Ana Jurinjak Tušek

Modeling and kinetic parameter estimation of alcohol dehydrogenase-catalyzed hexanol oxidation in a microreactor

A mathematical model for hexanol oxidation catalyzed by NAD+‐dependent alcohol dehydrogenase from bakers yeast in a microreactor was developed and compared with the model when the reaction takes place in a macroscopic reactor. The enzyme kinetics was modeled as a pseudo‐homogeneous process with the double substrate Michaelis–Menten rate expression. In comparison with the kinetic parameters estimated in the cuvette, a 30‐fold higher maximum reaction rate and a relatively small change in the saturation constants are observed for the kinetic parameters estimated in the continuously operated tubular microreactor (Vm1=197.275 U/mg, Kmhexanol=9.420 mmol/L, and Km1NAD+=0.187 mmol/L). Kinetic measurements performed in the microreactor, estimated from the initial reaction rate experiments at the residence time of 36 s, showed no product inhibition, which could be explained by hydrodynamic effects and the continuous removal of inhibiting products. The Fourier amplitude sensitivity test method was applied for global kinetic parameter analysis, which shows a significant increase in the sensitivity of Km1NAD+ in the microreactor. Independent experiments performed in the microreactor were used to validate and to verify the developed mathematical model.

Enhancement of phenolic compounds oxidation using laccase from Trametes versicolor in a microreactor

Laccases catalyse the oxidation of a wide range of substrates by a radical-catalyzed reaction mechanism, with a corresponding reduction of oxygen to water in a four-electron transfer process. Due to that, laccases are considered environmentally friendly enzymes, and lately there has been great interest in their use for the transformation and degradation of phenolic compounds. In this work, enzymatic oxidation of catechol and L-DOPA using commercial laccase from Trametes versicolor was performed, in continuously operated microreactors. The main focus of this investigation was to develop a new process for phenolic compounds oxidation, by application of microreactors. For a residence time of 72 s and an inlet oxygen concentration of 0.271 mmol/dm3, catechol conversion of 41.3% was achieved, while approximately the same conversion of L-DOPA (45.0%) was achieved for an inlet oxygen concentration of 0.544 mmol/dm3. The efficiency of microreactor usage for phenolic compounds oxidation was confirmed by calculating the oxidation rates; in the case of catechol oxidation, oxidation rates were in the range from 76.101 to 703.935 g/dm3/d (18–167 fold higher, compared to the case in a macroreactor). To better describe the proposed process, kinetic parameters of catechol oxidation were estimated, using data collected from experiments performed in a microreactor. The maximum reaction rate estimated in microreactor experiments was two times higher than one estimated using the initial reaction rate method from experiments performed in a cuvette. A mathematical model of the process was developed, and validated, using data from independent experiments.

Catechol removal from aqueous media using laccase immobilized in different macro- and microreactor systems

Laccase belongs to the group of enzymes that are capable to catalyze the oxidation of phenols. Since the water is only by-product in laccase-catalyzed phenol oxidations, it is ideally “green” enzyme with many possible applications in different industrial processes. To make the oxidation process more sustainable in terms of biocatalyst consumption, immobilization of the enzyme is implemented in to the processes. Additionally, when developing a process, choice of a reactor type plays a significant role in the total outcome.In this study, the use of immobilized laccase from Trametes versicolor for biocatalytic catechol oxidation was explored. Two different methods of immobilization were performed and compared using five different reactor types. In order to compare different systems used for catechol oxidation, biocatalyst turnover number and turnover frequency were calculated. With low consumption of the enzyme and good efficiency, obtained results go in favor of microreactors with enzyme covalently immobilized on the microchannel surface.

Lipase catalysed biodiesel synthesis with integrated glycerol separation in continuously operated microchips connected in series

Although the application of microreactors in different processes has been extensively explored in recent decades, microreactors continue to be underexplored in the context of the enzyme-catalysed process for biodiesel production. Due to their numerous advantages, microreactors could become the next step in the development of a biodiesel production process characterised by sustainability, cost-effectiveness and energy efficiency. In this investigation, biodiesel production was catalysed by lipase from Thermomyces lanuginosus (Lipolase L100). Edible sunflower oil was used as a model substrate in order to investigate the process. After optimal process conditions had been determined, waste-cooking oil was used for biodiesel production to make the production process more sustainable. Three different substrate-feeding strategies were investigated and finally an optimal strategy was proposed. In all the investigated systems, fatty acids methyl esters (FAME) content was higher than 95% and obtained in a significantly shorter time (less than 2 h) compared to the batch process in which biodiesel production was catalysed by lipase (C = 95%, t = 96 h). After the optimal biodiesel production system had been proposed, an integrated system with two microchips connected in series was developed. The first microchip was used for biodiesel production and the second for simultaneous purification i.e. glycerol separation. Finally, purified biodiesel was produced with glycerol content below the detection limit.

Mathematical modelling of polyphenol extraction by aqueous two-phase system in continuously operated macro- and micro-extractors

ABSTRACT An aqueous two-phase system (ATPS) in combination with macro- and micro-extractors was used for polyphenol extraction from a model solution (gallic acid) and three real samples (red and white wine, and orange juice). The objective of the present study was to apply a mathematical model that successfully describes and predicts performances of macro- and micro-extractors. The micro-extractor system was selected as the most suitable for the polyphenol extraction because the same extraction efficiency was obtained for two levels of magnitude shorter residence time compared to the macro-extractor. Based on good agreement between model predictions and experimental results, the obtained simulations could be used for further process optimization and improvement.

Systems Biology Markup Language: Case Study of T-Cell Signal Transduction Network

Application of system theory in biology has led to development of computer languages for mathematical modeling which are in open source and apply common standards for model trans-platform exchangeability and integration with extensive model banks, databases, numerical and statistical methods and graphical tools. Standardization has led to systems biology Markup language (SBML) based on XML format, while open source policy enables integration of computer resources and provides synergistic integration of scientific results and methodologies from various scientific fields. Different software projects have been initiated resulting in software such as: SB Toolbox, J Designer, SB Workbench (SBW), and Cell Designer. The main use of the systems biology software is to investigate biochemical networks, gene regulatory networks, signal transduction, cell communication, dynamical complex behavior, and most importantly to lead to understanding of life regulation. In this work is presented use of systems biology markup language and modeling tools applied for analysis of signal transduction in T-cell as apart of a human immune system.

Integrated approach for bioactive quality evaluation of medicinal plant extracts using HPLC-DAD, spectrophotometric, near infrared (NIR) spectroscopy and chemometric techniques

ABSTRACT To establish a universal analytical tool that could be used as a bioactive quality determination procedure on medicinal plant extracts, a range of spectrophotometric assays, HPLC, near infrared reflectance spectroscopy and chemometric analysis were employed for determination of the bioactive quality of 16 widely spread medicinal plants. Macro-constituents (total carbohydrates, soluble polysaccharides, proteins, amino acids) and secondary plant metabolites (total phenols, flavonoids, hydroxycinnamic acids, flavons, and flavonols) were determined, and HPLC method for the simultaneous determination of phenolic acids and flavonoids was developed, and its linearity, limits of detection and quantification, precision, and accuracy were validated. The evaluated medicinal plant extracts were characterised by a marked protein (marigold – 4.22 g/kg dw), amino acid (marigold – 61.14 g/kg dw), and carbohydrate content (dandelion – 113.5 g/kg dw), while Lamiaceae plants were distinguished as the predominant sources of polyphenolic bioactives (<2.26 g GAE/L). The developed HPLC method enabled separation of 24 polyphenolic compounds within a short analysis time (30 min) and revealed rosmarinic and chicoric acids as the prevalent polyphenolic constituents. NIR spectroscopy coupled with chemometric analysis of all determined analytical parameters indicated the suitability of NIR analysis for amino acids, carbohydrates, and polyphenols determination in medicinal plant extracts.

Analysis of diffusivity of the oscillating reaction components in a microreactor system

When performing oscillating reactions, periodical changes in the concentrations of reactants, intermediaries, and products take place. Due to the mentioned periodical changes of the concentrations, the information about the diffusivity of the components included into oscillating reactions is very important for the control of the oscillating reactions. Non-linear dynamics makes oscillating reactions very interesting for analysis in different reactor systems. In this paper, the analysis of diffusivity of the oscillating reaction components was performed in a microreactor, with the aim of identifying the limiting component. The geometry of the microreactor microchannel and a well defined flow profile ensure optimal conditions for the diffusion phenomena analysis, because diffusion profiles in a microreactor depend only on the residence time. In this paper, the analysis of diffusivity of the oscillating reaction components was performed in a microreactor equipped with 2 Y-shape inlets and 2 Yshape outlets, with active volume of V = 4 μL at different residence times.

Properties of medicinal plants related to NIR spectroscopy

The aim of the study on Big Data in Public Health, Telemedicine and Healthcare is to identify applicable examples of the use of Big Data in Health and develop recommenda-tions for their implementation in the European Union. Examples of Big Data in Health were identified by a systematic literature review, after which the added value of twenty selected examples was evaluated. Based on the as-sessment of the added value and the quality of the evidence, ten priority examples were selected. Furthermore, potential policy actions for the implementation of Big Data in Health were identified in the literature, and a SWOT analysis was conducted to check the feasibility of the proposed actions. Based on this analysis, and with the help of renowned experts, the study team developed ten policy recommendations in the field. These recommendations were validated through public consultations at three relevant conferences in Europe and were again reviewed by the Expert Group. The recommendations aim to benefit European citizens and patients in terms of strengthening their health and improving the performance of Member State’s health systems. They should be seen as suggestions for the European Union and its Member States on how to utilise the strengths and exploit the opportunities of Big Data for Public Health without threatening privacy or safety of citizens. Recommendations were developed for ten relevant fields: awareness raising, education and training, data sources, open data and data sharing, applications and purposes, data analysis, governance of data access and use, standards, funding and financial resources, as well as legal aspects and privacy regulation.P molecules found in plants are utilized currently for improving prostate health. The botanicals with the highest recognized profiles are Resveratrol and the isoflavonoids. Prostate health is compromised with aging, especially beginning in the mid-40s, when testosterone levels decline and estrogenic hormonal actions increase along with enhanced expression of the prostatic 5α-reductase enzyme that result in prostate enlargement commonly known as benign prostatic hyperplasia (BPH). There is a linear increase in BPH incidence with increasing age in men over 60 years old. Approximately 1 in 5 men with BPH had a clinical event (prostatectomies), within 1 year of initiating treatment for BPH. The symptoms of BPH include nocturia, incomplete emptying, urinary hesitancy, weak stream, frequency and urgency that negatively impact the quality of life. Current pharmaceuticals are somewhat effective, but can have serious side effects. Interest in complementary and alternative medicines (CAM) for BPH has increased during the last two decades. CAM agents include polyphenolic compounds such as Resveratrol and the isoflavonoids. Resveratrol is effective in vitro, but, in vivo administration presents efficacious challenges. Genistein was first thought to be responsible for improved prostate health, however, since the equal hypothesis was proposed in the late1990s, there has been increased focus on this isoflavonoid molecule. This presentation will briefly review Resveratrol and some isoflavonoid molecules where clinical studies have shown improvement of BPH symptoms in men using standardized laboratory biomarkers and survey indexing parameters. Clearly there is an opportunity for an efficacious and cost-effective approach for the treatment of BPH with botanicals.

Adaptation of CHO cells in serum-free conditions for erythropoietin production: application of EVOP technique for process optimization.

Mammalian cell cultures are the preferred expression systems for the production of biopharmaceuticals requiring posttranslational processing. Usually, cell cultures are cultivated in medium supplemented with serum, which supports cell proliferation, viability, and productivity. However, due to scientific and regulatory concerns, serum‐free conditions are required in recombinant protein production. Cell lines that are intended for commercial recombinant protein production have to adapt to serum‐ or protein‐free conditions early in their development. This is a labor‐ and time‐consuming process because of the specific cell requirements related to their adaptation in new microenvironment. In the present study, a Chinese hamster ovary (CHO) cell line producing glycosylated recombinant human erythropoietin (rhEPO) was adapted for growth and rhEPO production in serum‐ and protein‐free conditions. The physiology, growth parameters, and morphology of the CHO cells and rhEPO biosynthesis and structure were closely monitored during the adaptation process to avoid unwanted selection of cell subpopulations. The results showed that the CHO cells were successfully adapted to suspension growth and rhEPO production in the protein‐free conditions and that the structure of rhEPO remained nearly unchanged. In addition, during rhEPO production in the protein‐free suspension conditions, the agitation rate seem to be significant for optimal process performance in contrast to the initial cell concentration, evaluated through evolutionary operation method.