Pressurized carbon dioxide in biocatalysis enhanced catalytic activity of lipase

Pressurized Carbon Dioxide in Biocatalysis: Enhanced Catalytic Activity of Lipase Thesis submitted for the degree of Doctor of Engineering by Hoang Nam Hai Matsuda Laboratory Department of Bioengineering Graduate School of Bioscience and Biotechnology Tokyo Institute of Technology Yokohama, July 2018 Abstract Abstract Solvents, which play an important role in chemical and pharmaceutical industries, have often been claimed as a major source of waste generation and associated environmental and economic burdens With the eco-environmental urge of developing sustainable processes, academic and industrial intentions have been focused both on minimizing the overall solvent usage and on replacing traditional organic solvents by more environmentally friendly alternative solvents On the other hand, the scope of biocatalysis, particularly in asymmetric synthesis, has been extended through the use of enzymes in organic solvents as well as in ‘green’ anhydrous media, such as ionic liquids and supercritical fluids Carbon dioxide, which has a number of positive impacts on green chemistry as a nonflammable, nontoxic, abundant, and generally chemically inert source, has been intensively studied in its supercritical phase as an attractive solvent for enzymatic reactions due to its greenness, low viscosity, high diffusivity, and ease of recovery of the reaction products However, supercritical CO2 remains drawbacks that hinders its utilization in many industrial applications, given its high pressure operation, low solubility power to polar or high molecular-weight compounds In this study, I have utilized pressurized CO2 as in liquid CO2 and CO2-expanded bio-based liquids for biocatalysis In particular, lipase-catalysed reactions resulted in significantly increased activity and broaden substrate specificity to bulkier substrates In addition, by taking advantage of this in situ solvent tunability, the lipase activity was successfully modulated for organic synthesis of wide range chiral compounds The molecular dynamics simulation revealed novel changes in the lipase structure that the enzyme became more tolerant to wider range of bulky substrates in pressurized CO2 Keywords: Lipase-catalysed transesterification, Kinetic resolution, Substrate specificity, Liquid carbon dioxide, Bio-sourced solvents, CO2-expanded bio-based liquids, Green solvent engineering i Table of content Table of content Abstract i Table of content ii Acknowledgements vi Declaration vii List of abbreviations xi List of figures xii List of tables xiv Introduction 1.1 Overview of biocatalysis and Candida antarctica lipase B catalysed kinetic resolution 1.1.1 Enzymes as biocatalysts for organic synthesis 1.1.2 Biocatalysis in non-aqueous solvents 1.1.3 Lipase catalysis in enantioselective synthesis 1.1.4 Structure and catalytic mechanism of Candida antarctica lipase B 1.1.5 Substrate selectivity and chiral recognition by Candida antarctica lipase B 1.2 Pressurized carbon dioxide as reaction solvents 10 1.2.1 Supercritical and liquid CO2 11 1.2.2 CO2-expanded liquids 12 1.3 Use of pressurized CO2 solvents in biocatalysis to promote sustainable chemistry: Scope of the study 13 Liquid carbon dioxide: Broadening substrate scope of lipase catalysis 16 2.1 CALB catalyzed KR of 1-phenylethanol in organic solvents and in liquid CO2 16 2.2 The use of liquid CO2 in continuous flow reactors for large-scale biosynthesis of optically pure compounds 20 ii 2.2.1 CO2 Table of content KR of 1-phenylethanol in a continuous-flow stirred-tank reactor using liquid 21 2.2.2 CO2 KR of 1-phenylethanol in a continuous packed – bed reactor using liquid 23 2.2.3 Comparison of the productivity of continuous-flow and batch reactors using liquid CO2 as an medium 25 2.3 CO2 Expanding substrate scope of lipase-catalysed KR of sec-alcohols by using liquid 27 2.3.1 Substrate specificity of CALB catalyzed transesterification 27 2.3.2 Expanding substrate scope of CALB using liquid CO2 28 2.3.3 Substrate specificity of Burkholderia cepacia lipase catalyzed transesterification 30 2.3.4 2.4 Preparative scale synthesis of chiral compounds in liquid CO2 31 Conclusion 31 CO2-expanded bio-based liquids: Modulating lipase activity by controlling physicochemical properties of solvent 32 3.1 CALB CO2-expanded bio-based liquids as novel solvents for KR of sec-alcohols by 32 3.1.1 Volumetric expansion of several bio-based liquids with CO2 32 3.1.2 KR of rac-1-phenylethanol by CALB in CO2-expanded MeTHF and other CO2- expanded bio-based liquids 33 3.1.3 The substrate specificity of CALB towards secondary alcohols in neat MeTHF and in CO2-expanded MeTHF 36 3.1.4 3.2 Scale-up synthesis 39 Modulating lipase catalysis in CO2-expanded bio-based liquids by tuning the physicochemical properties 40 3.2.1 Lipase-catalysed KR of rac-1-adamantylethanol 40 iii 3.2.2 Table of content KR of rac-1-adamantylethanol in different CO2-expanded bio-based liquids 41 3.2.3 Effect of substrate bulkiness on conversion of CALB catalysis in neat MeTHF and in CO2-expanded MeTHF 44 3.2.4 Activity of CALB as a function of CO2 mole fraction (XCO₂) and dipolarity/polarizability (π*) 45 3.3 Conclusion 47 Molecular behaviour of Candida antarctica lipase B in pressurized carbon dioxide 48 4.1 Experimental investigation 48 4.1.1 Effect of acyl donor 48 4.1.2 Effect of solvents and reaction pressure 49 4.1.3 Effect of pre-treatment of CALB with liquid CO2 50 4.1.4 Effect of CO2 content on substrate specificity of CALB 50 4.2 Molecular dynamics simulation 52 4.2.1 Overall structural conformation 53 4.2.2 The entrance conformation of the active site 53 4.2.3 The cavity conformation of the active site 55 4.3 Discussion 56 4.4 Conclusion 57 Conclusion 58 Experimental methods 58 6.1 Chemicals and instrument 58 6.2 General procedure for preparation of rac-alcohols 59 6.3 General procedure for preparation of acetates 61 6.4 General procedure for enzymatic reaction in organic solvents 65 6.5 General procedure for enzymatic reaction in pressurized carbon dioxide 65 iv 6.5.1 Table of content General procedure for enzymatic reaction in liquid CO2 65 6.5.2 General procedure for enzymatic reaction in CO2-expanded liquids 66 6.6 General procedure for determination of absolute configuration of products 66 6.7 Data analysis 69 References 72 Appendix 79 v Acknowledgements Acknowledgements I would like to express my great appreciation to: As first and foremost, my supervisor, Professor Tomoko Matsuda for accepting me as a graduate student in the interesting field of biocatalysis in her wonderful group I have learned a lot during these years under her excellent guidance and encouragement, devoted care and patience I will always treasure all supports and opportunities I received from you, Sensei! Financial supports from the Japanese Government (MEXT) Scholarship and the Sasakawa Scientific Research Grant from the Japan Science Society have made this work possible Those the current and former members of Matsuda laboratory, for sharing with me their knowledge, for creating a nice working environment, for their friendly attitude and nice discussions I will miss all those happy moments we have been through All friends from Japan, Vietnam or other countries over the world, for making my life more joyful and worthy! Last but not the least, my family, for always supporting and loving me vi Declaration Declaration The work presented herein was performed at Tokyo Institute of Technology (Japan) between October 2013 and September 2018, during the time which I enrolled in the Integrated Doctoral Education Program of Graduate School of Bioscience and Biotechnology, and which I was supervised by Professor Tomoko Matsuda I hereby declare that this thesis original work and I am the sole author All additional contributions by collaborators and coworkers are fully acknowledged below Collaborator Institution Chapter Contribution Prof Shuichi Mori and Prof Hiroyuki Kagechika Emanuel GraneroFernandez and Prof Yaocihuatl Medina-Gonzalez Dr Hassan Monhemi and Prof Mohammad Reza Housaindokht Yoshihiro Nagashima and Shinjiro Yamada Tokyo Medical and Dental University (Japan) Université de Toulouse, CNRS (France) Synthesizing and characterizing boron cluster alcohols Measuring physicochemical properties of CO2-expanded MeTHF Ferdowsi University of Mashhad (Iran) Tokyo Institute Technology (Japan) of Simulating molecular dynamics of Candida antarctica lipase B and analysing of the protein structure Enzymatic activity screening Materials, techniques and equipment support were provided by:  Yosuke Sugiyama, Ayaka Masuda, Eri Yokoyama, Ayana Hirukawa, Yusuke Koike, Kawaguchi, Dr Chen Cao at Matsuda laboratory, for general training of laboratory techniques;  Ms Mayumi Tamura (Tokyo Institute of Technology), for training and general laboratory support;  Dr Duy Phuoc Tran (Tokyo Institute of Technology), for dynamic dynamics simulation training;  Prof Akio Kitao (Tokyo Institute of Technology), for collaboration of computational resource;  Prof Yuichi Kobayashi (Tokyo Institute of Technology), for polarimeter usage support;  Amano Pharmaceutical Co Ltd (Japan), for Lipase PS-C sample;  Novozymes Japan Co., Ltd (Japan), for free from of CALB sample vii Declaration Useful discussions and intellectual input were provided by:  Prof Kaoru Nakamura;  Prof Shuichi Mori (Tokyo Medical and Dental University, Japan);  Prof Yaocihuatl Medina-Gonzalez (Université de Toulouse, France);  Dr Hassan Monhemi at Ferdowsi University of Mashhad (Iran); The research was partly funded by the Sasakawa Scientific Research Grant from the Japan Science Society (grant no 27-343 to Hai Nam Hoang), and Novozymes Japan Research Fund 2016 Parts of the results discussed in this thesis have been published in academic journals or book chapters and presented at conferences or seminar, those are listed as below Journal articles Hoang N H., Granero-Fernandez E., Yamada S., Mori S., Kagechika H., Medina-Gonzalez Y., Matsuda T (2017) Modulating biocatalytic activity toward sterically bulky substrates in CO2-expanded biobased liquids by tuning the physicochemical properties ACS Sustainable Chemistry & Engineering, (11), 11051-11059 Hoang N H., Nagashima Y., Mori S., Kagechika H., Matsuda T (2017) CO2-expanded biobased liquids as novel solvents for enantioselective biocatalysis Tetrahedron, 73 (20), 2984-2989 Hoang, H N., Matsuda, T (2016) Expanding substrate scope of lipase-catalyzed transesterification by the utilization of liquid carbon dioxide Tetrahedron, 72 (46), 7229– 7234 Hoang, H N., Matsuda, T (2015) Liquid carbon dioxide as an effective solvent for immobilized Candida antarctica lipase B catalyzed transesterification Tetrahedron Letters, 56 (4), 639-641 viii Declaration Book chapters Hoang, H N.; Are, K R A., Matsuda, T (in press) Biocatalysis in supercritical and liquid carbon dioxide and carbon dioxide expanded liquids In Supercritical and Other Highpressure Solvent Systems; Green Chemistry Series Attard, T M., Hunt, A H., Eds.; The Royal Society of Chemistry: London Hoang, H N.; Matsuda, T (2017) Biotransformation using liquid and supercritical CO2 In Future Directions in Biocatalysis; 2nd ed.; Matsuda, T., Ed.; Elsevier: Amsterdam, Chapter 1, pp 3−25 DOI: 10.1016/B978-0-444-63743-7.00001-9 International conferences Hoang, H N., Matsuda, T (2018, July) Utilize pressurized carbon dioxide as novel and sustainable reaction platforms for biocatalysis Speaker at the Gordon Research Seminar on Biocatalysis and poster presentation at the Gordon Research Conference on Biocatalysis, Maine, USA Hoang, H N., Matsuda, T (2017, May) CO2-expanded bio-based liquids: Feasible media for biocatalysis of unexpected bulky compounds Oral and poster presentation at the 4th International Symposium on Green Chemistry (ISGC 2017), La Rochelle, France Hoang, H N., Matsuda, T (2016, September) Utilizing liquid CO2 as a superior solvent for biotransformation: Acceleration of lipase-catalytic activity toward sterically bulky substrates Poster presentation at the 5th International Conference on Cofactors (ICC-05) joint with Active Enzyme Molecule (AEM 2016), Toyama, Japan Hoang, H N., Matsuda, T (2015, December) Expansion of substrate scope of Candida antarctica lipase B toward bulky phenylalkanols by liquid carbon dioxide Poster presentation at the 7th International Chemical Congress of Pacific Basin Societies (PACIFICHEM 2015), Hawaii, USA Hoang, H N., Matsuda, T (2015, July) Expansion of substrate specificity of lipasecatalyzed transesterification using liquid carbon dioxide Poster presentation at the 12th BIOTRANS 2015, Vienna, Austria ix ... optimisation of biotransformation processes falls under biocatalysis engineering, which includes different aspects: substrate engineering, medium engineering, protein (enzyme) engineering, biocatalyst... protein consisting of one or more folded chains of amino acid residues linked by peptide bonds.1 Although a typical enzyme can comprise hundreds of amino acid residues, their catalytic activity. .. University of Mashhad (Iran) Tokyo Institute Technology (Japan) of Simulating molecular dynamics of Candida antarctica lipase B and analysing of the protein structure Enzymatic activity screening Materials,