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Mung bean (Vigna radiata L.) is a leguminous plant cultivated mainly in south-east Asia and used as an ingredient in local cuisine. Its principal nutritional value is contained in its constituents such as starch, proteins, (poly)phenols, and natural antioxidants. Fluorescence spectroscopy is increasingly used as a method of choice for food analysis; due to the presence of different fluorophores originating from aromatic amino acids and secondary metabolites, it is useful for proteins and phenolics detection. In this study, the total protein and phenolic contents of mung bean seed extracts were determined using the Bradford method and Folin–Ciocalteu (FC) reagent, respectively. Antioxidant activity was determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Fluorescence spectra were recorded for a series of excitation-emission wavelengths. Further, we used the multivariate analysis on the recorded excitation-emission fluorescence matrix of the studied samples. The results showed the presence of three different fluorescence components, with the position of the emission maximum corresponding to the fluorophore of proteins (component 1 with excitation/emission peak maxima at Ex 290/Em 345 nm) and phenolics (component 2 - Ex 295/Em 395 nm and component 3 - Ex 350/Em 450 nm). This fluorescence-based method could be a useful approach for estimating the nutrient properties of leguminous food.

Honey bees have a very important role in pollinating plants, in addition to honey production. Bee diseases are one of the reasons of a significant decrease in bee colonies in the world in recent years. Honey samples originating from hives with different infestations with N. ceranae were analyzed. Total phenolic content and total protein content were determined by the Folin-Ciocalteu assay and the Bradford assay, respectively. Fluorescence spectroscopy combined with PARAFAC was used to determine the spectral components originating from proteins (PFSC1) and phenolics (PFSC2) in honey samples, and their ratios were calculated. Phenols and proteins content in the honey samples, obtained by spectrophotometric quantification, decreases with increasing infestation levels in the respective hives infected with N. ceranae. A negative correlation was obtained for the ratio of PARAFAC components PFSC2/PFSC1 and the level of infestation with N. ceranae in honey samples. These results indicate that fluorescence spectroscopy combined with PARAFAC could be used as an optical tool for non-invasive and rapid screening of honey to estimate bee health status.

Environmental safety may be compromised by the presence of hazardous chemical compounds, such as bisphenol A (BPA), which is commonly used in the production of certain types of plastics. BPA is an emerging organic contaminant that could be found in many matrices, such as drinking water, due to anthropogenic activities. Within this study, we used fluorescence spectroscopy to analyse the photoluminescent characteristics of BPA in various solutions and drinking water. The emission spectra of BPA in various solvents are recorded from 280 nm to 380 nm, after excitation wavelength at 230 nm. These results imply that the fluorescence approach can be used for rapid detection and estimation of the level of BPA in water samples and, hence, for non-invasive monitoring of BPA in drinking water is an important concern for public health and environmental protection.

Varroa destructor is a parasitic mite responsible for the loss of honey bee (Apis mellifera) colonies. This study aimed to find a promising marker in honey for the bee colony infestation level using fluorescence spectroscopy and biochemical analyses. We examined whether the parameters of the honey samples’ fluorescence spectra and biochemical parameters, both related to proteins and phenolics, may be connected with the level of honey bee colonies’ infestation. The infestation level was highly positively correlated with the catalase activity in honey (r = 0.936). Additionally, the infestation level was positively correlated with the phenolic spectral component (r = 0.656), which was tentatively related to the phenolics in honey. No correlation was found between the diastase activity in honey and the colonies’ infestation level. The results indicate that the catalase activity in honey and the PFC1 spectral component may be reliable markers for the V. destructor infestation level of the colonies. The obtained data may be related to the honey yield obtained from the apiaries.

Varroa destructor is a parasitic mite responsible for the loss of honey bee (Apis mellifera) colonies. This study aimed to find a promising marker in honey for the bee colony infestation level using fluorescence spectroscopy and biochemical analyses. We examined whether the parameters of the honey samples’ fluorescence spectra and biochemical parameters, both related to proteins and phenolics, may be connected with the level of honey bee colonies’ infestation. The infestation level was highly positively correlated with the catalase activity in honey (r = 0.936). Additionally, the infestation level was positively correlated with the phenolic spectral component (r = 0.656), which was tentatively related to the phenolics in honey. No correlation was found between the diastase activity in honey and the colonies’ infestation level. The results indicate that the catalase activity in honey and the PFC1 spectral component may be reliable markers for the V. destructor infestation level of the colonies. The obtained data may be related to the honey yield obtained from the apiaries.

Aflatoxin B1 (AFB1), is a naturally hazardous environmental contaminant, contained mainly in various cereal seeds. The maize (Zea mays L.) seeds containing various levels of the AFB1 was investigated using optical and electron paramagnetic resonance spectroscopies. The changes of lignin content and organic free radicals as indicators of the seeds' defense response to the AFB1-stressor were estimated, in the seeds' inner and outer fractions. The lignin content was determined using the Acetyl Bromide method. No correlation was found between the lignin content and the AFB1 concentration in the inner fraction (R of 0.030, p = 0.943), while a significant positive linear correlation (R of 0.9923, p= 0.00005) between the lignin content and the AFB1 concentration in the outer fraction was obtained. Employing electron paramagnetic resonance (EPR) spectroscopy, the different response of the fractions was observed, regarding the content of free radicals with increasing AFB1 concentrations. In addition, front-face fluorescence spectroscopy in combination with the deconvolution showed a positive linear correlation between the ratio of the green and blue spectral emission components (C4/C2) area and the AFB1 concentration in the outer fraction. Thus lignin content and the (C4/C2) area ratio may be reliable indicators for the screening of lignin changes related to the level of AFB1 in the seeds.

Semena kukuruza (Zea mais L.) mogu biti kontaminirana sa jednim od najpasnijih prirodnih kancerogenih kontaminanata kao što su aflatoksini. U ovoj studiji, proučavan je uticaj aflateritoksina na strukturne karakteristike strukture. Korišćena je fluorescentna spektropija u kombinaciji sa matematičkom analizom spektra. Primenjen je matematički model za analizu spektra asimetričnog modela (Log-normal) koji koristi Nelder-Mead algoritam (Matlab 6.5). Rezultati razlaganja fluorescentnih spektara pokazuju četiri glavne fluorescentne fluorescentne emisije sa 054 emisiona približna 050 komponenta. Ovaj rezultat može da se koristi za praćenje promene u semenu usled prisustva aflatoksina. Glavni pokazatelj uticaja aflatoksina na strukturu kukuružnog semena je pomeraj dugo-talasne emisione komponente (520 nm).

Leaning stems of woody plants form reaction wood, in hardwood trees termed tension wood (TW). Typical TW fibers, gelatinous fibers (G-fibers), are characterized by an inner gelatinous cell wall layer (G-layer). Xyloglucan endotransglycosylases (XETs) was proposed as the essential enzyme in cell wall modifications in TW, by making xyloglucan (XG) cross-links between G- and S2-layers in G-fibers, and thus maintaining their contact. The etermination of TW presence in a sample is of great importance for the forest products industry, biofuel production, and tree physiology studies. However, TW is not easy to detect visually. The colorimetric assay for XET activity determination as an indicator of TW presence in a sample was tested on powdered stem segments of juvenile Populus x euramericana trees exposed to severe long-term static bending. In parallel, histochemical and ultrastructural characterization of stem samples of bent and control trees was performed. The tested colorimetric assay for XET activity determination could be suggested as a useful and easily applicable tool for fast screening of powdered wood samples for the presence of TW.

The application of enzymes for phenol removal from polluted waters is an effective and environmentally favorable method and an ongoing worldwide research topic. Oxidoreductive enzymes, like Soybean peroxidase (SBP), catalyze oxidation and polymerization of phenolic compounds in the presence of H2O2. The industrial application, however, requires enzyme immobilization on various carriers to overcome the disadvantages of using the soluble form. Chemically modified pectin has been chosen as a carrier for entrapment of SBP inside a threedimensional polymeric network. Immobilization of SBP was performed in an emulsion polymerization reaction producing enzymes entrapped in covalently crosslinked tyramine-pectin in the shape of micro-beads. The specific activity of immobilized SBP was determined using pyrogallol as a substrate. In this study, the stability of the immobilized SBP onto modified pectin in three different molar ratios was tested to determine the carrier with the best performance. Immobilized peroxidase has potential for application as a biocatalyst for phenol removal from wastewater.

Plant secondary metabolism, as well as the growth and development of specific photomorphogenic responses, can be induced by UV light. The effects of UV-A and UV-B irradiations on the antioxidant activity and fluorescence characteristics of Glycine max L. seeds were investigated for two different exposure periods (1, 3 h). The antioxidant activity and photoluminescent characteristics of irradiated seeds were evaluated by the DPPH (2,2'-diphenyl-1-picrylhydrazyl radical) assay and excitation-emission matrices (EEMs) fluorescence spectroscopy, respectively. The DPPH scavenging activity significantly increased with exposure time in the UV-A treated seeds, while variations were observed after UV-B treatment. EEM showed three main peaks characterized with excitation/emission 280/355 nm (Peak A), 290/400 nm (Peak B), and 370/465 nm (Peak C). The most prominent change in fluorescence intensity was observed in peak B. Obtained results indicate that the type of UV radiation, as well as period of exposure, induce biochemical and biophysical changes in the soybean seeds.

Leaning stems of woody plants form reaction wood, in hardwood trees termed tension wood. Tension wood contains more cellulose and less lignin and it is anatomically different from normal wood. Fourier-transform infrared (FTIR) spectroscopy showed that there was no difference in lignin content in segments of bent trees compared to the controls, but the higher amounts of cellulose, xylan, and xyloglucan were observed in bent trees, corresponding to the rearrangements in the cellulose/xyloglucan network.

For the purpose of immobilization, one of the most commonly used enzymes is horseradish peroxidase (HRP). Different carriers can be used as supports for the immobilization of HRP: alginate, pectin, magnetic-beads, macroporous copolymers, silicas etc. Covalent binding of an enzyme to the carrier leads to the formation of strong linkage, thus preventing the enzyme leakage. Macroporous copolymers with different porous characteristics were used for the immobilization of horseradish peroxidase by employing periodate and glutaraldehyde method. Five and 25 mg of HRP were immobilized per gram of the copolymer. Increasing the amount of added enzyme leads to the increase of specific activity of immobilized enzyme. Copolymer with the pore diameter of 297 nm showed the most promising results in terms of specific activity. Immobilized enzymes can be used for the removal of phenolic compounds from waste effluents.

Alginate is a natural polymer present in the cell wall of brown algae. Due to its many advantages, it has been used extensively in the food industry, pharmacy, and biomedicine. To enhance properties, such as stability and biodegradability, alginate is often chemically crosslinked. In this study, alginate was crosslinked using N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and tyramine hydrochloride. Horseradish peroxidase was self-immobilized within hydrogel microbeads during the polymerization reaction. The glucose oxidase/glucose system generates H2O2 internally, which can prevent the detrimental effect of excess peroxide. A small amount of leaking enzyme shows potential for longer storage and reuse.

Silica is beneficial element for plants. Many studies of silica show the useful effects that Si can provide on plant-environment relationships in a wide variety of crops, as increase in growth and yield and improvement of resistance to metal toxicity, salt stress, drought resistance etc. The occurrence of Si within a plant is a result of its uptake in the form of soluble Si(OH)4 or Si(OH)3O ion from the soil, and its polymerization at a final location. Si is mostly accumulated in the cell walls (CWs), thus providing strengthening of CWs, increasing mechanical strength, and alleviating effects of various biotic and abiotic types of stress. Lignin, as one of the main components of the plant CW, is a natural phenolic polymer with high molecular weight and complex structure. Accumulation of silica in plants is directly connected with chemistry and structure of lignin. Silicon affects the composition of CWs by its binding to the phenolic and carbohydrate CW components in polymerization reactions by altering linkages of non-cellulosic polymers and lignin. We studied, in an in vitro system, the interaction of SiO2 with the peroxidase catalyzed polymerization of a lignin monomer into the lignin model compound (DHP). Fluorescence microscopy and spectroscopy combined with AFM technique showed that Si was bound to the final polymer, and the structure of the Si-DHP differed from pure DHP. We show that Si significantly influence on lignin structure. The results contribute to understanding of Si binding to lignin in cell walls, which may have impact on possible Si applications in agriculture.

Varroa infestation is considered a major threat to honeybees due to its strong impact on the colony, attacking larval cells, increasing bee mortality, and serving as a vector of pathogens. Coumaphos is an organophosphate-based acaricide insecticide which is frequently used to control Varroa (Varroa destructor) mites in the honey bee (Apis melifera) colonies. The accumulation of these lipophilic contaminants in honey and beeswax may affect honey bee health, mainly honey bee larvae which are the most sensitive, and negatively affect queen quality. In this study, we analyzed honey samples from hives of honey bees that were treated with Coumaphos, as well as the samples of pure Coumafos. The front-face fluorescence measurements of analyed honey samples were recorded using an Fl3-221 P spectrofluorimeter, with a 450 W Xenon lamp. Obtained results showed an emission peak with maximum around 400 nm, after excitation at 340 nm. It could be related to the presence of Coumaphos in honey samples, which showed the characteristic position of emission maximum at the same wavelength. These results showed that fluorescence spectroscopy may be useful as a fast and sensitive method, for the efficient and specific determination of toxic substances such as Coumaphos residues in honey. Due to the proven toxic effects on honey bees, as well as on humans, monitoring hazardous contaminants in honey is of crucial importance in the field of food and agriculture.

BACKGROUND Aflatoxin B-1 (AFB(1)) is the most dangerous of the mycotoxins that contaminate cereal seeds naturally. A stress lignin formation is linked with the accumulation of reactive oxygen species causing a change in the redox status and formation of stable organic radicals, constituting the first layer of defense. The relationship between AFB(1) and changes in lignin organic free radicals in seeds is not known, nor is the part of the seed that is more targeted. Using optical and electron paramagnetic resonance spectroscopy, we investigated AFB(1)-induced changes in lignin and organic free radicals in seeds, and whether the inner and outer seed fractions differ in response to increasing AFB(1). RESULTS Different changes in the content of lignin and free radicals with increasing AFB(1) concentrations were observed in the two seed fractions. There was a significant positive linear correlation (R = 0.9923, P = 0.00005) between lignin content and AFB(1) concentration in the outer fraction, and no correlation between the lignin content and the AFB(1) concentration in the inner fraction. We found a positive correlation between the area of the green spectral emission component (C4) and the AFB(1) concentration in the outer fraction. CONCLUSIONS To the best of our knowledge, the results showed, for the first time, that maize seed fractions respond differently to aflatoxin with regard to their lignin and organic free radical content. Lignin content and (C4) area may be reliable indicators for the screening of lignin changes against AFB(1) content in the seeds, and thus for seed protection capacity.

The UV-B represents the minor fraction of the solar spectrum, while UV-C is not contained in natural solar radiation, but both radiation types can cause damaging effects in plants. Cell walls (CWs) are one of the targets for external stressors. Juvenile P. omorika trees were treated either with 21 day-high doses UV-B or with 7 day- UV-C in open-top chambers. Using spectroscopic and biochemical techniques, it was shown that the response to UV radiation includes numerous modifications in needle CW structure: relative content of xylan, xyloglucan, lignin and cellulose decreased; cellulose crystallinity changed; yield of lignin monomers with stronger connection of C[dbnd]C in side chain with the ring increased; re-distribution of inter- and intra-polymer H-bonds occurred. The recovery was mediated by an increase in the activities and changes in isoform profiles of CW bound covalent peroxidases (POD) and polyphenol oxidases (PO) (UV–B), and ionic POD and covalent PO (UV–C). A connection between activities of specific POD/PO isoforms and phenolic species (m- and p-coumaric acid, pinoresinol and cinnamic acid derivatives) was demonstrated, and supported by changes in the sRNA profile. In vivo fluorometry showed phenolics accumulation in needle epidermal CWs. These results imply transversal connections between polymers and changed mechanical properties of needle CW as a response to UV. The CW alterations enabled maintenance of physiological functions, as indicated by the preserved chlorophyll content and/or organization. The current study provides evidence that in conifers, needle CW response to both UV-B and UV-C includes biochemical modifications and structural remodeling. © 2021 Elsevier Masson SAS

In the present study, we used the Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) algorithm to analyse the excitation-emission matrices (EEM's) of various cultivars of colored maize (Zea mays L.) seeds and its fractions. The EEMs were recorded as a set, with the excitation ranging from 280 nm to 380 nm and the emission spectra ranging from 300 nm to 550 nm. The MCR-ALS analysis yielded two major fluorescence components for all of the analysed samples. Both position and shape of the component 1 (C1), varied among the samples. On the other hand, the position and shape were similar for the component 2 (C2). C1 could be used as a marker for discrimination of colored seeds and their fractions. The observed variations in C1 between the analysed seeds, based on the presence of their individual fluorophores, can be assigned to anthocyanins, proteins, and phenolics. In conclusion, the MCR-ALS analysis of the seed emission spectra have a great potential for the rapid and non-expensive characterization of various cultivars of colored seeds.

Maize is the most commonly consumed cereal in the world. Its various compounds are associated with nutraceutical properties and health-promoting benefits. Therefore, maize seeds are considered to be functional food with high antioxidant activities. The drying conditions have a huge impact on maintaining the seed quality. This study aimed to evaluate the effect of temperature on the antioxidant activities of white and red maize (Zea mays L.) cultivars. A drying time of 1 h at a temperature of 45 °C was tested. The antioxidant activity expressed in percentages was evaluated as radical scavenging activity using the DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent. The results revealed that the antioxidant activity of the dried white seeds (74.08 %) was not significantly different (p>0.005) with respect to the un-dried seeds used as control (72.6 %). Contrary, drying at 45 °C caused a significant (p<0.005) rise of antioxidant activity in the red-coloured seeds' being 85.95 % and 92.25 % before and after the treatment, respectively. The obtained results show that the antioxidant activity may be a reliable indicator for the estimation of the cereal seeds’ quality, which is useful in the food industry and agriculture.

This paper investigates the effects of different treatment methods of rubber aggregates for self-compacting concrete (SCC) on compressive strength and modulus of elasticity. SCC mixtures with 10% replacement of fine aggregate with crumb rubber by total aggregate volume and with different aggregate treatment methods were investigated. The rubber aggregate was treated in three different methods: dry process, water-soaking, and NaOH treatment plus water soaking. Properties of SCC in a fresh and hardened state were tested and evaluated. Scanning electron microscope (SEM) analysis of three different SCC patches were made and discussed. It was observed that applying the proposed NaOH plus water soaking method resulted in the improvement of fresh and hardened concrete properties. It resulted in a more uniform distribution of rubber particles in the cement matrix, a better bond between rubber particles and the cement matrix, and higher compressive strength of SCC rubberized concrete.

UV-B light, plays a crucial role as a signal for inducing plant response and development of specific photomorphogenic responses. The UV radiation may have a damaging effect on cellular components and macromolecules in seeds, which may influence seed quality. We compared Glycine max L. seeds exposed to UV-B radiation for 1 h with non-irradiated seeds (control). The antioxidant activity was estimated using a DPPH assay. The seeds’ fluorescence characteristics were studied by fluorescence excitation-emission matrices. According to the obtained results the UV-B irradiated seeds possess a significantly higher free radical scavenging activity compared to the control. The fluorescence analysis showed differences in the spectral emission profiles of irradiated seeds compared to the control seeds. The results imply that 1h seed exposure to UV-B increases free radical content which may alter the structures of cellular macromolecules resulting in degradation of some of the fluorophores.

Enzyme immobilization onto support carriers is a method used to overcome some of the limitations of soluble enzymes in practical applications. Various different materials have been used as carriers, such as inorganic matrixes, natural and synthetic polymers. Production of carriers from natural biopolymers and their derivatives are the focus of numerous research worldwide, and a summary of their applications for enzyme immobilization is presented in this paper. Enzymes or cells are entrapped inside a three-dimensional polymeric network that is able to retain water in large amounts, called hydrogel. This network could be formed by chemical cross-linking, ionotropic gelling in the presence of cation or in thermo reverse polymerization, depending on the polymer in use and its characteristics. Most frequently used biopolymers as carriers for immobilization include alginate, cellulose, chitosan, collagen, xylan, pectin, starch, and others.

The quality of cereal seeds could be altered by the different temperature conditions. In this study, the influence of temperature on the antioxidant activities and the fluorescence characteristics of various cultivars of colored maize (Zea mays L.) seeds were estimated. For that purpose, the seeds were exposed to different temperatures (25°C (Control), 45°C, and 90°C), for 60 minutes. The antioxidant activities of the various colored seeds have been determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent. Our results showed that a rise in temperature caused an increase in the antioxidant activities in the yellow, light- and dark-red colored seeds. This was more pronounced in the dark-red colored seeds. However, no statistically significant differences were found in the antioxidant activities of the white-colored seeds at the different temperatures. The fluorescence analysis indicates differences in emission spectral parameters among the analyzed seed types and effect of various temperature conditions. Both methods have proven to be useful for monitoring changes caused by temperature treatment of the seeds but could also be applied for characterization and quality control of seeds after different types of treatments.

Legumes have multiple functions in sustainable agriculture, but also are a favourable ingredient of functional nutrition. Antioxidants in legumes have several beneficial physiological properties and provide protection against chronic diseases. In this study, we compared the antioxidant activities of the seed fractions (outer and inner) for two different legumes: mung bean (Vigna radiata L.) and soybean (Glycine max L). The antioxidant activity was estimated using a modified DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay in a 96-well microplate. We showed that mung bean hulls possessed significantly higher (p<0.05) DPPH free radical scavenging activity (80.80 % ± 0.19) compared to their inner fraction (10.94 % ± 0.23), as well as to both fractions of the soybean. On the other hand, the soybean seeds' inner fractions (21.00 % ± 0.32) exhibited a significantly (p<0.05) higher activity than the hulls (8.78 % ± 0.71) and the inner fraction of the mung bean. The obtained results indicated that in each of the two analysed legume species, inner and outer seed fractions exhibited different antioxidant activities regarding to the elimination of the free radicals. The obtained results indicate that antioxidant capacity may be a useful indicator in the estimation of the quality of legume seeds as food and feed.

Hemicellulose rich in xylan was isolated from corncobs. Functional groups were introduced first by carboxymethylation and then by coupling with tyramine via carbodiimide-mediated reaction. Obtained hydrogel was tested for horseradish peroxidase (HRP) immobilization within microbeads, formed in an emulsion based enzymatic polymerization reaction. This model system showed that modified hemicellulose isolated from corncob is a suitable candidate for enzyme or small molecule immobilization for different purposes.

INTRODUCTION: Alginate dressings are widely used in the treatment of exuding wounds29. The enzymatically synthesized lignin model compound dehydrogenate polymer (DHP) from coniferyl alcohol by the enzyme peroxidase, is the best lignin substitute used in various experiments30. In our previous work, we have shown that synthesized DHP has antibacterial and antibiofilm properties, and in combination with alginate has good potential for wound treatment31. OBJECTIVES: The objective of this paper was to study the sustained release of DHP from low and medium viscosity alginate beads.

Derivatives of xylans were synthesized from corncob xylan by carboxymethylation, oxidization with different molar ratios of periodate (5, 10 15 and 20 mol%) and by reductive amination with tyramine. Modifications of tyramine carboxymethyl xylans (Tyr-CMX) were confirmed by FTIR, UV and NMR spectra. Concentration of ionizable groups increased from 1.5 mmol/g for carboxymethyl xylan (CMX) to 5.4 mmol/g for Tyr-CMX oxidized with 20 mol% of periodate. All Tyr-CMXs were able to form hydrogels the cross-linking reaction with horseradish peroxidase and peroxide. Tyr-CMXs were tested for amyloglucosidase (AG) encapsulation within hydrogel microbeads obtained in a reaction of emulsion polymerization with peroxidase. Average diameter of Tyr-CMX hydrogel microbeads was 52±25 µm and after encapsulation optimization with respect to the extent of CMX modification with tyramine, the concentration of Tyr-CMX, and the amount of added AG, microbeads with AG specific activity of 2 U/mL and 20% yield of immobilization were obtained. The optimum pH of the immobilized AG was not changed compared to the soluble one, while half-life at 60 °C was increased around 10 times. The Michaelis-Menten constant for the immobilized enzyme, 1.03 mM, was significantly lower than that for the soluble one, 1.54 mM. After 5 cycles of repetitive use in batch reactor, the immobilized AG retained 68% of initial activity.

Horseradish peroxidase (HRP, E.C. 1.11.1.7) catalyzes oxidation of aqueous aromatic compounds using hydrogen peroxide. Enzymatic treatment methods for phenol removal from wastewaters has become an efficient and environmentally friendly alternative for the traditional methods. Carboxymethyl cellulose (CMC) derivative with tyramine attached via amide bond to carboxyl groups has been chosen as carrier for immobilization. In effort to overcome the main disadvantage of entrapment immobilization method, loss of enzyme activity due to washing out from the carrier, HRP was modified in a reductive amination reaction and tyramine was bound to the enzyme. Immobilization of tyramine-HRP onto tyramide-carboxymethyl cellulose carrier was carried in an emulsion polymerization reaction that produced carboxymethyl cellulose microbeads. The highest specific activity of the obtained biocatalyst was 0.227 U/ml and after 48h of storage 0.197 U/ml. Immobilized tyramine-HRP retained 87% of activity after 48 h. Immobilized HRP is a suitable candidate for wastewater treatment.

Steady state fluorescence spectroscopy in combination with Principal Component Analysis (PCA) for spectral analysis was used to differentiate multifloral honeys from different parts of Serbia. The emission spectra were recorded in the wavelength range 280 – 550 nm, after excitation in the 270 – 370 nm range. After normalization of the spectra, chemometric evaluation of the spectral data was carried out using principal component analysis (PCA). This study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of geographical origin of honey and may also be useful for determination of the botanical origin within the same unifloral honey type.

Phenolic compounds are one of the most common pollutants in aqueous systems, so their removal from water is of major interest. Among biocatalysts used for phenol removal, horseradish peroxidase is the most investigated for this purpose. Enzyme inactivation is a major problem which could be successfully overcome by immobilization of the enzyme onto different polymers. Tyramine-alginate micro-beads were tested for the immobilization of horseradish peroxidase. Different concentrations of tyramine-alginate were used and their influence on specific activity of the enzyme was tested. Increasing concentration of oxidized alginate results in increase of specific activity. Immobilized HRP was tested for phenol removal in a batch reactor. Presented results were obtained with HRP immobilized within 10 mol% tyramine-alginate micro-beads. These biocatalysts can be used up to three cycles.

Phenols are hazardous substances that enter into the environment as byproducts of various industries. The immobilizate of aminated horseradish peroxidase (HRP) onto oxidized alginate beads was tested for reusability in the removal of aqueous phenol. In the present study, the possibility of continuous phenol removal using immobilized aminated HRP on alginate beads was shown to be promising. After five repeated cycles, the efficiency of HRP immobilizate for phenol removal has decreased by 46%

Pectin is a natural biopolymer contained in the plant cell wall. Ability of pectin to form hydrogels has been widely investigated for various purposes from food industry to biomedical applications. The goal of our study was to create modified pectin with improved gelling properties, that could produce stable covalently cross-linked hydrogels. Aromatic groups were introduced into the previously oxidized pectin chains in a reductive amination reaction with tyramine and cross-linking was achieved through enzyme reaction with soybean peroxidase in the presence of hydrogen peroxide. Characterization confirmed that a series of obtained tyramine-pectins (oxidized from 2.5 to 20 mol%) had different degrees of modification. UV spectra confirmed the presence of tyramine group with the absorbance peak at 275 nm. Chemical shifts of modified pectin in 1 H NMR spectra correspond to newly formed functional groups. The presence of C-C in-ring stretching vibrations peaks at 1518 and 1417 cm-1 in FTIR spectra of modified pectin confirmed the presence of aromatic rings. Increased nitrogen percentage in elemental analysis and additional ionizable group on the titration curve of tyramine-pectin proved the introduction of positively charged amino group. Surface morphologies also showed certain differences under SEM-EDS. Tyramine-pectin has been successfully tested as a carrier for enzyme immobilization, but potential applications of this material could be in tissue engineering, drug delivery or wound healing.

Removal of phenolic compounds from wastewaters was previously studied using different enzymatic approaches. In the presence of hydrogen peroxide, peroxidases are able to oxidize phenol-like compounds and form non-soluble polymers that could be easily removed from aqueous phase. Horseradish peroxidase (HRP) is the most investigated peroxidase used for phenol removal from waste effluents, but it can be easily inactivated during this process by excess of hydrogen peroxide. In order to increase operational stability of the enzyme, immobilization on different materials and various peroxide delivery systems were tested. In our previous work, we studied bioinspired hydrogels based on natural cell wall polymers and enzymes, for efficient removal of phenols from water. In this work, tyramine-alginate hydrogels that we have previously developed were used for horseradish peroxidase encapsulation within micro-beads obtained in a coupled emulsion polymerization reaction. The aim of this research was to study the influence of tyramine-alginate concentration and hydrogen peroxide delivery system on operational stability and efficiency of phenol removal by immobilized peroxidase. The best result of 96% phenol removal from water solution was achieved by peroxidase immobilized within 20% (w/v) tyramine-alginate micro-beads using delivery system for hydrogen peroxide composed of 0.187 U mL-1 of glucose oxidase and 4 mmol L-1 of glucose. The reusability studies showed that these biocatalysts can be used up to five cycles with slight decrease in their catalytic performance.

Peroksidaza iz soje (SBP, E.C. 1.11.1.7) je enzim koji pripada klasi III biljnih peroksidaza i katalizuje oksidaciju i polimerizaciju različitih fenolnih jedinjenja u prisustvu vodonik peroksida kao supstrata. Nalazi se u semenom omotaču soje koji predstavlja jeftini nusproizvod i polazni materijal za izolovanje velike količine sirovog enzima.Izolovana SBP je imobilizovana primenom dve kovalentne metode na seriju makroporoznih kopolimera poli(GMA-co-EGDMA) različitih dijametara pora. Glutaraldehidni metod imobilizacije se pokazao boljim od perjodatnog a najveća specifična aktivnost od 23 IU/g dobijena je na kopolimeru veličine pora od 120 nm. Imobilizovana peroksidaza pokazala je veću termalnu stabilnost i stabilnost u organskom rastvaraču, aktivnost u širem opsegu pH i 2,86 puta veću Km vrednost za pirogalol u odnosu na rastvorni enzim.Sintetisana je serija modifikovanih pektina oksidacijom perjodatom u molarnom odnosu od 2,5 do 20 mol% i naknadnom reduktivnom aminacijom tiraminom i cijanoborhidridom. Dobijeni tiramin-pektini prave stabilne hidrogelove umrežavanjem fenolnih grupa u reakciji katalizovanoj peroksidazama u prisustvu vodonik peroksida. SBP je uspešno imobilizovana unutar mikro-kuglica hidrogela nastalih enzimskom polimerizacijom modifikovanog pektina u emulziji sa najvećom specifičnom aktivnošću postignutom na tiramin-pektinu oksidovanom 5 mol% perjodatom. Imobilizovana SBP je pored šireg pH optimuma pokazala i veću termalnu stabilnost i stabilnost u organskom rastvaraču u odnosu na slobodan enzim. Mikrokuglice sa imobilizovanom SBP zadržale su 50% početne aktivnosti nakon 7 ciklusa ponovne upotrebe za oksidaciju pirogalola u bač reaktoru.Pod optimalnim uslovima sa unutrašnjom dostavom vodonik peroksida postignuto je uklanjanje fenola od 64% imobilizovanom SBP na poli(GMA-co-EGDMA) veličine pora od 120 nm., Soybean peroxidase (SBP, E.C. 1.11.1.7) is an enzyme that belongs to class III of plant peroxidases that catalyses oxidation and polymerization of various phenolic compounds in the presence of hydrogen peroxide as substrate. It's located in the soybean seed hulls, an inexpensive byproduct and excellent material for isolation of large amounts of crude enzyme.SBP was immobilized by using two covalent methods onto a series of macroporous copolymers of poly(GMA-co-EGDMA) with various pore diameters. Glutaraldehyde immobilization method proved to be better than periodate with highest specific activity of 23 IU/g obtained with copolymer with pore size of 120 nm. Compared to the soluble enzyme, immobilized peroxidase showed increased thermal and organic solvent stability, broader pH activity range and 2.86 times higher Km value for pyrogallol.Series of modified pectins have been synthesized by oxidation with periodate in molar ratios from 2.5 to 20 mol% and subsequent reductive amination with tyramine and cyanobohrhydride. Obtained tyramnine-pectine made stable hydrogels by crosslinking phenol groups in a peroxidase catalysed reaction in the presence of hydrogen peroxide. SBP was immobilized within hydrogel microbeads created in enzyme polymerization of modified pectin in emulsion with highest specific activity achieved with tyramine-pectin oxidized by 5 mol% of periodate. Immobilized SBP in addition of having wider pH optimum showed higher thermal and organic solvent stability compared to the free enzyme. SBP bound in microbeads retained 50% of the original activity after 7 cycles of repeated usage for pyrogallol oxidation in batch reactor.Under optimal conditions with internal hydrogen peroxide delivery phenol removal of 64% has been achieved with SBP immobilized onto poly(GMA-co-EGDMA) with pore size of 120 nm.

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 mu mol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K (m) value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

Toxic aromatic pollutants that are found in various industrial wastewaters pose a serious environmental threat. Current methods for phenol removal have certain disadvantages, such as low efficiency, high cost or generation of even more toxic products. On the other hand enzyme-based treatments are highly selective and efficient. Soybean hull peroxidase (SHP) as well as other class III peroxidases catalyzes oxidation reaction in the presence of hydrogen peroxide, resulting in phenol polymerization and formation of less hazardous phenolic polymers. As a by-product of the food industry, soybean hulls are inexpensive and readily available source of large quantities of crude peroxidase. The aim of our research was to isolate SHP from soybean hulls and immobilize it onto a glycidyl methacrylate based carriers using glutaraldehyde method and characterize the resulting product. Immobilized SHP showed dependence upon the pore size of the carrier matrix,with the highest obtained specific activity of 22.8 U g-1 of carrier. Immobilized enzyme proved as an effective phenol removal alternative method with improved thermal and organic solvent stabilities compared to the free form. It also showed greater stability and tolerance to pH fluctuations, showing higher specific activities over a wider pH range. Operational stability was tested by repeated pyrogallol oxidation cycles in a batch reactor. After three cycles, immobilized SHP retained over 60% of the initial activity.