biostudies-literatureYu JNatural Science Foundation of Zhejiang ProvinceNational Natural Science Foundation of China2357-2364https://www.ebi.ac.uk/biostudies/studies/S-EPMC9077389biostudies-literatureUnknown8(5)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 <i>N</i>-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 reacRSC advancesEfficient promiscuous Knoevenagel condensation catalyzed by papain confined in Cu₃(PO₄)₂ nanoflowers.PMC9077389LY18B06000921576062LY15B060011Wang AYang LWu SGPei XChen XYu JJiang MZhang PfalseEfficient 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 <i>N</i>-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 reac2018-01-01T00:00:00Z2018 Jan2025-04-18T20:24:11.279Z2025-04-07T08:22:22.026ZS-EPMC90773893554149010.1039/c7ra12940h