Current Projects
Project One
Plastics degradation by enzymes
(based in TU Dublin, Grangegorman Dublin 7)
There is a need for methods to hasten the degradation of certain persistent plastics in the environment. As the gradual phasing out of traditional consumer plastics is occurring, a new generation of bioplastics is rapidly gaining ground. Worldwide production of polylactide (PLA), for example, has more than trebled in the last few years. Polylactide is a persistent plastic that despite its biodegradability can persist in landfill for many years due to low numbers of PLA degrading organisms in soils. The need for enhancement of this process is evident. Similar enhancements are required for other biodegradable plastics. Specific hydrolytic enzymes are an attractive solution. Critically, this route allows the recovery of plastic monomers and the reusing of these monomers to synthesise new plastics or other materials of value.
We recently reported the 3D structure of a novel cutinase (AML) with plastic degrading activity. The specificity of this enzyme was unusual in degrading certain plastics such as polybutylene succinate (PBS) and polycaprolactone (PCL). However, it did not degrade PLA. Lipases capable of PLA degradation are known. This leads to the exciting prospect of being able to degrade mixed plastic waste. Thus, treatment with one enzyme (AML) would allow for the degradation of PBS/PCL while another would degrade PLA and so on. The prospect of being able to catalyse the sequential degradation of mixed waste (without the need for waste segregation) is highly attractive for a host of industrial uses. It allows for speed of degradation, recovery of monomers, treatment of mixed waste (without need for segregation) and for use of recovered monomers as building blocks for further plastics synthesis. All with virtually no environmental impact. Applications in delayed release drug delivery can also be envisaged.
Project Two
β-Glucosidase for Novel Glycoside Synthesis
(based in TU Dublin, Grangegorman Dublin 7)
In this laboratory, we discovered, cloned, and expressed, an exceptionally robust Glucosidase enzyme (β-glucosidase from Streptomyces griseus (SgBgl)). Preliminary experiments showed that this enzyme could synthesise certain alkyl glucosides (biodegradable detergent made from glucose and alcohols) with unusually high yield, under mild conditions, in the presence of Deep Eutectic Solvents (DES).
However, this reaction was not optimized or extended to long chain derivatives. Reactions with alternative glycosyl donors or acceptors were not explored. This exciting finding opens a host of possibilities for Green Chemistry applications. Our preliminary studies show that this enzyme can be developed into a broadly useful biocatalyst by appropriate modification. Such a catalyst will have important applications in research and industry. We plan to optimize this enzyme and use it to synthesize biodegradable glycosides of various kinds. We will extend this to synthesis of industrially important bioactive glycoside derivatives. By varying the glycosyl donor or acceptor a range of glycosylated products can, in principle, be synthesised. We are particularly interested in glycosylation of drugs, peptides and bioactives such as Quercetin, hydroxytyrosol, Daidzin and related molecules.
Project Three
Redox Biocatalysis in Deep Eutectic Solvents
(based in TU Dublin, Grangegorman Dublin 7)
Redox reactions in Deep Eutectic Solvents have received relatively little attention. The huge potential to explore the activity of redox enzymes in eutectic solvents is the focus of this project. The initial part of the project will focus on the use of amine oxidases and will subsequently extend to use of alcohol dehydrogenases. Dehydrogenases have a history of applications in fine chemistry for enantiomeric resolution. The potential to examine both enzymes in tandem as part of a cascade will also be explored.
The conversion of an amine to the corresponding aldehyde is synthetically useful in organic chemistry provided the enzyme can utilise a wide range of substrates. The use of DES can lead to enhanced “promiscuity” of enzyme active sites thereby facilitating the use of a wide range of substrates. The extent of this broadening of specificity will be explored in this study. To date, few attempts to examine the effects of ionic liquids on these enzymes have been reported.
Dehydrogenases have similarly been neglected due to their need for expensive cofactors although some examples have been reported. A little known dismutation reaction which uses very little cofactor can be exploited. This allows aldehyde oxidation to the carboxylate. The advantage of this reaction is the ability of the enzyme to regioselectively modify an aldehyde group and to exhibit enantioselectivity for chiral synthesis while recycling the cofactor. This reaction is especially interesting since its aldehyde substrate is the product of amine oxidase oxidations allowing for the development of cascade transformations where enzymes can work in sequence to synthesise specific chemicals enantioselectively. The dismutation will produce an alcohol as well as an aldehyde. Basic questions surround the use of this reaction in low water media such as DES remain unanswered.
Project Four
Biocatalysis: Lipases in Green Chemistry
(based in TU Dublin, Grangegorman, Dublin 7)
This project proposes to clone and express two lipases from Pseudomonas reinekei (PRL) and Pseudomonas brenneri (PRB) and explore their applications in organic synthesis. These enzymes were recently identified, by screening of soil samples, to be of interest as biocatalysts. Preliminary work suggests these enzymes are particularly robust and “promiscuous”. We have previously employed lipases in synthesis of sugar esters and flavour compounds and we are interested in the application of these methodologies to synthesis of lipopeptides and liponucleotides. Lipopeptides are known for their action as antibacterial agents and more recently as potent anti-cancer compounds. They are also effective in bioremediation applications as biosurfactants. Liponucleotides have been shown to dramatically alleviate the symptoms associated with Flu and Covid 19 [20]. Synthesis of these compounds will be used as demonstration projects. The ability of these enzymes to synthesis fatty acid sugar esters will be evaluated as an initial step to explore substrate specificity of these new enzymes.
Project Five
Flow Biocatalysis Platforms for Green Chemistry
(based in Dublin City University, Dublin 9).
Biocatalysis for the synthesis of a range of compounds such as synthesis intermediates, therapeutics, flavourings, cosmeceuticals etc., has been undergoing a revolution. However, in many cases the scaling up of bench reactions to process level has encountered problems such as mass transfer, heat transfer, mixing and foaming issues. These difficulties have driven the emergence of Flow Biocatalysis. The rapid growth in this field is due to advances in molecular biology and biotechnology and the emergence of the broader field of flow chemistry in chemical synthesis in manufacturing industries. In this project, we will develop novel flow biocatalytic processes in nonconventional media known as Deep Eutectic Solvents (DES).
Typical approaches to continuous flow biocatalysis involve the immobilsation of the enzyme of interest onto a support material. Most supports are typically packed bed systems involving either monoliths (polymers polymerised within the channels) or inert particles such as silica, solgels that are packed within the channel. Consequently, undesirable fluctuating back pressures can be observed using these packed channels. Given that the chemistry we are exploring is being carried out in DES, it is expected that the viscosity of the DES will cause significant issues with respect to backpressures. A solution to this problem has been recently employed by the Nolan group at Dublin City University: to eliminate backpressure: a monolithic porous layer open tubular (monoPLOT) capillary system was used. With the monoPLOT capillary, the polymer synthesised within the channel is controlled so that only a layer of the monolith exists on the walls of the capillary leaving the centre of the capillary open. In this way, backpressure is eliminated allowing for a continuous flow process for synthesis. Exploring novel enzyme immobilization strategies such as the use of monoPlot columns and 3-D printed reaction chambers is the focus of this project. This will be carried out in close collaboration with the postgraduates engaged in developing new Biocatalytic processes which will be migrated to Flow platforms.