Enzymatic conversions in highly concentrated reaction systems
Introduction
Pressure is an important process parameter, which is not yet used for synthetic purposes in biocatalytic processes. Enzyme performance at extremely high pressure (up to 1000 MPa) is being studied with the aim to develop improved processes for enzymatic synthesis.
Theory
For a chemical reaction, the equilibrium constant (K) is related to pressure by the reaction volume change of the system (∆V).
(∂lnK/∂P)T = -∆V/RT
This equation implicates that the application of pressure to a system in equilibrium will force the equilibrium to the state with the smallest volume. This means that a negative reaction volume shifts the equilibrium to the product side of the reaction.
We calculated hypothetical yields for a synthetic equilibrium reaction A + B → C. For the formation of chemical bonds reaction volumes around –10 cm3/mol are found1. The figure below shows the exponential improvement of the equilibrium constant at different pressures. Further calculations on yield and some examples have recently been published2.
At these high pressures, more stable enzymes are needed. We performed experiments on gluco-oligosaccharide synthesis, catalysed by the hyperthermophilic β-glycosidase from Pyrococcus furiosus. Oligosaccharide yields decreased by pressure. This process could be described with a positive reaction volume of 2.3 mol/cm.
Pressure is an important parameter in synthesis reactions that influences the equilibrium of a reaction. To study this effect, relatively low pressures can be applied (up to 200 MPa) at which enzyme inactivation is not yet important. Our calculations show that for process optimisation, much higher pressures (up to 1,000 MPa) have to be used. At these high pressures, more stable enzymes are needed. We show that with the use of a thermophilic enzyme, higher pressures can be applied. We will continue to study equilibrium reactions under pressure at our laboratory, taking advantage of the ongoing discovery of more stable enzymes.
Project information
The project was financed by a VENI-grant from the Netherlands Organisation for Scientific Research (NWO-STW) and was running till august 2008. The project leader is Marieke Bruins, who started work as a Post-doc since September 1st 2004 at Process Engineering. In 2003 she finished her PhD on the topic of oligosaccharide production with thermophilic enzymes at the same laboratory.
Literature
[1] Asano, T., and J. Lenoble. 1978. Activation and Reaction Volumes in Solution. Chemical Reviews 78:407-489.
Output
Pressure aided proteolysis of β-casein. M.E. Bruins, N. Creusot, H. Gruppen, A.E.M. Janssen, R.M. Boom. Submitted
Inactivation and conformational studies of β-glucosidase from Pyrococcus furiosus in the pressure-temperature-plane. M.E. Bruins, F. Meersman, A.E.M. Janssen, K. Heremans, R.M. Boom. (2008) Accepted in FEBS journal
Methylation in methanol-water mixtures: the effect of solvent quality and high-pressure. M.E. Bruins, K.M. Bekers, A.E.M. Janssen, R.M. Boom. (2008) Biophysical Chemistry. 134: 207-213. Full Text
The effect of pressure and temperature on the gelatinisation of starch at various starch concentrations. T. Baks, M.E. Bruins, A.E.M. Janssen, R.M. Boom. (2008) Biomacromolecules. 9(1): 296-304. Full Text
Effect of gelatinisation and hydrolysis conditions on the selectivity of starch hydrolysis with α-amylase from B. licheniformis. T. Baks, M.E. Bruins, A.M. Matser, A.E.M. Janssen, R.M. Boom. (2008) Journal of Agricultural and Food Chemistry. 56(2): 488-495. Full Text
Gelatinization and hydrolysis of starch suspensions at high pressures. T. Baks, M.E. Bruins, A. Warmerdam, A.M. Matser, A.E.M. Janssen, R.M. Boom. Oral presentation: Joint 21st AIRAPT & 45th EHPRG Conference on High Pressure Science and Technology, 2007, Catania, Italy.
Enzymatische reacties bij hoge druk. M.E. Bruins, A.M. Matser, A.E.M. Janssen, R.M. Boom. (2007) NPT 2: 11-13.
Buffer selection for HP treatment of biomaterials and its consequences for enzyme inactivation studies. M.E. Bruins, A.M. Matser, A.E.M. Janssen, R.M. Boom. (2007) High Pressure Research. 27(1): 97-104. Full Text
A thermozyme at high pressure. M.E. Bruins, A.E.M. Janssen, R.M. Boom. Poster and abstract: Biocatalysis in the food and drinks industry 2006, Wageningen
HP and pH. M.E. Bruins, A.M. Matser, A.E.M. Janssen, R.M. Boom. Oral presentation: EHPRG 2006 conference, Prague, Czech Republic.
High pressure and its use in enzymatic synthesis. M.E. Bruins, A.E.M. Janssen, R.M. Boom. Poster and abstract: NBC 2006 symposium, Ede
Equilibrium shifts in enzyme reactions at high pressure. Bruins, M.E., Janssen, A.E.M., Boom, R.M., (2006) J. Mol. Catal. B. Enzymatic.39: 124-127 Full Text
High pressure and its use in enzymatic synthesis. M.E. Bruins, A.E.M. Janssen, R.M. Boom. Poster and abstract Biotrans 2005 symposium, Delft