Directed Evolution of Bacillus gibsonii Alkaline Protease towards Washing Applications: a Study of Protein Adaptation by Ronny Ernesto Martínez Moya A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemical Engineering Approved, Thesis Committee Prof. Dr. Ulrich Schwaneberg, RWTH Aachen University Prof. Dr. Danilo Roccatano, Jacobs University Bremen Prof. Dr. Karl-Heiz Maurer, Henkel AG & Co KGaA Date of Defense: September 10, 2010 School of Engineering and Science Table of contents Table of contents Table of contents ............................................................................................................................... i Acknowledgments........................................................................................................................... iii Abstract ........................................................................................................................................... iv Abbreviations .................................................................................................................................. vi Part I: General introduction.............................................................................................................. 1 1. Protein engineering and Directed Evolution ............................................................................ 1 2. Cold adaptation and thermal stability of enzymes ................................................................... 5 2.1. - Thermostable enzymes ................................................................................................... 6 2.1. - Cold-adapted enzymes.................................................................................................... 7 3. - Aim of the project.................................................................................................................. 9 Part II: Directed Evolution of Bacillus gibsonii Alkaline Protease towards washing applications 11 1. – Introduction ............................................................................................................................. 11 1.1. - Aim of this work ............................................................................................................... 11 1.2. –Proteases............................................................................................................................ 11 1.2.1. – Serine proteases and subtilisins................................................................................. 13 1.2.2. –Subtilisin proteases applications in industry.............................................................. 16 1.2.3. – Protein engineering in subtilisin proteases................................................................ 18 1.2.4. – B. gibsonii Alkaline Protease (BgAP)....................................................................... 21 1.3. - SeSaM............................................................................................................................... 23 1.3.1. – SeSaM principle........................................................................................................ 23 1.3.2. - SeSaM Features ......................................................................................................... 24 2. - Materials and methods ............................................................................................................. 26 2.1. – Materials........................................................................................................................... 26 2.1.1. – Chemicals.................................................................................................................. 26 2.1.2. – Bacterial strains......................................................................................................... 26 2.1.3. – Plasmids .................................................................................................................... 26 2.1.4. – Oligonucleotides ....................................................................................................... 26 2.1.5. – Cell culture media and cultivation ............................................................................ 28 2.2. - Methods ............................................................................................................................ 28 2.2.1. - Cloning ...................................................................................................................... 28 2.2.2. - Random mutagenesis library generation.................................................................... 28 2.2.3. - Protein expression...................................................................................................... 29 2.2.4. - Screening for improved activity at 15°C ................................................................... 29 2.2.5. - Screening for improved thermostability .................................................................... 30 2.2.6. - Site-directed mutagenesis and site saturation mutagenesis of BgAP variants........... 30 2.2.7. - Protein purification .................................................................................................... 30 2.2.8. - Proteolytic activity: Suc-AAPF-pNA ........................................................................ 31 2.2.8. - Enzyme activity: Azocasein....................................................................................... 31 2.2.9. - Temperature dependence of the specific activity ...................................................... 32 2.2.10. - Thermal inactivation................................................................................................ 32 2.2.11. - Thermal shift assay.................................................................................................. 32 2.2.12. - Homology modeling ................................................................................................ 33 3. – Results ..................................................................................................................................... 34 3.1. – Directed Evolution to improve activity at low temperatures ........................................... 34 3.1.1. - BgAP expression system ........................................................................................... 34 3.1.2. - Skim milk microtiter plate assay ............................................................................... 35 3.1.3. - SeSaM library generation .......................................................................................... 36 3.1.4. - Screening of BgAP SeSaM library round 1............................................................... 37 3.1.5. - Screening of BgAP SeSaM library round 2............................................................... 40 3.1.6. - Screening of BgAP SeSaM library round 3............................................................... 43 i Table of contents 3.1.8. - Purification and characterization of selected clones from Directed Evolution.......... 47 3.1.9. – Characterization using the suc-AAPF-pNA substrate............................................... 50 3.1.10. – Characterization using the macromolecular substrate Azo-casein.......................... 51 3.2. - Directed Evolution of BgAP for improved thermostability.............................................. 52 3.2.1. – Re-screening of SeSaM libraries............................................................................... 55 3.2.2. – Site-directed mutagenesis to generate variant 39IC1+N253D.................................. 57 3.2.3. - Site-directed mutagenesis and combination of amino acid substitutions .................. 58 3.2.4. - Purification and characterization of variants with improved thermostability............ 59 3.3. - Homology modeling of BgAP. ......................................................................................... 65 4. - Discussion and conclusions ..................................................................................................... 66 4.1.- Analysis of the mutations generated by SeSaM ................................................................ 66 4.2. - Directed Evolution of BgAP............................................................................................. 68 4.2.1. – Cold adaptation of BgAP .......................................................................................... 68 4.3.2. – Increasing thermal stability of BgAP........................................................................ 71 4.3. - Conclusions of Part I......................................................................................................... 75 Part III: Temperature Effects on Dynamics of Psychrophilic Protease S41 and its Thermostable Mutants in Solution. ....................................................................................................................... 76 1. – Introduction ............................................................................................................................. 76 1.1. - Aim of this work ............................................................................................................... 76 1.2. – Molecular dynamics simulations...................................................................................... 76 1.3. – Extremophiles and protein adaptation.............................................................................. 77 1.4.