Masters Theses

Ziran Sun

Date of Award

8-1999

Degree Type

Thesis

Degree Name

Master of Science

Major

Chemical Engineering

Major Professor

Paul Frymier

Committee Members

Gary Saylor Charles Moore

Abstract

The experimental and mathematical analyses in this study have furthered knowledge in batch production of 2,5-diketo-gluconic acid by Pantoed citrea (ATCC37410). This study was driven by three objectives: (a) to derive a mathematical model based on Michaelis-Menten enzyme kinetics to describe the batch production of 2,5-DKG, (b) to investigate the role central cellular metabolism played in the batch production of 2,5-DKG, and (c) to compare the relative activity of the major internal pathways for glucose catabolism.Central carbon metabolism, growth rates, and cell yields were determined in P.citrea using glucose, fructose, gluconate, or pyruvate as growth substrates. Key Enzymatic activities of the Embden-Meyerhoff-Pamas (BMP), Entner-Douderoff (ED),and pentose phosphate (PP) pathways were examined using Escherichia coli as a reference. All cells were grown on minimal medium containing 28mM glucose, fructose,gluconate, or pyruvate at 28 C. The cell yield on glucose was 0.089 compared to 0.17 for fructose or gluconate. Cells grown on glucose achieved half the concentration of those grown on fructose. The key EMP enzyme, phosphofructokinase, showed a low activity(0.21 umts/mg protein) relative to E. coli (0.42 units/mg protein). 6-phosphogluconate dehydratase and KDPG aldolase, two key enzymes of the ED pathway, also showed lower activities in P. citrea than in E. coli. 6-phosphogluconate dehydrogenase, the key enzyme of the pentose phosphate pathway, exhibited a higher activity in P. citrea than in E. coli. 2-keto-gluconate dehydrogenase, a membrane-bound enzyme in P. citrea, was also assayed. Results show a high level of activity for 2-ketogluconate dehydrogenase.The low activity of phosphofructokinase, coupled with poor growth rate on glucose, andhigh activity of the membrane-bound dehydrogenases suggested that the glucose is channeled through the outer membrane dehydrogenases and thus prevented from entering central metabolism in P. citrea.Batch productions using both viable and nonviable cells were performed to show the effect of central .metabolism on the batch production of 2,5-DKG. Batch runs with viable cells yielded a final conversion of glucose to 2,5-DKG of 73% in 10 hours. Two Forms of nonviable cells were used: (1) cellular membrane fractions obtained by lysing viable cells using a French press, yielding 70% conversion in 12 hours and (2)permeabilized cells obtained by exposure to toluene, yielding over 90% conversion in 20 hours. While elimination of central metabolism by permeabilization of the cells resulted in an overall higher overall conversion from glucose to 2,5-DKG, the time it took to reach the final conversion was much longer. However, elimination of central metabolism by French pressing the cells did not result in an overall conversion,A mathematical model based on the Michaelis-Menten kinetics of the three oxidation enzymes responsible for 2,5-DKG production glucose dehydrogenase,gluconate dehydrogenase, and 2-KDG dehydrogenase, was developed to describe the 2,5-DKG concentration profile in the batch case. Kinetic parameters for each of these dehydrogenases were determined experimentally.The model consisted of a set of 4 coupled, nonlinear ordinary differential equations, each one describing the concentration of a species involved in the 2,5-DKGpathway. The input parameters needed were the volumetric activities of each enzymes,glucose dehydrogenase (El), gluconate dehydrogenase (E2), and 2-KDG dehydrogenase(E3), cell density (X), and a glucose uptake rate constant, Ut- Of these parameters, only the volumetric E3 activity and cell density were measurable. The other three parameters were determined by fitting the experimental data for one case of batch experiments performed with whole cells. These fit parameters were volumetric El activity =volumetric E2 activity = volumetric E3 activity, and Ut = 0.08 mmol/g/min. This model,with the fit parameters, successfully predicted the 2,5-DKG profile from another experiment with whole cells.A sensitivity analysis on how the overall conversion of glucose to 2,5-DKG(79 %) and time required to reach that nominal conversion (11 hr) would change with perturbations in the volumetric activities of El and E2, and Uj revealed that small perturbations of the volumetric activities of El and E2, and Ut did not affect greatly either the overall nominal conversion of glucose to 2,5-DKG or the amount of time required to achieve that conversion. The overall nominal conversion was most sensitive to perturbations in the volumetric El activity and Ut, and the time required to achieve the nominal conversion was most sensitive to the perturbations in the volumetric E2 activity.

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