Mining biological diversity to optimize chlorite dismutase for water treatment

Perchlorate;Chlorite Dismutase;Catalytic Inactivation

Mobilia, Kellen

关键词: Perchlorate;    Chlorite Dismutase;    Catalytic Inactivation;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/78685/MOBILIA-THESIS-2015.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Enzymes are powerful, natural catalysts that could be harnessed to remove harmful microcontaminants, like perchlorate, from drinking water. As of now, such technologies are only theoretical, but they could eventually be employed as sustainable alternatives to current methods or complementary approaches, where specialized applications are required. In this work, enzymatic treatment of perchlorate-contaminated drinking water is examined as a test case. A diverse superfamily of enzymes, known as chlorite dismutases (Clds), catalyzes the final reaction of biological perchlorate reduction. The goal of this work was to mine the diversity within the Cld superfamily to identify a homolog that is well suited for water treatment. To find an optimal Cld, a head-to-head comparative analysis was performed on seven Cld homologs from a variety of bacterial species. A kinetic assay was developed to track product formation using a chloride ion selective electrode. This assay was used to quantify and compare the kinetics, shelf life, and catalytic life of the seven homologs. Additionally, a custom polyclonal antibody was developed to quantify the concentration of Cld. Unfortunately, attempts to quantify the concentration of Cld using this custom antibody were unsuccessful due to issues with unequal binding affinity across the homologs. Therefore, throughout this work, the concentration of heme b was used as an approximation of the Cld concentration.With these methods, I replicated the only previous study that quantified the catalytic life of Cld and assessed the catalytic life of an additional six previously untested homologs. Cld from Ideonella dechloratans was identified as the homolog best suited for water treatment. Most importantly, this homolog was able to catalyze approximately 140,000 reactions, a catalytic life at least double that of the other tested homologs. At the other extreme, Cld from Candidatus Nitrospira defluvii was found to be poorly suited for treatment, given its relatively short catalytic life. This analysis also found that Cld has an exceptional storage half-life, greater than 2-months when stored at 4 °C. With a solid candidate for treatment identified, future work could fall under applied development or fundamental investigation. Even at 140,000 reactions, the catalytic life of Cld is still relatively short. This could be further improved by generating random mutations in the sequence of Cld and then applying directed evolution to select for beneficial mutations. If successful, this type of engineering could also further the limited understanding of the mechanisms by which Cld inactivates. In summary, this work has brought enzymatic perchlorate treatment technologies one step closer to being a viable and sustainable alternative.