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Development of Immobilized Biocatalysts for Continuous-Flow

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Development of Immobilized Biocatalysts for Continuous-Flow Budapest University of Technology and Economics Faculty of Chemical Technology and Biotechnology Department of Organic Chemistry and Technology George A. Olah Doctoral School DEVELOPMENT OF IMMOBILIZED BIOCATALYSTS FOR SELECTIVE BIOTRANSFORMATIONS PHD THESIS Emese Abaházi Supervisor: Prof. Dr. László Poppe 2018 2 DECLARATION 3 DECLARATION I, Emese Abaházi hereby declare, that this PhD thesis is my own work and I have used in this thesis only references listed in the list. Any parts that I have taken literally or in the same content but reworded from other sources are clearly indicated. Budapest, 02 September 2018. ……………………………… NYILATKOZAT Alulírott Abaházi Emese kijelentem, hogy ezt a doktori értekezést magam készítettem és abban csak a megadott forrásokat használtam fel. Minden olyan részt, amelyet szó szerint, vagy azonos tartalomban, de átfogalmazva más forrásból átvettem, egyértelműen, a forrás megadásával megjelöltem. Budapest, 2018. szeptember 2. ……………………………… 4 TABLE OF CONTENTS DEVELOPMENT OF IMMOBILIZED BIOCATALYSTS FOR SELECTIVE BIOTRANSFORMATIONS ............ 1 DECLARATION ..................................................................................................................................... 3 NYILATKOZAT ..................................................................................................................................... 3 TABLE OF CONTENTS ........................................................................................................................... 4 ACKNOWLEDGEMENT / KÖSZÖNETNYILVÁNÍTÁS............................................................................... 7 ABBREVIATIONS .................................................................................................................................. 8 1. INTRODUCTION ........................................................................................................................ 10 2. LITERATURE OVERVIEW .......................................................................................................... 11 2.1. Significance of chirality in pharmaceutical industry ............................................................ 11 2.2. Enzymes – catalytic biopolymers ............................................................................................ 14 2.3. Enzyme structure and classification ....................................................................................... 14 2.4. Mechanism of enzyme catalyzed reactions ............................................................................. 16 2.5. Enzyme kinetics ....................................................................................................................... 17 2.6. Enzyme-catalyzed kinetic resolutions ..................................................................................... 20 2.7. Lipases- structure and mechanism of catalysis ...................................................................... 23 2.8. ω-Transaminases- valuable biocatalysts for chiral amine synthesis .................................... 26 2.8.1. Transaminase catalysis - Mechanism of transamination ......................................................... 29 2.9. Oxidoreductases ....................................................................................................................... 30 2.10. Cascade reactions involving transaminases and ketoreductases .......................................... 31 2.11. Biocatalyst immobilization techniques ................................................................................... 33 2.11.1. Covalent enzyme immobilization ..................................................................................... 35 2.11.2. Sol-gel entrapment of enzymes or whole-cells ................................................................ 37 2.11.3. Molecular imprinting ...................................................................................................... 38 2.11.4. Immobilization of lipases ................................................................................................. 39 2.11.5. Immobilization of ω-transaminases ................................................................................ 40 2.12. Continuous-flow biocatalysis .................................................................................................. 42 3. MATERIALS AND METHODS ..................................................................................................... 44 3.1. Materials .................................................................................................................................. 44 3.1.1. Additives ................................................................................................................................... 44 3.1.2. Enzymes and whole-cells .......................................................................................................... 44 3.1.3. Supports .................................................................................................................................... 45 3.2. Analytical methods .................................................................................................................. 46 3.2.1. Scanning electron microscope analysis ................................................................................... 46 3.2.2. Gas chromatography analysis .................................................................................................. 46 3.3. Surface activation of aminoalkyl polymer resins with bisepoxides or glutaraldehyde ......... 47 TABLE OF CONTENTS 5 3.4. Immobilization of lipases (BcL or CaLB) ............................................................................... 48 3.4.1. Immobilization of BcL by adsorption on mesoporous silica gel with mixed grafting .............. 48 3.4.2. BcL immobilization by adsorption followed by cross-linking on mesoporous silica gel with mixed grafting ........................................................................................................................... 48 3.4.3. Immobilization of CaLB on bisepoxide-activated aminoalkyl resins ....................................... 48 3.5. Immobilization of ω-transaminase on bisepoxide-activated aminoalkyl resins .................... 49 3.6. Immobilization of E. coli and LeKRED whole-cells in templated sol-gel matrices .............. 49 3.7. Enantiomer selective acetylation of rac-1a and rac-1b .......................................................... 50 3.8. Kinetic resolution of racemic amines catalyzed by immobilized transaminase preparations in batch mode ........................................................................................................................... 50 3.9. Continuous-flow mode biotransformations ............................................................................ 51 3.9.1. Kinetic resolutions of rac-1a with BcL- or CaLB-filled continuous-flow bioreactors ............. 51 3.9.2. Continuous-flow immobilization of CvTAW60C and kinetic resolution of racemic amines ........ 52 4. GOALS OF THE THESIS .............................................................................................................. 54 5. RESULTS AND DISCUSSION ........................................................................................................ 56 5.1. Systematic study of the effect of additives enhancing catalytic properties of lipase from Burkholderia cepacia immobilized on mixed-function-grafted mesoporous silica gel ......... 56 5.1.1. Effect of additives on the biocatalytic properties of covalently immobilized BcL .................... 59 5.1.2. Thermal stability of BcLs adsorbed onto mixed-function-grafted mesoporous silica gel ........ 61 5.1.3. Reuse of adsorbed BcL biocatalysts in successive kinetic resolutions ..................................... 63 5.1.4. Continuous-flow kinetic resolutions of rac-1a with BcL / PVA 18-88 and BcL / PEG 20k ..... 63 5.2. Bisepoxide-activated aminoalkyl resins designed for covalent immobilization of Candida antarctica lipase B—Enhanced thermal stabilization of the enzyme in continuous-flow reactors ..................................................................................................................................... 66 5.2.1. Covalent immobilization of CaLB on bisepoxide-activated supports and kinetic resolution of rac-1a ........................................................................................................................................ 66 5.2.2. Optimization of the bisepoxide activation for covalent immobilization of CaLB ..................... 70 5.2.3. Operational stability of the CaLB biocatalysts......................................................................... 71 5.2.4. Long-term storage stability of the CaLB preparations ............................................................. 72 5.2.5. Continuous-flow kinetic resolution of rac-1a catalyzed by CaLB preparations on bisepoxide- activated resins—Substrate concentration and temperature effects ......................................... 73 5.3. Covalently immobilized Trp60Cys mutant of ω-transaminase from Chromobacterium violaceum for kinetic resolution of racemic amines in batch and continuous-flow modes .. 77 5.3.1. Bisepoxide activation of polymer resins for immobilization of CvTAW60C and batch-mode kinetic resolution of racemic amines ........................................................................................ 77 5.3.2. Recycling of the CvTAW60C immobilized on
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