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I USING ENZYME STRUCTURE-ENVIRONMENT-ACTIVITY RELATIONSHIPS TO USING ENZYME STRUCTURE-ENVIRONMENT-ACTIVITY RELATIONSHIPS TO ENHANCE BIOCATALYST UTILITY by Joel L. Kaar B.S. Chemical Engineering, University of Pittsburgh, 2001 Submitted to the Graduate Faculty of the School of Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2007 i UNIVERSITY OF PITTSBURGH SCHOOL OF ENGINEERING This dissertation was presented by Joel L. Kaar It was defended on September 11th, 2007 and approved by Dr. Mohammad M. Ataai, Professor, Departments of Chemical Engineering and Bioengineering Dr. Eric J. Beckman, Professor, Department of Chemical Engineering Dr. Harry C. Blair, Professor, Departments of Pathology and Physiology and Cell Biology Dr. Johnny Huard, Professor, Departments of Orthopaedic Surgery and Bioengineering Dissertation Director: Dr. Alan J. Russell, Professor, Departments of Surgery and Chemical Engineering ii Copyright © by Joel L. Kaar 2007 iii USING ENZYME STRUCTURE-ENVIRONMENT-ACTIVITY RELATIONSHIPS TO ENHANCE BIOCATALYST UTILITY Joel L. Kaar, PhD University of Pittsburgh, 2007 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 iv 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. v DESCRIPTORS Biocatalytic buffering Muscle fibrosis Bioremediation Nerve agents Diisopropylfluorophosphatase Nonaqueous enzymology Green chemistry Organophosphorous hydrolase Ionic liquids PEGylation Lipase Transesterification Matrix metalloproteinase-1 Urease vi TABLE OF CONTENTS LIST OF TABLES.......................................................................................................................XII LIST OF FIGURES ................................................................................................................... XIII NOMENCLATURE ................................................................................................................XVIII PREFACE.................................................................................................................................. XIX 1.0 INTRODUCTION ......................................................................................................... 1 2.0 BACKGROUND AND LITERATURE REVIEW........................................................ 6 2.1 ANHYDROUS ENZYMOLOGY......................................................................... 6 2.1.1 Enzyme Structure in Anhydrous Solvents ...................................................... 7 2.1.2 Influence of Water on Enzyme Activity......................................................... 8 2.1.3 Solvent Types.................................................................................................. 9 2.2 EFFECT OF pH ON ENZYME ACTIVITY AND STABILITY ....................... 12 2.3 PROTEASE STABILITY ................................................................................... 15 2.4 STRATEGIES FOR ENZYME STABILIZATION............................................ 16 2.4.1 Chemical Additives....................................................................................... 16 2.4.2 Chemical Modification ................................................................................. 19 2.4.3 Immobilization.............................................................................................. 23 2.4.4 Protein Engineering ...................................................................................... 28 2.4.5 Directed Evolution........................................................................................ 30 vii 3.0 SPECIFIC AIMS.......................................................................................................... 32 4.0 CHARACTERIZATION OF ENZYME ACTIVITY AND STABILITY IN IONIC LIQUIDS .............................................................................................................................. 35 4.1 INTRODUCTION ............................................................................................... 35 4.2 MATERIALS AND METHODS ........................................................................ 38 4.2.1 Materials ....................................................................................................... 38 4.2.2 Methods......................................................................................................... 39 4.2.2.1 Determination of Octanol-Water Partition Coefficients ....................... 39 4.2.2.2 Solvatochromic Characterization.......................................................... 40 4.2.2.3 Poly(Ethylene Glycol)-Modified Lipase Synthesis .............................. 41 4.2.2.4 Lipase-Containing Polyurethane Foam Synthesis ................................ 41 4.2.2.5 Lipase Stability in Ionic Liquids........................................................... 41 4.2.2.6 Water Activity Measurements in Ionic Liquids Using Humidity Sensor . ............................................................................................................... 42 4.2.2.7 Water Transfer Measurements in Ionic Liquids ................................... 42 4.2.2.8 Lipase-Catalyzed Transesterification of Methyl Methacrylate and 2- Ethylhexanol ......................................................................................... 43 4.3 RESULTS AND DISCUSSION.......................................................................... 44 4.3.1 Physical Characterization of Ionic Liquids................................................... 44 4.3.2 Lipase Activity in Ionic Liquids ................................................................... 48 4.3.3 Use of Modified and Immobilized Lipases in Ionic Liquids ........................ 51 4.3.4 Lipase Stability in Ionic Liquids................................................................... 53 4.3.5 Controlled Water Activity in Ionic Liquids.................................................. 56 viii 4.3.6 Impact of Water Activity on Lipase Activity in Ionic Liquids..................... 61 4.4 CONCLUSIONS ................................................................................................. 64 5.0 BIOCATALYTIC pH CONTROL IN ENZYMATIC REACTIONS ......................... 65 5.1 INTRODUCTION ............................................................................................... 65 5.2 THEORETICAL MODELING OF DYNAMIC pH EQUILIBRIUM IN OPH- UREASE COMBINED ENZYME SYSTEM ...................................................... 69 5.3 MATERIALS AND METHODS ........................................................................ 71 5.3.1 Materials ....................................................................................................... 71 5.3.2 Methods......................................................................................................... 72 5.3.2.1 OPH Activity Assay.............................................................................. 72 5.3.2.2 Urease Activity Assay........................................................................... 72 5.3.2.3 Preparation of Helicobacter Pylori Urease........................................... 73 5.3.2.4
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