Study Towards Carotenoid 1,2-Hydratase and Oleate Hydratase as Novel Biocatalysts Aida HISENI Study Towards Carotenoid 1,2-Hydratase and Oleate Hydratase as Novel Biocatalysts PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Technische universiteit Delft, op gezag van de Rector Magnificus prof. ir. K.C.A.M Luyben, voorzitter van het College voor promoties, in het openbaar te verdedigen op dinsdag 22 april 2014 om 10:00 uur door Aida HISENI Diplom-Biologin, Heinrich-Heine-Universität Düsseldorf geboren te Doboj, Bosnië en Hercegovina. Dit proefschrift is goedgekeurd door de promotor: Prof. dr. I.W.C.E Arends Samenstelling promotiecommissie: Rector Magnificus voorzitter Prof. dr. I.W.C.E. Arends Technische Universiteit Delft, promotor Prof. dr. U. Hanefeld Technische Universiteit Delft Prof. dr. J.H. de Winde Universiteit Leiden Prof. dr. G. Muijzer Universiteit van Amsterdam Prof. dr. R. Wever Universiteit van Amsterdam Dr. L.G. Otten Technische Universiteit Delft Dr. P. Dominguez De Maria Sustainable Momentum Prof. dr. S. de Vries Technische Universiteit Delft, reservelid This project is financially supported by The Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (http://www.b-basic.nl) through B-Basic, a public- private NWO-ACTS programme [Advanced Chemical Technologies for Sustainability (ACTS)]. ISBN Copyright © 2014 by Aida HISENI All rights reserved. No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without any prior permission of the copyright owner. To my father Ismet Nukičić Table of Contents 1 General introduction .................................................................................................. 1 1.1 Enzymes and biocatalysis ............................................................................................................... 2 1.2 Enzymes as industrial biocatalysts ................................................................................................. 3 1.3 Enzyme engineering ....................................................................................................................... 6 1.4 Hydro-Lyases ................................................................................................................................. 8 1.4.1 Non-enzymatic water addition to a carbon-carbon double bond ............................................... 8 1.4.2 Enzymatic water addition to a carbon-carbon double bond ....................................................... 9 1.4.3 Carotenoid 1,2-hydratase ......................................................................................................... 11 1.4.4 Oleate hydratase ...................................................................................................................... 14 1.5 Scope and objectives .................................................................................................................... 22 1.6 Supplementary figures.................................................................................................................. 24 1.7 References .................................................................................................................................... 26 2 Biochemical Characterization of the Carotenoid 1,2-Hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina ................................................... 33 Abstract ..................................................................................................................................................... 34 2.1 Introduction.................................................................................................................................. 35 2.2 Materials and methods ................................................................................................................. 36 2.2.1 Construction of pET15b_CrtCRg and pET15b_CrtCTr expression vectors ............................ 36 2.2.2 Expression and purification of recombinant proteins .............................................................. 37 2.2.3 Tandem MS analysis ............................................................................................................... 37 2.2.4 CrtC activity assay and analysis of the products ..................................................................... 38 2.2.5 Substrate specificity ................................................................................................................. 38 2.2.6 Effects of pH and temperature on CrtC activity ...................................................................... 39 2.2.7 Effects of inhibitors and metal ions on enzyme activity .......................................................... 39 2.2.8 Circular dichroism (CD) spectroscopy .................................................................................... 39 2.2.9 Metal analysis using USN-ICP-OES ....................................................................................... 40 2.3 Results .......................................................................................................................................... 40 2.3.1 Expression and purification of the carotenoid 1,2-hydratases ................................................. 40 2.3.2 Hydratase activity .................................................................................................................... 41 2.3.3 Enzyme kinetics ....................................................................................................................... 41 2.3.4 Substrate specificity ................................................................................................................. 43 2.3.5 Effect of pH and temperature on hydratase activity and stability ............................................ 43 2.4 Discussion .................................................................................................................................... 46 2.5 Acknowledgments ......................................................................................................................... 49 2.6 Supplementary information .......................................................................................................... 50 2.7 References .................................................................................................................................... 57 3 Structural Characterization of the Carotenoid 1,2-Hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina ................................................... 59 Abstract ..................................................................................................................................................... 60 vii 3.1 Introduction .................................................................................................................................. 61 3.2 Materials and methods ................................................................................................................. 63 3.2.1 In silico analysis ...................................................................................................................... 63 3.2.2 Cloning of carotenoid 1,2-hydratase genes .............................................................................. 63 3.2.3 Construction of CrtC mutants .................................................................................................. 64 3.2.3.1 Single point mutations .................................................................................................... 64 3.2.3.2 N-terminally truncated Rg- and TrCrtC’s ....................................................................... 65 3.2.4 Recombinant expression of CrtC’s .......................................................................................... 65 3.2.5 CrtC purification ...................................................................................................................... 66 3.2.6 Determination of enzyme activity ............................................................................................ 66 3.3 Results and discussion .................................................................................................................. 67 3.3.1 Comparative in silico analysis of crtC genes ........................................................................... 67 3.3.2 Production of recombinant wildtype and mutant CrtC’s and enzymatic activity .................... 72 3.4 Conclusion ................................................................................................................................... 80 3.5 Acknowledgements ....................................................................................................................... 81 3.6 References .................................................................................................................................... 82 4 Oleate hydratase as model enzyme to design and evaluate high-throughput screening assay for alcohol detection ............................................................................. 85 Abstract ..................................................................................................................................................... 86 4.1 Introduction .................................................................................................................................. 87 4.2 Materials and methods