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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Harmful cyanobacterial blooms in waters have become a global environmental problem, this mainly due to the production and release of various microalgal toxins, in which microcystins (MCs) are distributed widely. Here, we focused on the study of a typical form of microcystins called microcystin-YR (MC-YR). It was found that initial 14.8 mg/L of MC-YR could be completely eliminated within 10 hr by the crude enzymes (CEs) of Sphingopyxis sp. USTB-05, a promising bacterial strain we isolated and identified in our previous study. During the enzymatic biodegradation of MC-YR with time course, the peaks of two intermediate and two final products were observed on the profiles of HPLC at the wavelengths of 238 nm and 230 nm, respectively. Based on the analysis of m/z ratios of MC-YR and its four products by LC-MS/MS, we suggested that at least four enzymes were involved in the biodegradation of MC-YR by Sphingopyxis sp. USTB-05. The first enzyme microcystinase converted cyclic MC-YR to linear MC-YR as the first product. Then the second enzyme serine protease was found to cleave the target peptide bond between alanine (Ala) and tyrosine (Tyr) of linearized MC-YR, producing a tetrapeptide and a tripeptide as second products, which were Adda-Glu-Mdha-Ala and Tyr-Masp-Arg, respectively. Next, the third enzyme peptidase converted the tetrapeptide of Adda-Glu-Mdha-Ala to Adda. And the fourth enzyme cleaved the tripeptide of Tyr-Masp-Arg to produce Tyr and dipeptide (Masp-Arg), which has never been reported. These findings will help us better understand the biodegradation pathway of MC-YR by Sphingopyxis sp. USTB-05. [ABSTRACT FROM AUTHOR]