| dc.description.abstract | Enzymes are readily being adopted as industrial catalysts due to their specificity, selectivity, and high activity in mild conditions, highlighted by the more than 12-fold growth in the global enzyme market from 0.5 to 6.4 billion USD between 2000 and 2021. This research is focused on the use of lipases in the detergent industry, specifically for cleaning fabrics. Oil-based stains can be hydrolysed by lipases which enhances stain removal with lower concentrations of detergents and lower temperatures. However, most commercial lipases have optimal activity at 30 °C or higher. Psychrophilic organisms are adapted to cold environments and have had to adapt to have optimal metabolism, and thus their enzymes have activity at low temperatures. Therefore, lipase sequences from psychrophilic organisms are prime targets to discovering cold-adapted lipases which may facilitate lowering the temperature of washing clothes. This thesis focuses on developing and discovering cold-adapted lipases for commercial laundry applications at low temperatures.
Firstly, stabilisation of lipase from the gram-negative psychrophilic marine bacterium Psychromonas ingrahamii (PinLip) was attempted; this previously studied lipase was shown to have benefits for cleaning fat stains at cold temperatures compared to the then-available commercial lipase Lipex®® 16L from Novozymes, however, suffered from low expression yields, solubility, and stability. The TaKaRa chaperone system has been employed to optimise the recombinant expression soluble yield of the enzyme. As all previous attempts to generate a crystal structure of the enzyme were unsuccessful, a 3D structure was built using the ab initio protein modelling server Robetta. As the flexibility and length of protein loop regions are linked to protein thermostability and susceptibility to proteolysis, the enzyme model was superimposed with and compared to the closest structurally resolved homologues from the fungi Gibberella zeae, Rhizomucor miehei and Thermomyces lanuginosus, which all have better thermostability. The superimposed model was then used to direct mutagenesis towards loops of the enzyme, which were significantly extended compared to the homologues. Finally, all mutant targets were successfully expressed, purified, and biochemically characterised, wherein they were shown to exhibit more proteolysis and lose their lipolytic activity.
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Lipase from Psychromonas ingrahamii was used as a search query to discover a library of novel putative cold-adapted lipase sequences that potentially constitute a new lipase family. The sequences had < 16% sequence identity to their nearest structurally resolved homologue. All sequences were predicted to be closest to the Lipase 3 family based on the ESTHER classification of α/β-hydrolases. However, Lipase from Pseudomonas ingrahamii and the putative library were found to share only up to 15% sequence identity with any of the current members of the Lipase 3 family. In addition, multiple sequence alignments analysis revealed large regions of conservation only seen between PinLip and the putative library, and phylogenetic analysis showed a significant branching away from the Lipase 3 family. From the library, putative lipase from gram-negative psychrophilic marine bacterium Moritella viscosa (MorvLip) was discovered to exhibit lipolytic activity after being expressed in bacteria.
The lipase from Moritella viscosa, with confirmed lipolytic activity, has been recombinantly expressed, purified, biochemically and structurally characterised, and tested for potential consumer product applications. However, the enzyme exhibited poor solubility and low long-term activity retention. Therefore, a rapid screening method was developed using a combination of high-throughput activity retention and nanoDSF screening to overcome the stability issues in MorvLip. The improvement in the storage buffer allowed for large enough yields from purification and increased activity retention over time to perform extensive biochemical characterisation and wash studies. However, crystallisation trials were unsuccessful for the enzyme, and the 3D structures of this enzyme and its cold-adapted homologues remain elusive.
Biochemical studies revealed the enzyme to be a cold-adapted lipase as it preferred hydrolysing para-nitrophenyl-esters of chain length C8-12 between temperatures between 4-30 °C in neutral to mildly alkaline conditions between pH 7.5 – 9.0. Furthermore, the enzyme was also shown to have superior stability towards non-ionic detergents and exhibited better removal of margarine and sun tan oil from fabrics at low temperatures than the latest commercial lipase from Novozymes, Lipex® Evity® 200L. Overall, the findings in this thesis constitute an expansion of knowledge in the field of cold-adapted lipases. Their high activity at low temperatures is a crucial desirable feature for cold-laundry applications, yet their inherent instability remains a challenge to overcome. | en_GB |
