Kaar, Joel L
(2008)
USING ENZYME STRUCTURE-ENVIRONMENT-ACTIVITY RELATIONSHIPS TO ENHANCE BIOCATALYST UTILITY.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The overall objective of this work was to probe the fundamental relationship between enzyme structure, environment, and activity. These relationships were in turn exploited as the basis for developing effective strategies for stabilizing and thus improving the efficiency of biocatalysts. Initially, the utility of ionic liquids, which represent an environmentally green alternative to conventional solvents, as reaction media for anhydrous enzymatic reactions was explored. Solvatochromic and partition coefficient analysis suggest that ionic liquids are considerably more polar and hydrophilic than organic solvents. In model transesterification reactions, the enzyme lipase was found to be highly active in 1-butyl-3-methylimidazolium hexafluorophosphate, but inactive in more hydrophilic ionic liquids. Conventional approaches to preventing deactivating conformational changes in non-aqueous environments were ineffective in improving lipase activity in ionic liquids. Moreover, stability studies indicated that lipase are significantly more stable in ionic liquids compared to in organic solvents.To effectively control the pH of essential enzyme-bound water molecules, which is critical even in anhydrous enzymology, a novel buffering approach was developed. This approach was based on the hypothesis that simultaneous biocatalytic reactions that produce acid and base will create a dynamic pH equilibrium. The concept of biocatalytic pH control was successfully demonstrated by employing urease-catalyzed urea hydrolysis, which forms ammonia, to neutralize acid produced by the enzymatic degradation of organophosphorous toxins. Based on the pH-dependent activity profiles of the enzymes, the pH of the combined enzyme system is predictable and can be controlled by adjusting the relative ratio of enzyme activities.Lastly, poly(ethylene glycol)-modification was investigated as a method of enhancing the stability of therapeutic proteases in in vivo settings. Matrix metalloproteinase-1 (MMP-1), which has considerable therapeutic potential in the treatment of fibrotic conditions, was employed as a model protease for these studies. Comparison of the efficacy of native full-length enzyme, the truncated active enzyme, and a poly(ethylene glycol)-modified form of the truncated active enzyme in a laceration model in mice showed that the truncated active enzyme produced the greatest reduction in interfibrillar collagen. Structural characterization of the modified enzyme suggests that site-specific modification are key to retaining activity and to improving the enzyme's stability.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
30 January 2008 |
| Date Type: |
Completion |
| Defense Date: |
11 August 2007 |
| Approval Date: |
30 January 2008 |
| Submission Date: |
15 October 2007 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Swanson School of Engineering > Chemical Engineering |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Bioremediation; Diisopropylfluorophosphatase; Green chemistry; Ionic liquids; lipase; Matrix metalloproteinase-1; Muscle fibrosis; Nerve agents; Nonaqueous enzymology; Organophosphorous hydrolyase; PEGylation; Transesterification; Biocatalytic buffering; Urease |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-10152007-152622/, etd-10152007-152622 |
| Date Deposited: |
10 Nov 2011 20:03 |
| Last Modified: |
15 Nov 2016 13:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/9471 |
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