Projects per year
Following my formal retirement in September 2016, I am an honorary Research Professor in the Department of Pure & Applied Chemistry (part of the WestCHEM research school). I no longer run an experimental lab, but am continuing various research activities. These include some projects using computer- (and even paper-) based meta-analysis of data from other sources, and appropriate simulations. I also have some continuing collaborations with others who are doing experimental work. I still teach on the Applied Biocatalysis module of the inter-university (IBioIC) MSc in Industrial Biotechnology.
My research interests are mostly related to the application of biological catalysts (enzymes or whole cells) in industrial processes. Rather than being focussed on specific products, I try to concentrate on general problems relevant to a range of actual and potential processes. I favour such strategic research as I would like to think that my work has relevance to a known field of application. However, I usually prefer a relatively fundamental approach, in the hope of finding the most generic solutions through proper understanding of the systems involved. Most of the research involves a physico-chemical approach to the systems, particularly using principles of thermodynamics and kinetics. Following my formal retirement in September 2016, I no longer run my own experimental laboratory. However I am continuing research in collaboration with others who do have experimental facilities, as well as working on data analysis, simulation and related styles of research.
The problems that are important in this area, as in so many others, are best tackled with a multi-disciplinary approach. I believe that my experience is a good basis for the application of knowledge from a wide range of relevant disciplines to these problems. This also means that much of my research is collaborative, often involving people from other backgrounds, particularly bioscience and process engineering, both at Strathclyde and elsewhere.
For many years my main area of work was on biocatalysts acting in low water media, particularly those based predominantly on organic liquids. As recently as the 1980s, textbooks still said that enzymes acted only in dilute aqueous solution, but it is now clear that low-water media can be used successfully, under appropriate conditions. Furthermore, these systems offer attractions for some industrial processes: particularly higher reactant content/concentration and the use of readily available hydrolytic enzymes to catalyse synthetic reactions. My main past contribution has been related to the role of the small amounts of water still present. I introduced the use of the thermodynamic water activity to characterise multi- phase systems of this type, and have studied its effects, measurements and control. The usefulness of this parameter is now widely accepted. Subsequently we developed theoretical and experimental approaches to the treatment of acid/base effects in these systems, which have gained some attention. These effects are closely related to the role of counter-ions in low-water enzymes, which has led to more recent crystallographic studies. We also investigated the potential of studies in such systems at controlled, low water activity to give fundamental information about enzyme behaviour, such as the role of water in function.
I have also worked on a number of related topics. These include other low-water and unusual systems in which enzymes can act in practically useful ways. Enzymatic syntheses in mainly solid systems have considerable practical attractions, such as the very high volumetric product contents obtained (1 kg per litre or more). With just a small fraction of liquid phase it is possible for a mixture of solid reactants to be converted smoothly into mainly solid products. The thermodynamics are such that the reaction shows “switch-like” behaviour – if solid product is formed, it will continue to accumulate until at least one of the solid starting materials is completely consumed. My earliest involvement was showing that this was expected theoretically and found experimentally. The analysis also showed that the reaction would be equally favourable whatever solvent was added to form the liquid phase, including water. Hence we were able to demonstrate that these systems could offer not only the high yields in reverse hydrolysis characteristic of organic media, but also the high rates and green attributes found in water. A subsequent theme was models to predict whether the “solid-to-solid” transformation is thermodynamically favourable, based on molecular structure and available data like melting points. In these systems the enzyme usually has to act in a highly concentrated solution, which has spawned some work on enzymology in such media, largely neglected in the literature.
A second unusual system for enzyme action is attack on solid-supported substrates, which can be very different from the analogous reactions in solution. Such reactions are of interest for solid-phase synthesis, on-bead screening and other applications. In general it is desired to use enzymes that do not normally act on solid-phase substrates, so do not have the special adaptations of for example cellulases. At first sight it might be thought that equilibria would not be affected by immobilisation of reaction substrates. However, experiments show that, for example, peptide bond synthesis can be strongly favoured even in aqueous media. My involvement began with showing that this was in fact thermodynamically expected, because of two contributions: transfer of hydrophobic groups out of bulk water, and reduction in mutual repulsion of charged groups on the support surface. Subsequently we also looked at kinetics in these systems, in particular the use of two-photon microscopy for spatially resolved assay inside beads, including in real time as well.
Another theme has been the use of spectroscopic methods, especially circular dichroism (CD), to improve our understanding of proteins in solid particles, such as in immobilised enzymes. This started with an attempt to detect conformational changes that could cause loss of activity during use, using CD, fluorescence and IR. We had to modify the CD methods to make the first reported measurements on particles in the 100 micrometre size range. It then became clear that these methods were more widely applicable to protein-containing particles: not just immobilised enzymes, where they offer wider understanding of the basis for effects on catalytic activity; but also systems such as protein storage forms, pharmaceutical formulations and solid biological samples. This led to a study of how to correct for absorption flattening, a phenomenon which affects such measurements, and most recently an indication that known methods (including my own) do not actually work properly across the necessary wavelength range. We have also demonstrated the feasibility of measuring CD spectra from single solid particles containing protein, using the Diamond synchrotron source. All this CD work has also led on a more general study of issues in enzyme immobilisation – in part prompted by my wish to see the large body of work published in the 1970’s re-evaluated in a modern light, before it gets forgotten completely! I have also collaborated with a colleague, Aaron Lau, who is pioneering a novel immobilisation chemistry based on polyphenols.
One final theme has expanded from a very simple initial investigation of a topic that seemed neglected. Most screening in the pharmaceutical area involves mixing solutions of drug-like molecules in dimethyl sulfoxide (DMSO) with an excess of aqueous assay medium. Controls are done to check for effects of DMSO, but only recently has there been some concern about what happens to the organic test compounds themselves, with realisation that their precipitation cannot be ignored. So we made some studies of the physical chemistry of this process, precipitation from solution in the micromolar concentration range. I was very concerned about my lack of background knowledge on this topic, but then became more confident that no-one knows very much about what can happen – crystallisation is normally studied at concentrations a thousand or more times higher, as encountered in preparative applications.
Since the closure of my experimental lab, I have turned to topics that make use of data analysis, simulations and related approaches. This is not computational chemistry (as normally understood), but uses computer methods of other types. Three topics have had significant work so far:
• Optimal experimental design methods applied to biocatalysis. The field has seen quite extensive study of so-called statistical design methods like response surface methodology. But these methods completely ignore any knowledge about the underlying kinetic models that describe the system. By using such knowledge, more informative experiments can be planned, using the methods of optimal experimental design that are known from other fields where mathematical models of system behaviour can be formulated.
• A computer-run tool to validate data from enzyme studies as complete and free from obvious mistakes. Analysis of published papers, both in applied biocatalysis and enzymology more generally, reveal that a high proportion have key details missing. As a member of the Standards in Reporting Enzyme Data (STRENDA) Commission, I have contributed to the development and promotion of the STRENDA-DB electronic tool that checks data and metadata from enzyme experiments (https://www.beilstein-strenda-db.org/strenda/). Continuing work is extending the validation algorithms used by the system to spot many of the mistakes in data entry commonly found in the literature.
• Approaches to estimate initial rate from discontinuous reaction time course data. Researchers commonly make such estimates, often with only a small number of data points available, each of course subject to some experimental error. I want to establish how different methods compare with each other, and see if it is possible to identify an objective method that does not require human judgment.
Lectures, labs, tutorials and student exercises for the Applied Biocatalysis module of the inter-university (IBioIC) MSc in Industrial Biotechnology. I present a general introduction to the topic, then more details on biocatalyst kinetics, stability, and the behaviour of immobilised biocatalysts. I also act as coordinator for the whole module. The module runs on an intensive basis, with 3 blocks of about 1 week each, which in the last few years were between November and January.
23/03/18 → …
Project: Non-funded project
Research Output per year
Research output: Contribution to journal › Article
Research output: Contribution to journal › Article
Halling, P. (Creator), University of Strathclyde, 13 Mar 2020
Student thesis: Doctoral Thesis
Therapeutic protein and vaccine stabilisation technology with global reach across the pharmaceutical industry
Impact: Impact - for External Portal › Economic and commerce, Health and welfare - new products, guidelines and services