Removal of humic acid interference in soil enzymatic analysis using poly-γ-glutamic acid

Soil enzymes are biological indicators in environmental and agricultural monitoring. However, brownish humic acid (HA) in samples interferes significantly with various analytical methods, especially in optical-based techniques. Here, we implemented a coagulation-flocculation process to carry out continuously an enzymatic reaction without separation and transfer of a sample solution. The elimination of HA in a soil suspension using poly-γ-glutamic acid (PGA) by coagulation to minimize the HA interference in soil enzymatic analysis was investigated. As a result of the optimization of preliminary parameters, the removal efficiency of HA was > 92% in 100 mg L^-1 HA in neutral pH, using 100 mg L^-1 PGA and aluminum trivalent as a coagulant aid. However, the fluorescent intensity of the enzyme product (i.e., 4-methylumbelliferone) decreases by about 50% as HA was removed under the conditions used. A decrease in the enzymatic detection of 3,3',5,5'-tetramethylbenzidine (TMB) was not observed from treated samples even though the initial level of HA was different. The results suggested that the coagulation-flocculation approach is suitable for the reduction of HA interference, while maintaining target analyte detection. Therefore, the proposed sample treatment can be used to examine enzyme activity based on TMB product detection without regular standard addition calibration.