Activity and stability of catalase immobilized in alginate capsules in industrial applications

Efektywną metodą zahamowania rozwoju zanieczyszczeń mikrobiologicznych (bakteryjnych i grzybowych) obecnych w wodzie pitnej, mleku czy w sokach jest dodanie nadtlenku wodoru. Technika ta nie jest w Polsce powszechnie stosowana, jest jednak dopuszczona w wielu krajach pod warunkiem całkowitego usunięcia dodanego nadtlenku wodoru przed dopuszczeniem produktu do spożycia lub do dalszych etapów przetwarzania. Szybką metodą usunięcia nadtlenku wodoru może być zastosowanie enzymu – katalazy. W celu możliwości wielokrotnego użycia preparatu enzymatycznego, jak i łatwej jego separacji z mieszaniny reakcyjnej, zaproponowano immobilizację katalazy w kapsułkach alginianowych. Przeanalizowano możliwość zastosowania otrzymanego preparatu do usuwania H₂O₂ dodanego do różnych płynów: wody, mleka, soków warzywnych. Określono stabilność preparatu w warunkach procesowych (11 i 24 °C) i w temperaturze przechowywania preparatu (4 °C). Wyznaczone w buforach o danym pH stałe równania kinetycznego zweryfikowano w badaniach wymienionych płynów. Na podstawie parametrów kinetycznych określono warunki do przeprowadzenia procesu o założonej wydajności. Zaproponowano proces okresowy z separacją preparatu na sitach. Podano czas procesu dla poszczególnych szarży. W przypadku, gdy temperatura procesu zostanie obniżona do 11 °C, czas reakcji musi być znacząco dłuższy, niemniej liczba przeprowadzonych szarży (z uwagi na zachowaną aktywność preparatu) rekompensuje dłuższy czas procesu. Prowadzenie reakcji w obniżonej temperaturze jest uzasadnione w przypadku, gdy medium oczyszczane jest przetrzymywane w warunkach chłodniczych, co zwykle ma miejsce. Temp. 11 °C jest najniższą wartością, w której stosowany enzym wykazuje aktywność.The addition of hydrogen peroxide is an efficient method to inhibit the growth of microbial contaminants (bacterial and fungal) present in drinking water, milk, or juice. This technique is not commonly applied in Poland, but it is allowed in many countries provided that the hydrogen peroxide added to the product is completely removed from it prior to its release for consumption or for further processing. A rapid method of removing hydrogen peroxide could be the use of a catalase enzyme. In order to make it possible to repeatedly use that enzyme preparation and to easily separate it from the reaction mixture, it was suggested to immobilize the catalase in alginate capsules. The potentiality of using the produced preparation for H₂O₂ removal was analyzed through adding it to various liquids: water, milk, and vegetable juices. The stability of the preparation was determined under the process conditions (11 and 24°C) and at a temperature for storing the preparation (4 °C). The values of the kinetic equation constants, determined in the buffers of a given pH value, were verified through the analyses of the above named liquids. Based on the kinetic parameters, the processing conditions were determined for a planned process efficiency. A batch process including separation on sieves was proposed. The time of individual process charges was given. In the case of decreasing the process temperature to 11°C, the reaction time must be significantly longer; however, the number of performed charges (because of the maintained activity of the preparation) balances the effect of a longer process time. Performing the reaction at a lower temperature is also valid in the case of keeping the medium being purified under the refrigeration conditions, which is usually the case. A temperature of 11°C is the lowest value, at which the enzyme applied is active

Enzymatic synthesis of hydrophobic compounds integrated with membrane separation

The enzymatic synthesis of a highly hydrophobic product (dipeptide precursor) in which the reaction is accompanied by the mass transfer of the reaction product to the organic phase and the substrates to the water phase is considered. Equations describing both continuous and batch processes are formulated. The range of variability in the operating parameters of such a bioreactor is specified, and the correlations reported in the literature to describe mass transfer in the membrane contactor are validated. The proposed process was verified experimentally, and good agreement between the determined and calculated concentrations was obtained in both phases

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

The size of the gel preparation, the concentration of the encapsulated enzyme and the ratio of the preparation volume to the volume of the reaction mixture influence the reaction efficiency with encapsulated biocatalysts. A model of first order enzymatic reaction with substrate diffusion is presented and validated by the decomposition reaction of hydrogen peroxide by catalase. The Thiele modulus (Ф) contains the modified (including the enzyme concentration) enzymatic reaction constant (k′). Based on the model analysis, the Thiele modulus should not exceed a value of 2 (optimally less than 0.5). This value can be controlled by appropriate selection of the enzyme concentration inside and the size of the capsule. A lower Ф value gives a flat substrate concentration profile inside the gel capsule and all the enzyme molecules are involved in the reaction. The optimal diameter of the gel capsule with respect to their separation from the reaction mixture is 1–2 mm

Biodegradation of whey waste in a continuous stirred-tank bioreactor

Post-production whey has a high concentration of proteins and lactose that has to be decreased 500-fold before disposal in the environment. The paper describes goat whey biodegradation in a continuous stirred tank reactor (CSTR) using a Bacillus licheniformis strain. The data obtained emphasize the effect of excess organic compounds on growth rate. Accordingly, the Luong equation was applied to describe the batch process while accounting for substrate inhibition; the constants vmax = 2.29·10–4 1/s, Km = 4.79 g/dm3, Ki = 6.03 g/dm3, and n = 1.1 were experimentally obtained. According to accepted theory, the continuous process can be expressed using Monod’s equation (only for the rising side of the kinetic graph) in a restricted range of substrate concentrations (for glucose, up to 2.16 g/dm3). The estimated values of the constants were vmax = 8.52·10–5 1/s and Km = 0.45 g/dm3. The selected strain effectively decreased the lactose content in whey. At residence times above 30 h, almost total decrease in lactose content was observed that met the requirements of the Polish Ministry of Environmental Protection. The biodegradation of proteins was much slower, but their content does not greatly influence the required BOD and COD values

Two-step treatment of harmful industrial wastewater: an analysis of microbial reactor with integrated membrane retention for benzene and toluene removal

Standards for highly toxic and carcinogenic pollutants impose strict guidelines, requiring values close to zero, regarding the degradation of such pollutants in industrial streams. In many cases, classic bioremoval processes fail. Therefore, we proposed a stream leaving the microbial membrane bioreactor (MBR) that is directed to an additional membrane separation mode (NF/RO). Under certain conditions, the integrated process not only benefits the environment but may also increase the profitability of the bioreactor operation. An appropriate model was developed and tested in which the bioremoval of benzene and toluene by Pseudomonas fluorescens was used as an example. This paper presents equations for selecting the operation parameters of the integrated system to achieve the expected degree of industrial wastewater purification

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

Biodegradable polylactide and thermoplastic starch blends as drug release device – mass transfer study

Four different compositions of polylactide/thermoplastic starch blends (PLA/TPS blends) for application as drug carriers were examined. Initially, using cyanocobalamin (1.355 kDa) as a model compound, the blend with the highest starch content (wt. 60%) was selected for further research of mass transfer phenomenon. In this case, different concentrations of acetaminophen (0.151 kDa), doxorubicin hydrochloride (0.580 kDa) and cyanocobalamin (1.355 kDa) were used for determination of particular releasing profiles. Besides from the comparative analysis of obtained results, the values of the overall mass transfer coefficient (K) were calculated for each of tested drug molecules. Depending on the size and properties of used compound, determined values of the coefficient range from 10−11 to 10−13 m/s. Based on these outcomes, it could be stated that PLA/TPS blend selected in preliminary research, seems to be preferred material for fabrication of long-term drug delivery systems, which could be successfully applied for example in anti-cancer therapy