Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in enzymatic removal of pharmaceuticals from wastewater 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 Universität Greifswald vorgelegt von Katarzyna Litwińska geboren am 22.03.1990 in Wrocław, Polen Greifswald, 2020 Dekan: Prof. Dr. Gerald Kerth 1. Gutachter: Prof. Dr. Hans-Joachim Schüller 2. Gutachter: Prof. Dr. Vlada Urlacher Tag der Promotion: 07.07.2020 TABLE OF CONTENTS TABLE OF FIGURES ............................................................................................................. IV TABLE OF TABLES ............................................................................................................... VI LIST OF ABBREVIATIONS ................................................................................................. VII SUMMARY ............................................................................................................................. IX ZUSAMMENFASSUNG ......................................................................................................... XI 1. INTRODUCTION .............................................................................................................. 1 1.1. Pharmaceuticals in the environment ............................................................................ 1 1.2. Removal of pharmaceuticals in wastewater treatment plants ...................................... 8 1.3. Laccases ..................................................................................................................... 14 1.4. Cytochromes P450 ..................................................................................................... 17 1.5. The yeast Arxula adeninivorans ................................................................................ 20 1.6. Aim of the work ......................................................................................................... 23 2. MATERIALS AND METHODS ...................................................................................... 25 2.1. Media and cultivation methods .................................................................................. 25 2.1.1. Cultivation media ................................................................................................ 25 2.1.2. Cultivation of E. coli XL1-Blue.......................................................................... 26 2.1.3. Cultivation of A. adeninivorans .......................................................................... 26 2.1.4. Cultivation of white-rot fungi ............................................................................. 26 2.1.5. Dry cell weight (DCW) measurement ................................................................ 26 2.1.6. Optical density (OD600) measurement ................................................................ 27 2.1.7. Glucose concentration measurement .................................................................. 27 2.1.8. Fed-batch fermentation of A. adeninivorans ...................................................... 27 2.2. Molecular biology techniques .................................................................................... 28 2.2.1. Primers, plasmids, and cloning strategies ........................................................... 28 2.2.2. Polymerase chain reaction (PCR) ....................................................................... 33 2.2.3. Sequencing .......................................................................................................... 34 2.2.4. Reverse transcription polymerase chain reaction (RT-PCR) .............................. 34 2.2.5. DNA and RNA electrophoresis .......................................................................... 34 2.2.6. Determination of nucleic acid concentrations..................................................... 35 2.2.7. DNA cleavage with restriction endonucleases ................................................... 35 2.2.8. DNA fragments’ extraction from agarose gel ..................................................... 35 2.2.9. Gibson Assembly ................................................................................................ 35 I 2.2.10. Plasmid DNA isolation from E. coli .................................................................... 35 2.2.11. Transformation of E. coli ..................................................................................... 36 2.2.12. Transformation of A. adeninivorans .................................................................... 36 2.2.13. Genomic DNA isolation form A. adeninivorans ................................................. 36 2.2.14. RNA isolation ...................................................................................................... 37 2.2.15. cDNA synthesis ................................................................................................... 37 2.2.16. Preparation of A. adeninivorans cell extracts ...................................................... 37 2.2.17. Microsomes isolation methods ............................................................................ 38 2.3. Biochemical techniques .............................................................................................. 40 2.3.1. SDS-PAGE analysis and molecular mass determination .................................... 40 2.3.2. SDS-PAGE analysis of membrane proteins ........................................................ 40 2.3.3. Western blotting analysis ..................................................................................... 41 2.3.4. Protein purification .............................................................................................. 41 2.3.5. Native gel electrophoresis ................................................................................... 41 2.3.6. Glycosylation staining of PAA gels .................................................................... 42 2.3.7. Deglycosylation reaction ..................................................................................... 42 2.3.8. Determination of protein concentration ............................................................... 42 2.3.9. Determination of laccase activity ........................................................................ 42 2.3.10. Determination of kinetic parameters ................................................................... 43 2.3.11. Estimation of temperature optimum and stability ............................................... 43 2.3.12. Estimation of pH optimum and stability .............................................................. 43 2.3.13. Determination of influence of ionic strength ....................................................... 44 2.3.14. Determination of metal ions influence ................................................................ 44 2.3.15. Optimization of laccase production ..................................................................... 44 2.3.16. Determination of CYP450 activity ...................................................................... 45 2.3.17. Laccase immobilization methods ........................................................................ 45 2.3.18. Degradation tests for carbamazepine, diclofenac, sulfamethoxazole .................. 46 2.3.19. Growth of yeast cells on medium supplemented with carbamazepine ................ 47 2.3.20. Determination of laccase mediators influence on pharmaceuticals degradation .......................................................................................................... 47 2.4. Analytical techniques .................................................................................................. 47 2.4.1. Thin layer chromatography ................................................................................. 47 2.5. Bioinformatics tools .................................................................................................... 48 3. RESULTS .......................................................................................................................... 49 II 3.1. Laccase gene expression ............................................................................................ 49 3.2. Analysis of laccases sequences .................................................................................. 57 3.3. Cultivation condition and Tvlcc5 enzyme purification.............................................. 58 3.4. Biochemical characterization of recombinant laccase Tvlcc5 ................................... 64 3.5. Increase of laccase production ................................................................................... 70 3.6. Laccase immobilization ............................................................................................. 75 3.7. Cytochrome P450 gene expression ............................................................................ 78 3.8. Analysis of CYP450 synthesis in A. adeninivorans .................................................. 79 3.9. Degradation of pharmaceuticals................................................................................. 86 3.9.1. Degradation of pharmaceuticals by laccase ........................................................ 86 3.9.2. Degradation of carbamazepine by whole cell catalyst .......................................