The presented work harnessed the high activity of fungal laccases and developed a novel enzyme biocatalyst for treating persistent organic contaminants in water. Enzyme biocatalysis has high catalytic efficiency under ambient conditions of temperature and pressure. However, using enzymes in solution as additives for water treatment is not suitable and economical due to short enzyme lifetimes and non-reusability. Our study overcomes these challenges by immobilizing the fungal laccase on the outer surface of yeast cells using synthetic biology methods, resulting in a new class of renewable biocatalyst (referred to as surface display laccase (SDL)).  This study investigated a critical fundamental question relevant to using the SDL for contaminant degradation: whether and how surface display would influence the reaction kinetics and reaction mechanism of contaminant degradation by laccase. A widely detected pharmaceutical contaminant acetaminophen (APAP) was used as the target contaminant. Results showed that transformation of APAP by the SDL followed second-order reaction while APAP degradation by free laccase followed first-order reaction. The enzymatic reaction by both SDL and free laccase fit well into the Michaelis-Menten model. However, the two forms of biocatalysts had distinct values for K<sub>m</sub> (62.16 μM for SDL and 337.66 μM for free laccase) and V<sub>max</sub> (0.081 μM/min for SDL and 1.044 μM/min for free laccase), indicating that cell surface display could substantially affect the catalytic properties of laccase. Mass spectrum analysis of degradation products suggested that surface display of laccase did not change the mechanism of APAP degradation compared to that by free laccase. Particularly, the degradation pathway involved enzymatic oxidization of APAP to highly reactive radical intermediates and the following chemical coupling of the intermediates to form dimers and trimers, leading to detoxification of APAP. Furthermore, the SDL biocatalyst could effectively degrade APAP in actual secondary wastewater effluent and the reaction rate was improved compared to that in acetate buffer. Kinetic characterization suggested that there might be some electron mediating compounds in the secondary effluent to facilitate APAP degradation. Results from this study provided insights into the factors affecting the efficacy of the SDL and laid the scientific basis for developing and implementing innovative surface display enzyme biocatalysts for remediation applications.

Engineering surface-display laccase biocatalyst for treating emerging organic contaminants: Kinetics and enzyme catalytic properties in acetaminophen degradation

2019 Spring National Meeting

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