Date of Award

Spring 5-4-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. George Pierce

Second Advisor

Dr. Sidney Crow

Third Advisor

Dr. Eric Gilbert

Abstract

Acrylamide (AMD) is an important industrial chemical used in coagulators, water treatment, soil conditioners, mineral refining, paper treatment, adhesives, paints, and petroleum recovering agents. It is typically shipped in an aqueous solution of 30-50% w/w acrylamide, with 40% being preferred where transportation distances may be significant. 20% w/w is employed where the AMD is used to make PAM at the point of manufacture. To bypass the cost of shipping a solution that is 80-50% w/w water, one can locally produce AMD or poly-AMD using microorganisms to convert acrylonitrile (AN) to AMD. Induced cells of Rhodococcus rhodochrous DAP 96253 produce high levels of the enzyme nitrile hydratase, which is capable of converting acrylonitrile to acrylamide. In this study, whole cells, immobilized whole cells, lysate, free purified enzyme, and immobilized purified enzyme derived from induced cells of R. rhodochrous DAP 96253 were used to compare the production of 20% to 40% AMD from AN.

Acute lymphoblastic leukemia (ALL) is a rare blood cancer of the bone marrow. While ALL is less than 1% of adulthood cancers, it comprises 25% of childhood cancers and 80% of leukemia in children. The regression of acute lymphoblastic leukemia has been linked to the enzyme L-asparaginase. This enzyme catalyzes the degradation of L-asparagine into aspartic acid and ammonia. While this enzyme is readily found in bacteria, plants, and animals, it is not readily found in humans. The cancerous cells of ALL rely on asparagine from human cells to supplement their metabolism in order to grow and divide. Adding L-asparaginase to a patient’s plasma and depleting L-asparagine leads to cell apoptosis, killing the cancerous cells. The survival rate for patients 14 and younger has increased to >90%. However, 50% of patients showing recurrence within 10 years with the majority of those patients showing relapse within the first 2 years. Induced cells of Rhodococcus rhodochrous DAP 96253 exhibit commercially comparable asparaginase activity with decreased levels of endotoxins. Further research on the expression, purification, and pegylation of nitrile hydratase with asparaginase activity was conducted with the immediate goal of preserving enzymatic activity in pegylated, i.e. immobilized, cells.

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