Abstract Our understanding of how depolymerase sequence and structure determine substrate specificity is fragmentary due to the limited number of experimentally characterized enzymes. Here we show DepoCatalog - an experimentally validated collection of 129 recombinantly prepared Klebsiella phage depolymerases (90 enzymes produced in this study and 39 homologs from the literature), with specificity spanning 75 KL-types. Enzymes originated from podo-, sipho-, myo-, jumbo phages, and prophages.