Date of Award

5-4-2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. George E. Pierce

Second Advisor

Dr. Sidney Crow

Third Advisor

Dr. Eric S. Gilbert

Abstract

Bacterial L-Asparaginase has been utilized along with chemotherapy in the treatment of acute lymphoblastic leukemia. Current forms of treatment are fraught with undesirable outcomes in a significant number of cases. This has led to a continued investigation of other bacterial sources of L-asparaginase.

Rhodococcus rhodochrous strain DAP 96253 when induced on specialized media was found to present L-asparaginase and L-glutaminase activity. A purification scheme was developed evaluating the effect of the method of cell lysis, the use of a reducing agent and dialysis on the final product. A protein preparation was purified from the crude extract employing dialysis, anion exchange and size exclusion chromatography using a Hitrap®Q HP and a HiprepTM 16/60 SephacrylTM S500HR column respectively. The purified protein exhibited 1492 and 2173 IU/mg of L-asparaginase and L-glutaminase respectively.

Further kinetic studies of the purified preparation demonstrated an optimum reaction pH at 7.6, maximal enzyme activity at 37°C and stability exceeding 60 minutes. The Km for L-asparagine and L-glutamine were determined to be 13.6µM and 850µM respectively. The protein was determined to be stable in human serum at 37ºC with a T1/2 of approximately 53 hours. Endotoxins were 771 times less than an Escherichia coli sample, with the endotoxins detected being introduced during the purification process, the three subunits of the protein were estimated to be ~72, 28 and 23kDa versus a homotetrameric protein in E. coli, and the protein was determined to be effective in reducing the cell number and viability of Jurkat E6-1 clone and Molt-4 cells exhibiting an IC50 of 0.123 and 1.982 IU/mg respectively. This is the first attempt to characterize an L-asparaginase from R. rhodochrous.

File Upload Confirmation

1

Available for download on Thursday, April 07, 2022

Share

COinS