Doctoral Dissertations

Date of Award

8-2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemical Engineering

Major Professor

Douglas Gordon Hayes

Committee Members

Paul D. Frymier, Tsewei Wang, Qixin Zhong

Abstract

Purification of proteins by microemulsion was improved through use of a 3-phase (“Winsor-III”) microemulsion system instead of the traditionally employed 2-phase (“Winsor-II”) water-in-oil microemulsion system. As a consequence, two of the main problems of the traditional method were improved: the low and slow rate of recovery of proteins encapsulated by the microemulsions and the low protein solubilization capacity of the microemulsions.

Microemulsion systems employed a mixture of surfactants, two pH-degradable “cyclic ketal” alkyl ethoxylates (O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl) methoxy]–O’- methoxy poly(ethylene glycol)n , where n, the average degree of polymerization for the ethoxylate group, equaled 3.0 or 5.45, CK3 and CK7, respectively) and Aerosol-OT (AOT). CK7’s ethoxylate size has broad molecular weight distribution. Partitioning behavior of CK7 molecules as a function of their ethoxylate size, temperature, and the addition of a second surfactant (AOT, CK3, or octyl β-D-glycoside) were investigated. A semi-empirical thermodynamic mathematical model was developed to calculate the phase inversion temperature (PIT) for a surfactant mixture of a specified composition. This information is useful to find the optimal Winsor-III for protein extraction.

Finally different Winsor-III systems formed by water, isooctane and surfactant mixtures of CK7, CK3 and AOT were tested in forward extraction of α-chymotrypsin, cytochrome-c, lysozyme, BSA and pepsin. Three approaches were used to obtain Winsor- III systems suitable for forward extraction: employment at 40°C, addition of a more hydrophobic surfactant (CK3, 25°C), and the addition of 1.5 wt. % NaCl (aq) to increase the ionic strength (25°C). Protein concentrations achieved in microemulsion phase were 10 times higher than values reported in the literature for extraction by Winsor-II microemulsion systems.

Back extraction was tested for α-chymotrypsin, cytochrome-c, and lysozyme. Aqueous striping solutions used for back extraction contained either a high ionic strength (5 wt. % NaCl) or a high pH (12.0). Back-extraction was fast and total recovery of the activity for α-chymotrypsin was achieved. Other proteins have smaller percentage of mass recovery in the conditions tested but comparable with yields reported in the literature for microemulsion-based extraction.

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