Role of Ionic Liquids in Enzyme Catalysis

Role of Ionic Liquids in Enzyme Catalysis

Introduction 

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Enzymes are efficient catalysts with rate enhancements under near ambient conditions Enzymes are enantioselective catalysts due to their chiral structure Many economically profitable processes are in operation in fine chemistry, in bulk chemistry, pharmaceuticals that are based on enzyme catalysis

Enzyme Catalysis Aqueous solvent

Non-aqueous Nonsolvent Organic solvent

Ionic liquid Supercritical fluids

Ionic liquids 

Ionic liquids (molten salts) are defined as material containing only ionic species without any neutral molecules.

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Exists in liquid form at room temperature or near room temp.

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Are nonvolatile and nonflammable

Cations:

Anions: Anion

Full Name

Abbreviation

BF4-

tetrafluroborate

[BF4]

PF6-

hexaflurophosphate

[PF6]

NO3-

nitrate

[NO3]

CH3CO2-

acetate

[Ac]

CF3CO2-

trifluroacetate

[TFA]

CH3SO4-

methylsulphate

[MeSO4]

CF3SO3-

trifluromethylsulfonate

[TfO]

(CF3SO2)2N-

Bis[(trifluromethyl)sulfonyl]amide

[Tf2N]

Properties of Ionic Liquids      

Polarity Hydrophobicity Solubility Viscosity Thermal stability Designer solvents

Enzyme activity in ionic liquids 

Active only when suspended.

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Dissolved forms are inactive.

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Higher activity than observed in conventional organic solvents.

Effect of solvent properties of ionic liquids 

Hydrophobicity

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Viscosity

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Polarity

Effect of water Rate of enzyme catalyzed reaction with low and high water content

Effect of pH and impurities Contradictory reports of enzyme activity in ionic liquids Ionic liquid

Enzyme Reaction

Activity Reference

[BMIM][PF6] CaLB

Transesterification Yes No

Itoh et al., 2001 Schofer et al., 2001

[BMIM][PF6] CRL


Kaar et al., 2003 Itoh et al., 2001

[BMIM][PF6]


Husum et al., 2001 Schofer et al., 2001

MML

CaLB, Candida antarctica lipase B; CRL, Candida rugosa lipase; MML Mucor miehei lipase.

Horseradish peroxidase 

Activity studied in [BMIM][BF4]-water mixtures

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Avctivity only above pH 9.0

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At neutral and acidic pH activity decreases

Enzyme inactivation in ionic liquids 

Denaturation is related to ionic nature of ionic liquid

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Active in ionic liquids containing BF4-, PF6-, and Tf2N- anions

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Inactive in NO3-, CH3CO2-, CF3CO2- and CF3SO3-

Examples of using enzymes in ionic liquids Biocatalyst

Reaction

Ionic liquid

Lipase

Transesterification Alcoholysis, ammoniolysis, perhydrolysis, Kinetic resolution of chiral alcohols Resolution of amino acid ester Esterification of carbohydrates Synthesis of polyesters Enantioselective reduction of 2octanone Synthesis of Z-aspartame Transesterification Transesterification Resolution of amino acid ester Synthesis of N-acetyllactosamine Oxidation of guaiacol Oxidation of anthracene Regeneration of NADH

[BMIM]PF6] [BMIM][PF6], [BMIM][BF4]

Alcohol dehydrogenase Thermolysin α-Chymotrypsin Esterase Subtilisin β-Galactosidase Peroxidase Laccase Formate dehydrogenase

[BMIM][Tf2N] [EPy][BF4], [EMIM][BF4] [MOEMIM][BF4] [BMIM][PF6] [BMIM][Tf2N] [BMIM][PF6] [EMIM][Tf2N],[MTOA][Tf2N] [BMIM][PF6] [EPy][TFA]–H2O (15:85, v/v) [MMIM][MeSO4]–H2O [BMIM][PF6] [BMIM][PF6] [MMIM][MeSO4]–H2O(25:75, v/v)

Enzyme stability in ionic liquids 

Enzyme stability is higher.

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Stability of esterase from Bacillus stearothermophilus in [BMIM][BF4] and [BMIM][PF6].

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α-chymotrypsin shows increase in stability in ionic liquids at 50oC(increase in half life).

Thermal stability of enzymes in ionic liquids

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Spectroscopic study of stability of α-chymotrypsin in [EMIM][Tf2N] at elevated temperatures showed that: Melting temp. and heat capacity of enzyme enhanced Structural compaction of enzyme Changes in secondary structure of enzyme

Stability in presence of substrate  Stability can be increased by incubating the enzyme in presence of substrate  Reuse of free lipase (CaLB) in [BMIM][PF6] showed increase in half life by 2300 times than in absence of substrate  Association of substrate to enzyme causes conformational change

Enzyme selectivity in ionic liquids 

Enantioselectivity

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Regioselectivity

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Suppression of side reactions

Enantioselectivity 

Kinetic resolution of 1-phenylethanol catalyzed by lipase in [BMIM][Tf2N]

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E-value decreased from 200 to 150 at temp. between 25-90oC

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E-value in MTBE decreased to 4 at 55oC

Cont… 

Subtilisin used in resolution of amino acid esters in [EPy][TFA]-water (15:85)

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Increase in enantioselectivity as compared to that in acetonitrile-water (15:85)

Regioselectivity 

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Lipase catalyzed acetylation of glucose in [MOMEMIM][BF4] Yield of monoacetylated product 93% Lipase catalyzed acetylation of glucose in [BMIM][PF6] Yield of monoacetylated product 29%

Suppression of side reactions 

Galactosylation of N-acetylglucosamine with lactose by β-galactosidase

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In aqueous solution yield less than 30%

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In ionic liquid [MMIM][MeSO4] yield is 58%

Effect of ionic liquids on enzyme activity and the Hofmeister series 

Hofmeister series- sequence of ion ability in stabilizing proteins

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Kosmotropic ions: strongly hydrated ions

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Chaotropic ions: weakly hydrated ions

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Kosmotropic anions stabilize protein while cations destabilize protein

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Chaotropic cations stabilize protein while anions destabilize protein

Cont… 

In hydrophobic ionic liquids enzyme activity does not follow Hofmeister series

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BF4-, PF6- are chaotropic anions but still stabilize enzyme in ionic liquid

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In hydrophilic ionic liquids enzyme activity follows Hofmeister series

Hydrophobicity and water activity

The relationship of log P values of three ionic liquids and the optimal hydration of Candida rugosa lipase

Nature of Effect

Order of effectiveness

Hydrophobic or anhydrous ILs Stability of Novozym 435 in ILs at 30ºC

Anions: CH3COO- >PF6- >NO3([BMIM]+ or [MMEP]+ based ILs) Cations: [MMEP]+ > [BMIM]+ (CH3COO, PF6-, or NO3- salts)

Initial reaction rates of PEG-lipase catalyzed alcoholysis in ILs

Cations: [OMIM]+ > [HMIM]+ > [BMIM]+ (PF6- based ILs)

Enantioselective acylation of 1phenylethanol by lipase CaLB

Anions: [(CF3SO2)2N]-, [CF3SO3]- >BF4>PF6- ([BMIM]+ based ILs) Cations: [OMIM]+ > [HMIM]+ > [BMIM]+ (BF4-based ILs)

Enantioselectivity of the acetylation of 1phenylethanol with vinyl acetate by lipase from Pseudomonas cepacia in ILs (purification method B, no additive)

Anions: BF4− >PF6− ([BMIM]+ based ILs) Cations: [EMIM]+ > [PrMIM]+, [BuPy]+ > [PrPy]+ > [BMIM]+ (BF4- based ILs)

Enantioselectivity of Candida rugosa lipase in the esterification of 2-substitutedpropanoic acids and 1-butanol in ILs

Anions: PF6− >BF4− ([BMIM]+ based ILs) Cations: [BMIM]+ > [OMIM]+ (PF6− based ILs)

Activity of Candida antarctica lipase B in transesterification of ethyl butanoate and 1butanol

Anions: BF4− >PF6− > [lactate]− >NO3− ([BMIM]+ based ILs)

Nature of effect

Order of effectiveness

Hydrophilic ILs containing water Specific activity of esterase from Anions: [(CF3SO2)2N]− >BF4− Bacillus >PF6− ([BMIM]+ based ILs) stearothermophilus in the kinetic resolution of 1-phenylethanol (10 mM) with vinyl acetate (200 mM) at 40ºC, aw = 0.11 Stability of α-chymotrypsin in ILs (2% water, v/v) at 50ºC

Anions: PF6− >BF4− ([BMIM]+ based ILs)

Stability of Candida antarctica lipase B in ILs (2% water, v/v) exhibited by incubation without substrates

Anions: BF4− >PF6− > [(CF3SO2)2N]− ([EMIM]+ and [BMIM]+ based ILs) Cations: [MMIM]+ > [BMIM]+ ([(CF3SO2)2N]− based ILs)

Activity of cellulase from Trichoderma reesei in salt solutions containing 20–100% water

Sodium citrate buffer > sodium dodecylsulfate > NaCl > [BMIM]Cl

Thermolysin 

Dissolved thermolysin is inactive in [BMIM][PF6]

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Suspended form is active

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Yield of 95% obtained in ionic liquid compared to that in ethyl acetate

Cont…..

Lipases 

Anhydrous organic medium is an absolute requirement with non-hydrolytic reactions in the presence of lipases

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Lipases are used in alcoholysis, ammoniolysis and perhydrolysis reactions in ionic liquid

Transesterification by lipase CaLB R1CO2Et

+ R2OH

R1CO2R2

+

[bmim][PF6] or [bmim][BF4] 40oC

Increased reaction rate as compared to that in tert-butyl alcohol

EtOH

Ammoniolysis by lipase

Complete conversion in 4 days in IL while 17 days are required in methylisobutylketone

Kinetic resolution of 1-phenylethanol

Increase enantioselectivity than in organic solvents

Enantioselective esterification of menthol       

Candida rugosa lipase (CRL) Ionic liquid- [BMIM][PF6] and [BMIM][BF4] Comparison with organic solvent hexane Propionic anhydride - acylating agent Ionic liquid uses less propionic anhydride CRL activity 2.5 times higher in Ionic Liquid In hexane activity decreased to less than 60%

Ibuprofen anchored ionic liquid for the lipase catalyzed kinetic resolution 

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Substrate- ibuprofen anchored with ionic liquid CaLB catalyzed hydrolysis to give S-enantiomer This facilitated post-resolution isolation of enantiomer Ionic liquids can be recycled

Cont…

Ionic liquid anchored substrate

Enzymatic condensation reactions in ionic liquids 

Glycosidase and peptide amidase hydrolyze glycosidic bonds or amides, respectively

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In ionic liquids reverse reaction is possible

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β-galactosidase in [MMIM][MeSO4] favours the formation of disaccharides

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Peptide amidase in [BMIM][MeSO4] favours the formation of amide

Ionic liquids allow unconventional reaction techniques 

Catalyst recovery

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Product evaporation

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Two-phase system with supercritical CO2

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Two phase aqueous systems

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Two phase systems with organic solvent

Conclusion 

Increased stability, activity and selectivity

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Ionic liquids can be used as a reaction medium for wide range of substrates

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Solvent properties can be tuned

Future prospectus 

Investigation to understand the fundamentals

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Investigation to find reasons behind better activity, stability and selectivity.

Thank You!