Efficient promiscuous Knoevenagel condensation catalyzed by papain confined in Cu₃(PO₄)₂ nanoflowers

To develop an efficient and green immobilized biocatalyst for promiscuous catalysis which has a broad scope of applications, hybrid nanoflower (hNF) confined papain as a biocatalyst has been proposed and characterized in this study. hNFs were firstly prepared through mixing CuSO₄ aqueous solution with papain in phosphate saline (PBS) at room temperature. The resulting hNFs were characterized by SEM and verified through a hydrolysis reaction with N-benzoyl-dl-arginine amide as substrate. Under optimal conditions, this nano-biocatalyst demonstrated a 15-fold hydrolytic activity compared with papain of free form, along with better thermal stability. A series of reaction factors (reaction temperature, time, and solvent) have been investigated for Knoevenagel condensation reactions with hNFs as catalyst. At optimal conditions, product yield of the hNFs catalyzed reaction was 1.3 fold higher than that of the free enzyme with benzaldehyde and acetylacetone as substrates. A few aldehydes and methylene compounds have also been used to test the generality and scope of this new enzymatic promiscuity. To sum up, the obtained hNFs demonstrate better catalytic properties than free papain and the inorganic metal-salt crystal can function as both support and promotor in biocatalysis.