Skip to Content
Merck
  • The effect of thermodynamic properties of solvent mixtures explains the difference between methanol and ethanol in C.antarctica lipase B catalyzed alcoholysis.

The effect of thermodynamic properties of solvent mixtures explains the difference between methanol and ethanol in C.antarctica lipase B catalyzed alcoholysis.

Journal of biotechnology (2015-09-02)
Francesco Sasso, Tobias Kulschewski, Francesco Secundo, Marina Lotti, Jürgen Pleiss
ABSTRACT

Kinetic modelling, molecular modelling, and experimental determination of the initial reaction velocity of lipase-catalyzed alcoholysis were combined to study the effect of the alcohol substrate to catalytic activity. The model system consisted of methanol or ethanol at varying concentrations, vinyl acetate as ester substrate 15.2% (v/v), toluene as organic solvent, water at a controlled thermodynamic activity of 0.09, and C. antarctica lipase B as enzyme. For both alcohol substrates, the initial reaction velocity increased sharply at low concentrations and reached a maximum at 0.7% (v/v) for methanol and 2% (v/v) for ethanol. For higher concentrations, the reaction rate decreased to a level of 74% and 60% of the peak value, respectively, due to substrate inhibition. The concentration dependency was described by a kinetic model, including a ping-pong bi-bi mechanism and competitive inhibition by the alcohol, and confirmed previous observations that methanol is more efficiently inhibiting the enzyme than ethanol. However, if the initial reaction velocity was expressed in terms of thermodynamic activity of the two alcohol substrates, the maximum of initial reaction velocity was similar for methanol (a MeOH(max)=0.19) and ethanol (a EtOH(max)=0.21). This was confirmed by molecular modelling which resulted in similar KM (0.22 and 0.19) and Ki values (0.44 and 0.49) for methanol and ethanol, respectively, if expressed in thermodynamic activities. Thus, the experimentally observed difference between methanol and ethanol is not due to differences in interaction with the enzyme but is a consequence of the thermodynamics of the substrate-solvent mixture. For low concentrations in toluene, the activity coefficient of methanol is 40% higher than the activity coefficient of ethanol (γ MeOH=8.5, γ EtOH=6.1).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Lithium-7Li chloride, 99 atom % 7Li, 99% (CP)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, NMR reference standard
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, ACS spectrophotometric grade, ≥99.9%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, puriss., meets analytical specification of Ph Eur, ≥99.7% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, BioReagent, ≥99.93%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, Laboratory Reagent, ≥99.6%
SKU
Pack Size
Availability
Price
Quantity
Supelco
Ethanol solution, certified reference material, 2000 μg/mL in methanol
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, anhydrous, 99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Methanol, Absolute - Acetone free
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, for molecular biology
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, for molecular biology
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, ReagentPlus®, ≥99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Vinyl acetate, contains 3-20 ppm hydroquinone as inhibitor, ≥99%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, anhydrous, 99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, ≥99%, FCC, FG
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Toluene, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.7% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, biotech. grade, ≥99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, ACS reagent, ≥99.5%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Toluene, Laboratory Reagent, ≥99.3%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, natural, ≥99%, FCC, FG
SKU
Pack Size
Availability
Price
Quantity
Supelco
Vinyl acetate, analytical standard
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, ACS reagent, ≥99.5%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, puriss., meets analytical specification of Ph. Eur., BP, NF, ≥99.5% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl acetate, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.5% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Toluene, ACS reagent, ≥99.5%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, meets USP testing specifications
SKU
Pack Size
Availability
Price
Quantity