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Edited by
Sergio Riva and
Wolf-Dieter Fessner
Cascade Biocatalysis
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Edited by Sergio Riva and Wolf-Dieter Fessner
Cascade Biocatalysis
Integrating Stereoselective and Environmentally FriendlyReactions
The Editors
Dr. Sergio RivaIstituto di Chimica del RiconoscimentoMolecolareCNRVia Mario Bianco 920131 MilanoItaly
Prof. Wolf-Dieter FessnerTU DarmstadtInstitut fur Organische Chemie undBiochemiePetersenstr. 2264287 DarmstadtGermany
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V
Contents
List of Contributors XIIIPreface XXI
1 Directed Evolution of Ligninolytic Oxidoreductases: from FunctionalExpression to Stabilization and Beyond 1Eva Garcia-Ruiz, Diana M. Mate, David Gonzalez-Perez, PatriciaMolina-Espeja, Susana Camarero, Angel T. Martınez, Antonio O.Ballesteros, and Miguel Alcalde
1.1 Introduction 11.2 Directed Molecular Evolution 11.3 The Ligninolytic Enzymatic Consortium 31.4 Directed Evolution of Laccases 61.4.1 Directed Evolution of Low-Redox Potential Laccases 71.4.2 Directed Evolution of Medium-Redox Potential Laccases 71.4.3 Directed Evolution of Ligninolytic High-Redox Potential Laccases
(HRPLs) 81.5 Directed Evolution of Peroxidases and Peroxygenases 111.6 Saccharomyces cerevisiae Biomolecular Tool Box 151.7 Conclusions and Outlook 16
Acknowledgments 17Abbreviations 17References 18
2 New Trends in the In Situ Enzymatic Recycling of NAD(P)(H)Cofactors 23Erica Elisa Ferrandi, Daniela Monti, and Sergio Riva
2.1 Introduction 232.2 Recent Advancements in the Enzymatic Methods for the Recycling of
NAD(P)(H) Coenzymes and Novel Regeneration Systems 242.2.1 In Situ Regeneration of Reduced NAD(P)H Cofactors 242.2.1.1 Formate Dehydrogenase and Glucose Dehydrogenase 242.2.1.2 Phosphite Dehydrogenase 262.2.1.3 Hydrogenase 27
VI Contents
2.2.1.4 Glucose 6-Phosphate Dehydrogenase 292.2.1.5 Alcohol Dehydrogenase 292.2.2 In Situ Regeneration of Oxidized NAD(P)+ Cofactors 312.2.2.1 Lactate Dehydrogenase 312.2.2.2 NAD(P)H Oxidase 322.2.2.3 Alcohol Dehydrogenase 342.2.2.4 Mediator-Coupled Enzyme Systems 352.3 Conclusions 37
Acknowledgments 38References 38
3 Monooxygenase-Catalyzed Redox Cascade Biotransformations 43Florian Rudroff and Marko D. Mihovilovic
3.1 Introduction 433.1.1 Scope of this Chapter 433.1.2 Enzymatic Oxygenation 433.1.3 Effective Cofactor Recycling 443.1.4 In Vitro Multistep Biocatalysis 463.1.5 Combined In Vitro and In Vivo Multistep Biocatalysis 483.1.6 In Vivo Multistep Biocatalysis 513.1.7 Chemo-Enzymatic Cascade Reactions 563.1.8 Conclusion and Outlook 60
References 61
4 Biocatalytic Redox Cascades Involving 𝛚-Transaminases 65Robert C. Simon, Nina Richter, and Wolfgang Kroutil
4.1 Introduction 654.2 General Features of ω-Transaminases 664.2.1 Cascades to Shift the Equilibrium for Amination 674.3 Linear Cascade Reactions Involving ω-Transaminases 694.3.1 Redox and Redox-Neutral Cascade Reactions 704.3.2 Carbonyl Amination Followed by Spontaneous Ring Closure 754.3.3 Deracemization of Racemic Amines Employing Two
ω-Transaminases 784.3.4 Cascade Reactions of ω-TAs with Lyases and C–C
Hydrolases/Lipases 804.4 Concluding Remarks 82
References 83
5 Multi-Enzyme Systems and Cascade Reactions Involving CytochromeP450 Monooxygenases 87Vlada B. Urlacher and Sebastian Schulz
5.1 Introduction 875.1.1 Multistep Cascade Reactions 875.1.2 Cytochrome P450 Monooxygenases 885.1.3 General Overview of presented cascade types 91
Contents VII
5.2 Physiological Cascade Reactions Involving P450s 925.2.1 Multistep Oxidations Catalyzed by a Single P450 925.2.2 Multistep Oxidations Catalyzed by Multiple P450s 1025.3 Artificial Cascade Reactions Involving P450s 1085.3.1 Cascade Reactions Involving P450s and Cofactor Regenerating
Enzymes 1085.3.1.1 Cofactor Regeneration in Cell-Free Systems (In Vitro) 1085.3.2 Cofactor Regeneration in Whole-Cell Biocatalysts 1145.3.3 Artificial Enzyme Cascades Involving P450s and Other Enzymes 1155.3.3.1 Artificial Multi-Enzyme Cascades with Isolated Enzymes 1165.3.3.2 Artificial Multi-Enzyme Cascades In Vivo 1205.4 Conclusions and Outlook 124
References 125
6 Chemo-Enzymatic Cascade Reactions for the Synthesis ofGlycoconjugates 133Ruben R. Rosencrantz, Bastian Lange, and Lothar Elling
6.1 Introduction 1336.1.1 Impact of Glycoconjugates and Their Synthesis 1336.1.2 Biocatalysts for the Synthesis of Glycoconjugates 1346.1.2.1 Glycosyltransferases 1346.1.2.2 Glycosidases and Glycosynthases 1366.1.3 Definition of Cascade Reactions 1376.2 Sequential Syntheses 1396.2.1 Nucleotide Sugars 1396.2.2 Glycoconjugates 1416.3 One-Pot Syntheses 1466.3.1 Nucleotide Sugars 1466.3.2 Glycan Structures 1486.4 Convergent Syntheses 1516.5 Conclusion 153
Acknowledgment 153References 153
7 Synergies of Chemistry and Biochemistry for the Production of 𝛃-AminoAcids 161Josefa Marıa Clemente-Jimenez, Sergio Martınez-Rodrıguez, FelipeRodrıguez-Vico, and Francisco Javier Las Heras-Vazquez
7.1 Introduction 1617.2 Dihydropyrimidinase 1637.3 N-Carbamoyl-β-Alanine Amidohydrolase 1667.4 Bienzymatic System for β-Amino Acid Production 1737.5 Conclusions and Outlook 174
Acknowledgments 174References 174
VIII Contents
8 Racemizable Acyl Donors for Enzymatic Dynamic KineticResolution 179Davide Tessaro
8.1 Introduction 1798.2 The Tools 1808.2.1 The Enzymes 1808.2.2 The Racemization of Acyl Compounds 1828.3 Applications of DKR to Acyl Compounds 1838.3.1 Base-Catalyzed Racemization 1838.3.2 DKR of Oxoesters 1858.3.3 DKR of Thioesters 1888.4 Conclusions 193
Acknowledgments 194References 194
9 Stereoselective Hydrolase-Catalyzed Processes in Continuous-FlowMode 199Zoltan Boros, Gabor Hornyanszky, Jozsef Nagy, and Laszlo Poppe
9.1 Introduction 1999.1.1 General Remarks on Reactions in Continuous-Flow
Systems 1999.1.1.1 Stereoselective Reactions in Continuous Flow Systems 2029.1.1.2 Analytical Applications 2039.1.2 Nonstereoselective Enzymatic Processes 2049.2 Enzyme-Catalyzed Stereoselective Reactions in Continuous-Flow
Systems 2049.2.1 Stereoselective Processes Catalyzed by Nonhydrolytic
Enzymes 2049.2.2 Stereoselective Processes Catalyzed by Hydrolases 2079.2.2.1 Applicable Types of Selectivities 2079.2.2.2 Stereoselective Hydrolytic Reactions 2079.2.2.3 Stereoselective Acylations 2119.2.2.4 Effects of the Operation Conditions and the Mode of Enzyme
Immobilization 2209.3 Outlook and Perspectives 222
References 222
10 Perspectives on Multienzyme Process Technology 231Paloma A. Santacoloma and John M. Woodley
10.1 Introduction 23110.2 Multienzyme System Classification 23310.3 Biocatalyst Options 23310.3.1 Transport Limitations 23510.3.2 Compartmentalization 23710.4 Reactor Options 237
Contents IX
10.5 Process Development 23910.5.1 Recombinant DNA Technology 24010.5.2 Process Engineering 24110.6 Process Modeling 24110.7 Future 24410.8 Concluding Remarks 245
References 245
11 Nitrile Converting Enzymes Involved in Natural and Synthetic CascadeReactions 249Ludmila Martınkova, Andreas Stolz, Fred van Rantwijk, NicolaD’Antona, Dean Brady, and Linda G. Otten
11.1 Introduction 24911.2 Natural Cascades 25011.2.1 Nitrile Hydratase – Amidase 25011.2.2 Aldoxime Dehydratase–Nitrile Hydratase–Amidase 25511.2.3 Other Natural Cascades 25611.3 Artificial Cascades 25711.3.1 Nitrile Hydratase–Amidase 25711.3.2 Nitrilase–Amidase 25811.3.3 Hydroxynitrile Lyase–Nitrilase 25911.3.4 Hydroxynitrile Lyase–Nitrilase–Amidase 26111.3.5 Hydroxynitrile Lyase–Nitrile Hydratase 26111.3.6 Oxygenase–Nitrilase 26211.3.7 Lipase–Nitrile Hydratase–Amidase 26311.4 Conclusions and Future Use of These Enzymes 264
Acknowledgments 265References 265
12 Mining Genomes for Nitrilases 271Ludmila Martınkova
12.1 Strategies in Nitrilase Search 27112.2 Diversity of Nitrilase Sequences 27212.2.1 Nitrilases in Bacteria 27412.2.2 Nitrilases in Fungi 27412.2.3 Nitrilases in Plants 27512.3 Structure–Function Relationships 27512.3.1 Sequence Clustering 27512.3.2 Analysis of Specific Regions 27612.3.3 Analysis of Enzyme Mutants 27612.4 Enzyme Properties and Applications 27712.4.1 Arylacetonitrilases 27712.4.2 Aromatic Nitrilases 27812.4.3 Aliphatic Nitrilases 27812.4.4 Cyanide-Transforming Enzymes 279
X Contents
12.5 Conclusions 279Acknowledgment 279References 280
13 Key-Study on the Kinetic Aspects of the In Situ NHase/AMase CascadeSystem of M. imperiale Resting Cells for Nitrile Bioconversion 283Laura Cantarella, Fabrizia Pasquarelli, Agata Spera, LudmilaMartınkova, and Maria Cantarella
13.1 Introduction 28313.2 The Temperature Effect on the NHase–Amidase Bi-Enzymatic
Cascade System 28413.3 Effect of Nitrile Concentration on NHase Activity and Stability 28713.4 Effect of Nitrile on the AMase Activity and Stability 28913.5 Concluding Remarks 293
Acknowledgments 293References 293
14 Enzymatic Stereoselective Synthesis of 𝛃-Amino Acids 297Varsha Chhiba, Moira Bode, Kgama Mathiba, and Dean Brady
14.1 Introduction 29714.2 Preparation of β-Amino Acids 29814.2.1 Chemical Methods for Generating β-Amino Acids 29814.2.2 Biocatalytic Preparation of Enantiopure β-Amino Acids 29914.2.2.1 Lipases and Aminoacylases 29914.2.2.2 Transaminases 30014.2.2.3 Nitrile Converting Biocatalysts 30014.3 Nitrile Hydrolysis Enzymes 30114.3.1 Nitrilase 30114.3.1.1 Nitrilase Structure and Mechanism 30114.3.1.2 Nitrilase Substrate Selectivity 30214.3.2 Nitrile Hydratase 30214.3.2.1 Nitrile Hydratase Structure and Mechanism 30314.3.3 Amidases 30414.3.3.1 Amidase Structure and Mechanism 30414.3.4 Nitrile Hydratase and Amidase Cascade Substrate Selectivity 30414.4 Conclusion 308
Acknowledgments 309References 309
15 New Applications of Transketolase: Cascade Reactions for AssayDevelopment 315Laurence Hecquet, Wolf-Dieter Fessner, Virgil Helaine, and FranckCharmantray
15.1 Introduction 31515.2 Cascade Reactions for Assaying Transketolase Activity In Vitro 317
Contents XI
15.2.1 Coupling with Other Enzymes as Auxiliary Agents 31715.2.1.1 Coupling with NAD(H)-Dependent Dehydrogenases 31715.2.1.2 Coupling with Bovine Serum Albumin 31915.2.1.3 Coupling with BSA and Polyphenol Oxidase 32115.2.2 Coupling with a Nonprotein Auxiliary Agent 32515.2.2.1 Chemoenzymatic Cascade Reaction Based on Redox
Chromophore 32515.2.2.2 Phenol Red as pH Indicator 32615.3 Cascade Reactions for Assaying Transketolase Activity by In Vivo
Selection 32915.3.1 Biocatalyzed Synthesis of Probes 16a,b 33015.3.2 In Vitro Studies with Wild-Type TK and Probes 16a,b by LC/MS 33015.3.3 Detection of TK Activity in E. coli Auxotrophs from Amino Acid
Precursors 33115.4 Conclusion 334
References 335
16 Aldolases as Catalyst for the Synthesis of Carbohydrates andAnalogs 339Pere Clapes, Jesus Joglar, and Jordi Bujons
16.1 Introduction 33916.2 Iminocyclitol and Aminocyclitol Synthesis 34016.3 Carbohydrates and Other Polyhydroxylated Compounds 35116.4 Conclusions 355
Acknowledgments 356References 356
17 Enzymatic Generation of Sialoconjugate Diversity 361Wolf-Dieter Fessner, Ning He, Dong Yi, Peter Unruh, and Marion Knorst
17.1 Introduction 36117.2 A Generic Strategy for the Synthesis of Sialoconjugate Libraries 36317.2.1 Synthesis of Sialic Acid Diversity 36817.2.1.1 Neuraminic Acid Aldolase 36817.2.1.2 Neuraminic Acid Synthase 37117.2.2 Nucleotide Activation of Sialic Acids 37217.2.2.1 Kinetics of Sialic Acid Activation 37317.2.2.2 Substrate Binding Model 37317.2.2.3 Engineering of Promiscuous CSS Variants 37617.2.3 Sialic Acid Transfer 37717.3 Cascade Synthesis of neo-Sialoconjugates 37817.3.1 Choice of Sialyl Acceptor 37817.3.2 One-Pot Two-Step Cascade Reactions 37917.3.3 One-Pot Three-Step Cascade Reactions 38317.3.4 Metabolic Diversification 38517.3.5 Post-Synthetic Diversification 386
XII Contents
17.3.6 Biomedical Applications of Sialoconjugate Arrays 38817.4 Conclusions 388
Acknowledgments 389References 389
18 Methyltransferases in Biocatalysis 393Ludger Wessjohann, Martin Dippe, Martin Tengg, and MandanaGruber-Khadjawi
18.1 Introduction 39318.2 SAM-Dependent Methyltransferases 39518.2.1 Substrates 39618.2.2 Cofactors 40018.2.3 Higher Homologs and Derivatives of SAM 40318.2.4 Cofactor (Re)Generation 40618.2.5 Cascade Applications 41018.3 Conclusion and Outlook 415
Abbreviations 417Acknowledgement 417References 418
19 Chemoenzymatic Multistep One-Pot Processes 427Harald Groger and Werner Hummel
19.1 Introduction: Why Chemoenzymatic Cascades and Why One-PotProcesses? 427
19.2 Concepts of Chemoenzymatic Processes 42719.3 Combination of Substrate Isomerization and their Derivatization with
Chemo- and Biocatalysts Resulting in Dynamic Kinetic Resolutionsand Related Processes 429
19.4 Combination of Substrate Synthesis (Without Isomerization) andDerivatization Step(s) 438
19.4.1 One-Pot Processes with an Initial Biocatalytic Step, Followed byChemocatalysis or a Noncatalyzed Chemical Process 439
19.4.2 One-Pot Process with an Initial Chemo Process, Followed byBiocatalysis 443
19.4.2.1 Combination of Noncatalyzed Organic Reactions andBiocatalysis 443
19.4.2.2 Combination of Metal Catalysis and Biocatalysis 44519.4.2.3 Combination of Organocatalysis and Biocatalysis 44919.5 Conclusion and Outlook 453
References 453
Index 457
XIII
List of Contributors
Miguel AlcaldeInstitute of Catalysis, CSICDepartment of BiocatalysisC/Marie Curie n◦2Cantoblanco28049 MadridSpain
Antonio O. BallesterosInstitute of Catalysis, CSICDepartment of BiocatalysisC/Marie Curie n◦2Cantoblanco28049 MadridSpain
Moira BodeUniversity of the WitwatersrandMolecular Sciences InstituteSchool of ChemistryPO Wits2050 JohannesburgSouth Africa
Zoltan BorosBudapest University ofTechnology and EconomicsDepartment for OrganicChemistry and TechnologySzt Gellert ter 4H-1111 BudapestHungary
Dean BradyUniversity of the WitwatersrandSchool of Chemistry, MolecularSciences InstitutePO Wits2050 JohannesburgSouth Africa
and
CSIR BiosciencesScientia campusCSIR Building 18Meiring Naude RoadPretoria, 0184South Africa
Jordi BujonsInstituto de Quımica Avanzada deCataluna IQAC-CSICDept Chemical Biology andMolecular ModelingBiotransformation and BioactiveMolecules GroupJordi Girona 18-2608034 BarcelonaSpain
XIV List of Contributors
Susana CamareroCSIC, Centro de InvestigacionesBiologicasRamiro de Maeztu 9E-28040 MadridSpain
Laura CantarellaUniversity of Cassino and ofLazio MeridionaleDepartment of Civil andMechanical Engineeringvia Di Biasio 4303043 Cassino (FR)Italy
Maria CantarellaUniversity of L’AquilaDepartment of Industrial andInformation Engineering andEconomicsvia Giovanni Gronchin.18-Nucleo industriale di Pile67100 L’AquilaItaly
Franck CharmantrayUniversite Blaise PascalInstitut de Chimie deClermont-Ferrand (ICCF)UMR CNRS 6296BP 10448, F-63177 AubiereFrance
Varsha ChhibaCSIR BiosciencesScientia campusCSIR Building 18Meiring Naude RoadPretoria, 0184South Africa
Pere ClapesInstituto de Quımica Avanzada deCataluna IQAC-CSICDept Chemical Biology andMolecular ModelingBiotransformation and BioactiveMolecules GroupJordi Girona 18-2608034 BarcelonaSpain
Josefa Marıa Clemente-JimenezUniversidad de AlmerıaDepartamento de Quımica yFısicaCarretera de Sacramento S/NEdificio C.I.T.E. ILa Canada de San Urbano04120 AlmerıaSpain
Nicola D’AntonaCNR National ResearchCouncil of ItalyInstitute of BiomolecularChemistryVia P. Gaifami 1895126 CataniaItaly
Martin DippeLeibniz Institute of PlantBiochemistryWeinberg 3D-06120 HalleGermany
List of Contributors XV
Lothar EllingRWTH Aachen UniversityDepartment of Biotechnology andHelmholtz-Institute forBiomedical EngineeringWorringer Weg 152056 AachenGermany
Erica Elisa FerrandiIstituto di Chimica delRiconoscimento MolecolareC. N. R.Via Mario Bianco 920131 MilanoItaly
Wolf-Dieter FessnerTechnische UniversitatDarmstadtDepartment of OrganicChemistry and BiochemistryPetersenstr. 22D-64287 DarmstadtGermany
Eva Garcia-RuizInstitute of Catalysis, CSICDepartment of BiocatalysisC/Marie Curie n◦2Cantoblanco28049 MadridSpain
David Gonzalez-PerezInstitute of Catalysis, CSICDepartment of BiocatalysisC/Marie Curie n◦2Cantoblanco28049 MadridSpain
Harald GrogerBielefeld UniversityFaculty of ChemistryUniversitatsstr. 2533615 BielefeldGermany
Mandana Gruber-KhadjawiACIB GmbHc/o Graz University ofTechnologyInstitute of Organic ChemistryStremayrgasse 98010 GrazAustria
Ning HeTechnische UniversitatDarmstadtDepartment of OrganicChemistry and BiochemistryPetersenstr. 22D-64287 DarmstadtGermany
Laurence HecquetUniversite Blaise PascalInstitut de Chimie deClermont-Ferrand (ICCF)UMR CNRS 6296BP 10448, F-63177 AubiereFrance
Virgil HelaineUniversite Blaise PascalInstitut de Chimie deClermont-Ferrand (ICCF)UMR CNRS 6296BP 10448, F-63177 AubiereFrance
XVI List of Contributors
Gabor HornyanszkyBudapest University ofTechnology and EconomicsDepartment for OrganicChemistry and TechnologySzt Gellert ter 4H-1111 BudapestHungary
Werner HummelHeinrich-Heine-University ofDusseldorfResearch Centre JulichInstitute of Molecular EnzymeTechnologyStetternicher Forst52426 JulichGermany
Jesus JoglarInstituto de Quımica Avanzada deCataluna IQAC-CSICDept Chemical Biology andMolecular ModelingBiotransformation and BioactiveMolecules GroupJordi Girona 18-2608034 BarcelonaSpain
Marion KnorstTechnische UniversitatDarmstadtDepartment of OrganicChemistry and BiochemistryPetersenstr. 22D-64287 DarmstadtGermany
Wolfgang KroutilUniversity of GrazInstitute of ChemistryHeinrichstr. 288010 GrazAustria
Bastian LangeRWTH Aachen UniversityDepartment of Biotechnology andHelmholtz-Institute forBiomedical EngineeringWorringer Weg 152056 AachenGermany
Francisco Javier LasHeras-VazquezUniversidad de AlmerıaDepartamento de Quımica yFısicaCarretera de Sacramento S/NEdificio C.I.T.E. ILa Canada de San Urbano04120 AlmerıaSpain
Angel T. MartınezCSIC, Centro de InvestigacionesBiologicasRamiro de Maeztu 9E-28040 MadridSpain
Sergio Martınez-RodrıguezUniversidad de AlmerıaDepartamento de Quımica yFısicaCarretera de Sacramento S/NEdificio C.I.T.E. ILa Canada de San Urbano04120 AlmerıaSpain
Ludmila MartınkovaAcademy of Sciences of the CzechRepublicInstitute of MicrobiologyLaboratory of BiotransformationVidenska 1083CZ-142 20 PragueCzech Republic
List of Contributors XVII
Diana M. MateInstitute of Catalysis, CSICDepartment of BiocatalysisC/Marie Curie n◦2Cantoblanco28049 MadridSpain
Kgama MathibaCSIR BiosciencesScientia campusCSIR Building 18Meiring Naude RoadPretoria, 0184South Africa
Marko D. MihovilovicVienna University of TechnologyInstitute of Applied SyntheticChemistryGetreidemarkt 9/163-OCA-1060 ViennaAustria
Patricia Molina-EspejaInstitute of Catalysis, CSICDepartment of BiocatalysisC/Marie Curie n◦2Cantoblanco28049 MadridSpain
Daniela MontiIstituto di Chimica delRiconoscimento MolecolareC. N. R.Via Mario Bianco 920131 MilanoItaly
Jozsef NagyBudapest University ofTechnology and EconomicsDepartment for OrganicChemistry and TechnologySzt Gellert ter 4H-1111 BudapestHungary
Linda G. OttenDelft University of TechnologyDepartment of BiotechnologyBiocatalysisJulianalaan 1362628 BL DelftThe Netherlands
Fabrizia PasquarelliUniversity of L’AquilaDepartment of Industrial andInformation Engineering andEconomicsvia Giovanni Gronchin.18-Nucleo industriale di Pile67100 L’AquilaItaly
Laszlo PoppeBudapest University ofTechnology and EconomicsDepartment for OrganicChemistry and TechnologySzt Gellert ter 4H-1111 BudapestHungary
Nina RichterUniversity of GrazInstitute of ChemistryHeinrichstr. 288010 GrazAustria
XVIII List of Contributors
Sergio RivaIstituto di Chimica delRiconoscimento MolecolareC. N. R.Via Mario Bianco 920131 MilanoItaly
Felipe Rodrıguez-VicoUniversidad de AlmerıaDepartamento de Quımica yFısicaCarretera de Sacramento S/NEdificio C.I.T.E. ILa Canada de San Urbano04120 AlmerıaSpain
Ruben R. RosencrantzRWTH Aachen UniversityDepartment of Biotechnology andHelmholtz-Institute forBiomedical EngineeringWorringer Weg 152056 AachenGermany
Florian RudroffVienna University of TechnologyInstitute of Applied SyntheticChemistryGetreidemarkt 9/163-OCA-1060 ViennaAustria
Paloma A. SantacolomaTechnical University of Denmark(DTU)Department of Chemical andBiochemical EngineeringSøltofts Plads2800 Kgs. LyngbyDenmark
Sebastian SchulzHeinrich-Heine UniversityDusseldorfInstitute of BiochemistryUniversitatsstr. 140225 DusseldorfGermany
Robert C. SimonUniversity of GrazInstitute of ChemistryHeinrichstr. 288010 GrazAustria
Agata SperaUniversity of L’AquilaDepartment of Industrial andInformation Engineering andEconomicsvia Giovanni Gronchin.18-Nucleo industriale di Pile67100 L’AquilaItaly
Andreas StolzUniversity of StuttgartInstitute of MicrobiologyAllmandring 3170569 StuttgartGermany
Martin TenggACIB GmbHc/o Graz University ofTechnologyInstitute of MolecularBiotechnologyPetersgasse 148010 GrazAustria
List of Contributors XIX
Davide TessaroPolitecnico di MilanoDepartment of ChemistryMaterials and ChemicalEngineering ‘‘G. Natta”Piazza Leonardo da Vinci 3220131 MilanoItaly
and
The Protein Factory UniversityCenter for Protein Biotechnologyvia Mancinelli, 730131 MilanoItaly
Peter UnruhTechnische UniversitatDarmstadtDepartment of OrganicChemistry and BiochemistryPetersenstr. 22D-64287 DarmstadtGermany
Vlada B. UrlacherHeinrich-Heine UniversityDusseldorfInstitute of BiochemistryUniversitatsstr. 140225 DusseldorfGermany
Fred van RantwijkDelft University of TechnologyDepartment of BiotechnologyBiocatalysisJulianalaan 1362628 BL DelftThe Netherlands
Ludger WessjohannLeibniz Institute of PlantBiochemistryWeinberg 3D-06120 HalleGermany
John M. WoodleyTechnical University of Denmark(DTU)Department of Chemical andBiochemical EngineeringSøltofts Plads2800 Kgs. LyngbyDenmark
Dong YiTechnische UniversitatDarmstadtDepartment of OrganicChemistry and BiochemistryPetersenstr. 22D-64287 DarmstadtGermany
XXI
Preface
Sustainability is one of the key issues to enhance, or at least maintain, the qualityof life in our modern society. As it has been codified in 1987 in an official UNdocument, a ‘‘sustainable development is development that meets the needs of thepresent without compromising the ability of future generations to meet their ownneeds’’. Applied to chemical processes, sustainability has generated the concept ofGreen Chemistry, for which guidelines have been summarized as the well-knownTwelve Principles of Green Chemistry.1)
In Europe, this effort has been recognized at the institutional level:the European Technology Platform for Sustainable Chemistry (SusChem,http://www.suschem.org) was created in 2004 with the main objective to revitalizeand inspire the European chemistry research, development, and innovation in asustainable way. Industrial Biotechnology, also known as White Biotechnology, isone of the three pillars that support sustainable chemistry nowadays and that areexpected to support it even more profoundly in the future. It is defined as ‘‘theuse of enzymes and micro-organisms to make efficient and sustainable productsin sectors as diverse as chemicals, plastics, food and feed, detergents, paper andpulp, textiles or bioenergy.’’
Although long and reiterating, this introduction is meant to raise the aware-ness that the roots and the branches of biocatalysis – as well as its fruits! –are deeply embedded in modern synthetic chemistry. In fact, the majority of theabove-mentioned Principles of Green Chemistry (PGC) fit perfectly with the peculiarproperties and synthetic application of enzymes, which are Nature’s catalysts. Thecontributions collected in this book offer a convincing testimony that biocatalysisis highly qualified to contribute to the development of future sustainable tech-nologies. Enzymes are highly efficient catalysts offering superior selectivity (PGC#9), thereby meeting criteria for atom economy by maximizing the incorporationof starting materials into the final product (PGC #2) while avoiding unnecessaryand unproductive derivatization, such as the use of temporary protection groups(PGC #8). Such steps are unavoidable when using conventional synthetic chem-istry approaches and require additional reagents and generate waste materials,particularly when utilizing multifunctionalized, bio-based renewable feedstocks
1) P. Anastas, J.C. Warner, Green Chemistry: Theory and Practice, Oxford University Press (2008).
XXII Preface
(PGC #7). Inherently, enzymes are biodegradable (PGC #10) and innocuous tothe environment (PGC #3), not the least because they operate in water as a safesolvent (PGC #5) at ambient temperature and pressure, which minimizes energyconsumption (PGC #6).
Cascade Biocatalysis is an effort to imitate the style of chemical conversionsoccurring in living beings, which are totally different from the traditional useof single enzymes by synthetic chemists in the laboratory for catalyzing isolatedtransformations. Instead, cells apply multistep synthetic strategies, catalyzed byseveral enzymes acting sequentially along a pathway, in which a product formedin one reaction in situ becomes the substrate of the next catalyst. This is possiblebecause of the very similar mild reaction conditions under which most enzymesoperate, which facilitates their combination and allows effective strategies ofreaction engineering, for example, to shift unproductive equilibria by coupling tothermodynamically favored processes for overall high conversion and economicefficiency.
This concept has recently been recognized as the major focus for a series ofinternational symposia on Multistep Enzyme-Catalyzed Processes, the last symposiumhaving just been celebrated in Madrid in April 2014. Research in this areahas also been coordinated within the activities of the European Union fundedCOST network CM0701 entitled Cascade Chemoenzymatic Processes – New SynergiesBetween Chemistry and Biochemistry (2008–2012; http://www.cost-cascat.polimi.it).This handbook brings together contributions from scientists deeply involved inthe activities of this COST action as well as complementary chapters on relatedresearch from additional authors, who are well known for their seminal work in thiscontemporary research field. The topics covered in the chapters span from examplesrelated to integrated applications of cofactor-dependent oxidoreductases to theexploitation of transferases; from the multistep modification of the nitrile functionalgroup to the synthesis of complex carbohydrates; and from developments of newdynamic kinetic resolution processes to intricate examples of chemoenzymaticmultistep one-pot procedures.
We would like to thank all the authors who, despite their busy schedules, haveparticipated in this project to share their expertise with the future readers ofthis book. Thanks are also due to Elke Maase and Stefanie Volk at Wiley-VCHPublishers, for their careful editorial support and for their continuous goad inorder to meet assigned deadlines.
Finally, we hope that our readers will find this volume useful as a stimulatingsource of ideas for their own research and/or teaching activities.
Sergio RivaWolf-Dieter Fessner
1
1Directed Evolution of Ligninolytic Oxidoreductases: fromFunctional Expression to Stabilization and BeyondEva Garcia-Ruiz, Diana M. Mate, David Gonzalez-Perez, Patricia Molina-Espeja, SusanaCamarero, Angel T. Martınez, Antonio O. Ballesteros, and Miguel Alcalde
1.1Introduction
The ligninolytic enzymatic consortium, formed mainly by nonspecific oxidoreduc-tases (laccases, peroxidases, and H2O2-supplying oxidases), is a potentially powerfulmultipurpose tool for industrial and environmental biotechnology. In nature, theseenzymes are typically produced by basidiomycete white-rot fungi that are involvedin lignin decay. Thanks to their broad substrate specificity, high redox potential,and minimal requirements, these enzymes have many potential applications inthe field of green chemistry, including the production of biofuels, bioremediation,organic syntheses, pulp biobleaching, food and textile industries, and the designof bionanodevices. The implementation of this enzymatic armoury in differentbiotechnological sectors has been hampered by the lack of appropriate molecularinstruments (including heterologous hosts for directed evolution) with which toimprove their properties. Over the last 10 years, a wealth of directed evolutionstrategies in combination with hybrid approaches has emerged in order to adaptthese oxidoreductases to the drastic conditions associated with many biotechnolog-ical settings (e.g., high temperatures, the presence of organic co-solvents, extremepHs, the presence of inhibitors). This chapter summarizes all efforts and endeavorsto convert these ligninolytic enzymes into useful biocatalysts by means of directedevolution: from functional expression to stabilization and beyond.
1.2Directed Molecular Evolution
Enzymes are versatile biomolecules that exhibit a large repertory of functionsacquired over millions of years of natural evolution. Indeed, they are the fastestknown catalysts (accelerating chemical reactions as much as 1019-fold) and areenvironmentally friendly molecules, working efficiently at mild temperatures,in water, and releasing few by-products. Moreover, they can exhibit highenantioselectivity and chemoselectivity. Nonetheless, when an enzyme is removed
Cascade Biocatalysis: Integrating Stereoselective and Environmentally Friendly Reactions, First Edition.Edited by Sergio Riva and Wolf-Dieter Fessner.c© 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.
2 1 Directed Evolution of Ligninolytic Oxidoreductases
from its natural environment and introduced into a specific biotechnologicallocation (e.g., the transformation of a hydrophobic compound in the presence ofco-solvents or at high temperatures), its molecular structure may not tolerate theextreme operational conditions and may unfold becoming inactive. Unfortunately,the enzymes that cells use to regulate strict metabolic pathways and that promotefitness and survival in nature are not always applicable to the harsh requirementsof many industrial processes.
The development of the polymerase chain reaction (PCR) in the early 1980sheralded a biotechnological revolution for protein engineers, allowing us forthe first time to manipulate and design enzymes by site-directed mutagenesissupported by known protein structures: the so-called rational design. However,further advances were frustrated owing to the limited understanding of proteinfunction and the lack of protein structures available at the time. Nevertheless, thefollowing decade saw a second biotechnological revolution with the developmentof directed molecular evolution. This powerful protein engineering tool doesnot require prior knowledge of protein structure to enhance the known featuresor to generate novel enzymatic functions, which are not generally required innatural environments. The key events of natural evolution (random mutation,DNA recombination, and selection) are recreated in the laboratory, permitting
New
generation
Best mutant hit
(parent for next generation)
Parental genes
Recombination
Screening assay
Functional expression
in HT format
Random
mutagenesisMutation
Linearized
plasmid
Cloning and transformation
in heterologous host
Clone growth
DNA diversity
Figure 1.1 Directed molecular evolution.The basic premises to carry out a success-ful directed evolution experiment are (i)a robust heterologous expression system
(typically S. cerevisiae or E. coli); (ii) a reli-able high-throughput (HT)-screening assay;and (iii) the use of different molecular toolsfor the generation of DNA diversity.
1.3 The Ligninolytic Enzymatic Consortium 3
scientifically interesting and technologically useful enzymes to be designed [1–3].Diversity is generated by introducing random mutations and/or recombination inthe gene encoding a specific protein [4, 5]. In this process, the best performersin each round of evolution are selected and used as the parental types in a newround, a cycle that can be repeated as many times as necessary until a biocatalystthat exhibits the desired traits is obtained: for example, improved stability at hightemperatures, extreme pHs, or in the presence of nonconventional media suchas organic solvents or ionic fluids; novel catalytic activities; improved specificitiesand/or modified enantioselectivities; and heterologous functional expression [6–8](Figure 1.1). Of great interest is the use of directed evolution strategies to engineerligninolytic oxidoreductases while employing rational approaches to understandthe mechanisms underlying each newly evolved property.
1.3The Ligninolytic Enzymatic Consortium
Lignin is the most abundant natural aromatic polymer and the second most abun-dant component of plant biomass after cellulose. As a structural part of the plantcell wall, lignin forms a complex matrix that protects cellulose and hemicellulosechains from microbial attack and hence from enzymatic hydrolysis. This recalci-trant and highly heterogeneous biopolymer is synthesized by the dehydrogenativepolymerization of three precursors belonging to the p-hydroxycinnamyl alcoholgroup: p-coumaryl, coniferyl, and sinapyl alcohols [9]. As one-third of the carbonfixed as lignocellulose is lignin, its degradation is considered a key step in therecycling of carbon in the biosphere and in the use of the plant biomass forbiotechnological purposes [10, 11]. Lignin is modified and degraded to differentextents by a limited number of microorganisms, mainly filamentous fungi andbacteria. Lignin degradation by bacteria is somewhat limited and much slowerthan that mediated by filamentous fungi [12, 13]. Accordingly, the only organismscapable of completing the mineralization of lignin are the white-rot fungi, whichproduce a white-colored material upon delignification because of the enrichmentin cellulose [14, 15].
Through fungal genome reconstructions, recent studies have linked the for-mation of coal deposits during the Permo-Carboniferous period (∼260 millionyears ago) with the nascent and evolution of white-rot fungi and their lignin-degrading enzymes [16]. Lignin combustion by white-rot fungi involves a verycomplex extracellular oxidative system that includes high-redox potential laccases(HRPLs), peroxidases and unspecific peroxygenases (UPOs), H2O2-supplying oxi-dases and auxiliary enzymes, as well as radicals of aromatic compounds andoxidized metal ions that act as both diffusible oxidants and electron carriers [12, 13,15, 17]. Although the role of each component of the consortium has been studiedextensively, many factors remain to be elucidated (Figure 1.2).
Laccases typically oxidize the phenolic units of lignin. Lignin peroxidases (LiPs)oxidize both nonphenolic lignin structures and veratryl alcohol (VA), a metabolitesynthesized by fungi that helps LiP to avoid inactivation by H2O2 and whose radical
4 1 Directed Evolution of Ligninolytic Oxidoreductases
O H
O
O2
O2
OC
CHO
GLX
AAO
AAO
HCOH
HC
MOXH
2O
2
CH3OH
H2COH
VP
LaccaseVP
Laccase
LaccaseVP
Lignin
Lignin
Lignin Lignin
VP
OCH3
OCH3 OCH
3
OCH3
OCH3
Mn3+
Mn2+
O
H2COH
H2COHH
2COH
HC
HC
HC
HC
HC
HCOHAAO
OCH3
Hemicellulose
Cellulose
OCH3
MnP/VP
Laccase/VP
LiP
OH
OH
OCH3O
R
O
R
HC = O HCOH
HC
CH O Fe
2+
O2
−
OH O
O
OO
OH
OR
QR
CH2OH
Fe3+
OH
(16)
(14)
(15)
(13)
(12)
(11)(10)
(8)
(9)(7)
(17)
(4)
(2)
(5)
(1)
(3)
(19)
(6)
(18)
(20)
OH
O
OO
O
O
O
OO
O
O O
OO
O
OO
OOH
OH
OHOH
O
OO
H
C
C C
C C C
C C C
C
C
O
O
OO
O
O
O
OH
H
H H
HH
H
HOH
OH
OH
OH
OH
OH
OH
HO
H
H
HH
O
O
O
OO
OO
OO
OO
OO
OO O
OO
OO
O
O
O
O
O
O
O
O
O
C C C
O
OH
H
HH
H H
OHOH
H
H
HH
H
O
O
O
O
O
O
OO
O
OO
OO
O
O
O
O
CC
C
OH
OH
O
C
CH2
CH2
CC
C
C
CC
C CC
C CC
OH OH
H3CO
OCH3
OCH3
OCH3
OCH3
HCOH
HCOH
OCH3 OCH
3
OCH3
CHO
OCH3
CHO
CH
HC
OCH3
OCH3
CH2
H2C
OCH3
CH2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
HOCH2
H2
COH
H3
CO
H3CO
H2
COH
H2COH
H2
CO
H2COH
H2COH
H2
COH
H2COH
H3CO
H2COH
CH2
OH
CH2
OH
2OH
CHO
O
OO
OO
O O
O
O
OO
O
O
O
OH
OH
H
HH
OHHO
CH2
OH
H2COHH
2COH
H3CO
HOCH2
H3
CO
OH
CH
CH
COCHOCH
CH
HC
HC
HC
HCHC
HC
HC
HC
HC
HC
OHC
HC
HC
C
CH
CH OH
CH
CO
CH
CH
H
CH
CH
CH
OO
COCH
CH HC
HCO
O
OH
O
O
O
CH
O
O
O O
O
O
O
O
O
O
O
H3CO
OCH3
CH2
OCH3
CH2
OCH3
Hydrogen bonds Hemicellulose Hemicellulosecross link
Cellulose Lignin
CH2
H3CO
OH
HOCH2 HOCH
2
HOCH2
OCH3
OH
OCH3
O
H2COH
H3CO
OCH3
HC Lignin
Lignin
Lignin
Lignin
HCOH
HCHC
HC O
O
OC
CH HC
HCOH
1.3 The Ligninolytic Enzymatic Consortium 5
cation may act as a redox mediator [20]. Manganese peroxidases (MnPs) generateMn3+, which upon chelation with organic acids (e.g., oxalate synthesized by fungi)attacks phenolic lignin structures; in addition, MnP can also oxidize nonphenoliccompounds via lipid peroxidation [21]. Versatile peroxidases (VPs) combine thecatalytic activities of LiP, MnP, and generic peroxidases to oxidize phenolic andnonphenolic lignin units [22]. Some fungal oxidases produce the H2O2 necessaryfor the activity of peroxidases. Among them, aryl-alcohol oxidase (AAO) transformsbenzyl alcohols to the corresponding aldehydes; glyoxal oxidase (GLX) oxidizesglyoxal producing oxalate, which in turn chelates Mn3+; and then methanol oxidase(MOX) converts methanol into formaldehyde; all the above oxidations are coupledwith O2 reduction of H2O2. Other enzymes such as cellobiose dehydrogenase(CDH) have been indirectly implicated in lignin degradation. This is because ofCDH ability to reduce both ferric iron and O2-generating hydroxyl radicals viaFenton reaction. These radicals are strong oxidizers that act as redox mediatorsplaying a fundamental role during the initial stages of lignin polymer decay, whenthe small pore size of the plant cell wall prevents the access of fungal enzymes [23].The same is true for laccases, whose substrate spectrum can be broadened in thepresence of natural mediators to act on nonphenolic parts of lignin [24].
High-redox potential laccases and peroxidases/peroxygenases are of great biotech-nological interest [25, 26]. With minimal requirements and high redox potentials(up to +790 mV for laccases and over +1000 mV for peroxidases), these enzymescan oxidize a wide range of substrates, finding potential applications in a variety ofareas, which are as follows:
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1.2 General view of the plant cell walland the action of the ligninolytic enzymaticconsortium. The lignin polymer is oxidizedby white-rot fungi laccases and peroxidases,producing nonphenolic aromatic radicals (1)and phenoxy radicals (2). Nonphenolic aro-matic radicals can suffer nonenzymatic mod-ifications such as aromatic ring cleavage (3),ether breakdown (4), Cα–Cβ cleavage (5), anddemethoxylation (6). The phenoxy radicals (2)can repolymerize on the lignin polymer (7) orbe reduced to phenolic compounds by AAO(8) (concomitantly with aryl alcohol oxidation).These phenolic compounds can be re-oxidizedby fungal enzymes (9). In addition, phenoxyradicals can undergo Cα–Cβ cleavage to pro-duce p-quinones (10). Quinones promote theproduction of superoxide radicals via redoxcycling reactions involving QR, laccases, andperoxidases (11, 12). The aromatic aldehydesreleased from Cα–Cβ cleavage, or synthesizedby fungi, are involved in the production of H2O2via another redox cycling reaction involving
AAD and AAO (13, 14). Methanol resultingfrom demethoxylation of aromatic radicals (6)is oxidized by MOX to produce formaldehydeand H2O2 (15). Fungi also synthesize glyoxal,which is oxidized by GLX to produce H2O2and oxalate (16), which in turn chelate Mn3+
ions produced by MnP (17). The Mn3+ chelatedwith organic acids acts as a diffusible oxidantfor the oxidation of phenolic compounds (2).The reduction of ferric ions present in woodis mediated by the superoxide radical (18)and they are re-oxidized by the Fenton reac-tion (19) to produce hydroxyl radicals, whichare very strong oxidizers that can attack thelignin polymer (20). AAO, aryl-alcohol oxidase;AAD, aryl-alcohol dehydrogenase; GLX, glyoxaloxidase; LiP, lignin peroxidase; MnP, man-ganese peroxidase; MOX, methanol oxidase;QR, quinone reductase; VP, versatile peroxi-dase. (Figure adapted from [18, 19].) (Source:Bidlack, J.M. et al. 1992 [18], Fig. 1, p. 1.Reproduced with permission of the OklahomaAcademy of Science.)
6 1 Directed Evolution of Ligninolytic Oxidoreductases
• The use of lignocellulosic materials (e.g., agricultural wastes) in the production ofsecond-generation biofuels (bioethanol, biobutanol) or the manufacture of newcellulose-derived and lignin-derived value-added products.
• The organic synthesis of drugs and antibiotics, cosmetics and complex polymers,and building blocks.
• In nanobiotechnology as (i) biosensors (for phenols, oxygen, hydroperoxides,azides, morphine, codeine, catecholamines, or flavonoids) for clinical and envi-ronmental applications; and (ii) biofuel cells for biomedical applications.
• In bioremediation: oxidation of polycyclic aromatic hydrocarbons (PAHs),dioxins, halogenated compounds, phenolic compounds, benzene derivatives,nitroaromatic compounds, and synthetic organic dyes.
• The food industry: drink processing and bakery products.• The paper industry: pulp biobleaching, pitch control, manufacture of mechanical
pulps with low energy cost, and effluent treatment.• The textile industry: remediation of dyes in effluents, textile bleaching (e.g.,
jeans), modification of dyes and fabrics, detergents.
A few years ago, the engineering and improvement of ligninolytic oxidoreductaseswas significantly hampered by the lack of suitable heterologous hosts to carry outdirected evolution studies. Fortunately, things have changed and several reliableplatforms for the directed evolution of ligninolytic peroxidases, peroxygenases,and several medium-redox potential laccases and high-redox potential laccases(HRPLs) have been developed using the budding yeast Saccharomyces cerevisiae.These advances have allowed us, for the first time, to specifically tailor ligninolyticoxidoreductases to address new challenges.
1.4Directed Evolution of Laccases
Laccases (EC 1.10.3.2) are extracellular glycoproteins that belong to the bluemulticopper oxidase family (along with ascorbate oxidase, ceruloplasmin, nitritereductase, bilirubin oxidase, and ferroxidase). Widely distributed in nature, theyare present in plants, fungi, bacteria, and insects [27, 28]. Laccases are greencatalysts, which are capable of oxidizing dozens of compounds using O2 fromair and releasing H2O as their sole by-product [29–31]. These enzymes harborone type I copper (T1), at which the oxidation of the substrates takes place,and a trinuclear copper cluster (T2/T3) formed by three additional coppers,one T2 and two T3s, at which O2 is reduced to H2O. The reaction mechanismresembles a battery, storing electrons from the four monovalent oxidation reactionsof the reducing substrate required to reduce one molecule of oxygen to twomolecules of H2O. Laccases catalyze the transformation of a wide variety ofaromatic compounds, including ortho- and para-diphenols, methoxy-substitutedphenols, aromatic amines, benzenothiols, and hydroxyindols. Inorganic/organicmetal compounds are also substrates of laccases, and it has been reported thatMn2+ is oxidized by laccase to form Mn3+, and organometallic compounds such