Abstract/Summary

Food processing at low temperatures has many advantages as it minimises undesirable chemical reactions as well as lowers the risk of microbial contamination, which is higher when processes are carried out at higher temperatures. Cold adapted enzymes have higher catalytic efficiencies than their analogous mesophilic enzymes, allowing the use of lower concentrations, which subsequently can be inactivated by relatively low heat processing and therefore reduce heat consumption. Consequently, cold adapted enzymes are becoming increasingly important and can help considerably in developing clean label foods and sustainable processes. Whey protein is incorporated in a variety of foods and beverages due to its proven beneficial effects on bone health, improved cognitive performance and even prevention of cancer. These whey protein hydrolysates (WPH) are produced by enzymatic digestion followed by heat inactivation of active enzymes. There is high market demand for coldadapted and thermolabile exopeptidases that could be inactivated at <50°C, following WPH production. Low temperature inactivation of active peptidases is essential to overcome the high-temperature-induced gelling of the resulting WPH peptides and successive loss of commercial product. Additionally, exopeptidases shape the resultant flavour profile of the WPH product, by eliminating terminal hydrophobic amino acids responsible for bitterness effect. The aim of this work was to understand the fundamental structure-function relationships underpinning the thermal properties of a protease enzyme. The main focus was toward engineering the thermolability of leucine aminopeptidase A (LapA), a glycoprotein originating from Aspergillus oryzae. To this end, a recombinant expression platform, utilizing P. pastoris X33 yeast strain for secretory expression of LapA into culture supernatant, was created. The thermolability engineering strategy, developed in the course of this work, was successfully tested using LapA. By reducing the number of rigidifying residues and/or intramolecular interactions, site-directed mutants were found that showed a 50% decrease in half-life at 60 °C, compared to wild-type protein. These thermolabile mutants displayed comparable specific activity and kinetic parameters to the wild-type enzyme and maintained their structural and functional integrity. In addition, three dimensional structures of the wild-type LapA precursor and mature protein were solved during this work, which are the first of their kind. These structures have provided a detailed understanding of the potential role for the LapA pro-domain in premature inactivation and its role as an intra-molecular chaperone in LapA proteogenesis. Furthermore, high resolution structures for six LapA thermolability mutants were determined, which helps provide a deeper structural understanding of highly compromised thermostability for these mutants. This work embodies a novel structure-to-function approach to unconventional engineering of thermal lability of industrially relevant peptidases and lays essential foundations for further work in this area.