Development of an enzyme-based method for production of food with low purine content I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Dagmara Jankowska geboren am 27. Juni 1984 in Wrocław, Polen - Greifswald, 2013 - Dekan: Prof. Dr. Klaus Fesser 1. Gutachter : Prof. Dr. habil. Rϋdiger Bode Universität Greifswald, Institut für Biochemie 2. Gutachter: Prof. Dr. Raffael Schaffrath Universität Kassel, Institut für Biologie Tag der Promotion: 14.04.2014 Table of contents TABLE OF CONTENTS Summary ............................................................................................................................ i Zusammenfassung ............................................................................................................ iii List of abbreviations .......................................................................................................... v 1 Introduction .................................................................................................................. 1 1.1 Purine degradation pathway ........................................................................................ 1 1.1.1 Purines .............................................................................................................. 1 1.1.2 Purine degradation ........................................................................................... 2 1.2 Why do humans lack urate oxidase? ........................................................................... 4 1.3 Purines in food products .............................................................................................. 5 1.4 Uric acid and hyperuricemia ........................................................................................ 5 1.5 Gout .............................................................................................................................. 7 1.6 Treatment of hyperuricemia and gout ......................................................................... 9 1.6.1 Acute gout treatment ..................................................................................... 10 1.6.2 Uricostatic drugs ............................................................................................. 12 1.6.3 Uricosuric drugs .............................................................................................. 12 1.6.4 Uricolytic drugs ............................................................................................... 13 1.6.5 Prevention of gout .......................................................................................... 13 1.7 Adenine deaminase .................................................................................................... 14 1.8 Xanthine oxidoreductase ........................................................................................... 15 1.9 Arxula adeninivorans .................................................................................................. 16 1.10 Xplor®2 transformation/expression platform ............................................................ 17 2 Aim of the thesis ......................................................................................................... 20 3 Materials and methods ............................................................................................... 21 3.1 Microorganisms .......................................................................................................... 21 3.2 Oligonucleotides ......................................................................................................... 21 3.3 Plasmids and vectors .................................................................................................. 22 3.4 Culture media ............................................................................................................. 22 3.4.1 Bacterial media ............................................................................................... 22 3.4.2 Yeast media .................................................................................................... 23 3.5 Enzymes and chemicals .............................................................................................. 24 3.6 Cultivation of microorganisms ................................................................................... 24 3.6.1 Cultivation of E. coli cells ................................................................................ 24 3.6.2 Cultivation of yeast cells ................................................................................. 25 3.7 Methods used for work with DNA .............................................................................. 25 3.7.1 Agarose gel electrophoresis ........................................................................... 25 3.7.2 DNA fragment extraction from agarose gel ................................................... 25 Table of contents 3.7.3 Mini isolation of plasmid DNA from E. coli cells ............................................ 26 3.7.4 DNA restriction ............................................................................................... 26 3.7.5 DNA ligation ................................................................................................... 26 3.7.6 Ethanol precipitation of DNA ......................................................................... 26 3.7.7 Transformation of E. coli cells ........................................................................ 27 3.7.8 Preparation of E. coli competent cells ........................................................... 27 3.7.9 Polymerase Chain Reaction (PCR) .................................................................. 27 3.7.10 Southern blotting ........................................................................................... 28 3.8 Methods used for work with RNA ............................................................................. 29 3.8.1 RNA isolation from yeast cells ....................................................................... 29 3.8.2 Electrophoresis of RNA .................................................................................. 30 3.8.3 Removal of genomic DNA from RNA samples ............................................... 30 3.8.4 cDNA synthesis ............................................................................................... 31 3.8.5 Real-time quantitative PCR ............................................................................ 31 3.9 Methods used for work with proteins ....................................................................... 31 3.9.1 Determination of protein concentration ....................................................... 31 3.9.2 Protein electrophoresis in a polyacrylamide gel (SDS-PAGE) ........................ 32 3.9.3 Protein electrophoresis in a native polyacrylamide gel................................. 33 3.9.4 Western blotting (immunoblotting) .............................................................. 33 3.10 Protein purification .................................................................................................... 35 3.10.1 Cell wall disruption ......................................................................................... 35 3.10.2 Affinity chromatography (Ni-NTA chromatography) ..................................... 35 3.10.3 Ion exchange and gel filtration ...................................................................... 36 3.11 N-terminal sequencing............................................................................................... 36 3.12 MALDI-TOF ................................................................................................................. 37 3.13 Methods used for work with yeast ............................................................................ 37 3.13.1 Transformation of yeast cells ......................................................................... 37 3.13.2 Preparation of competent A. adeninivorans G1212 cells .............................. 38 3.13.3 Stabilization of insert in the yeast genomic DNA .......................................... 38 3.13.4 Isolation of yeast chromosomal DNA ............................................................ 39 3.14 Expression of wild-type proteins, Aadap and Axorp, in A. adeninivorans LS3 .......... 39 3.15 Cultivation of transgenic strains for protein expression ........................................... 40 3.15.1 E. coli .............................................................................................................. 40 3.15.2 A. adeninivorans ............................................................................................. 40 3.16 Subcellular fractionation ............................................................................................ 40 3.17 Yeast cells microscopy ............................................................................................... 41 3.18 Cell growth monitoring .............................................................................................. 41 3.18.1 Optical density of the culture ........................................................................ 41 3.18.2 Dry cell weight ................................................................................................ 41 3.19 Enzymatic assays .......................................................................................................