High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis
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High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis. Hanzi Li Comprehensive oral presentation Advisor: Dr. Scott Calabrese Barton Department of Chemical Engineering and Materials Science Michigan state University. Nov , 2011. Introduction and background.

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High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

Hanzi Li

Comprehensive oral presentation

Advisor: Dr. Scott Calabrese Barton

Department of Chemical Engineering and Materials Science

Michigan state University

Nov , 2011


Introduction and background

Introduction and background


Dehydrogenase based electrochemical conversion

Dehydrogenase-based electrochemical conversion

  • Dihydroxyaceton(DHA): Sunless tanning cream; Precursor to pharmaceuticals

  • Mannitol: Natural sugar alcohol sweetener; Additive to food and pharmaceuticals

    • Why electrode: Cofactor electrochemical regeneration

    Dual Chamber Catalysis

    e-

    NAD+

    NADH

    Anode

    GlyDH

    Power supply

    Glycerol

    DHA

    MtDH

    Cathode

    Fructose

    Mannitol

    NADH

    NAD+


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Cofactor electroregeneration

    • Thermodynamically, NADH oxidation should be observed at low potential.

    Enzyme

    NAD+

    Product

    -0.49 V/Ag|AgClat pH 6

    NADH

    NAD+

    Substrate

    CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Cofactor electroregeneration

    • Direct NADH oxidation requires high overpotential; Reaction rate is low.

    Glassy carbon Electrode

    Typical planar electrode:

    Glassy carbon electrode ( 3 mm diameter)

    NADH

    NAD+

    E0’ = -0.49 V/Ag|AgClat pH 6

    • Cyclic voltammograms in 0.5 mM NADH at glassy carbon electrode, 50 mV/s, 0.1 M PBS, pH 6

    CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    High-performance cofactor regeneration

    • Achieve high-rate kinetics for NADH oxidation by electrode modification

    NADH

    NAD+

    Electrode

    • Analyze the conversions in NADH oxidation using modified electrode as working electrode


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Bioelectrocatalysis involving cofactor regeneration

    NAD+

    NADH

    • Evaluate bioelectrocatalysis based on NADH electrocatalysis

    catalyst red

    catalyst ox

    Substrate

    Anode

    Enzyme

    Product

    • Model glycerol oxidation and fructose reduction coupled with cofactor regeneration


    Electropolymeried azine electrodes modified with carbon nanotubes for nadh oxidation

    Part 1

    Electropolymeried azine electrodes modified with carbon nanotubes for NADH oxidation

    May 13th, 2011


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Electrode modification

    NADH

    • High-surface area material to increase active site density

    NAD+

    NADH

    NAD+

    Glassy carbon Electrode

    NADH

    NAD+

    Catalyst ox

    NADH

    NAD+

    Catalystox

    • Electrocatalyst

    • to decrease activation energy

    High surface area material

    High-surface area material

    Glassy carbon Electrode

    Glassy carbon Electrode

    Glassy carbon Electrode

    Catalystred

    Catalystred

    Gorton, L.; Dominguez, E. J Biotechnol 2002, 82, 371.

    Zhao, X.; Lu, X.; Tze, W. T. Y.; Wang, P. Biosensors and Bioelectronics 2010, 25, 2343.

    Villarrubia, C. W. N.; Rincon, R. A.; Atanassov, P.; Radhakrishnan, V.; Davis, V. ECS Meeting Abstracts 2010, 1001, 443.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Modify electrode with CNT

    • CNT-GC: CNT were coated on glassy carbon electrode surface (3 mm diameter RDE) by drop-casting 5 µl CNT ink on the surface of GC electrode and drying in vacuum.

    Drop Casting CNT ink

    SEM image of CNT on electrode surface

    Carboxylated CNT (Nanocyl)

    Glassy carbon Electrode

    http://www.nanocyl.com/

    Li, H.; Wen, H.; Calabrese Barton, S. In Electroanalysis, 2011.

    Wen, H.; Nallathambi, V.; Chakraborty, D.; Calabrese Barton, S. Microchim. Acta, 1.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    CNT-GC: High-surface area material

    Active surface area / Geometric surface area

    (Assuming 25 µF/cm2)

    Capacitance (mF/cm2)

    in 1 M sulfuric acid

    Barton, S. C.; Sun, Y.; Chandra, B.; White, S.; Hone, J. Electrochemical and Solid-State Letters 2007, 10, B96.

    Kinoshita, K.; Carbon: Electrochemical and Physicochemical Properties; 1st ed.; Wiley-Interscience, 1988.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Coat electrocatalyst: Electropolymerization

    Toluidine Blue O

    Methylene Green

    Poly(azine) ox

    Poly(azine) red

    Glassy carbon Electrode

    CNT

    Cyclic voltammograms of PTBO (Right: Top) and PMG (Right: Bottom) electropolymerization on 0.85 mg cm2- CNT-coated GC, 20 cycles, 50 mV/s, 0.4 mM TBO, 0.01 M borate buffer pH 9.1, 0.1M NaNO3, 30 ºC

    Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553.

    Zeng, J.; Wei, W.; Wu, L.; Liu, X.; Liu, K.; Li, Y. Journal of Electroanalytical Chemistry 2006, 595, 152.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    NADH electrocatalysis

    NADH

    NAD+

    Poly(azine) ox

    Poly(azine) red

    Glassy carbon Electrode

    CNT

    Kar, P.; Barton, S. C. ECS Meeting Abstracts 2010, 1001, 405.

    Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    NADH electrocatalysis

    • a&c: PTBO ; b&d: PMG

    • 1: Bare GC; 2: 0.21 mg/cm2 CNT-GC; 3: 0.85 mg/cm2CNT-GC

    NADH concentration study of PTBO-CNT-GC (a) and PMG-CNT-GC (b) at 50 mV/Ag|AgCl; Polarization curves of PTBO-CNT-GC (c) and PMG-CNT-GC (d) in 0.5 mM NADH. 0.1 M phosphate buffer pH 6.0, 900 rpm, 30 ºC. Markers: Experimental data; Solid line: Fitting using mass-transport corrected model; Dash line: Simulation for mass-transport corrected curves.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    NADH electrocatalysis


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Part 2

    • Analysis of the bulk rate of cofactor electroregeneration


    Cnt modified carbon paper toray

    CNT modified carbon paper (Toray)

    Active surface area / Geometric surface area

    (Assuming 25 µF/cm2)

    Capacitance was obtained in 0.01 M borate buffer pH 9.1, 0.1 M NaNO3, 30 ºC


    Nadh oxidation using pmg cnt toray

    NADH Oxidation Using PMG-CNT-Toray

    • CNT-Toray: CNT were coated on carbon paper surface (2.5×2.5 cm2) by air-brushing 2 mg ml-1 CNT ink on the surface and drying in vacuum.

    • 1.2×0.8 cm2 (Exposed surface area 1.0×0.8 cm2 , CNT loading 0.9 ± 0.1 mg/cm2) CNT-Toray was used for further modification and working electrode.

    NADH

    NAD+

    Batch reactor to study the conversion

    CNT-PMG

    PMG-CNT acts as electrocatalyst for NADH oxidation

    Carbon Paper

    NADH oxidation was performed with initial NADH concentration 0.94 mM in 20 ml pH 6 phosphate buffer, constant applied potential 0.5 V/ Ag|AgCl, 1200 rpm magnetically stirred, 30 ºC.


    Conversions in nadh bulk oxidation

    Conversions in NADH bulk oxidation

    Electrocatalysis:

    NADH consumption:

    Decay

    k=(1.0± 0.1 ) ×10-3 min-1

    NADH concentration profile can be simulated.


    Conversions in nadh bulk oxidation1

    Conversions in NADH bulk oxidation

    NADH concentration was measured using UV-Vis spectra during NADH bulk oxidation


    Enzyme cycling assay for detecting bioactive nad

    Enzyme cycling assay for detecting bioactive NAD+

    • During electraocatalysis and after electrocatalysis, enzyme assay was employed for bulk solution

    NAD+

    NADH

    MTTox

    Pyruvate

    Diaphorase

    Initially: LDH, Lactate, Diaphorase, MTTox

    LDH

    Lactate

    MTTred

    Very fast

    Relatively slow

    www.bioassaysys.com

    in the solution


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Bioactive NAD+


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Part 3

    • Immobilization of enzymes and cofactors on poly(azine)-CNT modified electrodes to achieve high-performance bioelectrocatalysis


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    N6 –linked-NAD+/NADH by Vieille Lab

    NAD+

    Aryl amine

    Lindberg, M.; Larsson, P.-O.; Mosbach, K. European Journal of Biochemistry 1973, 40, 187


    Typical rde set up

    Electrochemical activity of N6-linked NADH

    Typical RDE Set-up

    40 µl - Electrolyte Set-up

    ω

    electrolyte

    electrode

    electrode

    40 µl, Room temperature

    0.02 µmoles NADH is needed for 0.5 mM solution

    electrolyte

    • 900 rpm,30 °C, At least 10 ml solution, Purged Ar

    • 5 µmoles NADH is needed for 0.5 mM solution


    Polarization curves

    Electrochemical activity of N6-linked NADH

    Polarization curves

    Steady-state data from chronoamperometry , pH 6 PBS, Standard NADH solution: 0.5 mM

    • Can be fixed by

      • Compare RDE data in 0 rpm and in air

      • (Experiment in N2 or Ar)

    • The lower activity may due to

      • Limited mass transport

      • O2 present


    Biosensor based on electronic interface

    Biosensor based on electronic interface

    NADH

    NAD+

    Reference

    electrode

    catalyst red

    catalyst ox

    Anode

    Malate

    MDH

    Kinetics:

    Oxaloacetate

    • Step 1 relectro

    • Step 2 renzyme

    • Evaluate the whole process by monitoring the responding current


    Biosensor towards malate concentration

    Biosensor towards malate concentration

    • Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH

    • (immobilization method by Worden Lab)

    PMG-CNT-GC, chronoamperometry , E=0.4 V vs. Ag|AgCl, 900 rpm, pH 6 PBS, 30 oC, MDH 0.83 µM, initially NAD+ 10 mM


    Back up plan for cofactor enzyme immobilization

    Back-up plan for cofactor/enzyme immobilization

    • Cofactor is non-covalently attached to CNT via π-πstacking interaction

    Zhou, H.; Zhang, Z.; Yu, P.; Su, L.; Ohsaka, T.; Mao, L. Langmuir 2010, 26, 6028.

    CVs obtained at the MWCNT-modified Pt electrodes in 0.1 PBS buffet before (blue curve) and after (black curve) at the electrodes were first immersed into the aqueous solution of 10 mM NAD+ for 1 h and then thoroughly rinsed with distilled water. Scant rate: 50 mV/s. Inset: structure of NAD+ cofactor.


    Model glycerol oxidation and fructose reduction coupled with cofactor regeneration

    Part 4

    Model glycerol oxidation and fructose reduction coupled with cofactor regeneration


    Linear model

    Linear model

    NADH

    NAD+

    X=0

    X=l

    Mass balance involving kinetics and diffusion within film :

    catalyst red

    Glycerol

    catalyst ox

    Anode

    GlyDH

    Boundary conditions:

    Dihydroxyacetone

    (DHA)

    Steady-state within film :


    Non dimensionalization

    Non-dimensionalization

    Damkohler numbers

    Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.


    Porous model

    Porous model

    Mass balance:

    Boundary conditions:


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Parameters

    a: parameter values regarding NADH electrocatalytic reaction have been shown in Project 1

    b: assumed to be the same as methanol

    Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.

    Nishise, H.; Nagao, A.; Tani, Y.; Yamada, H. Agricultural and Biological Chemistry 1984, 48, 1603.

    Gartner, G.; Kopperschlager, G. J. Gen. Microbiol. 1984, 130, 3225.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Simulation results

    Linear model:

    Porous model:

    • DaNAD+ = 16

    • Daglycerol= 0.0013;

    • DaaNAD+ = 406;


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Summary

    • Fabricated poly(azine)-CNT-GC demonstrates high-rate for NADH electrocatalysis.

    • NADH bulk oxidation shows 80% conversion of 1 mM NADH in 1 hr. Bioactive NAD+ was verified.

    • Calibration curve for immobilized cofactor evaluation and dehydrogenase-based biosensor are proposed

    • NondimensionalDamkohler numbers can provide useful approach to simulate, predict and evaluate performance of bioreactor.


    Thank you

    Thank you.


    Supplemental information

    Supplemental information


    Biosensor towards malate concentration1

    Biosensor towards malate concentration

    • Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH

    • (immobilization method by Worden Lab)

    PMG-CNT-GC, chronoamperometry , E=0.4 V vs. Ag|AgCl, 900 rpm, pH 6 PBS, 30 oC, MDH 0.83 µM, initially NAD+ 10 mM


    Cystein

    Cystein


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    • The decay of NADH in 0.1 M phosphate buffer pH 6.0, magnetic stirred speed 1200 rpm, 30 ºC. a. At varies NADH initial concentrations, NADH decay was monitored using UV-Vis spectra at 340 nm; b. The slopes in a. varying with NADH initial concentration.


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Acknowledgements

    • Collaborators

      • Dr. Mark Worden

      • Dr. Claire Vieille

      • Justin Beauchamp

    • The National Science Foundation

    • (Award CBET-0756703)


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Principle of LDH-MTT Assay

    NAD+

    NADH

    Initially: LDH, Lactate, Diaphorase, MTTox

    MTTox

    Pyruvate

    Diaphorase

    LDH

    www.bioassaysys.com

    Lactate

    MTTred

    Very fast

    Relatively slow

    When NAD+ presents in the sample, it is converted to NADH in LDH and lactate.

    MTTox uses NADH to oxidize into MTTred. The NADH is thus converted back to NAD+.

    The enzyme cycle starts over.

    Once the cycle starts, NADH concentration in the assay is not changing = [NAD]+[NADH] in the sample


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Kinetics assay using LDH-MTT Assay Kit

    www.bioassaysys.com

    NAD+

    NADH

    Initially: LDH, Lactate, Diaphorase, MTTox

    MTTox

    Pyruvate

    Diaphorase

    LDH

    Lactate

    MTTred

    • Linear kinetics within 15 mins

    in the sample


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Modified electrodes

    High-surface area electrodes for NADH electrocatalysis


    Why mannitol

    Why Mannitol?

    • Mannitol is a natural sugar alcohol sweetener.

    • Mannitol is especially useful as an additive to food and pharmaceuticals

      • It has low caloric and cariogenic properties

      • It is not metabolized by the body

      • It has a cool sweet taste

    • Currently mannitol is produced by hydrogenating a 1:1 fructose/glucose syrup

      • Very high temperatures, pressure and a Raney nickel catalyst

      • Needs highly purified substrates

      • Energy intensive

      • Costly purification

      • Low yield (15%)

    • Enzymatic catalysis reducing fructose to mannitol

      • Potential applications to other dehydrogenases


    Overall objective

    Overall Objective

    • Glucose  fructose using a thermostable glucose isomerase

      • Triple mutant of Thermotoga neapolitana xylose isomerase (TNXI 1F1)

        • Optimized for high activity at 60°C, and high activity at pH 6.0 while maintaining glucose activity

    • Fructose  mannitol

    • NADH regeneration from cathodic current pulls reaction towards mannitol production


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Nicotinamide

    Dinucleotide

    Adenine


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Literature review about NADH electrocatalytic oxidation: The reported steady-state current densities for NADH oxidation were far less than 1 mA cm-2 under low overpotential


    For the reduction of u in polarization curves

    For the reduction of U in polarization curves

    Take one PTBO-0.85 mg/cm2 CNT-GC and PTBO-GC as an example:

    Polarization curve: 0.5 mM NADH , 900 rpm, pH 6 PBS, 30 oC

    Proposed reason: Impact of Mass-transport

    Controlled by mass-transport

    (not controlled by applied potential)

    Mixed Control

    (By both applied potential and mass-transport)

    Controlled by electron-transfer rate (controlled by applied potential)


    Cnt gc

    CNT-GC

    • High-surface area of CNT-GC: Good utilization of CNT

    • 115 m2/g (capacitive surface area) vs. 80-140 m2/g (BET)

      • Carboxylated multiwall carbon nanotubes (CNT) instead of untreated CNT were used: hydrophilic property of COOH-CNT make it possible to utilize the good properties of CNT for electrochemical experiments

      • Dimethylformamid (DMF) is used as solvent to form CNT-ink:

        • Organic solvent, disperse CNT well; Can evaporate; Miscible in water

    Wen, H.; Nallathambi, V.; Chakraborty, D.; Barton, S. C. ECS Meeting Abstracts 2010, 1002, 366.


    Characterization of ptbo and pmg films

    Characterization of PTBO and PMG films

    • CV in pH 6 0.1 M PBS, 50 mV/s 30 oC

    Poly(azine) ox

    Poly(azine) red

    Glassy carbon Electrode

    CNT


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    • DMF: Dimethylformamid(CH3)2NC(O)H


    Process of catalytic reaction of nadh

    Process of catalytic reaction of NADH

    Thecatalysis efficiency varies with polymers. Even though the mechanisms are not well developed, it is reported that the differences of azine chemical structures affect the electrocatalytic activity toward NADH oxidation. For instance, the additional electron acceptor groups in the aromatic ring always lead to higher electrocatalytic activity, while the additional proton donor groups cause lower electrocatalytic activity. [13, 36] Methylene green has an additional –NO2 group and toluidine blue has an additional -CH3 group. Thus PMG-modified electrodes tend to show higher activity especially at high positive potential region and high NADH concentration.

    1. Qi-Jin, C. and D. Shao-Jun, Journal of Molecular Catalysis A: Chemical, 1996. 105(3): p. 193-201.

    2. Cooney, M.J., et al., Energy & Environmental Science, 2008. 1(3): p. 320-337.

    Or


    Cnt modified gc electrode capacitance measurement

    CNT-modified GC electrode: Capacitance measurement

    • Carbon nanotube is coated on glassy carbon electrode surface (3 mm diameter RDE) by drop-casting:

    • Each CNT layer: 5ug 1mg/ml CNT-DMF suspension

    Example of capacitance measurement: 0.50 mg/cm2 CNT loaded on GC

    Drop Casting CNT

    Glassy carbon Electrode

    Capacitance data were obtained by cyclic voltammetry in the 0.3 to 0.4 V vs. Ag/AgCl at 0.01 M borate buffer pH 9.1, 0.1 M NaNO3, 30 oC


    Proposed structure of poly mb

    Proposed structure of Poly (MB)

    Karyakin, A.A., et al., 1999. 11(8): p. 553-557.


    Imax after mt correction

    Imax after MT correction


    High rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

    Effect of Mass transport

    MT Correction

    Mass balance:

    Electrochemical experiments:

    Obtain i’maxand K’s for pure kinetic control

    Solid lines : kinetic control

    Dotted lines : partially MT limited


    Bioreactor based on electronic interface

    Bioreactor based on electronic interface

    Power supply

    NADH

    NAD+

    Reference

    electrode

    catalyst red

    catalyst ox

    Glycerol

    Anode

    GlyDH

    • Step 2 r2

    • Step 1 r1

    Dihydroxyacetone

    (DHA)

    Kinetics:


    Concentration profile for substrate conversion

    Concentration profile for substrate conversion

    For the whole batch reactor:

    • Initial values: t=0, [NADH] = [NADH]0 ;

    • [NAD+]=0;

    • [Glycerol] = [Glycerol]0

    • [NADH]0=20 mM;

    • [Glycerol]0 =100 mM;

    • V = 20 cm3;

    • S = 1 cm2;

    • E = 10 µM;

    • KNADH =7.0 mM;

    • kcat= 9.1s-1;

    • Kglycerol= 11 mM

    • KNAD+ = 25 µM;

    • 3.02 hrs

    • Key parameters:

      • Sk1/nFV ( µM/s ) 2. [Enzyme] (µM or mM)


    Fabrication of pmg cnt toray

    Fabrication of PMG-CNT-Toray

    CNT-Toray: Spray-coat (air-brushing) CNT ink on Toray paper surface and dry in vacuum.

    Toray paper: 3.5 cm × 3.5 cm; 100 µm thickness

    CNT ink: 20 mg CNT dispersed in 10 ml DMF

    Exposed surface area of Toray to CNT ink: 2.5 cm × 2.5 cm

    Resulted loading: 1.1 mg ± 0.11 CNT/ cm2

    Bare Toray: 0.16 m2/cm3

    For 0.9 cm2 bare Toray, active surface area: 14.4 cm2

    Capacitance: 608 uF/cm2


    How sk 1 nfv or and enzyme impact time constant

    How Sk1/nFV or/and [Enzyme] impact Time constant?

    • Zoom in


    Nondimensionalization

    Nondimensionalization

    • DEQs

    • Boundary conditions:

    • t=0, x=1, y=0, f=1

    • Important parameters:

    • Variables:

    • Parameters:

    • Equilibrium constant, representing key operation conditions

    • Time constants for step 1, step 2 and their ratio


    Current work enzyme kinetics of mtdh

    Current work: Enzyme kinetics of MtDH

    • Ordered bi bi kinetics

    NADH

    Fructose

    Mannitol

    NAD

    k1

    k2

    k3

    k4

    k-1

    k-2

    k-3

    k-4

    A: NADH

    B: Fructose

    P: Mannitol

    Q: NAD

    1. Segel, Irwin H. (1993). New York: Wiley


    Enzyme kinetics of mtdh

    Enzyme kinetics of MtDH

    • Definition of parameters

    A: NADH

    B: Fructose

    P: Mannitol

    Q: NAD

    • The values of 10 parameters I extracted based on experimental data

    Vf and Vr: U/mg; All Km’s and Ki’s: mM; Keq: dimensionless 2. For 60 oC, pH 6.1

    1. Segel, Irwin H. (1993). New York: Wiley 2. SeungHoon, S., N. Ahluwalia, et al. (2008). "Applied Microbiology and Biotechnology: 81 (3) 485-495 81(3): 485-495.


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