Preparation and investigation of novel endopeptidase-exopeptidase co-immobilized nanoflowers with improved cascade hydrolysis

Enzymatic hydrolysis is a promising approach for protein and food processing. However, the efficiency of this approach is constrained by the self-hydrolysis, self-agglomeration of free enzymes and the limited applicability resulted from enzymes' selectivityt. Here, novel organic-inorganic hybrid nanoflowers (AY-10@AXH-HNFs) were prepared by coordinating Cu2+ with both endopeptidase of PROTIN SD-AY10 and exopeptidase of Prote AXH. The results indicate that the AY-10@AXH-HNFs exhibited 4.1 and 9.6 times higher catalytic activity than free Prote AXH and PROTIN SD-AY10, respectively, for the enzymatic hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE). The kinetic parameters of Km, Vmax and Kcat/Km by AY-10@AXH-HNFs were determined to be 0.6 mg/mL, 6.8 mL·min/mg and 6.1 mL/(min·mg), respectively, surpassing the values obtained from free endopeptidase and exopeptidase. Furthermore, the ability of AY-10@AXH-HNFs to retain 41 % of their initial catalytic activity after undergoing 5 cycles of repeated use confirmed their stability and reusability. This study introduces a novel approach of co-immobilizing endopeptidase and exopeptidase on nanoflowers, resulting in significantly enhanced stability and reusability of the protease in catalytic applications.