Introduction of a Glycine Linker Connecting the Heavy and Light Chains in Synthetic Cardosin B‑Derived Rennet Changes the Specificity of Subpocket S3′

The development of plant-based synthetic rennets is of high commercial interest, due to the current great consumer demand for animal product alternatives. A previously developed recombinant form of the aspartic protease cardosin B with a three-glycine linker showed great potential due to its good performance in milk coagulation. This enzyme was found to be more specific and less proteolytically active than the native form for milk clotting, but the underlying structural causes for these activity changes were not completely clear. Here, we have performed molecular dynamics simulations with the recombinant enzyme with and without the linker. Our results showed that the introduction of the linker changes the subpocket S3′, which is located more than 4 nm away. These results showcase how small modifications in proteins can have significant effects in distant regions in the protein structure that affect their biotechnological applications.