’s intermediate host, the snail.
Our involvement in the project is to study the structure of liver fluke proteins involved in the mode of action, metabolism and resistance to the anthelmintic triclabendazole, in addition to potential vaccine candidate molecules. We have solved the structure of thioredoxin, which was found to have the active site cysteine residues in both the reduced and disulfide oxidation states (Line et al., 2008, Mol. Biochem. Parasitol. 161, 44-48). Studies are also ongoing to obtain the structure of the typical 2-Cys peroxiredoxin enzyme. We have also recently solved the structure of a novel sigma class GST and a Mu class GST, both implicated in metabolism of the drug triclabendazole. To further our understanding of what liver fluke proteins interact with the drug triclabendazole we have initiated a collaborative project with Dr Chris Hamilton (UEA) to synthesis derivatives of the drug molecule that can be immobolised and used to 'fish' out binding proteins from liver fluke extract.
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