Synergistic precipitant powered self-assembly of papain for cross-linked enzyme crystals preparation

In this study, we prepared cross-linked enzyme crystals (CLECs) of papain to further broaden the application of the enzyme with high activity in extreme environments. Initially, papain crystals were successfully obtained based on the micro-batch, batch, and expanded batch crystallization experiments. Specifically, ammonium sulfate and polyethylene glycol 6000 (PEG6000) were synergistically used as the precipitants, while l-cysteine was applied to enhance the activity of papain. Furthermore, the interaction between l-cysteine and papain was modeled by molecular docking technique. It was found that l-cysteine could form a hydrogen bond with aspartic acid residue (Asp) at site 158, and the electrostatic attraction with lysine residue (Lys) at site 156 was also quite obvious. Then the enzyme crystals were cross-linked by glutaraldehyde at optimized conditions. The papain CLECs were identified by various methods, and it was found that the thermal stability and enzymatic activity both increased compared to the raw enzyme. More importantly, it could be applied at more rigorous conditions, for example, pH of 4.