Biochemical studies of enzymes involved in glycolysis of the thermoacidophilic crenarchaeon Sulfolobus solfataricus Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften – Dr. rer. nat. – vorgelegt von Patrick Haferkamp geboren in Essen Institut für Molekulare Enzymtechnologie und Biochemie der Universität Duisburg-Essen 2011 Die vorliegende Arbeit wurde im Zeitraum von Januar 2008 bis August 2011 im Arbeitskreis von Prof. Dr. Bettina Siebers am Institut für Molekulare Enzymtechnologie und Biochemie der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 18.11.2011 Gutachter: Prof. Dr. Bettina Siebers Prof. Dr. Daniel Hoffmann Table of contents 1 INTRODUCTION .................................................................................................. 1 1.2 Aims of this study .......................................................................................................................................... 10 2 MATERIALS AND METHODS ........................................................................... 11 2.1. Materials ....................................................................................................................................................... 11 2.1.1 Laboratory equipment .............................................................................................................................. 11 2.1.2. Chemicals and Plasmids .......................................................................................................................... 13 2.1.3 Antibiotics ................................................................................................................................................ 13 2.1.4 Enzymes ................................................................................................................................................... 13 2.1.5 Oligonucleotide sets for mutagenesis PCR .............................................................................................. 14 2.1.6 Kits ........................................................................................................................................................... 15 2.1.7 Strains and growth conditions .................................................................................................................. 15 2.1.8 Bioinformatics .......................................................................................................................................... 16 2.1.9 Internet databases and tools...................................................................................................................... 17 2.2 Techniques Molecular Biology ..................................................................................................................... 17 2.2.1 Qualitative and quantitative analysis of DNA .......................................................................................... 17 2.2.2 Purification of DNA fragments ................................................................................................................ 17 2.2.3 Agarose gel electrophoresis of DNA........................................................................................................ 18 2.2.4 Amplification of genomic and plasmid DNA via polymerase chain reaction (PCR) ............................... 18 2.2.5 Amplification of genomic DNA and plasmid DNA by PCR ................................................................... 19 2.2.6 DNA and vector restriction ...................................................................................................................... 19 2.2.7 5`-Dephosphorylation of the linearized vector DNA ............................................................................... 20 2.2.8 Ligation of vector DNA and insert ........................................................................................................... 20 2.2.9 Preparation of competent E. coli cells ...................................................................................................... 20 2.2.10 Electrocompetent cells: .......................................................................................................................... 21 2.2.11 Transformation of competent E. coli strains DH5α and Rosetta (DE3)-RIL ......................................... 21 2.2.12 Colony PCR ........................................................................................................................................... 22 2.2.13 Plasmid preparation ................................................................................................................................ 22 2.2.14 DNA sequencing .................................................................................................................................... 22 2.2.15 Heterologous gene expression in E. coli Rosetta (DE3) pRIL ............................................................... 23 2.3 Techniques Biochemistry.............................................................................................................................. 24 2.3.1 Determination of protein concentration ................................................................................................... 24 2.3.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)........................................................................... 24 2.3.5 Preparation of S. solfataricus crude extracts (Zaparty et al 2010) ........................................................... 25 2.3.4 Crude extracts and heat precipitation ....................................................................................................... 25 2.3.6 Dialysis .................................................................................................................................................... 26 2.3.7 Ion exchange chromatography ................................................................................................................. 26 2.3.8 Gel filtration ............................................................................................................................................. 26 2.4 Enzyme assays ............................................................................................................................................... 28 2.4.1 Glucose dehydrogenase (GDH) ............................................................................................................... 28 2.4.2 Enolase (ENO) ......................................................................................................................................... 28 2.4.3 Pyruvate kinase (PK) ................................................................................................................................ 29 2.4.4 Phosphoglycerate mutase (PGAM) .......................................................................................................... 29 2.4.5 Phosphoenolpyruvate synthase (PEPS) .................................................................................................... 30 2.4.6 Pyruvate, phosphate dikinase (PPDK) ..................................................................................................... 30 2.4.7 Metabolite stabilities ................................................................................................................................ 31 2.4.8 Calculation of specific activity ................................................................................................................. 32 2.4.9 Michaelis Menten kinetics and Hanes-Woolf plot ................................................................................... 32 2.4.10 Sequence analysis................................................................................................................................... 34 3 RESULTS ........................................................................................................... 35 3.1 Heterologous Expression of the S.solfataricus ED Proteins in E.coli ........................................................ 35 3.2 Enzyme Enrichment and Purification ......................................................................................................... 35 3.3 Biochemical Characterization ...................................................................................................................... 36 3.3.1 Glucose dehydrogenase ............................................................................................................................ 36 3.3.2 Phosphoglycerate Mutase ......................................................................................................................... 41 3.3.3 Pyruvate kinase: ....................................................................................................................................... 46 3.3.4 Enolase ..................................................................................................................................................... 51 3.3.5 Phosphoenolpyruvate synthase and pyruvate dikinase ............................................................................. 55 3.4 Crude Extracts Studies ................................................................................................................................. 58 3.5 Metabolite stabilities ..................................................................................................................................... 60 4 DISCUSSION ....................................................................................................