Date of Award

12-16-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

George E. Pierce

Second Advisor

Sidney A. Crow Jr.

Third Advisor

Eric S. Gilbert

Abstract

Rhodococcus rhodochrous is a Gram-positive, aerobic, non-pathogenic bacterium ubiquitous in soil. The bacterium exhibits pleomorphic growth and produces orange to red-orange pigmented colonies on selective media. Rhodococcus rhodochrous can utilize multiple carbon and nitrogen sources and is capable of growth at a range of 28°C and 40°C. Environmentally derived Rhodococcus strains have been used for over 70 years in industrial fermentation to produce pharmaceutical and industrial-grade products. The bacterium has a broad metabolic and physiological diversity that allows for numerous therapeutic, industrial, and agricultural applications.

In this study, flask cultures and pre-pilot fed-batch fermentations (20L) of multiple R. rhodochrous DAP 96253 substrains were conducted to study the effects of Co2+ media supplementation on biomass production and enzyme activity potential of the bacterium. It was shown that the introduction of the metal ion, in conjunction with amides, upregulated the production and activity of nitrile hydratase and allowed up to 8.6x107 IU of crude nitrile hydratase to be synthesized from every liter of fermentation harvest that can be used for the biotransformation of nitriles. The use of Co2+ also increased the activity of asparaginase and glutaminase of the crude cell-free lysate (CFL) from harvested cells. This significant increase in activity allowed for the fermentation production of up to 5.5x105 IU of purified asparaginase per harvested liter. Asparaginase is an enzyme that is currently used for the treatment of juvenile acute lymphoblastic leukemia and has shown promising in vitro anti-cancer results in our lab.

In addition to high enzyme production, this study also showed that immobilized whole-cells and specific purified enzymes, such as monooxygenases, can be used as a contact-independent application for the delayed fruit ripening of climacteric fruit and as an antifungal catalyst towards phytopathogenic fungi of peaches and strawberries.

These findings indicate that the use of supplemental Co2+ in conjunction with pre-pilot fed-batch fermentation of R. rhodochrous DAP 96253 allows for the large-scale production of highly active enzymes and could be a potential biomanufacturing candidate for therapeutic and agricultural products.

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