Biocatalysis and Enzyme Stability in Ionic Liquids

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Biocatalysis and Enzyme Stability in Ionic
Liquids

• Jason Berberich, Joel Kaar, Anita Mesiano, Markus
  Erbeldinger, Alan Russell
• University of Pittsburgh
• McGowan Institute for Regenerative Medicine,
• Department of Bioengineering

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Outline

• Ionic Liquids for Biocatalysis
• Structure
• Physical Properties
• Biocatalytic Synthesis of Acrylates
• Biocatalytic Polyester Synthesis
• Conclusions

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Hydrophilic Ionic Liquids Physical Properties

• Hydrophobic ionic liquids
• Water immiscible
• BMIMPF6
• Hydrophilic ionic liquids
• Water miscible
• Good solvents for very polar substrates
• Sugars

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Structures of the Ionic Liquids Used in this Study
Cations
Anions
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Solvatochromatic Characterization of Solvent
Polarity

• Reichardts dye
• Normalized scale
• 0 nonpolar solvent (tetramethylsilane)
• 1 polar solvent (water)

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Interpretation of Biocompatibility
Octanol-Water Partition Coefficient
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Log P for Ionic Liquids

• Log P determined using the shake flask method
• Concentration determined using UV spectroscopy
• Ionic liquids have very low log P values

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Physical Characterization -Conclusion

• Solvatochromatic characterization suggests that
  ionic liquids have polarity similar to ethanol
• The very low log P values for the ionic liquids
  suggests that these solvents are very hydrophilic
  and may compete with the enzyme for water

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Biocatalytic Synthesis of Acrylates
100 mM Methylmethacrylate 100 mM
2-Ethyl-1-hexanol 20 mg/ml Candida rugosa
Lipase Temperature 30oC Agitation 300 rpm
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Effect of Solvent on Lipase Activity

• Lipase activity is higher in BMIMPF6 than in
  hexane
• No activity in polar organic solvents
• No activity in hydrophilic ionic liquids
• Lipase dissolves in some ionic liquids

C. rugosa lipase
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Can We Stabilize Lipase for use in Hydrophilic
Ionic Liquids?

• Methods investigated to stabilize lipase for use
  in hydrophilic ionic liquids
• Lipase adsorbed on acrylic support
• Novozyme 435 (Candida antarctica lipase)
• PEG-modified lipase
• PEG-NCO and Candida rugosa lipase
• Lipase-immobilized polyurethane foam
• Hypol 3000 and Candida rugosa lipase

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Results Immobilized Lipase
mM/hr-mg

• No activity was detected in hydrophilic ionic
  liquids with immobilized lipases

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Biocatalytic Polyester Synthesis
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Monomer Solubilities in BMIMPF6 (50C)

• Monomers of interest show appreciable solubility
  in the ionic liquid

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Polymerization Studies
Reaction DVA BD Conditions 50C, 24 hours,
2 Novozym 435 Results Mw Mn PD
I 2887 2401 1.20 Polymer precipitates out
of ionic liquid
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Polyester results
Reaction DVA HFPD DVA
OFOD Conditions 50C, 24 hours,
2 Novozym 435 Results Mw Mn PDI HFPD 1254 1
113 1.13 OFOD 962 866 1.11
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Stability of Novozyme 435in BMIMPF6
Incubate Enzyme in Ionic Liquid
Remove Enzyme from Ionic Liquid
Assay Enzyme Activity in Buffer
Olive Oil
Fatty Acids Diglycerides
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Stability of Novozym 435 at 50C
BMIMPF6
THF
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Stability of Novozym 435 at 50Cin the presence
of Substrates
BMIMPF6
THF
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Stability of Novozym 435 at 70C
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Conclusions

• Solvatochromatic characterization and
• log P measurements indicate that these solvents
  are polar and hydrophilic.
• Enzymatic synthesis of
  2-ethylhexylmethacrylate occurred
  efficiently in BMIMPF6 and hexane.
• Lipase dissolves in hydrophilic ionic liquids.
• Standard methods of stabilization were not
  affective in protecting the lipase from
  deactivation in hydrophilic ionic liquids.

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Conclusions

• The enzymatic polymerization of DVA and
  1,4-butanediol has been achieved in BMIMPF6.
• Enzymes of interest show enhanced stability in
  the BMIMPF6 when compared to a traditional
  organic solvent.

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Acknowledgements

• Sachem USA, Inc.
• Environmental Protection Agency
• (Grant R-82813101-0)

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Stability of Novozyme 435 in Various Ionic Liquids
BMIMAcetate
MMEPAcetate
MMEPCH3SO3
BMIMPF6
MMEPNO3
BMIMNO3
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Stability of Novozyme 435 in Conventional Solvents
Hexane
THF
ACN
DMSO
Butanol