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  • Enzyme Engineering4. Enzyme Reaction Kinetics

    4.1 Enzyme Reaction Kinetics & Reactor Design

    4.2 Cofactor Regeneration

  • Enzyme KineticsIt provides valuable information for enzyme mechanism

    It gives an insight into the role of an enzyme under physiological conditions

    It can help show how the enzyme activity is controlled and regulated

  • One Substrate ReactionsOne substrate reactionsEquilibrium assumptionSecond reaction is slower than first reverse reaction(k-1>>k2)Michaelis-Menten eqn

  • Parameter EstimationLineweaver-Burk equationSlope : Intercept :

  • One Substrate ReactionsSignificance of the result1. kcat/Km is the substrate specificity2. kcat/Km can be used for the applicability of equilibrium or steady state assumption3. Km is [S] where the rate is (1/2) Vmax - Km may be the affinity of an enzyme to the substrate(not always)

  • Batch KineticsThe time course of variation of [S] in a batch enzymatic reaction can be determined by integrating equationintegrationOr,A plot of 1/t([ln[S0]/[S]) versus {[S0]-[S]}/t results in a line of slope -1/Km with a y-intercept of Vm/Km

  • InhibitionInhibition of enzyme activityCompetitive inhibition Binding into active siteNon-competitive inhibition Binding outside active site

  • InhibitionCompetitive inhibitionNon-competitive inhibition

  • Substrate InhibitionSubstrate inhibitionHigh substrate concentrations may cause inhibition in some enzymatic reactions

  • Substrate InhibitionSubstrate inhibition

  • Effect of TemperatureEffect of temperature

    Effect on enzyme structureDenaturation(unfolding)Structural change in active site

    Effect on enzyme catalysis- Catalytic rate generally increase with temperature

  • Effect of pHEffect of pHpH optimum and specific range of activity

  • Parameter Estimation

  • Bioreactor Design Eqn

  • 4.2 Cofactor Regeneration

  • Nature 2001, 409, 258-268Biocatalysis cycle

  • [a]Many flavin- and metal porphyrin complexes dependent mono- or dioxygenases require additional NAD(P)H as an indirect reducing agent.

    Common cofactors required for biotransformation and their representative in situ regeneration methodsBiotransformations in Organic Chemistry, 2004Current Opinion in Biotechnology, 2003,14(6), 583-589

    CofactorEnzymeReaction typeRepresentative regeneration methodNAD+/NADHOxidoreductaseRemoval or addition hydrogenGlutamate dehydrogenase with -ketoglutarate/ Formate dehydrogenase with formateNADP+/NADPHOxidoreductaseRemoval or addition hydrogenGlutamate dehydrogenase with -ketoglutarate/ Glucose dehydrogenase with glucoseATPKinease, synthasePhosphorylationAcetate kinase with acetyl phosphateSugar nucleotidesKinease, synthaseGlycosyl transferBacterial couplingAcetyl CoADehydrogenase, Transferase, SynthaseAcyl transfer (C2-alkylation)Phosphotransacetylase with acyl phosphatePAPSTransferase Sulfuryl transferAryl sulfotransferase IV with p-nitrophenyl sulfateSAMDehydrogenase, Transferase, SynthaseMethyl transfer (C1-alkylation)No demonstrated methodFlavins[a]Oxygenase, hydroxylaseOxygenationSelf-regenerationPyridoxal phosphateTransaminaseTransaminationSelf-regenerationBiotinCarboxylase, DecarboxylaseCarboxylationSelf-regenerationMetal porphyrin complexes[a]Monooxygenase, Peroxidase, MutasePeroxydation, oxygenationSelf-regeneration

  • Oxidoreductases are valuable enzymes which have potential in synthesizing many kinds of chemicals used in pharmaceutical applications, food additives, etc. (amino acids, chiral alcohols, ketones, steroids, etc.)

    Especially, enzymatic oxidation by oxidoreductase is attractive due to myriad of applications for the organic synthesis as well as analytical purpose including clinical diagnosis and fuel generation.Oxidoreductase

  • Structure of nicotinamide cofactors

  • Table. Cost of nicotinamide cofactor.http://www.sigmaaldrich.com (2009)The Chemical Record 2004, 4, 254-265Necessity of cofactor regenerationCosts of redox equivalents

    Cost (US dollar) / gCost (US dollar) / molNAD+5637,150NADH9768,599NADP+344263,298NADPH1,080900,018

  • Strategies for cofactor regeneration

    MethodAdvantageDisadvantageEnzymatic method High selectivityCompatibility High enzyme costEnzyme instabilityComplexity of product isolationElectrochemical methodLow cost of electricityNo stoichiometric regenerating reagentEasy product isolationClean processComplex apparatus and procedureRequirement in many systems for mediating redox agent Chemical methodCommercially available reagentsNo requirement for added enzyme IncompatibilityComplexity of product isolationLow product yieldLow TTN Photochemical methodNo stoichiometric regenerating reagent in some systemsNo requirement for added enzymes Complex apparatusIncompatibilityLimited stabilityRequirement for photosensitizer and redox dyes

  • Adv. Synth. Catal. 2008, 350, 2305-2312Tetrahedron: Asymmetry 2004,15(18), 2933-2937Enzymatic regeneration of NAD+

  • Strategies for cofactor regeneration

    MethodAdvantageDisadvantageEnzymatic method High selectivityCompatibility High enzyme costEnzyme instabilityComplexity of product isolationElectrochemical methodLow cost of electricityNo stoichiometric regenerating reagentEasy product isolationClean processComplex apparatus and procedureRequirement in many systems for mediating redox agent Chemical methodCommercially available reagentsNo requirement for added enzyme IncompatibilityComplexity of product isolationLow product yieldLow TTN Photochemical methodNo stoichiometric regenerating reagent in some systemsNo requirement for added enzymes Complex apparatusIncompatibilityLimited stabilityRequirement for photosensitizer and redox dyes

  • Direct oxidation of NADHCofactors to be regenerate itself on the electrode surfaceRequires high overpotentialLead to undesired side-reactionsMediated oxidation of NADHLow overpotentialRedox mediators catalyzing the electron transferUsed as soluble mediators as well as immobilized on the electrode surfaceIndirect electroenzymatic oxidation of NADHAccelerate electron transfer kinetics from NAD(P)H to the oxidized mediator by enzymatic catalysisElectrochemical regeneration of NAD+

  • Electroenzymatic synthesis

  • Enzyme(cofactor)Mediator/enzymeSubstrateProductElectrodeEapplRemarksReferenceMediator-free systemNAD+-glucose dehydrogenase-glucosegluconateCylinder type of RVC+0.8 V vs. SCETTNcof>10 000TOFcof=2257 h-1Yield=99.99%Bonnefoy et al., 1988NAD+-lactate dehydrogenase-L-lactateD-lactateCarbon felt (anode)Mercury (cathode)+0.5 V vs. SCE (anode)-1.75 V vs. SCE (cathode)97 % conversionBiade et al., 1992NAD(P)+-glucose dehydrogenase-glucosegluconateGraphite felt+0.7 V vs. Ag/AgClMaximum substrate consumption rate (rs)rs=32 mol min-1 (w/o PEI)rs=59 mol min-1 (w/ PEI)Obn et al., 1997NAD+-alanine dehydrogenase-L-alanineD-alannineCarbon felt (anode)Hg pool (cathode)+0.5 V vs. SCE (anode)-1.350 V vs. SCE (cathode)100% conversion (140 h)Anne et al., 1999NADP+-alcohol dehydrogenase-2-propanolAcetoneAnodic tin oxide-0.5 V vs. Ag/AgCl91% conversion (51 h)Kim et al., 2009NAD+-alcohol dehydrogenase-(rac)-2-pentanol(R)-2-pentanolAnodic tin oxide-0.5 V vs. Ag/AgCl50% conversion (9 h)ee>99% Productivity = 0.03 g l-1 h-1Max. productivity = 0.16 g l-1 h-1Kim and Yoo, 209Mediated systemNAD+-alcohol dehydrogenaseFe(tmphen)32+2-hexen-1-ol2-hexenalGraphite felt+0.63 V vs. Ag/AgClCprod=1.77 mM (60 min)TTNcof=181TTNmed=36Current efficiency=90%Komoschinsk and Steckhan, 1988NADP+-alcohol dehydrogenaseFe(tmphen)32+2-butanol2-butanoneGraphite felt+0.63 V vs. Ag/AgClCprod=4.1 mM (150 min)TTNcof=41 TTNmed=82Current efficiency=95%Komoschinsk and Steckhan, 1988NAD+-alcohol dehydrogenaseTris(1,10-phenanthroline-5,6-dione) ruthenium (II) perchloratecyclohexanolcyclohexanoneCarbon foil+0.1 V vs. Ag/AgClTOF=35 h-1 (aerobic, 60 min)TOF=28 h-1 (anaerobic)Hilt and Steckhan, 1993NAD+-alcohol dehydrogenase[Co(tren)(phendi)](BF4)2cyclohexanolcyclohexanoneCarbon foil+0.1 V vs. Ag/AgClTOF=81 h-1 (aerobic, 60 min)Hilt and Steckhan, 1993NAD+-alcohol dehydrogenaseABTS2-meso-3,4-dihydroxymethylcyclohex-1-ene(3aR,7aS)-3a4,7,a-tetrahydro 3H-isobenzofurane-1-oneCarbon felt+0.585 V vs. Ag/AgClee>99.5% 93.5 % conversion (46.5 h)Productivity=3.24 g l-1 d-1TTNmed=30.4Schrder et al., 2003NAD+-glycerol dehydrogenaseABTS2-1-phenyl-1,2-ethanediol(S)- 1-phenyl-1,2-ethanediolCarbon felt+0.585 V vs. Ag/AgCl-Degenring et al., 2004Electroenzymatic system NADP+-isocitrate dehydrogenaseCAV/AMAPOR(rac)-isocitrate(2S, 3S)-isocitrategraphite-0.2 V vs. SCEProductivity number=13 000 mmol kg-1 h-1 (3.8 h)ee>99 %Schulz et al., 1995NADP+-isocitrate dehydrogenaseAQ-S/AMAPOR(rac)-isocitrate(2S, 3S)-isocitrategraphite-0.2 V vs. SCEProductivity number=14 000 mmol kg-1 h-1 (3.6 h)ee>99 %Schulz et al., 1995NADP+-6-phosphogluconate dehydrogenaseCAV /AMAPOR6-phosphogluconateribose 5-phosphategraphite-0.2 V vs. SCE80% conversion (2.3 h) (crude extract AMAPOR)98% conversion (2.5 h) (partially enriched AMAPOR)Schulz et al., 1995

  • Enzymes can be easily deactivated on the electrode surface.

    The overall reaction is often limited by the cofactor regenerate rate which is usually much slower than the enzymatic reaction rate.

    Used materials for mediator are often toxic to enzyme causing enzyme deactivation.

    The stability of the mediator is affecting the performance and stability of the process.

    Current problems of electrochemical regeneration system

  • Figure. Cyclic voltammograms of tin oxide in the absence (---) and presence () of 0.5 mM NADH. Conditions: 100 mM potassium phosphate buffer (pH 7.5), scan rate=20 mV sec-1, electrode size=5 cm2.Metal oxide electrodeImprovement o

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