Unraveling the substrate specificity of a thermostable and highly active isoamylase for one-pot starch upgrading via enzymatic cascade

The debranching of α-1,6-glucose branch-points in starch by isoamylase is essential for in vitro enzymatic cascades containing α-glucan phosphorylase (αGP) that convert starch into valuable compounds. Herein, a highly active isoamylase, GK2827, from thermophilic Geobacillus kaustophilus was enzymatically characterized with a half-life of 46.6 h at 65°C. GK2827 exhibits a specific activity of 848 U/mg toward maltodextrin (dextrose equivalent 8.6), which is 31.8-fold higher than that toward pullulan. Molecular dynamics simulations revealed that a combination of favorable electrostatic interactions, stronger covalent binding, and stable van der Waals interactions enhance the binding affinity of GK2827 for maltodextrin compared to its affinity for pullulan. In an enzymatic cascade comprising αGP, α-glucosidase and cellobiose phosphorylase for cellobiose synthesis in one-pot, the inclusion of GK2827 increased the maltodextrin utilization rate from 52.6% to 80.9%. This excellent heterologously expressed isoamylase facilitates αGP-based starch phosphorylation in biomanufacturing various functional saccharides via in vitro enzymatic cascade.