ENZYME IMMOBILIZATION INTO POLYMERS AND COATINGS
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`
`by
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`Géraldine F. Drevon
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`BS, Chimie Physique Electronique Lyon, 1997
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`
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`Submitted to the Graduate Faculty of
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`School of Engineering in partial fulfillment
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`of the requirements for the degree of
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`Doctor of Philosophy
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`
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`University of Pittsburgh
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`2002
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`UNIVERSITY OF PITTSBURGH
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`School of Engineering
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`This dissertation was presented
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`by
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`
`
`Géraldine Drevon
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`
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`It was defended on
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`
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`November, 2002
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`and approved by
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`Eric J. Beckman, Professor, Chemical and Petroleum Engineering Department
`
`Toby M. Chapman, Associate Professor, Department of Chemistry
`
`William Federspiel, Professor, Chemical and Petroleum Engineering Department
`
`Krzysztof Matyjaszewski, Professor, Department of Chemistry, Carnegie Mellon
`University
`
`Douglas A. Wicks, Professor, Department of Polymer Science, University of Southern
`Mississippi
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`Dissertation Advisor: Alan J. Russell, Professor, Chemical and Petroleum Engineering
`Department
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`ABSTRACT
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` ENZYME IMMOBILIZATION INTO POLYMERS AND COATINGS
`
`
`
`Géraldine F. Drevon, PhD
`
`University of Pittsburgh, 2002
`
`
`
`In this study, we have developed strategies to immobilize enzymes into various
`
`polymer and coatings. Three categories of bioplastic matrices were investigated. The
`
`first
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`type of bioplastics was prepared by
`
`irreversibly
`
`incorporating di-
`
`isopropylfluorophosphatase (DFPase) into polyurethane (PU) foams. The resulting
`
`bioplastic retained up to 67 % of the activity for native enzyme. The thermostability of
`
`DFPase was highly affected by the immobilization process. Unlike native enzyme,
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`immobilized DFPase had biphasic deactivation kinetics. Our data demonstrated that the
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`initial rapid deactivation of immobilized DFPase lead to the formation of a hyper-stable
`
`and still active form of enzyme. Spectroscopic studies enabled a structural analysis of
`
`the hyper-stable intermediate.
`
`Biopolymers were also prepared via atom transfer radical polymerization
`
`(ATRP) using acrylic and sulfonate-derived monomers. ATRP ensured the covalent and
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`multi-point immobilization of enzyme within polymer matrices. However, this approach
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`was only partially successful, as no activity retention was obtained after polymerization
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`Enzyme-containing PU- and Michael adduct (MA)-based coatings correspond to
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`the last category of bioplastics that was investigated. DFPase was irreversibly
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`incorporated into PU coatings. The distribution of immobilized DFPase as well as
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`activity retention were homogeneous within the coating. The resulting enzyme-
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`containing coating (ECC) film hydrolyzed DFP in buffered media at high rates retaining
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`approximately 39% intrinsic activity. DFPase-ECC had a biphasic deactivation profile
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`similar to that of bioplastic foams. The synthesis of enzyme-containing MA coatings
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`was performed in a two-step process using carbonic anhydrase (CA, E.C. 4.2.1.1). CA
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`was first covalently immobilized into NVF-based water-soluble polymer (EP). The
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`resulting EP was further entrapped into the matrix of MA coating. The so-formed
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`ECC’s exhibited approximately 7% apparent activity. CA-ECC showed good stability
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`under ambient conditions and retained 55% activity after 90 days of storage.
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`TABLE OF CONTENTS
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`Page
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`1.0
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`2.0
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`INTRODUCTION ................................................................................................ 1
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`BACKGROUND AND LITERATURE REVIEW .............................................. 5
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`2.1
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`Biocatalyst Deactivation and Regeneration ...................................... 5
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`2.1.1 Reversible Denaturation.................................................................... 6
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`2.1.2 Modes of Inactivation........................................................................ 7
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`2.1.2.1 Thermoinactivation. ................................................................... 7
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`2.1.2.2 Oxidation ................................................................................. 19
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`2.1.2.3
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`Inactivation by pH ................................................................... 21
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`2.1.2.4
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`Inactivation by Organic Solvents............................................. 22
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`2.1.2.5 Metal Chelators........................................................................ 26
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`2.1.2.6 “Salting-in” Effect ................................................................... 26
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`2.1.2.7 Sulfhydryl-Reducing Agents. .................................................. 27
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`2.1.2.8 Mechanical Modes of Inactivation .......................................... 28
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`2.1.2.9 Radiation.................................................................................. 29
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`2.1.2.10 Cold, Freezing and Lyophilization.......................................... 29
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`2.1.3 Strategies to Minimize Inactivation ................................................ 31
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`2.1.3.1 Soluble Stabilizers ................................................................... 31
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`2.1.3.2 Chemical Modification............................................................ 35
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`2.1.3.3
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`Immobilization......................................................................... 39
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`2.1.3.4 Protein Engineering ................................................................. 44
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`2.1.3.5 Directed Evolution. .................................................................. 46
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`2.2
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`Immobilization of Agentases .......................................................... 47
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`2.2.1 Agentases ........................................................................................ 47
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`2.2.2 Nerve-Agent Degrading Biomaterials............................................. 49
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`2.3
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`2.4
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`2.5
`
`Incorporation of Enzyme into Polyurethane Foams........................ 54
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`Polymers Prepared Using ATRP..................................................... 57
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`Enzyme Immobilization into Coatings............................................ 58
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`2.5.1 Waterborne Two-Component (2K) Polyurethane (PU) Coatings as
`Support ............................................................................................ 85
`
`2.5.2 Michael Adduct-Based Coating as Support .................................... 86
`
`3.0
`
`4.0
`
`SPECIFIC AIMS ................................................................................................ 88
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`IRREVERSIBLE IMMOBILIZATION OF DFPASE IN PU POLYMERS ..... 92
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`4.1
`
`4.2
`
`Introduction..................................................................................... 92
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`Materials and Methods.................................................................... 96
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`4.2.1 Materials .......................................................................................... 96
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`4.2.2 Methods........................................................................................... 97
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`4.2.2.1 Protein-Containing Polymer Synthesis .................................... 97
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`4.2.2.2 Activity of DFPase Polyurethanes. .......................................... 98
`
`4.2.2.3 Product and Substrate Partitioning .......................................... 99
`
`4.2.2.4 Determination of Kinetic Constants ...................................... 100
`
`4.2.2.5 Protein Concentration Determination.................................... 101
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`4.2.2.6 PEGylation of DFPase........................................................... 101
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`4.2.2.7 Characterization of DFPase Modification ............................. 101
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`4.2.2.8 Preparation of apo-DFPase .................................................... 102
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`4.2.2.9 Thermostability of Native DFPase ........................................ 102
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`4.2.2.10 Thermostability of Native DFPase in Presence of PEG-
`Amine. ................................................................................... 102
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`4.2.2.11 Thermostability of PEG-Modified DFPase ........................... 103
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`4.2.2.12 Thermostability of Immobilized DFPase............................... 103
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`4.2.2.13 CD Spectroscopy. .................................................................. 103
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`4.3
`
`Results and Discussion.................................................................. 104
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`4.3.1 Reversibility of DFPase Attachment............................................. 104
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`4.3.2 Substrate and Product Partitioning................................................ 104
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`4.3.3 Activity in Absence of Surfactant ................................................. 105
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`4.3.4 Activity with Surfactants............................................................... 106
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`4.3.5 Effect of Surfactant on Polymer Morphology............................... 111
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`4.3.6 Effect of Salt removal on Enzyme Activity.................................. 111
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`4.3.7 Thermoinactivation of Native DFPase.......................................... 114
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`4.3.8 Thermostability of DFPase-Containing Polyurethane .................. 116
`
`the Thermostability of Native and
`4.3.9 Effect of Calcium on
`Immobilized DFPase ..................................................................... 120
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`4.3.10 Thermostability of PEG-Modified DFPase................................... 128
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`4.3.11 Structural Basis for Deactivation.................................................. 133
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`4.4
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`Conclusion..................................................................................... 134
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`5.0
`
`IMMOBILIZATION OF CA INTO POLYMERS USING ATRP .................. 136
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`Introduction................................................................................... 136
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`Materials and Methods.................................................................. 137
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`5.1
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`5.2
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`5.2.1 Materials ........................................................................................ 137
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`5.2.2 Activity Assay............................................................................... 138
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`5.2.3 CA Stability in the Presence of Reagents for ATRP..................... 138
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`5.2.4 Chemical Modification with 2-Bromo-Propionyl Chloride .......... 139
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`5.2.5 Chemical Modification with Bromoisobutyric Acid..................... 140
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`5.2.6 Characterization of Bio-Macroinitiator ......................................... 140
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`5.2.7 ATRP Polymerization................................................................... 141
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`5.2.8 Biopolymer Characterization ........................................................ 143
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`5.3
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`Results and Discussion.................................................................. 144
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`5.3.1 Synthesis of Bio-Macroinitiator.................................................... 144
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`5.3.2 CA Stability in the Presence of Reagents for ATRP..................... 145
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`5.3.3 Enzyme Immobilization................................................................ 149
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`5.4
`
`Conclusion..................................................................................... 150
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`6.0
`
`2K-PU
`INTO WATERBORNE
`IMMOBILIZATION OF DFPASE
`COATING ........................................................................................................ 153
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`6.1
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`6.2
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`Introduction................................................................................... 153
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`Material and Methods.................................................................... 154
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`6.2.1 Material ......................................................................................... 154
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`6.2.2 ECC Synthesis............................................................................... 154
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`6.2.3 Protein Concentration Determination............................................ 156
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`6.2.4 Synthesis of Enzyme/Gold Conjugates......................................... 156
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`6.2.5 Localization of Gold-DFPase Conjugate in Coating..................... 157
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`6.2.6 Activity of ECC’s.......................................................................... 157
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`6.2.7 Determination of Kinetic Constants .............................................. 158
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`6.2.8 Diffusion Cell Experiments........................................................... 158
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`6.2.8.1 Determination of Susbtrate Effective Diffusion Coefficient,
`Deff.......................................................................................... 158
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`6.2.8.2 Activity Measurements.......................................................... 159
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`6.2.9 Enzyme Modification with Desmodur N3400. ............................. 162
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`6.2.10 ECC Thermostability..................................................................... 163
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`6.3
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`Results and Discussion.................................................................. 165
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`6.3.1 Reversibility of DFPase Attachment to ECC’s ............................. 165
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`6.3.2 Enzyme Distribution in ECC’s...................................................... 165
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`6.3.3 Activity of ECC’s.......................................................................... 166
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`6.3.4 Effective Diffusivity of DFP in ECC, Deff .................................... 167
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`6.3.5 Desmodur N3400-Modified ECC’s .............................................. 171
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`6.3.6 Thermostability of ECC’s ............................................................. 175
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`6.4
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`Conclusion..................................................................................... 178
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`7.0
`
`IMMOBILIZATION OF CA INTO MICHAEL-BASED COATING ............ 181
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`7.1
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`7.2
`
`Introduction................................................................................... 181
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`Material and Methods.................................................................... 182
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`7.2.1 Material ......................................................................................... 182
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`7.2.2 Michael Adduct Synthesis............................................................. 182
`
`3-(N-vinylformamido)
`N—hydroxyethyl
`of
`7.2.3 Synthesis
`propionamide................................................................................. 183
`
`7.2.4 Polymerization of CA with Michael Adducts............................... 183
`
`3-(N-
`N—hydroxyethyl
`of
`7.2.4.1 Activation
`vinylformamido)propionamide .............................................. 183
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`7.2.4.2 CA- and Neurotensin-MANVF ............................................. 183
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`7.2.4.3 EP Synthesis .......................................................................... 183
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`7.2.4.4 ECC’s Synthesis .................................................................... 184
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`7.2.5 Immobilization of CA onto Coating Surface. ............................... 187
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`7.2.5.1 Direct coupling Between CA and Coating Surface ............... 187
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`7.2.5.2
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`Immobilization onto Coatings by Glow-Discharge and
`Treatment with Glutaraldehyde ............................................. 187
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`7.2.5.3
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`Immobilization onto Partially Hydrolyzed Coating .............. 188
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`7.2.6 Activity Assays ............................................................................. 188
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`7.2.6.1 Native CA and EP’s............................................................... 188
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`7.2.6.2 ECC Activity ......................................................................... 188
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`7.2.6.3 ECC Stability......................................................................... 189
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`7.2.6.4 Enzyme Kinetics.................................................................... 189
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`7.2.6.5 Thermostability. ..................................................................... 190
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`7.2.7 Characterization of Neurotensin- and CA-MANVF. .................... 190
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`7.2.8 EP Characterization. ...................................................................... 190
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`7.2.8.1 Aqueous GPC ........................................................................ 190
`
`7.2.8.2 Analytic Ultracentrifugation.................................................. 191
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`7.3
`
`Results and Discussion.................................................................. 191
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`7.3.1 CA and Neurotensin Modified with Activated MANVF .............. 191
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`7.3.2 Activity and Stability of EP .......................................................... 191
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`7.3.3 Activity of ECC’s.......................................................................... 197
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`7.3.4 Immobilization of CA onto Pre-Formed Coatings ........................ 198
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`7.3.5 Thermostability of ECC’s ............................................................. 202
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`7.4
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`Conclusion..................................................................................... 202
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`BIBLIOGRAPHY ......................................................................................................... 204
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`LIST OF TABLES
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`Table No.
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`KINETIC PROPERTIES OF ENZYME-CONTAINING COATINGS ...........................64
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`THERMOSTABILITY OF ENZYME-CONTAINING COATING...............................77
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`DFP PARTITIONING INTO BIOPLASTICS.......................................................107
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`KINETIC PARAMETERS FOR DFPASE-CONTAINING POLYMERS AND SOLUBLE
`DFPASE.....................................................................................................109
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`KINETIC PARAMETERS FOR THERMOINACTIVATION OF NATIVE DFPASE....117
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`KINETIC PARAMETERS FOR THERMOINACTIVATION OF PEG-MODIFIED AND
`IMMOBILIZED DFPASE...............................................................................121
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`SECONDARY STRUCTURE OF NATIVE AND MODIFIED DFPASE IN THE PRESENCE
`OF EGTA AND DIFFERENT FREE CALCIUM CONCENTRATIONS....................125
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`SECONDARY STRUCTURE OF NATIVE AND MODIFIED DFPASE DURING
`DENATURATION AT 65 OC...........................................................................132
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`STABILITY OF CA IN THE PRESENCE OF REAGENTS FOR ATRP ..................152
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`KINETIC PARAMETERS FOR DFPASE-CONTAINING COATINGS AND SOLUBLE
`DFPASE.....................................................................................................172
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`KINETIC PARAMETERS FOR CA IMMOBILIZED INTO NVF- AND MANVF-
`DERIVED POLYMER, ECC’S, AND NATIVE CA ............................................196
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`LIST OF FIGURES
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`Figure No.
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`DECOMPOSITION OF DISULFIDE BRIDGE .......................................................13
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`DEAMIDATION OF ASPARAGINES RESIDUES ..................................................15
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`LYSINE RESIDUES CROSS-LINKING ...............................................................16
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`RACEMIZATION OF AMINO ACIDS.................................................................18
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`MAILLARD REACTION ..................................................................................18
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`OXIDATION OF METHIONINE ........................................................................20
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`OXIDATION OF CYSTEINE.............................................................................21
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`REACTION SCHEMATIC OF BIOPOLYMER SYNTHESIS.....................................56
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`PRINCIPLES FOR ATRP. ...............................................................................59
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`10
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`IRREVERSIBLE IMMOBILIZATION OF ENZYME INTO WATERBORNE 2K-PU
`COATINGS ....................................................................................................87
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`11 MODEL MECHANISM FOR THE DFPASE-CATALYZED HYDROLYSIS OF DFP..94
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`EFFECT OF DFPASE CONCENTRATION ON DFPASE-CONTAINING POLYMER
`EFFICIENCY. ...............................................................................................108
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`EFFECT OF SURFACTANT ON DFPASE-POLYMER EFFICIENCY. ....................112
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`SCANNING ELECTRON MICROGRAPHS OF POLYMERS PREPAred without
`surfactant and with L62. ...........................................................................113
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`EFFECT OF DFPASE LOADING ON DFPASE-CONTAINING POLYURETHANE
`EFFICIENCY IN THE ABSENCE OF SALT. .......................................................115
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`CONFORMATION OF NATIVE DFPASE AT VARIOUS REMAINING ENZYMATIC
`ACTIVITIES DURING THERMOINACTIVATION...............................................118
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` EFFECT OF CALCIUM ON DFPASE SECONDARY STRUCTURE. .....................127
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`18 MALDI SPECTRA OF PEG-DFPASE PREPARED WITH A 1/100 PROTEIN TO PEG-
`NCO MOLAR RATIO. ..................................................................................130
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`PEGYLATION OF DFPASE..........................................................................131
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`ENZYME COUPLING TO 2-BROMO-PROPIONIC CHLORIDE............................139
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`COUPLING OF CA TO BROMOISOBUTYRIC ACID..........................................141
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`COUPLING OF NEUROTENSIN WITH 2-BROMO PROPIONYL CHLORIDE..........146
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`COUPLING OF NEUROTENSIN WITH 2-BROMO ISOBUTYRIC ACID.................147
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`COUPLING OF CA WITH BROMO-INITIATORS..............................................148
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`SCHEMATIC OF THE DFP CONCENTRATION PROFILE IN THE CASE OF
`SIMULTANEOUS DIFFUSION AND ENZYMATIC REA CTION IN THE DFPASE-
`CONTAINING POLYURETHANE COATING. ....................................................164
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`ENZYME DISTRIBUTION IN POLYURETHANE COATING ................................168
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`EFFECT OF DFPASE CONCENTRATION ON DFPASE-CONTAINING COATING
`EFFICIENCY. ...............................................................................................169
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`EFFECTIVE DIFFUSION OF DFP THROUGH COATINGS..................................173
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`PROFILES FOR DFP CONSUMPTION IN DIFFUSION CELLS.............................174
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`PROFILES FOR DFP CONSUMPTION IN DIFFUSION CELLS.............................176
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`THERMOINACTIVATION OF DFPASE-CONTAINING COATING. .....................179
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`THERMOINACTIVATION OF DFPASE-CONTAINING COATING AT ROOM
`TEMPERATURE ...........................................................................................180
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`DIAGRAM OF ENZYME MODIFICATION WITH MANVF ...............................185
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`STEPS LEADING TO THE PR EPARATION OF ECC’S . ..................................... 186
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`COVALENT COUPLING OF ENZYME TO THE COATING SURFACE VIA SCHIFF’S
`BASES .........................................................................................................188
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`EFFECT OF UV IRRADIATION TIME ON THE ACTIVITY OF NATIVE CA AND ON
`THE APPARENT ACTIVITY RETENTION OF EP’S ...........................................194
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`THERMOINACTIVATION OF NATIVE CA AND EP AT 65 OC..........................195
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`EFFECT OF CA CONCENTRATION ON CA-CONTAINING COATING APPARENT
`EFFICIENCY. ...............................................................................................199
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`ELECTRON MICROGRAPHS OF MICHAEL ADDUCT DERIVED-COATINGS. ......200
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`ECC’S REUSABILITY FOR ACTIVITY ASSAYS ..............................................201
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`THERMOINACTIVATION OF DRY CA-CONTAINING COATING UNDER AMBIENT
`CONDITIONS ...............................................................................................203
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`ATRP
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`bpy
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`BTP
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`CA
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`DFP
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`DFPase
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`ECC
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`EP
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`MANVF
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`MA
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`NHS
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`NVF
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`NaSS
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`PEG
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`PEG-NCO
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`PEG-(NCO)2
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`PU
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`TMATf
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`Tris-HCl
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`NOMENCLATURE
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`Atom transfer radical polymerization
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`Bipyridine
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`Bis-tris propane
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`Carbonic anhydrase
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`Diisopropylfluorophosphate
`
`Diisopropylfluorophosphatase
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`Enzyme-containing coating
`
`Enzyme-polymer
`
`Michael adduct from N-vinylformamide and methyl
`acrylate
`
`Michael adduct
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`N-Hydroxysuccinimide
`
`N-Vinylformamide
`
`Sodium styrene sulfonate
`
`Poly(ethylene glycol)
`
`Poly(ethylene glycol)-monoisocyanate
`
`Poly(ethylene glycol)-diisocyanate
`
`Polyurethane
`
`2-(N,N,N-trimethylammonio)ethyl
`trifluoromethanesulfonate
`
`
`methacrylate
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`Tris(hydroxymethyl)aminomethane-HCl
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`1.0 INTRODUCTION
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`Enzymes are attractive catalysts as they are highly effective and specific under
`
`ambient conditions. A major drawback is their short lifetimes. Research over the last
`
`four decades has focused on understanding the modes of enzymatic deactivation, as
`
`well as developing methods to overcome this shortcoming of the biocatalytic approach.
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`Most enzymes are stable when stored at low temperatures and neutral pH’s in aqueous
`
`media. This state is fragile and can be easily disturbed by means of external stresses
`
`such as high pressures and temperatures, extreme pH’s, organic solvents, freezing,
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`drying, and by oxidative, chelating, or denaturing agents. Conformational changes as
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`well as chemical processes at the level of the polypeptide chains may be induced,
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`leading to the enzyme inactivation. The resulting activity loss may be reversible or
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`irreversible. The severity of these effects varies with the type of enzyme and the nature
`
`and the intensity of the stress. The main structural and covalent mechanisms in
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`biocatalyst inactivation include the disulfide intra- and inter-exchanges, the deamidation
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`of asparagine residues, the decomposition of disulfide bridges by b-elimination, the
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`hydrolysis of peptide chains, the b-isomerization of asparagine and aspartic acid
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`residues, and amino acid racemization.
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`A first approach to prevent deactivation consists of changing the enzyme
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`environment, for example, by means of soluble additives such as metals, surfactants,
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`polyols, PEGs and sugars. Stabilization results from the non-covalent interactions
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`between the additive, the biocatalyst and the solvent. Stabilizing effects of similar origin
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`have often been observed for enzymes that are entrapped within a solid matrix or
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`physically adsorbed onto a support. Other strategies rely on the alteration of the enzyme
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`primary structure using one of the following methods: chemical modification; covalent
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`immobilization; protein engineering; or directed evolution.
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`Immobilization refers to the preparation of insoluble biocatalytic derivatives and
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`involves the coupling of enzymes to solid supports that are either organic or inorganic.
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`It has been increasingly used in industrial applications as it facilitates the separation of
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`biocatalysts from the effluents and, hence, the recovery and purification of the products.
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`Moreover, solid biocatalysts offer the major advantage of being reusable. The large
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`variety of matrices that can be used ranges from natural and synthetic polymers to silica
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`beads. Covalent
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`immobilization often proceeds by
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`the
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`reaction of specific
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`functionalities at the support surface with amino acid side chains that are readily
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`available on the enzyme surface. The covalent coupling may induce drastic changes in
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`the enzymatic kinetics especially when it occurs near the active site. Another important
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`effect is to reduce the enzyme flexibility. As the number of linkages between the
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`enzyme and the support increases, so does the enzyme rigidity. By providing a
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`maximum rigidity, multi-point covalent immobilization is likely to prevent enzyme
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`unfolding upon heating or in the presence of a denaturant. A non-conventional strategy
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`to achieve multi-point covalent immobilization within a polymer network is by
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`copolymerizing the enzyme with monomers capable of a chemical reaction with
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`specific functionalities on its surface. During polymerization, the enzyme acts as a
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`monomer and is, thus, expected to be uniformly distributed within the resulting
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`biopolymer.
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`A
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`large range of polymer matrices have been employed for enzyme
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`immobilization. In this study, enzymes were inserted into various polymer networks. To
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`optimize the catalytic efficiency and stability of the resulting biopolymers, the attempt
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`was made
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`to understand
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`the
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`immobilization effects on enzymatic properties.
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`Polyurethane foams are attractive supports, and can be used to prepare highly active and
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`stable bioplastics via multi-point and covalent immobilization. Therefore, their potential
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`as matrices for the immobilization of the agentase diisopropylfluorophosphatase
`
`(DFPase) was investigated. Atom transfer radical polymerization (ATRP) is another
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`potential method for the mutli-point and covalent immobilization of enzymes, as it is
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`compatible with a large variety of functionalized monomers and can be performed
`
`under mild conditions. It was determined whether ATRP could be used for the
`
`incorporation of biocatalysts into polymer matrices. The third category of polymers that
`
`were tested includes polyurethane- and Michael adduct-based coatings. One important
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`challenge in preparing enzyme-containing coatings (ECC’s) was to reduce the internal
`
`diffusional limitations and, hence, assure good apparent activity retentions. A method to
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`facilitate the access of immobilized enzyme to substrate for bio-PU coatings was
`
`developed. When working with Michael adduct-based coatings, the main aim was to
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`incorporate homogeneously the enzyme within the hydrophobic blend used for the
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`casting of films. Given the enzyme hydrophilicity, the enzyme properties had to be
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`altered to enable its dispersion in the coating bulk.
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`2.0 BACKGROUND AND LITERATURE REVIEW
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`2.1 Biocatalyst Deactivation and Regeneration
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`Biocatalysts offer major advantages over traditional metallic and organometallic
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`catalysts such as high specificity and high efficiency under mild conditions of
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`temperature, pressure and pH. However, their storage and operational instability limit
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`considerably their use for industrial applications. Therefore, important efforts have been
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`made to develop stabilization strategies, which rely on the rational understanding of the
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`causes and pathways involved in their deactivation.
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`In this chapter, we will consider the reversible and irreversible deactivation of
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`biocatalysts. At this point, it is necessary to specify that we will preferably use terms
`
`such as enzyme, protein, proteinase and protease instead of the more general term
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`biocatalyst. We will describe the various modes of irreversible inactivation, and more
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`especially, the deactivation induced by heat, extreme pH, organic solvents, freezing,
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`antagonist additives such as detergents, salts, specific chemicals and metal ions. We
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`will further examine the techniques that have been developed to prevent or minimize
`
`biocatalyst deactivation. Stabilization in aqueous media can be achieved by altering the
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`water–protein interactions by means of external excipients, and by chemically or
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`genetically modifying specific amino acids. Protein immobilization is another major
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`strategy.
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`2.1.1 Reversible Denaturation
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`The denaturation of an enzyme corresponds to the unfolding of the enzyme by
`
`disruption of noncovalent intramolecular interactions. This can be induced by any
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`change in the enzyme environment such as an increase in temperature or the addition of
`
`a denaturing agent. The process is reversible when the native conformation of enzyme
`
`(E) and, hence, the original biological activity are spontaneously recovered by simply
`
`returning to the initial external conditions. After extensive unfolding, an enzyme is
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`predominantly randomly coiled, and hence inactive. The highest extent of protein
`
`reversible unfolding has been obtained at room temperature in the presence of
`
`denaturant chemicals such as urea, guanidine hydrochloride (GdnH+, GdnHCl) and
`
`guanidine thiocyanate. Heat results also in extensive loss of ordered secondary and
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`tertiar