Bacteriophages, viruses that only infect bacteria, are Earth’s most abundant and diverse biological entities. Their genomes represent the largest untapped source of novel enzymes that function under extreme conditions. Among these, endolysins (phage-encoded peptidoglycan hydrolases) are of huge interest as eco-friendly antimicrobial agents. Endolysins can be utilised in the food, agricultural and sanitation industries as alternatives to harsh chemicals and detergents, owing to their antimicrobial activities over a range of conditions. New cold-adapted endolysins offer the potential of energy-saving low-temperature cleaning, whilst thermophilic variants have potential use in fermentation, food processing and as low-resistance alternatives to antibiotics. In this webinar I will discuss research from my PhD project, in which I combined AlphaFold and other predictive methods with biophysical and experimental techniques to investigate putative cold-adapted and thermophilic endolysins. Sequences of interest were obtained from metagenomic sources of Icelandic hot springs and Arctic seawater. I will highlight how these investigations revealed unexpected and novel structural features within the enzyme class, the evolutionary implications of this, and the potential for biotechnological applications and rational design.