Oxidase-based Biocatalytic Processes

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Downloaded from orbit.dtu.dk on: Jun 15, 2021Oxidase-based biocatalytic processesRamesh, HemalataPublication date:2014Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Ramesh, H. (2014). Oxidase-based biocatalytic processes. DTU Chemical Engineering.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyrightowners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portalIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Oxidase-basedBiocatalytic ProcessesHemalata RameshPh.D. ThesisSeptember 2014

Oxidase-based BiocatalyticProcessesPhD. ThesisHemalata RameshDepartment of Chemical and Biochemical EngineeringTechnical University of DenmarkSeptember 20141

Copyright : Hemalata RameshSeptember 2014Address:CAPEC-PROCESSComputer Aided Process Engineering/Process Engineering and Technology centerDepartment of Chemical and Biochemical EngineeringTechnical University of DenmarkBuilding 229DK-2800 Kgs. LyngbyDenmarkPhone: 45 4525 2800Fax: 45 4593 2906Web:www.capec-process.kt.dtu.dkPrint:J&R Frydenberg A/SKøbenhavnApril 2015ISBN:978-87-93054-66-0 version 22

AbstractBiocatalytic processes are gaining significant focus in frontiers where they offer unique advantages(selectivity and mild operating conditions) over chemical catalysts. It is therefore not surprising that therehave been many industrial biocatalytic processes implemented.Despite past successes, the implementation of a new biocatalytic process still presents some challenges(demands placed on the biocatalyst) in terms of the requirements to make a viable industrial process. Inorder for a biocatalytic process to be economically successful, it is necessary that certain a set of targetmetrics (product titre, biocatalyst yield or space time yield and reaction yield) are achieved. Hence, thebiocatalyst must be able to work at high substrate and product concentrations. Such constraints that arisefrom the biocatalyst are classified as biocatalyst-related limitations. In addition, other limitations can arisefrom the reaction species (substrate and product volatility for example) and the process (such as oxygensupply, ability to control pH) and are classified as reaction-related and process-related constraintsrespectively. Although the development of biocatalyst and process engineering tools offers a number ofsolutions to overcome the limitations, it is often complicated to identify the key limitation of the systemthat prevents economic scale-up. Hence, development of a systematic method for identifying thelimitations during early-stage development of a biocatalytic process and potentially the order in which theyneed to be tackled would offer a valuable tool for process development.Biocatalytic oxidations are potentially of great value because of the selective chemistry that they offer,resulting in higher yields compared to those achievable through chemical catalysis. Oxidases areparticularly interesting biocatalysts because they use a mild oxidant (oxygen) as a substrate as opposed totheir chemical counterparts which use strong oxidants such as permanganates. A class of oxidases calledmonoamine oxidases has been used as the central case study for the thesis. The rationale for choosing thissystem is that it has been shown to exhibit the potential for resolution of racemic amines, and is capable ofproducing industrially interesting imines which are rather difficult to synthesize by chemical routes.An important aspect for biocatalytic reactions would be the implementation of monitoring and controlsystems that allow for rapid data collection to gain process knowledge. For oxidase-based biocatalysis,oxygen is consumed in stoichiometric amounts for the reaction. Therefore, oxygen sensors which canmeasure the oxygen concentration can be a valuable tool for monitoring of the process. The thesisexemplifies the use of novel solvent-resistant oxygen sensors as supporting technology for oxidase-basedreactions using a glucose oxidase reaction system as an example.i3

Implementation of biocatalytic oxidation at scale still requires process knowledge which includes thelimitations of the system and the knowledge about the potential solutions available to alleviate theselimitations. This thesis presents a methodology for development of oxidase-based biocatalytic processes. Aparticularly important aspect of the methodology includes the use of in silico analysis where propertyprediction tools have been used to identify the potential limitations to the reaction system prior toexperimentation. Such an analysis presents the opportunity to direct experimental work and thereforereduce the time and effort spent on process development, by eliminating unfeasible routes. The examplechosen for the development of the methodology was a specific monoamine oxidase-based syntheses forthe production of a pharmaceutical intermediate. This particular reaction system was chosen because ofthe potential use of the product of the biocatalytic reaction as a pharmaceutical intermediate. However,there was little information on the reaction system in the literature for the use of this biocatalyst forsynthesis of chemicals. Therefore, early stage process understanding was required. The chapters of thethesis identify the potential limitations for the reaction system by systematic evaluation of the reactionsystem through the use of property prediction tools as well as experiments. The results obtained from theexperiments are then used to identify the bottleneck for the implementation at scale. Furthermore, adiscussion of the limitations and the order which they need to be tackled is presented.ii4

Dansk ResuméFokus på biokatalytiske processer er øget betydeligt på grund af deres unikke fordele (selektivitet og mildereaktionsbetingelser) i forhold til kemiske katalysatorer. Det er derfor heller ingen overraskelse at mangebiokatalytiske processer allerede er blevet implementeret industrielt.Til trods for den hidtidige succes er det stadig en udfordring at sikre en bæredygtig industriel proces, når enny biokatalytisk reaktion skal implementeres på industriel skala. Det er nødvendigt at en række mindstemålfor processen (mht. produkt koncentration, biokatalysator udbytte, volumetrisk udbytte og reaktionudbytte) er opnået for at sikre økonomisk succes for en biokatalytisk proces. Det er derfor nødvendigt at engiven biokatalysator fungere selv ved høje substrat- og produktkoncentrationer. Proces restriktioner derfremkommer på grund af biokatalysatoren er klassificeret som biokatalysator-relaterede begrænsninger.Ydermere kan der fremkomme begrænsninger grundet reaktions specier (for eksempel substrat og produktflygtighed) samt selve processen (så som oxygen tilførsel eller evnen til at kontrollere pH), hvilke erklassificeret som henholdsvis reaktions- og proces-relaterede begrænsninger. Ingeniørværktøjer tilforbedring af både biokatalysatorer og kemiske processer gør det muligt at afhjælpe systembegrænsningerne, men det er oftest kompliceret at identificere nøgle-begrænsningerne der forhindre enøkonomisk forsvarlig opskalering. Det er derfor nødvendigt at udvikle systematiske metoder der gør detmuligt at identificere disse begrænsninger i en tidlig udviklingsfase af en biokatalytisk proces og potentieltogså rækkefølgen som disse begrænsninger skal afhjælpes i.Biokatalytiske oxidationer er potentielt meget værdifulde på grund af den høje kemiske selektivitet detilbyder, hvilket resulterer i højere udbytte sammenlignet med det opnåeligt med kemiske katalysatorer.Oxidaser er særligt interessante biokatalysatorer, da de udnytter et mildt oxidationsmiddel (oxygen) somsubstrat modsat deres kemiske modstykker der bruger stærke oxidationsmidler så som permanganater.Monoaminoxidaser, en klasse af oxidaser, er i denne afhandling brugt som det primære case study.Rationalet for valget af netop dette system er at enzymklassen kan bruges til resolution af racemiskeaminer samt industriel produktion af interessante iminer, som ellers er meget svære a syntetisere viakemiske ruter.Implementering af måle- og kontrolsystemer der muliggør hurtig data indsamling vil være et vigtigt aspektfor biokatalytiske reaktioner. Oxidase-baseret biokatalyse forbruger oxygen i støkiometriskmængde.Oxygensensorer der kan måle oxygenkoncentrationer kan derfor udgøre et værdifuldt måleinstrument til atfølge processen. Afhandlingen giver eksempler på brugen af moderne oxygensensorer der eriii5

modstandsdygtige overfor organiske solventer der kan benyttes til at følge eksempelvis en glukoseoxidasekatalyseret reaktion.Implementeringen af biokatalytisk oxidation på industrielskala kræver proceskendskab som inkluderersystembegrænsninger og viden der gør det muligt afhjælpe disse begrænsninger. Denne afhandlingpræsenterer en metodologi for udviklingen af oxidase-baserede biokatalytiske processer. Et specielt vigtigtaspekt i metodologien inkluderer brugen af in silico analyse hvor værktøjer til forudsigelse af kemiskeegenskaber er brugt til at identificere de potentielle systembegrænsninger før eksperimenter. En sådananalyse giver muligheder for at give retningslinjer for efterfølgende eksperimentelt arbejde og dervedreducere mængden a tid og ressourcer brugt på procesudvikling ved at udelukke urealiserbare alternativer.En monoaminoxidase baseret syntese af et intermediært lægemiddel blev benyttet som eksempel iudviklingen af metodologien. Dette reaktionssystem blev valgt specifikt på grund af den potentielleanvendelse af produktet som et vigtigt intermediært lægemiddel. Informationer i den videnskabeligelitteratur omkring brugen af denne biokatalysator og det pågældende reaktionssystemet var dog yderstbegrænsede, hvilket gjorde tidlig procesforståelse uundværligt. Kapitlerne i denne afhandling identificererde potentielle begrænsninger i reaktionssystemet ved hjælp af systematisk evaluering af systemet gennembrugen af værktøjer til forudsigelse af kemiske egenskaber samt eksperimenter. Resultaterne opnåetgennem det eksperimentelle arbejde er derefter brugt til at identificere flaskehalsene forimplementeringen af processen på industrielskala. Ydermere bliver systembegrænsninger og rækkefølgende skal afhjælpes i diskuteret grundigt.iv6

Abbreviations and NomenclatureAbbreviationAAOACAAMBACECPMEDOE.C.E. SKPiLC/MSLBLEDMMAOMAO-NMTBEMTQN.A.N.D.NMRNIRpET 16PQQrpmDescriptionAmino acid oxidaseAmino cephalosporanic acidAlpha methylbenzyl amineCrude-enzymeCyclopentylethyl etherDissolved oxygenEnzyme commissionEscherichia colienantiomeric excessFlavin adenine dinucleotideFood and drug administrationFormate dehydrogenaseFlow injection analysisFlavin mononeucleotideFourier transform spectroscopyGas chromatographyGram cell dry weightGenetically modified organismGlucose oxidaseHour(s)Hepatitis C virusIn situ product removalIn situ substrate supply strategyPotassium phosphate bufferLiquid chromatography/mass spectrometryLuria BertaniLight emitting diodeMolarMonoamine oxidaseMonoamine oxidase from Aspergillus nigermethyl tertbutyl ether1-methyl-1,2,3,4-tetrahydroisoquinolineNot applicableNot determinedNuclear magnetic resonanceNear infraredVector containing N-terminal His Tag and a T7 expression regionPyrroloquinoline quinoneRotations per minutev7

TBTTNWCWTTerrific brothTotal turnover numberWhole-cellsWild typeNomenclature DescriptionUnitccritaqCritical concentration of inhibitor in aqueous phaseMfiLogPConstant ( 25 for liquids at room temperature)Octanol-water partition water partition coefficient of inhibitorDimensionlessOD600pKaOptical density at wavelength of 600 nmAcid dissociation constantDimensionlessDimensionlesssiaqTAqueous solubility of inhibitorTemperatureM Cvi8

PrefaceThe work presented in this thesis was prepared at the Department of Chemical and BiochemicalEngineering (KT), at the Technical University of Denmark (DTU) in partial fulfilment of the requirements forthe PhD. Degree in Engineering.The work was conducted at the CAPEC-PROCESS center (former Centre for Process Engineering andTechnology, PROCESS, DTU Chemical Engineering) from October 2011 to September 2014. The project wassupervised by Prof. John M. Woodley and Dr. Ulrich Krühne.The project was partially funded by BIONEXGEN, financed by the European Union through the 7 thFramework people Programme (grant agreement no.: 266025). Collaborations were extended to otherUniversities and industries (in particular Technical University of Slovakia, University College of London andCLEA Technologies) in the framework.Kgs. Lyngby,September 2014Hemalata Rameshvii9

ContentsAbstract .iDansk Resumé . iiiAbbreviations and Nomenclature . vPreface . viiContents . viii1.Thesis Introduction . 11.1 Background . 11.2 When to use biocatalysis for chemical synthesis . 21.3 Biocatalytic oxidations . 41.3.1 Alcohol oxidases . 51.3.2 Amine oxidases . 81.4 Scope of the thesis . 81.5 Structure of the thesis . 91.6 Contributions . 11Journal articles . 11Published . 11In preparation . 122.Introduction to the target reaction system . 152.1 Amine oxidases . 152.1.1 Types of amine oxidases . 162.2 Monoamine oxidase-N (MAO-N) . 172.2.1 MAO-N structure . 192.3 MAO-N Application. 192.3.1 Synthesis of optically pure amines using MAO . 192.3.2 Synthesis of aldehyde from amines. 202.3.3 Synthesis of imine from amines . 222.5 Motivation for case study 1 . 233.Oxygen sensors as supporting tools for oxidase based biocatalysis . 253.1 Introduction . 25viii10

3.1.1 Online sensors for oxygen measurements . 263.1.2 Applications for advanced oxygen sensors . 264.Measurement of oxygen transfer in organic solvents using optical sensors . 314.1 Introduction . 314.2. Materials and methods . 334.2.1 Reactors . 334.2.2 Calibration of oxygen sensor . 334.2.3 Kla measurements . 334.2.4 Biocatalysis . 344.3 Results and discussion . 344.3.1 Substrate inhibition . 354.3.2 Solvents as vectors for oxygen transfer . 364.3 Applications for oxygen sensor in oxidase-based processes . 404.4 Conclusions . 415.In silico Analysis of Potential Limitations. 455.1 Introduction . 455.2.1 Process limitations . 465.3 Biocatalyst-related limitations . 475.3.1 Non-specific inactivation of the biocatalyst . 485.3.2 Specific interaction of the substrate and product with the biocatalyst . 495.3.3 Inactivation (toxicity) by reactive co-product . 505.3.4 Inactivation by oxygen . 505.4 Reaction species-related limitations . 515.4.1 pH shift during the reaction . 515.4.2 Degradation of the substrate and product by hydrogen peroxide . 515.5 Process-related limitations . 525.5.1. pH control . 525.5.2 Oxygen supply strategies . 525.6 Conclusions . 536.Reaction Species-Related Limitations . 556.1 Introduction . 556.1.1 pH shift. 556.2 Materials and methods . 57ix11

6.2.1 Biocatalyst preparation . 576.2.2 pH shift. 586.2.3 Test for presence of trimer and product volatility . 596.3 Results and discussion . 606.3.1 pH optima for MAO . 606.3.2 Effect of pH shift on the reaction profile . 606.3.3 Product volatility and trimerization. 626.3.4 Trimer formation . 656.4 Conclusions . 667.Biocatalyst-related limitations –Characteristics of the biocatalyst . 69Summary. 697.1 Introduction . 697.1.1 Mass transfer across the cell membrane . 707.1.2 Stability of the biocatalyst . 707.1.3 Overcoming product inhibition and toxicity . 717.2 Materials and methods . 737.2.1 Plasmid . 737.2.2 LB broth and plates. 747.2.3 Preparation of competent cells . 747.2.4 Plasmid transformation . 747.2.5 Fermentation. 747.2.6 Biocatalysis. 757.2.7 Sample preparation . 787.2.8 Gas Chromatography . 787.3 Results and discussion . 787.3.1 Mass transfer across the membrane . 787.3.1 Inhibition by the substrate (amine) . 807.3.2 Inhibition by product (imine). 837.3.3 Inhibition by co-product (hydrogen peroxide) . 847.3.4 Biocatalyst stability (Inactivation of the biocatalyst) . 857.3.5 Overcoming product toxicity . 897.3.6 Test for oxygen limitation . 907.3.7 Constraints identified . 91x12

7.4 Conclusions . 938.Analysis of process limitations . 978.1 Introduction . 978.2 Materials and methods . 1008.2.1 Plasmid and fermentation . 1008.2.2 Biocatalysis. 1008.2.3 Sample preparation . 1008.2.4 Gas chromatography. 1018.3 Results and discussion . 1018.3.1 Product-time profiles at different biocatalyst concentration . 1018.3.2 Reaction rate and oxygen transfer limit . 1048.3.3 Product concentration limit . 1058.3.4 Implication of the results . 1078.3.5 Effect of adopting a successful ISPR . 1088.3.6 Effect of improving protein expression . 1108.3.7 Improvement on process metrics achieved . 1118.4 Conclusion . 1129.Biocatalyst washing . 1139.1 Introduction .

Downloaded from orbit.dtu.dk on: May 07, 2021 Oxidase-based biocatalytic processes Ramesh, Hemalata Publication date: 2014 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Ramesh, H. (2014). Oxidase-based b

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