Hepatotoxic microcystins (MCs) are produced and released by the harmful bloom-forming cyanobacteria, which severely threaten drinking water safety and human health due to their high toxicity, widespread distribution, and structural stability. The linearized microcystinase (MlrB) further hydrolyses the poisonous linearized MCs produced by the microcystinase-catalysed MCs to form tetrapeptides. Here, the purification and activity of MlrB were investigated. The results showed that the linearized products generated by 12.5 mg/L MC-LR and MC-RR were removed by purified recombinant MlrB at a protein concentration of 1 mg/L within 30 min. The high catalytic activity of MlrB can be obtained via heterologous expression and affinity purification, which lays the foundation for further studies on the properties and mechanism of MCs biodegradation enzymes.

Cyanobacterial hepatotoxins, including microcystins (MCs) and nodularins (NODs), are widely produced, distributed and extremely hazardous to human beings and the environment. However, the catalytic mechanism of microcystinase for biodegrading cyanobacterial hepatotoxins is not completely understood yet. The first microcystinase (MlrA) catalyzes the ring opening of cyclic hepatotoxins, while being further hydrolyzed by the third microcystinase (MlrC). Based on the homology modeling, we postulated that MlrC of Sphingopyxis sp. USTB-05 was a Zn²⁺-dependent metalloprotease including five active sites: Glu56, His150, Asp184, His186 and His208. Here, the active recombinant MlrC and five site-directed mutants were successfully obtained with heterologous expression and then purified for investigating the activity. The results indicated that the purified recombinant MlrC had high activity to catalyze linearized hepatotoxins. Combined with the biodegradation of linearized NOD by MlrC and its mutants, a complete enzymatic mechanism for linearized hepatotoxin biodegradation by MlrC was revealed.