2 Apr 2025

Bacterial Remediation of Microsystin-HAB Toxins Utilizing Microcystinase (MlrA)

Abstract: Microcystins are a class of hepatotoxins produced by some harmful algal bloom–associated cyanobacteria and are the most reported tox-ins in freshwaters. Their cyclic structure makes them resistant to conventional methods used in water treatment operations (boiling, chlorination, and UV treatment). Some bacteria can naturally degrade microcystins via the mlrABCD cluster, a pathway initiated by the primary enzyme microcystinase (MlrA). MlrA linearizes the cyclic microcystin, greatly reducing its toxicity. Protein fusion was employed to produce a recombinant MlrA enzyme fused to maltose-binding protein ([MBP] MBP-MlrA) and to evaluate long-term enzymatic stabilization and reconstitution for future applications. MBP-MlrA degraded cyclic microcystin in vitro and demonstrated stability across a range of biological pHs. At a concentration of 0.61 ng/µl in buffer, MBP-MlrA achieved and maintained an average degradation rate of approximately 101.95 µM/h/ng of protein across fifteen freeze/thaw cycles. Stability assays demonstrated that enzyme activity was preserved over 5 months at −20°C. Results also demonstrated the effectiveness of MBP-MlrA to linearize microcystin upwards of 55.59 µM/h/ng of protein at the bench scale in both buffer and various freshwater matrices. The presence of the linear metabolite is of concern regarding intermediate toxicity, and future studies to incorporate the MlrB peptidase are discussed.