Anna Trusek-Holownia

Synthesis of ZAlaPheOMe, the precursor of bitter dipeptide in the two-phase ethyl acetate–water system catalysed by thermolysin

Enzymatic synthesis of ZAlaPheOMe, the precursor of a bitter dipeptide due to a very strong hydrophobic character of this compound was carried out in a two-liquid-phase ethyl acetate-0.1 moll(-1) Tris-HCl buffer system. The selected system and process conditions (60 degrees C, pH 7.0) ensured high activity and stability of thermolysin, a catalyst of this reaction. Owing to an almost complete extraction of the product to organic phase it was possible to obtain conversion degrees close to unity. The reaction phase was the aqueous phase in which almost whole catalyst mass was accumulated. The amount of protein adsorbed on the interface corresponded to 4-5% of protein dissolved in the aqueous phase. In the description of the enzymatic process rate the equation suitable for the mechanism of tetrahedral complex formation at a random order sequence of substrate bonding was used. The values of constants k=0.222 s(-1), K(mA)=11.5 mmoll(-1), K(mB)=149.3 mmoll(-1) were obtained.

Catalytic membrane preparation for enzymatic hydrolysis reactions carried out in the membrane phase contactor

Abstract Two concepts of the localisation of hydrolytic enzymes with the use of a membrane phase contactor are presented in the paper. Because of the participation of water molecules in the process, the enzyme should be immobilized so that it could be in contact with the aqueous phase. Thus, enzyme immobilisation on the hydrophobic membrane surface and in the hydrophilic membrane pores was considered. The lipase from Candida antarctica and from pancreatic porcine were selected for experiments. Both enzymes were adsorbed on the polypropylene membrane surface, packed in the pores of nitrocellulose and cellulose membranes and bound within the pores of the polyamide membrane after its activation with glutaraldehyde. On the basis of the amount of immobilized protein, enzyme activity and stability, the most appropriate preparation of each immobilized lipase was chosen having on mind its further application in the biphase organic solvent—water system.

A system for cleaning condensates containing ammonium nitrate by the reverse osmosis method

Abstract A process in which membrane techniques are used to obtain clean technology of ammonium nitrate production is described. The possibility of integration of this traditional chemical industry technology with membrane separations is revealed. Original technology has been patented and implemented. The aim of the system is to obtain a concentrate with ammonium nitrate concentration ranging from 40 to 80 g/dm 3 . The system consists of three elements-centers: preparation of raw material and preliminary filtration, three stages of reverse osmosis, and final degassing of the permeate. Very good results obtained during the 3-year operation of the system initiated further projects in this industry.

A membrane phase contactor for enzymatic synthesis of ZAlaPheOMe, the precursor of bitter dipeptide

Abstract Two process solutions were proposed for biochemical reactions carried out in the biphase organic solvent–water system where a membrane played the role of sustaining an interface. One of the two solutions with a passive- or active-type membrane was selected on the basis of the kinetic or diffusional regime of a particular process. Enzymatic synthesis of ZAlaPheOMe, the precursor of bitter dipeptide was discussed. On the basis of analysis of relative rates of enzymatic conversion and extraction of hydrophobic (a limiting substrate)—ZAlaOH from organic to aqueous phase, the range of the process was determined using the Hatta model. Very low values of the Hatta number were obtained which indicated that the process was carried out in the kinetic regime. Such a process solution was selected in which the enzyme was dissolved in the whole volume of the aqueous phase (passive character of the membrane). The process of ZAlaPheOMe synthesis confirmed a proper choice of the process solution; during the process the substrate concentrations in the aqueous phase were close to equilibrium concentrations. An intensive extraction of a hydrophobic product to the organic phase and high stability of the applied catalyst were observed.

Modeling of enzymatic conversion in the catalytic gel layer located on a membrane surface

Equations describing a multi-phase bioreactor with a catalytic membrane located on the boundary of phases, of which one is a substrate reservoir, were formulated. A model analysis of the process was carried out and the effect of main operating parameters of such a reactor, i.e., the catalytic layer thickness and the coefficients of diffusion mass transport in both phases, was determined. Two periods with different methods of supplying the catalytic layer can be distinguished in the process. There is also a characteristic thickness of the catalyst layer: when it is exceeded, the process duration is no longer shortened although full substrate conversion from the supply stream not always has a place. It is recommended to apply thin layers because then the catalyst activity is fully used. The analyses of the influence of mass transfer showed that substrate mass transfer in the water phase to the catalyst layer has a slight effect on the process rate when the intensification of its transfer in the organic phase might have a significant influence on the process rate.

Efficient utilisation of hydrogel preparations with encapsulated enzymes – a case study on catalase and hydrogen peroxide degradation☆

Graphical abstract

Mass transfer in the membrane phase contactor with an enzyme gel layer immobilized on a membrane

In the membrane phase contactor, the rate of reagent mass transfer from organic phase (a reservoir) to water phase (a reaction phase) depends on the three different resistances: the organic phase resistance, the resistance generated by the membrane and the water phase resistance. In the case when the enzyme is immobilized on the membrane surface and forms a gel layer, an additional mass transfer resistance is generated that could have an important influence on total mass transfer rate. Its value is directly proportional to the layer thickness and assumes particularly high values in the system where the distribution coefficient is much higher than unity. Since the gel layer thickness affects simultaneously the process yield, the decision concerning the amount of protein immobilized on the surface should be considered in detail.

Advanced treatment of wastewater with BTEX

Abstract Standards for concentrations of pollutants in wastewaters discharged to the environment are especially restrictive for BTEX, the compounds which are recognized to be particularly toxic. There is a need to lower the BTEX concentration in the stream coming out of the bioreactor. For this purpose, a high-pressure membrane process was proposed. A series of salts and benzene/toluene membrane separation processes on Nanomax 95 membrane were carried out. On the basis of these experiments, a pressure in the field 20–25 bar at 20°C was shown as the most efficient and effective for BTEX retention in the biodegradation zone.

Enhanced production of polygalacturonase in solid-state fermentation: selection of the process conditions, isolation and partial characterization of the enzyme.

Polygalacturonase (PG) production by Penicillium chrysogenum during solid-state fermentation was accompanied by decomposition of orange peels. A leaching procedure was developed through the selection of solvent, time and intensity of stirring. A maximum PG activity was observed after 48 h peel inoculation. Further cultivation decreased the enzyme activity significantly, up to 60% of the maximum PG activity. During fermentation, a rapid acidification of the solid medium which inhibited the pectinolytic enzyme, was observed. Buffering agents with different pH values and different ionic strengths were examined to identify the most suitable medium to avoid this problem. Buffer addition counteracted acidification and enhanced active protein production, which was observed for all of the applied pH values (6.5-8.0) of the buffering agent. The most satisfactory results were obtained when using the highest pH at 8.0. The protein content and PG activity increased from 3.5 mg/g and 1.09 U/g to 7.7 mg/g and 7.11 U/g during cultivation, with uncontrolled and pH-controlled medium, respectively. Measurements at wide pH and temperature ranges indicated an optimum for PG activity at pH 5.0 and 43°C; however, high thermal stability corresponded to lower temperatures, and a temperature of 37°C is thus recommended. Under these conditions, the operational stability was determined to be t1/2=570 h.

Dairy wastewater utilization: separation of whey proteins in membrane and chromatographic processes

AbstractDairies are obligated to utilize whey after cheese production. From an environmental protection point of view, the high content of lactose and proteins in post-production wastes, like whey, is harmful for the environment. From another point of view, whey is a source of very valuable, active proteins, particularly lactoferrin and serum albumin. Their modulatory potential is exhibited in their pure form and improves after partial, controlled hydrolysis. Unfortunately, the fractionation of this multicomponent medium is not an easy task. The paper describes an integrated process of fractionation of whey proteins. After the first step of treatment based on membrane techniques, the concentrated, most valuable whey proteins were subjected to a few steps of chromatographic separation. The separation properties of the ultrafiltration membranes were unexpected. The typical cut-off boundary was shifted in the direction of components having a lower molecular weight. After laboratory-scale testing, a concept f...