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Table of Contents Development of Transaminases for the Synthesis of Enantiomerically Pure Chiral Amines A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences. 2012 Jennifer Hopwood School of Chemistry Table of contents Table of contents ............................................................................................................. 4 Abstract ............................................................................................................................ 9 Declaration ..................................................................................................................... 10 Copyright statement...................................................................................................... 10 Acknowledgements ........................................................................................................ 11 Abbreviations ................................................................................................................ 12 1. Introduction ........................................................................................................... 14 1.1 Biocatalysis ...................................................................................................... 14 1.2 Enzymatic synthesis of chiral amines .............................................................. 18 1.2.1 Chiral amines ............................................................................................ 18 1.2.2 Biocatalytic synthesis of chiral amines ..................................................... 19 1.2.3 Kinetic resolution ...................................................................................... 19 1.2.4 Dynamic kinetic resolution ....................................................................... 22 1.2.5 Deracemisation .......................................................................................... 26 1.2.6 Asymmetric synthesis ............................................................................... 28 1.3 Transaminases .................................................................................................. 30 1.3.1 Pyridoxal 5’-phosphate and the transaminase active site .......................... 30 1.3.2 Transaminase mechanism ......................................................................... 32 1.3.3 Methods to overcome transaminase challenges ........................................ 32 1.3.4 Screening of transaminase enzymes.......................................................... 36 1.3.5 Transaminases for chiral amine synthesis ................................................. 38 1.4 Aims of the project ........................................................................................... 42 2. Results and discussion- Colourimetric assay ...................................................... 43 2.1 Path length determination ................................................................................. 44 2.2 Determination of extinction coefficient of pyrogallol red ................................ 45 2.3 Background activity of Pyrogallol red ............................................................. 46 4 Table of contents 2.4 Screening of amine substrates .......................................................................... 47 2.5 Varying ATA-117 concentration ...................................................................... 48 2.6 Effect of adding external pyridoxal-5’-phosphate ............................................ 51 2.7 Comparison of forward and reverse transamination reaction........................... 52 2.8 Determination of enantioselectivity ................................................................. 54 2.9 pH Stability ....................................................................................................... 55 2.10 Screening of whole cell transaminases ......................................................... 56 2.11 Transaminase activity with purified enzyme ................................................ 57 2.12 Conclusion .................................................................................................... 59 3. Results and discussion- Cloning identification and characterisation of two transaminase enzymes .................................................................................................. 61 3.1 Initiation of screen ............................................................................................ 62 3.2 Solid phase screen ............................................................................................ 66 3.3 Identification of unknown microorganisms...................................................... 67 3.4 Protein identification ........................................................................................ 67 3.5 Cloning and expression of Ochrobactrum anthropi ......................................... 69 3.6 Cloning and expression of C. violaceum (CMCC 104818). ............................. 72 3.7 Homology modelling of the Chromobacterium violaceum transaminases ...... 76 3.8 Conclusion ........................................................................................................ 78 4. Results and discussion- Biotransformations using transaminases ................... 80 4.1 4-Bromoacetophenone ...................................................................................... 81 4.2 Cascade reactions ............................................................................................. 85 4.2.1 Galactose oxidase/transaminase cascade .................................................. 85 4.2.2 Monoamine oxidase and transaminase cascade ........................................ 87 4.3 3 Methyl-3,4-dihydroisoquinoline .................................................................... 95 4.4 Conclusion ........................................................................................................ 97 5. Summary, conclusion and future work ............................................................. 100 5.1 Summary and conclusion ............................................................................... 100 5 Table of contents 5.2 Future work .................................................................................................... 101 6. Experimental ....................................................................................................... 102 6.1 Materials ......................................................................................................... 102 6.1.1 Chemicals, reagents and kits. .................................................................. 102 6.1.2 Enzymes .................................................................................................. 102 6.1.3 Strains ...................................................................................................... 102 6.1.4 Plasmids .................................................................................................. 103 6.1.5 Buffer solutions ....................................................................................... 103 6.1.6 Growth media .......................................................................................... 104 6.1.7 Antibiotics ............................................................................................... 106 6.1.8 IPTG ........................................................................................................ 106 6.1.9 Enzyme stocks for solution phase assay ................................................. 106 6.1.10 Sample preparation for colony PCR ....................................................... 107 6.1.11 DNA and protein gel markers ................................................................. 107 6.2 Methods .......................................................................................................... 108 6.2.1 Colourimetric transaminase assay with PGR .......................................... 108 6.2.2 Colourimetric transaminase assay with TBHBA 4-AAP ........................ 109 6.2.3 Path length determination ....................................................................... 109 6.2.4 Extinction coefficient of pyrogallol red .................................................. 110 6.2.5 Genomic DNA purification ..................................................................... 110 6.2.6 16S rRNA sequencing PCR recipe ......................................................... 111 6.2.7 Plasmid DNA purification....................................................................... 111 6.2.8 PCR of C. violaceum ............................................................................... 112 6.2.9 PCR of Ochrobactrum sp genomic DNA. .............................................. 113 6.2.10 Gel extraction .......................................................................................... 113 6.2.11 DNA quantification ................................................................................. 113 6.2.12 Cloning (Zero blunt TOPO) .................................................................... 114 6.2.13 Restriction Enzyme digests ..................................................................... 114 6 Table of contents 6.2.14 Transformation of E. coli ........................................................................ 115 6.2.15 Expression of PJH2 in BL21 (DE3) ........................................................ 115 6.2.16 Expression of PJH3
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