Contaminants deriving from human activities represent a constantly growing threat to our environment and have a direct impact on plant and animal health. To alleviate this ecological imbalance, biocatalysis offers a green and sustainable alternative to conventional chemical processes. Due to their broad specificity, laccases are enzymes possessing excellent potential for synthetic biotransformations in various fields as well as for the degradation of organic contaminants. Herein, we produced laccases in submerged cultures of P. ostreatus and T. versicolor in three different media. The fungi/medium combination leading to the highest enzymatic activity was malt extract (2%) + yeast extract (3%) + glucose (0.8%). Laccase production was further increased by supplementing this medium with different concentrations of Cu2+, which also provided a better understanding of the induction effect. Additionally, we disclose preliminary results on the interaction of laccases with mediators (ABTS and violuric acid - VA) for two main applications: lignin depolymerisation with guaiacylglycerol-β-guaiacyl ether (GBG) as lignin model and micropollutant degradation with Remazol Brilliant Blue (RBB) as enzymatic bioremediation model. Promising results were achieved using VA to increase depolymerization of GBG dimer and to enhance RBB decolorisation.

Three institutes of the Universities of Applied Sciences that are active in Chemistry and Life Sciences present a selection of their activities in the field of environmental sciences. These projects include analytical monitoring, removal of micropollutants, waste reduction and valorization.

This article provides an overview of activities in the fields of continuous processes, flow chemistry and microreactors at the Universities of Applied Sciences in Switzerland.

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.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are biobased and biodegradable alternatives to petrol-derived polymers, whose break-through has been prevented by high production cost. Therefore we investigated whether wastes from the food industry (nine types of fruit pomace including apricots, cherries and grapes, and waste frying oil) could replace the costly sugars and fatty acids typically used as carbon substrates for the bacterial fermentations. A selection of enzyme preparations was tested for converting the residual polysaccharides from the pomaces into fermentable monosaccharides. From the pomace of apricots, cherries and Solaris grapes, 47, 49 and 106gL−1 glucose were recovered, respectively. Solaris grapes had the highest sugar content whereas apricots contained the fewest growth inhibitors. These two pomaces were assessed for their suitability to produce mcl-PHA in bioreactor. A 2-step fermentation was established with Pseudomonas resinovorans, hydrolyzed pomace as growth substrate and WFO as mcl-PHA precursor. Solaris grapes proved to be a very promising growth substrate, resulting in the production of 21.3gPHA(Lpomace)−1 compared to 1.4g PHA (L pomace)−1 for apricots. Finally, capillary zone electrophoresis analyses allowed monitoring of sugar and organic acid uptake during the fermentation on apricots, which led to the discovery of reverse diauxie in P. resinovorans.

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

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.

A micro-reactor coupled to a microfluidic system and an online UV/VIS spectrometer is described. The enzymatic reaction studied is the hydrolysis of the N-benzoyl-L-tyrosine ethyl ester (BTEE) to N-benzoyl-L-tyrosine (BT) and ethanol, catalyzed by chymotrypsin. The production is online monitored with UV spectroscopy at 256 nm. Three different immobilization methods of the enzyme are discussed: Eupergit® C, controlled-pore glass (CPG), and Sepharose.

Phospholipase A2 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.

Manothermosonication (MTS) treatments, the simultaneous application of heat and ultrasound under moderate pressure, of milk during 12 s at 20 kHz ultrasound amplitude, 2 kg pressure, and 40 degrees C allowed elaboration of yoghurts with rheological properties superior to those of control yoghurts elaborated with untreated milk. Measurements performed on intact samples (compression tests, relaxation tests, and texture profile analysis) and on slowly stirred samples (flow curves, apparent viscosity, yield stress, and viscoelastic properties) showed that MTS yoghurts had stronger structures, which resulted in higher values of almost all of the many relevant rheological parameters. Homogenization of fat globules brought about by MTS treatments is not responsible for the superior properties of MTS yoghurts, because the control yoghurt was also elaborated with homogenized milk. These results show that MTS could be a useful tool to improve the texture of yoghurts.

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 1H 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.

ABSTRACT: In this study we investigated the inactivation of endogenous pectin methylesterase (PME) in tomato juice during combined high‐hydrostatic pressure (ambient to 800 MPa) and moderate temperature (60 to 75 °C) treatments under isobaric and isothermal processing conditions. PME inactivation rates increased with increasing processing temperature, with the highest rate obtained during processing at 75 °C and ambient pressure. Inactivation rates were dramatically reduced as soon as processing pressure was raised. High inactivation rates were again attained when processing pressure exceeded a value of about 700 MPa. Such a behavior was described by considering two parallel mechanisms of inactivation, each one following first order kinetics with its own kinetic parameters.

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.

The combined high pressure/thermal (HP/T) inactivation of tomato pectin methyl esterase (PME) and polygalacturonase (PG) was investigated as a possible alternative to thermal processing classically used for enzyme inactivation. The temperature and pressure ranges tested were from 60 degreesC to 105 degreesC, and from 0.1 to 800 MPa, respectively.. PME, a heat-labile enzyme at ambient pressure, is dramatically stabilized against thermal denaturation at pressures above atmospheric and up to 500-600 MPa. PG, however, is very resistant to thermal denaturation at 0.1 MPa, but quickly and easily inactivated by combinations of moderate temperatures and pressures. Selective inactivation of either PME or PG was achieved by choosing proper combinations of P and T. The inactivation kinetics of these enzymes was measured and described mathematically over the investigated portion of the PIT plane. Whereas medium composition and salinity had little influence on the inactivation rates, PME was found less sensitive to both heat and pressure when pH was raised above its physiological value. PG, on the other hand, became more labile at higher pH values. The results are discussed in terms of isoenzymes and other physicochemical features of PME and PG.

The impact of HHPT on tomatoes, carrots, potatoes and broccoli was investigated regarding enzyme activities as well as mechanical texture alteration. Pectin methylesterase, polygalacturonase and peroxidase were affected at pressures above 500 MPa. Extensive tissue damage occurred however already at 200 MPa, leading to a dramatic softening and liquid exudation during cutting/compression tests. As a consequence of tissue disruption, potatoes and carrots also turned brown very quickly. Tomato juice viscosity, on the other hand, was durably increased by HHPT.