Water Resources Research Act Program

Details for Project ID 2020AR109B

Mechanisms, Kinetics and Toxicity of Microcystin-LR Biodegradation by Free and Immobilized Enzymes

Institute: Arkansas
Year Established: 2020 Start Date: 2020-03-01 End Date: 2021-02-28
Total Federal Funds: $25,000 Total Non-Federal Funds: $50,000

Principal Investigators:

Project Summary: Microcystin-LR (MC-LR) is among the most problematic algal toxins in America, with several outbreaks and appearances in freshwater supplies in recent years. Microcystins are cyclic peptides with seven amino acids, and the congener with leucine (L) at position 2 and Arginine (R) at position four is known as MC-LR [1, 2], which makes traditional methods for management of MC-LR to have significant drawbacks or limited effectiveness. Emerging strategies, such as photocatalysis, ozonation, biofiltration, and membrane filtration, have been attempted to eradicate MC-LR, but there are drawbacks with each technology. To address their drawbacks, we propose a system combining full MC-LR removal and destruction via an enzymatic reactive membrane, through which the membrane is a physical barrier for the MC-LR, and the enzymes biodegrade it. The novelty here is the use of an enzyme triad to completely and expediently destroy the toxin. Various bacteria contain an mlr gene cluster for the expression of a protein triad, which consists of MlrA, MLB, and MlrC, responsible for the degradation of MC-LR [2][3]. Furthermore, these individual proteins cleave the MC-LR toxin at different bond locations [3]. While Mlr enzymatic catalysis is known to be non-sequential, the Mlr protein pathway and any partnering enzymes remain unidentified. The overall objective of this proposal is to investigate the kinetics and toxicity of biocatalytic degradation of MC-LR and to immobilize the enzymes on functionalized surfaces. Our central hypothesis is that the concerted three-dimensional orientation of the enzyme triad, when immobilized on a membrane surface, will bring the reaction pathway of MC-LR decomposition to completion. This hypothesis was formulated on the basis that the proteins have different substrates and need to be in the vicinity of byproducts to be effective. We propose using site-directed mutagenesis not only to immobilize a protein to a membrane but control orientation.