Molecular mechanisms of interaction between protease and humic acid of waste activated sludge

Humic acids were reported to inhibit the hydrolysis step in anaerobic digestion process. However, the influence mechanism of HA on hydrolysis has not been thoroughly understood. Here, the exact role and structural changes of hydrolytic enzymes (e.g., proteases) under HA stress at the microscopic and molecular levels were investigated. Results showed that HA statically quenched the fluorescent components of protease, probably due to the typical aromatic heterocyclic structures in HA. A significant reduction of enzyme activity was observed during this process. High doses of HA reduced the ratio of α-helix/(β-sheet + random coil), leading to a loose structure of protease, which was conducive to the binding of HA and protease. Molecular docking revealed that protease would provide plentiful sites to bind with HA, through hydrogen bond interaction forces. Density functional theory (DFT) calculations indicated that the van der Waals forces interactions also could be developed between the HA and protease in addition to hydrogen bonds. The overall binding occurred both spontaneously and exothermically, with ΔH = -4.22 kJ/mol, ΔS = 71.76 J/mol.K, ΔG = -25.6 kJ/mol at 25 °C. The response order of protease functional groups was C=O > -COO- > -OH > C-O under the disturbance of HA. This study highlights knowledge gaps that might assist in resolving the essential hydrolysis restriction issue.