Exploration of strategies to altering thermal properties of industrial enzymes

Endoproteases and exopeptidases occupy a pivotal position with respect to their commercial applications in food (e.g. as additives in whey protein processing) and, as additives in detergent, textile and a number of other industries. Food processing at low temperatures by cold-active enzymes has many advantages as it minimises undesirable chemical reactions as well as the risk of microbial contamination. Cold-active enzymes were found to display higher specific activity and catalytic efficiency resulting in lower quantities of enzyme required and significantly shortened processing times. On the other hand, industrial hydrolysis typically occur at elevated temperatures due to the faster reaction rates, increased substrate solubility and thermophilic biocatalysts are required to maintain reactions at very high temperatures. The aim of our work is to exploit structure-function relationships of extremophilic enzymes that give rise to novel industrially useful proteases. We are using the high-throughput capability of the Oxford Protein Purification Facility (OPPF) to study a number of structural modifications leading to protein extremophilic functional behaviour. Several strategies to effectively alter the thermal properties of commercial serine endoproteases and aminopeptidases are being tested including; i) site directed mutagenesis targeted to reduce quantity of prolines, salt bridges, S-S bridges, and hydrophobic clusters, and ii) iterative saturation mutagenesis relying on residues with low B-factors (local rigidity) according to available 3D structures are currently being implemented. Our recent results reveal the potential for an emerging universal mechanism to modify the thermostability of any given enzyme.

Optimizing enzyme behaviour through protein engineering

Galactooligosaccharides (GOS) constitute an important class of prebiotic compounds used by the food industry as active ingredients with potential health benefits. GOS are enzymatically produced from lactose using β-galactosidases through a reaction known as transgalactosylation. Many studies have been conducted in an attempt to increase GOS yields by controlling the reaction conditions using β-galactosidases from a range of microorganisms. In this study, we have used high-throughput protein engineering for two GOS producing β-galactosidases, BbgIII and BbgIV from Bifidobacterium bifidum in an effort to enhance transgalactosylation activity (over hydrolysis) thus favouring GOS synthesis. A total of 36 and 11 C- and N-terminus deletion mutants were designed for BbgIII and BbgIV, respectively. The mutant constructs ranged from highly active to completely inactive enzymes. Selected constructs were tested for their transgalactosylation activity. An increase ranging between 5 and 10% (of total carbohydrates) was obtained with the mutant enzymes. Additionally, up to 2-fold increase in the higher degree of polymerization of GOS products was observed for selected mutants compared to the native enzyme. Structure determination of two highly active constructs at 2.0 Å resolution indicated that truncations affected the oligomeric state of the enzymes, which may have implications for activity.

Structure-function characterisation of Chlamydophila pneumoniae MOMP

The major outer membrane protein (MOMP) from Chlamydophila pneumoniae is a promising candidate antigen for chlamydophila vaccine development. MOMP is a 40kDa protein, encoded by the gene omp1, accountable for 60% of the total outer membrane mass of Chlamydophila pneumoniae. 2-3% of all Gram negative genomes encode for this particular protein class (porins), emphasising its importance and fueling intensive research into MOMPs structure and function. Our particular interest is in the established link between human infection, by micro-organisms such as Chlamydophila pneumoniae, and atherosclerosis - a multifactorial killer disease in developed nations. Evidence suggests a role for purified MOMP and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE-/- mice through dampening of MHC class II activity. We have used bioinformatics, SRCD and FTIR spectroscopies, and electrophysiology to reveal details of the structure, stability and function of MOMP. Our research to date demonstrates MOMP as a beta-barrel membrane protein containing putative 'hatch' and 'plug' domain helices, which may have implications in its function as a porin. Additionally, we show that MOMP exhibits significant increased thermal stability in the presence of fatty acids, highlighting a role for key 'NPA' and 'NPS' signature motifs present in MOMP in the transport of solutes. Our results proffer solutions to the long standing bottleneck in recombinant production of Chlamydophila MOMPs and their functional characterisation. This work has promising implications for structure-driven vaccine design against Chlamydial related diseases.