Tonči Rezić

Comparing soluble and co-immobilized catalysts for 2-ketoaldose production by pyranose 2-oxidase and auxiliary enzymes

The tri-enzyme system pyranose 2-oxidase (P2O), laccase, and catalase was used to study major parameters in the homogeneous and heterogeneous application of a multi-component enzymatic machinery. P2O oxidizes aldoses to 2-ketosugars, which are interesting intermediates in carbohydrate chemistry, and concomitantly reduces oxygen or alternative electron acceptors. The enzyme was immobilized on eleven agarose or acrylic resins using various coupling methods. The binding capacity was determined and an acrylic carrier with the most suitable properties selected for detailed studies. As P2O shows higher turnover numbers with the electron acceptor 1,4-benzoquinone than with oxygen, the use of this alternative electron acceptor was enabled by employing laccase for the continuous reoxidation of hydroquinone. The laccase regeneration system was found to increase the specific productivity up to 3-fold. Catalase was used to disproportionate the formed hydrogen peroxide in close proximity to the oxygen consuming enzymes and applied in different amounts to adjust the hydrogen peroxide concentration, which was found to be the main reason for enzyme deactivation under turnover conditions. In contrast to homogeneous catalysis, the specific productivity of heterogeneous catalysts under the applied experimental conditions was limited primarily by oxygen transfer, an effect significantly reduced by the laccase regeneration system.

Removal of Cr, Mn, and Co from textile wastewater by horizontal rotating tubular bioreactor.

Environmental pollution by industrial wastewaters polluted with toxic heavy metals is of great concern. Various guidelines regulate the quality of water released from industrial plants and of surface waters. In wastewater treatment, bioreactors with microbial biofilms are widely used. A horizontal rotating tubular bioreactor (HRTB) is a combination of a thin layer and a biodisc reactor with an interior divided by O-ring shaped partition walls as carriers for microbial biomass. Using a biofilm of heavy metal resistant bacteria in combination with this special design provides various advantages for wastewater treatment proven in a pilot study. In the presented study, the applicability of HRTB for removing metals commonly present in textile wastewaters (chromium, manganese, cobalt) was investigated. Artificial wastewaters with a load of 125 mg/L of each metal underwent the bioreactor treatment. Different process parameters (inflow rate, rotation speed) were applied for optimizing the removal efficiency. Samples were drawn along the bioreactor length for monitoring the metal contents on site by UV-vis spectrometry. The metal uptake of the biomass was determined by ICP-MS after acidic microwave assisted digestion. The maximum removal rates obtained for chromium, manganese, and cobalt were: 100%, 94%, and 69%, respectively.

Fungal secretomes enhance sugar beet pulp hydrolysis

The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g–1 protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1–17.5 mg g–1 SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load.

Optimization of the TLC Separation of Seven Amino Acids

A ternary mobile phase for thin-layer chromatographic separation and identification of seven amino acids on microcrystalline cellulose, with ninhydrin as detection reagent, has been optimized by use of the experimental design software packages Design-Expert 6 and Statistica. When the results from each product were compared the optimum mobile phase was found to be butanol-glacial acetic acid-water 60.14:18.77:21.09. The performance of this mobile phase was confirmed experimentally.

Monitoring of Cu, Fe, Ni, and Zn in wastewater during treatment in a horizontal rotating tubular bioreactor.

The most appropriate systems for treatment of metal-contaminated waters are bioreactors with microbial biofilms. A horizontal rotating tubular bioreactor (HRTB) was studied for its applicability for removing copper, iron, nickel, and zinc (Cu, Fe, Ni, and Zn) from wastewater. Monitoring of the concentrations of Cu, Fe, Ni, and Zn by a fast, simple, onsite method was needed to make decisions for further optimization. The UV-VIS spectrophotometric quantification of Cu, Fe, Ni, and Zn using sodium diethyldithiocarbamate, 1,10-phenathroline, dimethylglyoxime, and 2-{[alpha-(2-Hydroxy-5-sulfophenylazo)-benzylidene]-hydrazino}-benzoic acid monosodium salt (=zincon monosodium salt) as reagents, respectively, was optimized and validated. The limits of quantification were 0.14, 0.12, 0.21, and 0.03 mg/L for Cu, Fe, Ni and Zn, respectively. The recovery for all elements was between 98 and 104%, the uncertainty of measurement was less than 6%. Depending on the reactor parameters applied, metal removals from 40 to more than 90% could be obtained.

Metal nanoparticles and carbon nanotubes—perfect antimicrobial nano-fillers in polymer-based food packaging materials

Abstract Advances in food packages are oriented to obtain improved quality and safety. Incorporation of antimicrobial compounds in the form of nano-fillers into polymer-based food packaging materials has received considerable attention. These active packages are applied to extend shelf life while maintaining nutrition quality and ensuring microbiological safety. Antimicrobial nanocomposite films are desirable for application in the food industry due to their advantages: structural integrity and barrier properties imparted by the matrix, as well as the antimicrobial properties resulting from impregnated antimicrobial agents. Metal nanoparticles, metal oxide nanomaterials, and carbon nanotubes are mostly used in developing antimicrobially active polymer materials. This chapter deals with some of the most important questions related to metallic nanoparticles in food packaging (health and environmental considerations, performance, and costs), with the special emphasize on their applicable and toxicological properties.

Lignocellulose degradation: An overview of fungi and fungal enzymes involved in lignocellulose degradation

This review aims to present current knowledge of the fungi involved in lignocellulose degradation with an overview of the various classes of lignocellulose‐acting enzymes engaged in the pretreatment and saccharification step. Fungi have numerous applications and biotechnological potential for various industries including chemicals, fuel, pulp, and paper. The capability of fungi to degrade lignocellulose containing raw materials is due to their highly effective enzymatic system. Along with the hydrolytic enzymes consisting of cellulases and hemicellulases, responsible for polysaccharide degradation, they have a unique nonenzymatic oxidative system which together with ligninolytic enzymes is responsible for lignin modification and degradation. An overview of the enzymes classification is given by the Carbohydrate‐Active enZymes (CAZy) database as the major database for the identification of the lignocellulolytic enzymes by their amino acid sequence similarity. Finally, the recently discovered novel class of recalcitrant polysaccharide degraders‐lytic polysaccharide monooxygenases (LPMOs) are presented, because of these enzymes importance in the cellulose degradation process.

Application of thin-layer chromatography, X-ray fluorescence spectrometry, and Fourier transformed infrared spectroscopy in the analysis of binding media present on mummies of St. Giovanni Olini (1200 AD) and St. Nicolosa Bursa (1500 AD)

The main goal of this work was to apply thin-layer chromatography (TLC) in the investigation of different binding media (proteins, sugars, waxes, resins, and oils) found on samples of two mummified bodies of saints originating from 1200 AD and 1500 AD. The historical samples were compared by testing them for the presence of different inorganic and organic compounds. The chemical methods used were TLC, microscopy, X-ray fluorescence spectrometry, and Fourier transformed infrared spectroscopy. The detected similarities in the composition of the binding media coatings on two mummies indicated that those were not applied immediately after death but much later showing resemblance in their preservation treatments. Moreover, according to the composition of the materials detected, the coatings did not seem to have had considerable impact on the mummification of the bodies. The combination of TLC and other chemical methods proved to be an effective and low-cost tool for obtaining valuable information during the archeological investigations.

Monitoring of bioprocess by thin-layer chromatography in a horizontal rotating tubular bioreactor for removal of heavy metals

This paper presents an innovative idea of application of thin layer chromatography (TLC) to characterize the bioprocess in horizontal rotating tubular bioreactor (HRTB) during heavy metals removal ions (Co2+, Cr6+ and Mn2+) from wastewaters. HRTB was designed as a combination of thin-layer and biodisc reactor and therefore had characteristics of both reactors. Before analysis of waste water samples, optimization of chromatographic system for separating metal ions was performed: retention factors (RF values) were calculated and the best chromatographic system for separation and identification of analyzed metal ions was chosen.