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  • Developing Immobilized Biocatalysts with

    Advanced Interface for Applications in

    Organic Synthesis

    vorgelegt von

    M.Sc. Chemie

    Changzhu Wu

    aus Anhui (China)

    Von der Fakultt II -Mathematik und Naturwissenschaften der Technischen Universitt Berlin

    zur Erlangung des akademischen Grades

    Doktor der Naturwissenschaften -Dr. rer.nat.-

    genehmigte Dissertation

    Promotionsausschuss: Vorsitzender: Professor Dr. Michael Gradzielski Berichter: Professor Dr. Marion B. Ansorge-Schumacher Berichter: Professor Dr. Udo Kragl

    Tag der wissenschaftlichen Aussprache: 27.09.2011

    Berlin 2011

    D 83

  • Acknowledgement To mentor: Professor Dr. Marion B. Ansorge-Schumacher: Thank you so much to give me the

    chance to join your group at TU Berlin, and to finance and support me to study such interesting topics. I am deeply impressed by your keen insights and great passions to science, and your endless patience and trust to me are much appreciated. I will not forget that every time after discussion I left your office with being motivated and excited to my future research. Anyway, thank you to inspire me to be my best!

    To dissertation defense committee Professor Dr. Udo Kragl: I am really grateful that you could agree to come to Berlin for

    my PhD defense, and appreciate your time to correct my PhD dissertation. Professor Dr. Michael Gradzielski: Thank you to aggree to be the chairman in my PhD

    defense. Also I am grateful that you allowed me to make several experiments in your lab in the past years.

    To organizations UniCat: Many thanks to UniCat, the Cluster of Excellence, to finance me for three years

    to complete my PhD research. My special appreciation to those people who organized hundreds of lectures and activities in the past for Unifying Concepts in Catalysis.

    BIG-NSE: I am truly grateful to BIG-NSE, the outstanding Berlin International Graduate School of Natural Science and Engineering, to accept me as a fellow at the beginning of my PhD. Within BIG-NSE, I am lucky to know many bright PhD students, especially these from batch 2008: Subhamoy Bhattacharya, Sara Bruun, Manuel Harth, Kirstin Hobiger, Stanislav Jaso, Sardor Mavlyankariev, Sylvia Reiche, Carlos Carrero, and Lars Lauterbach. I thank Professor Dr. Reinhard Schomcker (TU Berlin) and Dr. Jean-Philippe Lonjaret (TU Berlin) for organization.

    To collaborators Professor Dr. Helmuth Mhwald (MPI Potsdam): Thank you to support your student

    and group leader to collaborate with us for two projects. Professor Dr. Regine von Klitzing (TU Berlin): Many thanks to you for valuable

    discussion, and appreciate your permission for me to use several instruments in your group.

    Professor Dr. Dayang Wang (University of South Australia): Thanks for your fruitful discussion, and your great supports to our collaborations.

    Dr. Shuo Bai (MPI Potsdam): It is my pleasure to cooperate with you to complete two projects. Especially thank you to implement some characterization of our samples.

    Kornelia Gawlitza (TU Berlin): Thanks for your contributions to our cooperation. It is my happy experience to complete the experiments, posters, presentations, and papers together with you.

    http://www.big-nse.tu-berlin.de/index.php?id=273

  • To team players Dirk Schlienz (FU Berlin): Thanks for your hard and creative diploma work to make

    one challenging project successful. Jonas Hanske and Erik Walinda (FU Berlin): Thanks for your choice to come to the

    group of Professor Ansorge-Schumacher, and believe me to guide your short but successful internship.

    To colleagues I thank all our former and current colleagues in the group of Professor Ansorge-

    Schumacher (TU Berlin) for your helps and discussion. Your helps made me easy to adapt and live in Berlin.

    Especially I am grateful to the kind friendship offered from you: Dr. Lars Wiemann, Dr. Nora Bieler, Dr. Andy Maraite, Dr. Valantina Yazbik, Juliane Ratzka, Ren Nieguth, Alexander Scholz, Shenay Sali, and Christoph Loderer. Particularly, I thank Juliane Ratzka and Christoph Loderer for German translation in this dissertation.

    To buddies: Dr. Tanguy Chau (MIT Boston): Thank you, Tanguy! Thanks for your countless

    suggestions and discussion to help me make several important decisions of my family. Jian Li, Mengjun Xue, Zhengfang Zhang, Dr. Jingyu Chen, Dr. Susan Kelleher (TU

    Berlin): Nice to meet all of you in Berlin. I enjoyed every moment together with you.

    To sister and brother: Thank you my elder sister (Mengzhi Wu) and my younger brother (Changbao Wu).

    Your love and supports to me will never be forgotten.

    To family: Wife: Thank you so much to come to Berlin with me. Your love and patience mean a lot

    to me! Son: You are the biggest result and the best present of Dads PhD period. I love you

    forever!

    To parents: Dad: Thank you Dad (Xiangui Wu) for your patience, understanding, and hard working

    to raise and support me all the time! Mom: Thank you Mom (Xiuhua Deng) for your unconditional and everlasting love.

    Your perseverance taught me to never give up anything important to us; your dedication inspired me to sacrifice for something we love. Thanks for all you gave to me!

  • I

    Table of Content

    Table of Content .................................................................................................. I

    Abstract ............................................................................................................. IV

    List of Schemes ................................................................................................. IX

    List of Figures ..................................................................................................... X

    List of Tables...................................................................................................XVI

    Symbols and Abbreviations......................................................................... XVII

    1. Introduction ...............................................................................................1

    1.1. Enzyme Immobilization ............................................................................................... 1

    1.1.1. History of Enzyme Immobilization.................................................................... 1

    1.1.2. Methods of Enzyme Immobilization.................................................................. 5

    1.2. Immobilized Biocatalysts for Organic Synthesis in Non-aqueous Media ............. 16

    1.2.1. General Considerations .................................................................................... 16

    1.2.2. Key Parameters of Immobilized Biocatalysts for Organic Synthesis .............. 19

    1.2.3. Lipase Immobilization for Esterification in Organic Solvents......................... 24

    1.3. Pickering Emulsions and Janus Catalysts................................................................ 28

    1.3.1. Basic of Pickering Emulsions........................................................................... 28

    1.3.2. Janus Catalysts and Janus Biocatalyts.............................................................. 30

    1.4. Research Motivation .................................................................................................. 32

    2. Experimental ...........................................................................................35

    2.1. Materials...................................................................................................................... 35

    2.1.1 Chemical Materials........................................................................................... 35

    2.1.2 Biological Materials ......................................................................................... 36

    2.2. Immobilization of Enzymes in Diverse Carriers ..................................................... 36

    2.2.1. In Hydrophilic Hydrogels................................................................................. 36

    2.2.2. In Hydrophobic Silicone BASE ....................................................................... 37

    2.2.3. In Pickering Emulsions .................................................................................... 38

  • II

    2.3. Characterization of Diverse Immobilization Systems............................................. 39

    2.3.1. Characterization of NPs and Enzymes in Hydrophilic Hydrogels ................... 39

    2.3.2. Characterization of Hydrophobic Silicone BASE............................................ 41

    2.3.3. Characterization of Enzyme Encapsulation by Pickering Emulsions .............. 42

    2.4. Catalytic Assay of Immobilized and Free Enzymes ................................................ 42

    2.4.1. Activity Assay of Immobilized CalB in Hydrogels or as Free Enzymes......... 42

    2.4.2. Determination of BASE Activity and Conversion ........................................... 43

    2.4.3. Catalytic Performance of Encapsulated Enzymes in Pickering Emulsions ..... 43

    3. Using Hydrogel Microparticles to Transfer Hydrophilic Enzymes to

    Organic Media via Stepwise Solvent Exchange ...................................46

    3.1. Introduction ................................................................................................................ 46

    3.2. Results and Discussion ............................................................................................... 49

    3.2.1. Using Hydrogel MPs for Phase Transfer and Encaps