6 enzyme reactions in non-conventional media.ppt [호환...
TRANSCRIPT
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Enzyme Engineering
6. Enzyme Reactions in Non-Conventional Media
6.1 Enzyme Reactions in Organic Solvents
6.2 Enzyme Reactions in Non-Conventional Media
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Enzyme Reactions in Non-Conventional Media
-In organic solvent
-In supercritical liquid
-In ionic liquid
-In high salt condition
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Background
• Enzymes in vivo – not aqueous always
• Enzymes in vitro
• Steroid bioconversion-1st stage – cell culture (intracellular enzyme)
-Permeabilization of cell wall
-2nd stage – 2-phase reaction
* Solubility of substrate, product in aqueous solution - low
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Background• Equilibrium Shift in organic solvent* Why equilibrium shift?
- Lipase : polyester
- Laccase : urushiol polymer (Polycardinol / Polyphenol / Polyaniline)
- HRP(Horseredish peroxidase) : polymer formation
- Protease : peptide degradation
• Product separation in situ- Good for product inhibition kinetics
• Selectivity change- Chiral compounds
Prof. Martinek(Moscow State Univ.)
→ Prof. Klivanov(MIT)
→ Prof. Dordick(PRI)
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6.1 Enzyme Reactions in Organic Solvents
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Organic Solvent System• 2-Phase reaction - Water-immiscible solvent
e.g. steroid bioconversion
• Nearly anhydrouse.g. lipase
• Water – miscible solvent
• Advantages : new reaction, less contamination
• Disadvantages : solvent is toxic to human and enzyme, low stability of enzyme
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Enzyme Activity in Solvent System
• Activity vs. log P(log P : log of partition coefficient in (octanol / water)
Activity vs. solubility parameter
Activity vs. dielectric constant
Activity vs. dipole moment
→ no correlation
E E1 E2
Log P
* High enzyme activity at hydrophobic condition
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Enzyme Activity in Solvent System• NMR study : changes of enzyme structure is negligible
Solvent molecule in enzyme – affect flexibility
• Activity vs. water activity- Nearly anhydrous organic solvent system
- Water activity → equilibrium constant* Proposed by Prof. S.B.Lee (POSTECH)
• pH memorypH of enzyme buffer solution(aqueous)
= pH characters of enzyme after resuspension in organic solvent
• Essential water layer
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How to Increase the Efficiency of Enzyme Reaction in Organic Solvent?
1) Solvent engineering- Selection of solvent(mixture), concentration
- Trial and error
2) Enzyme engineering- More hydrophobic residues at the surface
- More hydrogen bonds at the surface
3) IME
4) PEG modification- Support bead → hydrophobicity / hydrophilicity
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How to Increase the Efficiency of Enzyme Reaction in Organic Solvent?
5) Reversed micelle- Separate the enzyme from the solvent
E
Solvent
Surfactant
Advantages : activity, stability
Disadvantages : mass transfer, residual surfactant should be removed
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How to Increase the Efficiency of Enzyme Reaction in Organic Solvent?
6) Microemulsion system- Hexane / 2-propanol / water
E
hexane
2-Propanol
Enzyme in water
No surfactant is used
* High RPM → affect water layer
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Solvent SelectionNot to destruct hydration shell of enzyme by solvent
(how to maintain hydration shell?)
1) Water, glycerol, EG, formamide
2) Methyl formamide, dimethyl formate
3) Methanol, ethanol, toluene
E
1)
2)
3) •Polyol
- High viscocity - not good for mixing
- Low polarity – low substrate solubility
→ not much used
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Enzyme Engineering: Research Activity at SNU
• Increase activity and stability in organic solvent
• Examples from directed evolution; hydrogen bond become strengthened
- Solvent molecule in enzyme; change residues to reduce the space for solvent
- Increase flexibility* Lipase as a model enzyme
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Reaction System1) Biphase system
Sol
aq
S + P
E
Sol
aqS
EP
PS’→ Agitate →
Reaction Phase separation
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Reaction System
2) Membrane bioreactor 3) IME in solvent- Enzyme is more stable
S
P
IME
ES P
S in solvent
P in solvent
aqueous
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6.2 Enzyme Reactions in Non-Conventional Media
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(1) Supercritical Fluid
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1. Reactants (acetic acid, acetic anhydride 등)
2. Two different immobilized lipases
3. Esterification extent of 100%
4. CO2/substrates molar ratio
5. CO2 vs. Conventional organic solvent(n-hexane)
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Isoamyl alcohol Isoamyl acetate
Impact of acyl donor
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Impact of the type of immobilized enzyme
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g enzyme/mol alcohol
Reactant, enzyme 결정 à economical point of view
CO2/substrates molar ratio
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Effect of temperature
Reactant, enzyme 결정 à economical point of view à operating condition
Stability of Novozym 435
30일동안 100% 수율유지
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Effect of solvent
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ConclusionsNovozym 435, was very efficient in catalysing the esterification of isoamyl
alcohol.
Its catalytic activity was maximum at 313K.
Enzyme/substrate ratio of 6.25 g/mol alcohol was the optimum for maximizing reaction rate and percentage of esterification.
Acetic anhydride resulted to be better acyl donor than acetic acid.
Enzyme deactivation by acetic anhydride and/or produced acetic acid limited the solvent/substrates molar ratio to a maximum of 7.0.
The esterification extent achieved in n-hexane and supercritical carbon dioxide were similar but the initial reaction ratewas higher in SC-CO2 probably due to improved diffusivity of the reactants in this medium.
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(2) Biocatalytic Transformation in Ionic Liquid
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Solvent Properties of Ionic Liquids
• Ionic liquidsHighly polar solventsex) [BMIm][BF4], [MMIm][MeSO4]Miscibility of ionic liquids varies widely and
unpredictably
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Ionic Liquids and Enzymes
• Tolerance of lipasesHigher activity à [HMIm][PF6]Not active à methylsulfate, nitrate, lactate anion
• Ionic liquids seem to affect enzymes in much the same way that conventional organic solvents do
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Ionic Liquids and Enzymes
• Higher thermal stability
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* Continuous green biocatalytic processes using ionic liquids and supercritical carbon dioxide
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Biotransformations in Ionic Liquid Medium
• Enantioselective acylation à One of major industrial applications of lipases
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Reaction Systems
• Catalyst recycling
• Product evaporationà Ionic liquids lack a vapor pressure
• Two-phase system with supercritical CO2
• Two-phase aqueous systems
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EnzymeProduct
Reactant Enzyme
ProductReactant
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Conclusion
• Activities are generally comparable with or higher than conventional organic solvents
• Enhanced thermal and operational stabilities
• Higher enantioselectivities
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