Atlantic cod (Gadus morhua) trypsin – isoenzyme diversity and antiviral activity

• Main Author: Sandholt, Gunnar Birgir
• Other Authors: Ágústa Guðmundsdóttir, Matvæla- og næringarfræðideild (HÍ), Faculty of Food Science and Nutrition (UI), Heilbrigðisvísindasvið (HÍ), School of Health Sciences (UI), Háskóli Íslands, University of Iceland
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: University of Iceland, School of Health Sciences, Faculty of Food Science and Nutrition 2019
• Subjects: Þorskur; Fiskirannsóknir; Matvælafræði; Doktorsritgerðir; Valda; atlantic cod; Gadus morhua
• Online Access: https://hdl.handle.net/20.500.11815/1027

The aim of the thesis work was to identify and characterise different trypsin isoenzymes from a benzamidine-purified Atlantic cod (Gadus morhua) trypsin isolate. Another aim was to test the ability of cod trypsin to deactivate upper respiratory tract viruses. Initially, the stability of the trypsin isoenzymes I, X and ZT, in terms of temperature and pH, was characterised along with their sensitivity to various inhibitors. Multiplex substrate profiling by mass spectrometry of trypsin ZT and trypsin I revealed a difference in substrate specificity between the two trypsin isoenzymes. Furthermore, trypsin X, I and ZT showed a difference in sensitivity to inhibitors. The deactivation potential of the cod trypsin isolate was tested on several upper respiratory tract viruses. The results demonstrated that cod trypsin can deactivate rhinovirus A and coxsackievirus B. Furthermore, cod trypsin deactivated coronavirus. This was shown by using a Renilla luciferase assay and confirmed with a viability test. Furthermore, Western blot analysis showed that cod trypsin degraded recombinant coronavirus spike protein. The SARS coronavirus spike protein was treated with cod trypsin isoenzymes I and ZT to test the contribution of each isoenzyme to the cleavage. Both isoenzymes cleaved the SARS coronavirus spike protein at low trypsin concentrations. In conclusion, the results show that cod trypsin X and cod trypsin ZT have interesting characteristics differing from the previously characterised cod trypsin I. Cod trypsin was also shown to deactivate different viruses and to cleave recombinant coronavirus spike proteins. Markmið doktorsverkefnisins var að einangra og skilgreina mismunandi trypsín ísóensím úr bensamidín hreinsaðri trypsínblöndu. Jafnframt að rannsaka hvort þorskatrypsín geti óvirkjað veirur sem valda sýkingum í efri öndunarvegi. Í grein I og II var byrjað á að greina trypsín isóensímin X og ZT úr benzamidine hreinsaðri trypsín blöndu. Stöðugleiki þeirra gagnvart breytingum á hitastigi, sýrustigi og ensímhindrum var ...