Simon Crelier

Inactivation of proteases and lipases by ultrasound

The inactivation of phospholipase A2, α-chymotrypsin, trypsin and porcine pancreatic lipase by heat and manothermosonication (MTS), and the simultaneous application of heat and ultrasound under moderate pressure, has been studied in different treatment media. MTS sensitivity varied strongly for the different enzymes. Whereas phospholipase A2 was almost insensitive to MTS treatments, α-chymotrypsin and porcine lipase MTS inactivation was much faster than heat inactivation. Trypsin heat inactivation was very different at low than at high temperatures. At low temperatures, it did not follow first order kinetics, contrary to what happened at high temperatures. MTS accelerated trypsin inactivation only at low temperatures. MTS changed also the inactivation order of trypsin (at low temperatures), α-chymotrypsin and porcine lipase; whereas heat inactivation of these enzymes did not follow first order kinetics, MTS inactivation fitted this well.

Coupling of a microfluidic mixer to a Fourier-transform infrared spectrometer for protein-conformation studies.

The biological properties of a protein critically depend on its conformation, which can vary as a result of changes in conditions such as pH or following the addition of various substances. Being able to reliably assess the quality of protein structures under various conditions is therefore of crucial importance. Infrared (IR) spectroscopy of the Amide I band of proteins is a powerful method for the determination of protein conformations and further allows the analysis of continuously flowing solutions of the target molecule. Here, a commercial Fourier-transform infrared spectrometer was coupled to a microfluidic mixer to allow the on-line monitoring of protein conformation under varying conditions. The validity of the concept was demonstrated by continuously recording the variations of the IR spectrum of poly-L-lysine resulting from repetitive, pH-induced conformational changes.

UV degradation of sesquiterpene lactones in chicory extract: Kinetics and identification of reaction products by HPLC-MS

Inulin, a valuable food ingredient, can be extracted from chicory roots (Cichorium intybus L.). However, the direct use of this material is limited, because of the presence of extremely bitter, co-extracted sesquiterpene lactones. Lactucin, a major component, has been degraded by UV irradiation, a process which showed neither temperature (293- 313 K) nor initial concentration dependence. An overall half life time of ca. 45 min was determined. The degradation product was identified by HPLC-MS and 1 H NMR and was the result of the addition of a water molecule on the lactucin C(1)-C(10) double bond, with the OH group located at the C(10) position.

Selective inactivation of phospholipase A2 in complex protein mixtures

Abstract Phospholipase A 2 is very resistant to thermal and manothermosonication treatments. It is a frequent contaminant of pancreatic proteolytic preparations. Its inactivation in such preparations can be achieved by dissolving the enzymes in buffers at neutral pH prior to its end use to allow the proteolytic action on the lipase. Alternatively, although much more complicated, the application of manothermosonication treatments makes phospholipase susceptible to proteolysis under conditions (buffer, pH and temperature) where the protease was not able by itself to hydrolyze PL.

Production of a Recombinant Catechol 2,3-Dioxygenase for the Degradation of Micropollutants

Phenolic compounds such as catechol represent a particular type of micropollutant whose high stability prevents rapid decay and metabolization in the environment. We successfully cloned a catechol 2,3-dioxygenase (C2,3O) from Pseudomonas putida mt-2 and expressed it in Escherichia coli BER2566. The biomass isolated from shake-flask fermentations was used to partially purify the enzyme. The enzyme proved unstable in clarified liquid fractions (50 mM Tris buffer, pH 7.6) and lost more than 90% of its activity over 7 h at 25 °C. In the presence of 10% acetone, the process was slowed down and 30% residual activity was still present after 7 h incubation. Storage of the enzyme in clear liquid fractions also proved difficult and total inactivation was achieved after 2 weeks even when kept frozen at -20 °C. Lowering the storage temperature to -80 °C preserved 30% activity over the same period. Only minor reactivation of the affected enzyme could be achieved after incubation at 20 °C in the presence of FeSO4 and/or ascorbic acid. Activity loss seems to be due mostly to Fe2+ oxidation as well as to subunit dissociation in the tetrameric structure. However, complete degradation of 1.0 mM catechol could be achieved at 20 °C and pH 7.6 over a 3 h period when using a suspension of whole cells or alginate-encapsulated cells for the biotransformation. Contrary to the clear liquid fractions, these forms of biocatalyst showed no significant sign of inactivation under the working conditions.

Sustainable Chemistry at the Universities of Applied Sciences

An overview of activities in the field of sustainable or ‘green’ chemistry at the Universities of Applied Sciences in Switzerland is presented.