| dc.description.abstract | The use of enzymes as industrial catalysts is a very promising alternative to conventional synthetic chemistry since enzymes are very specific, selective and display very high activity under mild experimental conditions. This research is focused on lipases, which are widely used in several industrial sectors and especially in the laundry industry. Enzymes in laundry formulation are exposed to alkaline pH, high concentrations of detergents and the presence of proteases. These harsh conditions have a negative effect on the stability and activity of the enzymes and can eliminate the benefits of adding enzymes to formulation entirely.
This study aimed to investigate the stabilisation of existing commercial lipases and the characterisation of a novel cold-adapted lipase for industrial applications.
The commercial lipase Lipex 16L is a variant of the Thermomyces lanuginosus lipase and is the current benchmark lipase for application in laundry products. Lipex 16L has been used to develop an improved method for carrier-free immobilisation, using Cross-Linked Enzyme Aggregates (CLEAs). The CLEAs production protocol has been modified by introduction of an activator step to obtain a higher number of individual lipase molecules in the "open lid" conformation and by the introduction of a terminator step to quench the cross-linking reaction at an optimal time to obtain smaller and more homogenous cross-linked particles. This improved immobilisation method has been compared to a commercially available immobilised enzyme and has been shown to be made up of smaller and more homogenous particles with higher activity than the Lipex 16L. The CLEAs produced show improved features for commercial applications such as an enhanced wash performance comparable with the free enzyme, improved stability to proteolysis and a higher activity after long-term storage.
The stabilisation of Lipex 16L has also been investigated through the introduction of two additional glycosylation sites on the protein surface. The commercial Lipex 16L has only one glycosylation site on asparagine 33. A tri-glycosylated mutant has been generated with the introduction of two further glycosylation sites on asparagine 37 and asparagine 99. This recombinant enzyme and a mono-glycosylated wild-type enzyme have been cloned and expressed in Pichia pastoris while a non-glycosylated variant has been expressed in Escherichia coli. The enzymatic activities of the glycosylated and non-glycosylated lipases have been compared under various conditions such as temperature, pH, detergents, and incubation with proteases. The results have demonstrated that while the additional glycans do not affect the lipase activity and cleaning performance, they do improve its resistance to proteases and its overall stability with an increase of the melting temperature of + 4 °C.
A novel lipase from the psychrophilic bacteria Psychromonas ingrahamii (PinLip) has been biochemically characterised. The enzyme shows activity towards short and medium chain fatty acids and has a good fat stain cleaning performance which makes it attractive for industrial applications. Structural characterisation of the PinLip has been attempted by using crystallisation trials for X-ray crystallography and NMR spectroscopy with limited success. A 3D homology model has been generated using the server I-TASSER using the most closely related known structures, Gibberella zeae, Rhizomucor miehei and Rhizopus microspores lipases, all with a sequence identity to PinLip between 20 and 24%.
The different lipases studied in this thesis have been tested for their stability in the presence of traditional laundry formulation ingredients and new novel biosurfactants using differential scanning fluorimetry (DSF). The results have shown an improved stability of all the Lipex variants in presence of mono-rhamnolipids based biosurfactant, while the cold-adapted PinLip was stabilised by a small concentration of a polymer (EPEI) and few other compounds (Tinopal CBS-CL, and Triethylamine).
The improved CLEAs method and the use of the PinLip enzyme have been patented (Patent no: WO2017/036901, WO2017/036902, WO2017/036915, WO2017/036916, and WO2017/036917). | en_GB |
