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Trypsin is a serine protease with important applications such as protein sequencing and tissue dissociation. Preserving protein structure and its activity during freeze-thawing and prolonging its shelf life is one of the most interesting tasks in biochemistry. In the present study, trypsin cryoprotection was achieved by altering buffer composition. Sodium phosphate buffer at pH 8.0 led to pH shift-induced destabilization of trypsin and formation of a molten globule, followed by significant activity loss (about 70%). Potassium phosphate and ammonium bicarbonate buffers at pH 8.0 were used with up to 90% activity recovery rate after 7 freeze-thaw cycles. The addition of non-ionic surfactants Tween 20 and Tween 80 led to up to 99% activity recovery rate. Amide I region changes, corresponding to specific secondary structures in the Fourier transform infrared (FTIR) spectrum, were modest in the case of Tween 20 and Tween 80. On the other hand, the addition of Triton X-100 led to the destabilization of α-helicoidal segments of trypsin structure after 7 freeze-thaw cycles but also increased protein substrate availability.

Papain is a proteolytic enzyme of great commercial value. It is a cysteine protease highly expressed in Carica papaya fruit latex, but also present in papaya leaves. Purification procedures mostly deal with the latex and include a combination of precipitation and/or chromatographic techniques. Due to its solubility, structure and activity characteristics, the pH and salt content play significant roles in handling papain extracts. Here we report a simple, rapid and easily scalable procedure for papain purification from papaya leaves, which contain different contaminants as compared to papaya latex. Sodium chloride precipitation of contaminants at pH 5 followed by ammonium sulphate precipitation resulted in the removal of other leaf proteins and protein fragments from papain solution and about a 3-fold purification. The procedure also benefits from the suppression of autoproteolysis and preservation of the native structure, as confirmed by FTIR analysis, and the high recovery of activity of over 80%.

Latex of common fig (Ficus carica) is a rich protein source with a high level of proteolytic activity contributing to its defensive role. The divergent group of cysteine proteases known as ficin (EC 3.4.22.3) represents the majority of latex protein content and shows activity towards fig parasites. Both classical and novel biochemical techniques suggest the intricate pattern of ficin expression and activity profiles. Even though structurally related, different ficin isoforms show some differences in pI values enabling their separation using ion-exchangers. A single alkaline isoform was purified and identified based on the available transcriptomic data as Ficin 1c. This isoform shows both general proteolytic and gelatinolytic activity suggesting a biological role in the degradation of a broad range of natural substrates. The insight into the Ficin 1c structure also provided some functional clues. The secondary structure content and the overall fold are similar to related proteases of the same and other plant sources resulting in similar unfolding routes. Stability assessment of Ficin 1c in comparison to ficin isoform mixture showed that isoform diversity might lead to increased protease stability. This is the peer-reviewed version of the article: Milošević, J.; Vrhovac, L.; Đurković, F.; Janković, B.; Malkov, S.; Lah, J.; Polović, N. Đ. Isolation, Identification, and Stability of Ficin 1c Isoform from Fig Latex. New J. Chem. 2020, 44 (36), 15716–15723. [https://doi.org/10.1039/D0NJ02938F]

Previously, the cytotoxic actions of five Pd(II) complexes with bidentate N-heteroaromatic chelators (complexes 1–5) on a palette of several cancer cell lines were investigated. However, the results of the cytotoxic activity did not correlate with the hydrophobic character of the complexes. To gain further insight into the structure–activity relationship, essential for the design of novel potential drugs, other factors, such as non-specific interactions with cellular proteins, have to be taken into account. To explore the potential non-specific influence of the complexes on protein structures, ovalbumin (OVA) was chosen as a model system to mimic cellular non-specific crowding environments with high protein concentrations. A Fourier-transform infrared spectroscopy study implied that the binding of 3 and 4 led to only moderate alternations in the secondary structures of the protein, without the possibility to penetrate into hydrophobic core of the protein and disruption of protein native fold. Contrary, the effect of complex 5 on OVA secondary structures was concentration-dependent. While the lower concentration of complex 5 had no effect on OVA structure, a doubled concentration of complex 5 led to complete disruption of the content native-like secondary structures. The concentration-dependent effect of complex 5 on the changes in secondary structures and considerable increase in the exposure of OVA hydrophobic surfaces to water may be related to a potential crosslinking that leads to OVA aggregation. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/5707]

Burkholderia cepacia is well known as the causative agent of infections in humans where often shares niche with other pathogens, like Pseudomonas aeruginosa. Clinical isolate Burkholderia sp. BCC4135 was selected due to its strong quorum quenching (QQ) activity. Whole genome sequencing unveiled this isolate as B. cepacia with unique sequence type ST1485 and a myriad of genes belonging to resistome and virulome. Two QQ lactonases YtnP and Y2-aiiA originated from B. cepacia BCC4135 were cloned, expressed, and functionally characterized. They were active against a broad substrate spectrum of the N-acyl-homoserine lactones (AHLs). The YtnP lactonase was inactive, while Y2-aiiA was active against N-tetradecanoyl-dl-homoserine lactone (C14-HSL) which could imply the difference in their biological roles from the aspect of its quorum sensing (QS) autoregulation and interference with the QS systems of bacteria residing within the same niche. Both YtnP and Y2-aiiA were able to attenuate virulence potential of P. aeruginosa MMA83 clinical isolate declining its biofilm formation and virulence factors production. B. cepacia BCC4135 lactonases interfered with the las, rhl, and even pqs QS circuit of P. aeruginosa MMA83 transcription and the effect of combined enzymes was even more prominent. B. cepacia BCC4135 also employs the CepI/R QS system for governing its own virulence traits and possibly self-regulates the QQ/QS network through the different expression and activity of YtnP and/or Y2-aiiA. Our findings pointed out that BCC4135 lactonases could be exploited as an effective antivirulence drugs against P. aeruginosa and gave us a new insight into B. cepacia QQ/QS machinery.

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4091]

Amyloid fibrils are highly ordered self-assembled (poly)peptide aggregates with cross-β structural pattern. Ovalbumin was used as a model for exploring the potential of infrared spectroscopy in detecting structural transitions and quantitative monitoring of amyloid fibrillation. Low pH (pH 2) and high temperature (90 °C) over the course of 24 h were conditions applied for amyloid formation. Fibrillation of ovalbumin was monitored by ThT and ANS fluorescence, and SDS PAGE. A significant increase in ThT fluorescence with a plateau reached after 4 h of incubation, without the lag phase, was detected. Structural transitions leading to amyloid fibrillation were analysed using all three Amide regions in ATR-FTIR spectra. Significant changes were detected in Amide I and Amide III region (decrease of α-helix and increase of β-sheet peaks). To establish a fast, precise and simple method for quantitative monitoring of amyloid fibrillation, the Amide I/Amide II ratios of aggregation specific β-sheets (1625 and 1695 cm−1, respectively) with 1540 cm−1 as internal standard were used, resulting in good correlation (R2 = 0.93 and 0.95) with the data observed by monitoring ThT fluorescence. On the other hand, assessing aggregation specific β-sheet contents by self-deconvolution showed lower correlation with ThT fluorescence (R2 = 0.75 and 0.64). Here we examined structural transitions during ovalbumin fibrillation in a qualitative and quantitative manner by exploiting the full potential of Amide regions simultaneously. Secondary structure distribution was monitored using second derivative spectra in Amide I region. A novel, simple mathematical calculation for quantitative monitoring of fibrils formation was presented employing that the increase in low and high frequency aggregation specific β-sheet in Amide I region compared to the internal standard in Amide II region is suitable for fibril formation monitoring. This is the peer-reviewed version of the article: (1) Milošević, J.; Petrić, J.; Jovčić, B.; Janković, B.; Polović, N. Exploring the Potential of Infrared Spectroscopy in Qualitative and Quantitative Monitoring of Ovalbumin Amyloid Fibrillation. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2020, 229. [https://doi.org/10.1016/j.saa.2019.117882] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3894]

Pseudomonas aeruginosa is one of the most common opportunistic pathogens that use quorum sensing (QS) system to regulate virulence factors expression and biofilm development. Delftia sp. 11304 was selected among 663 Gram-negative clinical isolates based on its QS inhibitory activity against P. aeruginosa MMA83 clinical isolate. Whole genome sequencing identified this isolate as D. tsuruhatensis and revealed genetic armamentarium of virulence factors and antibiotic resistance determinants. Ethyl acetate extract of D. tsuruhatensis 11304 culture supernatant (QSI extract) prevented biofilm formation of P. aeruginosa MMA83, but was unable to cause biofilm decomposition. QSI extract showed a synergistic effect in combination with meropenem and gentamycin, against P. aeruginosa MMA83. A dose-dependent reduction of the virulence factors: elastase, rhamnolipid and pyocyanin production by P. aeruginosa MMA83 and significant downregulation of lasI, lasR, rhlI, rhlR, pqs and mvfR expression were observed. Matrix-assisted Laser Desorption Ionization (MALDI) mass spectrometry of D. tsuruhatensis 11304 QSI extract revealed the presence of N-acyl homoserine lactones (AHL) with chain lengths of C12 to C18. The main ion peak was identified as N-octadecanoylhomoserine lactone (C18-HSL). Commercial C18-HSL (20 µM) reduced pyocyanin production as well as mRNA level of the lasI gene. A novel AHL species, dihydroxy-N-octadecanoylhomoserine lactone, was also described.

Proteolytic enzymes are used for proteolysis and peptide synthesis which can be run in various conditions including low pH value and the presence of ethanol. The most common cysteine protease applied in acidic-alcoholic conditions is well-characterized papain. Ficin, which is closely related to papain in terms of proteolytic activity and substrate specificity, could potentially be applied in the alcoholic beverage industry and peptide synthesis. The aim of this study was to compare papain and ficin stability in process conditions. Comparative stability study showed that ficin as a mixture of different isoforms has a broader range of stability in respect of pH and cold storage stability, in comparison to papain. It retains about 70% of initial activity after 3-week cold storage at low pH and in the presence of ethanol. Unlike ficin, papain loses about 70% of initial activity in the same incubation period as it is more prone to non-native aggregation that was confirmed by FTIR analysis. The presence of multiple isoforms of ficin stabilizes the protease against cold denaturation and aggregation, making it more suitable for biotechnological and laboratory usage than single papain isoform. It is more cold-stable in alcoholic-acidic and acidic conditions suggesting possible replacement of papain with even lower enzyme concentration. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3933]

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.

Pectins are a group of heterologous polysaccharides capable of forming hydrogels and applicable in many industrial processes. A new type of modified pectin was synthesized by periodate oxidation and reductive amination with dopamine and sodium cyanoborohydride. The success of modification was confirmed by UV–Vis,FTIR, and 1H NMR spectroscopy. The obtained dopamine-pectin could form hydrogels by ionic crosslinking of carboxyl groups with calcium or by crosslinking phenol groups with laccase. For enzymatic crosslinking with laccase from Streptomyces cyaneus expressed in E. coli, isolation and purification of the enzyme was done. Using emulsion-based enzymatic crosslinking polymerization, dopamine-pectin microbeads with immobilized laccase were made. The immobilized laccase showed improved thermal and pH stability in comparison to the free enzyme. The immobilized biocatalyst effectively decolorized various dyes: Amido Black 10B, Reactive Black 5, and Evans Blue. After ten cycles of repeated use, the microbead immobilized laccase could still decolorize 60% and 36% of Amido Black 10B and Reactive Black 5, respectively. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4494]

Removal of phenolic compounds from water is of major interest over the years, since they are one of the most common pollutants in aqueous systems. Horseradish peroxidase (HRP) is the most investigated biocatalyst for this purpose. Inactivation of the enzyme is a major issue which can be successfully overcome by the enzyme immobilization on different polymers. In this study, tyramine-alginate micro-beads were used as carriers for the immobilization of horseradish peroxidase. The effect of the oxidation degree of tyramine-alginates on a specific activity of the enzyme was tested. An increase in the concentration of oxidized alginate from 2.5 to 20% resulted in a gradual increase in the specific activity from 0.05 to 0.67 U/mL. HRP immobilized within these micro-beads was tested for the phenol removal in a batch reactor. Reaction conditions were optimized to achieve a high removal efficiency and substantial reusability of the system. In this study, for the first time, an internal generation of hydrogen peroxide from glucose and glucose oxidase was employed in the phenol removal process with HRP immobilized on tyramine-alginate. Within 6 h of repeated use 96% of phenol was removed when the system for internal delivery of H2O2, composed of 0.187 U/mL of glucose oxidase and 4 mmol/L of glucose was employed. A common straightforward addition of hydrogen peroxide provided the removal efficiency of only 42%, under the same reaction conditions. The highest efficiency of the phenol removal (96%) was obtained with HRP immobilized within 20 mol% oxidized tyramine-alginate micro-beads. Fifteen mol% oxidized tyramine-alginate showed lower removal efficiency in the first cycle of use (73%) but more promising reusability, since the immobilized enzyme retained 61% of its initial activity even after four consecutive cycles of use.

BACKGROUND The physiological role of fig latex is to protect the plant from pathogens. Latex is a rich source of proteases, predominantly ficin. Fig latex also contains collagenolytic protease and chitinolytic enzymes. Our aim was to investigate changes in protein composition, enzyme and antifungal activities of fig latex during fruit ripening. RESULTS Comparison of latex samples in different time periods showed a uniform increase of protein concentration in chronological order. The content of collagenolytic protease did not differ significantly in the latex samples, while the content of ficin decreased. Ficin-specific activity towards casein was the highest at the beginning of fruit development (about 80 U mg−1). Specific milk clotting activity increased as well as the abundance of casein band in the clots. Specific chitinolytic activity at the beginning of flowering was 6.5 times higher than the activity in the period when fruits are ripe. Antifungal activity is the most extensive in spring. CONCLUSION Ficin forms with different casein specificities are present in different proportions during fruit ripening, which is of importance for applications in the dairy industry. The protection mechanism against insects and fungi, which relies on chitinolytic activity, is the most important in the early phases of flowering and is replaced with other strategies over time. © 2015 Society of Chemical Industry

Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A.

Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes , Staphylococcus aureus , Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C 18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti , a bacteriocin with analogy to aureocin A. IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes , Staphylococcus aureus , Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers.

Collagenolytic proteases have many potential applications in different areas of science, industry, and medicine. The determination of the activity of such proteins is paramount to their application. Here, we describe methods which can be applied to determine the activity and some basic characteristics of potential collagenases. © 2017, Springer Science+Business Media LLC.

Novel metal-based compounds with therapeutic potential have become the subject of intense investigations in inorganic chemistry and biomedical science. Recently, strong dose-dependent cytotoxic activities of selenosemicarbazone metal complexes against several human cancer cell lines were demonstrated. The aim of the present study was to investigate in vitro antioxidative potential of Ni(II), Cd(II) and Zn(II) selenosemicarbazone complexes. All three investigated complexes exhibited high 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonic acid) radical cation (ABTS●+) scavenging capacity, comparable with ascorbic acid. In an acute toxicity study, administration of the compounds was performed orally to mice at single doses. The mice were observed for clinical signs, body weight effects and mortality for 14 days, after which they were sacrificed for gross organ necropsy. The body weight did not vary after administration, and the autoptic analysis failed to show appreciable macroscopic alterations of internal organs. Generally, the compounds exhibited low toxic effects as required for further in vivo therapeutic studies. Nova jedinjenja na bazi metala sa terapeutskim dejstvom postala su predmet istraživanja u neorganskoj hemiji i biomedicinskim naukama. Pokazana je jaka dozno-zavisna citotoksična aktivnost kompleksa selenosemikarbazona na većem broju ćelijskih linija humanih ćelija kancera. Cilj rada je ispitivanje in vitro antioksidativnog dejstva selenosemikarbazonskih kompleksa nikla, cinka i kadmijuma. Sva tri ispitivana kompleksa pokazala su jaku antioksidativnu aktivnost prema ABTS radikalu, uporedivu sa aktivnošću askorbinske kiseline. Tokom ispitivanja akutne toksičnosti, jedinjenja su oralno davana miševima u pojedinačnim dozama i praćeni su klinički znaci, težina tela i mortalitet nakon 14 dana, a potom su životinje žrtvovane radi autopsije organa. Težina tela nije varirala nakon aplikacije. Generalno, jedinjenja su pokazala mali toksični efekat što i zahteva buduće in vivo terapeutsko ispitivanje.

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. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3053]

Trypsin is a serine protease with widespread applications, including protein sequencing and typsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze-thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in beta-sheet content (band maximum detected at a frequency of 1634 cm in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of ayoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery ( gt 90%) and modest autolysis ( lt 10%). High activity recovery ( gt 90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life. (C) 2015 Elsevier B.V. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3586]

Food allergens must exhibit sufficient gastrointestinal stability to reach the intestinal mucosa where absorption and sensitization can occur. Therefore, investigation of protein stability within the gastrointestinal tract may provide a prospective test for the allergenic potential of novel proteins. The aim of this work was to examine the effect of the fruit matrix and purified pectin on the digestion in vivo of kiwifruit allergens in the rat gastrointestinal system. The major kiwi allergen, Act d 2, was quantified in several compartments of the gastrointestinal tract by a monoclonal antibody-based ELISA. Protein intactness was demonstrated by immunoblot. Under conditions of complex food digestion in vivo, a pepsin-labile protein survived passage from the stomach to the caecum during a 3-h period. Decay of Act d 2 in the rat gut exhibited an exponential pattern. Ingestion of kiwifruit was followed by a decrease in both total and specific pepsin activity. When purified, Act d 2 allergen was consumed together with pure apple pectin; both gastric acidity as well as specific and total pepsin activity declined and thus protected 23% of the ingested allergen from digestion for 90 min. In conclusion, ingestion of pectin-rich fruits and particularly pectin supplements may have a protective action on pepsin-labile allergens and prolong their survival in both gastric and duodenal juices, enabling efficient uptake and presentation to the immune system. © Springer Science+Business Media, LLC 2010.

Allergen specific immunotherapy, comprised of subcutanoues injections of increasing doses of allergen extracts, has been shown to be the only treatment able to influence the natural progression of allergic disease. Different forms of local immunotherapies, involving oral, sublingual and nasal routes of allergen adminstration, have also been considered in clinical practice. The inability of the protein to survive gastrointestinal physiological barriers is a generally encountered problem in oral administration of protein drugs. In order to overcome the problems of low allergen bioavailability and absorptivity, during oral immunotherapy, several stabilization strategies have been outlined in the recent years. This review focuses on interventions including: hexose monosaccharide, ethyl alcohol and water vehicles, oxygen-containing metal salt based preparations, particles with enteric coating, and poly (lactic-co-glycolic) acid microspheres. Regarding the enormous potential of oral responsiveness and/or oral tolerance, research that focuses on new and improved carriers or vehicles for safe allergen oral delivery has great potential in treating allergic diseases. This article also review some of the recent patent related to the field. © 2008 Bentham Science Publishers Ltd.

Background: Endotoxins, comprised of bacterial cell wall lipopolysaccharides (LPS), have been reported to have both protective and exacerbating effects on the development and maintenance of allergic disease in humans and on markers of allergic inflammation in animal models of allergy. In this study, we investigated the effect of low concentrations of LPS on human peripheral blood mononuclear cells (PBMC) stimulated with the major cat allergen Fel d 1. Methods: Extensive purification of recombinant (r) Fel d 1 yielded essentially endotoxin-free rFel d 1 (0.2 ng LPS /mg protein). PBMCs prepared from 15 subjects having IgE to cat (>0.7 kUA/l) and 8 subjects IgE negative to cat were stimulated with 2, 10 or 25 µg/ml of rFel d 1 in the presence or absence of 50 pg/ml LPS. Proliferation was measured after 7 days of culture and supernatants were analyzed for IFNγ, IL-5 and IL-10. Results: LPS (50 pg/ml) increased rFel d 1-stimulated proliferation of PBMCs both from subjects IgE-positive and subjects negative to cat allergens. PBMCs from 13 of the subjects did not proliferate in response to stimulation with 2 and 10 µg/ml rFel d 1 alone but did so in the presence of LPS. Moreover, LPS increased the levels of rFel d 1-stimulated IFNγ in cultures from cat-negative subjects, IL-5 from cat-positive subjects and IL-10 from both groups. Conclusion: Very low doses of LPS enhance proliferation and decrease the apparent threshold level for cell activation, prompting careful evaluation of allergen stimulated T cell activation in vitro.

Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 degrees C to 60 degrees C with Delta G degrees(321) of 13.9 +/- 0.3 kJ/mol and T-m value of 84 +/- 1 degrees C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular beta-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry. This is the peer-reviewed version of the following article: Rašković, B.; Popović, M.; Ostojić, S.; Ancrossed D Signelković, B.; Tešević, V.; Polović, N. Fourier Transform Infrared Spectroscopy Provides an Evidence of Papain Denaturation and Aggregation during Cold Storage. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2015, 150, 238–246. [https://doi.org/10.1016/j.saa.2015.05.061]

Chemically modified allergens (allergoids) have found use in both traditional and novel forms of immunotherapy of allergic disorders. Novel forms of immunotherapy include local allergen delivery, via the gastrointestinal tract. This study conveys the gastrointestinal stability of three types of mugwort pollen allergoids under simulated conditions of the gut. Allergoids of the pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, succinic and maleic anhydride. Gastrointestinal tract conditions (saliva, and gastric fluid) were simulated in accordance with the EU Pharmacopoeia. The biochemical and immunochemical properties of the derivatives following exposure to different conditions were monitored by determining the number of residual amino groups with 2,4,6-trinitrobenzenesulfonic acid, SDS PAGE, immunoblotting and inhibition of mugwort-specific IgE. Exposure to saliva fluid for 2 min did not influence the biochemical and immunochemical properties of the derivatives. In the very acidic conditions of the simulated gastric fluid, the degree of demaleylation and desuccinylation, even after 4 h exposure, was low, ranging from 10 to 30 %. The digestion patterns with pepsin proceeded rapidly in both the unmodified and modified samples. In all four cases, a highly resistant IgE-binding protein the Mwof which was about 28-35 kD, was present. Within the physiological conditions, no new IgE binding epitopes were revealed, as demonstrated by immunoblot and CAP inhibition of the mugwort specific IgE binding. An important conclusion of this study is the stability of the modified derivatives in the gastrointestinal tract of patients, within physiological conditions. The means that they are suitable for use in much higher concentrations in local forms of immunotherapy than unmodified ones. U ovom radu su prikazani rezultati ispitivanja stabilnosti tri tipa alergoida polena pelina u simuliranom želudačnom soku. Koristeći kalijum-cijanat anhidrid ćilibarne i anhidrid maleinske kiseline, napravljeni su alergoidi polena pelina (Artemisia vulgaris). Saliva i želudačni sok su simulirani na osnovu evropske farmakopeje. Biohemijske i imunohemijske osobine derivata posle izlaganja različitim uslovima, praćene su: određivanjem broja slobodnih amino grupa u reakciji sa TNBS, SDS PAG elektroforezom, imunoblotom i određivanjem pelin-specifičnog imunoglobulina E (IgE). Izlaganje salivi u trajanju od 2 minuta ne utiče na biohemijske i imunohemijske osobine derivata. U kiseloj sredini želudačnog soka ne dolazi do značajnog demaleilovawa i desukcinilovanja. Čak i posle četvoročasovnog izlaganja, taj procenat je u opsegu 10-30 %. Alergoidi pelina se trenutno digestuju pepsinom, sa izuzetkom visoko rezistentne proteinske trake molekulske mase 28-35 kD, koja odgovara važnom IgE-vezujućem proteinu polena pelina. Imunoblotom i CAP-inhibicijom je pokazano da, u okviru fizioloških uslova, ne dolazi do stvaranja novih IgE-vezujućih epitopa. Hemijska stabilnost modifikovanih derivata u simuliranim uslovima želudačnog soka omogućuje da se tokom imunoterapije mogu primenjivati veće doze alergoida nego nemodifikovanog ekstrakta polena pelina.

Food allergies, including kiwi fruit allergy, have been the subject of extensive research in the last few years. The aim of this study was to examine a possible relationship between the developmental stage of kiwi fruit and its allergenic potency. The protein and allergen patterns of kiwi fruit extracts in September, October, November and December fruit in the period from 2000-2002 were analysed. One of the factors that may contribute to the difficulties in proposing well-defined and standardized fruit extracts should also be the time of fruit harvesting. In this particular case, when the kiwi fruit was edible throughout November and December, we showed discrepancies in allergen content and potencies both in qualitative and quantitative terms. Two major allergens of kiwi fruit, Act c 1 and Act c 2, mainly accounted for the highest allergenic potential of November kiwi extract in vivo and in vitro. Not only the content of major allergens, but also the ratio of different proteins and even isoforms of the same allergen (Act c 2) change with fruit ripening. These findings should be taken into account during preparation of extracts for allergy diagnosis.

This study considers the influence of exposure to simulated gastrointestinal conditions (saliva, gut, intestine and acidic conditions of the gut) on IgG binding of unmodified allergens and three types of LMW allergoids of Artemisia vulgaris pollen extract obtained by means of potassium cyanate succinic and maleic anhydride. It also concerns the optimization of a self-developed ELISA assay for comparison of the specific IgG binding of mugwort pollen extract and modified mugwort pollen derivatives. The ELISA was conducted with a mugwort pollen extract coupled to the plate, using the sera from 12 mugwort- pollen allergic patients. The exposure to saliva fluid for 2 min did not influence the IgG binding properties of allergens and allergoids. Exposure of mugwort pollen allergens and LMW allergoids to the acidic conditions of the gut did not dramatically change their IgG binding properties. By exposing mugwort pollen extract and LMW derivatives to the SGF conditions for 1 h, the percent of IgG binding epitopes was reduced to a half of its starting value in the extract and to about 30%in all the allergoid samples. After prolonged exposure only the carbamyl derivative showed reduced IgG binding. Changes of the IgG binding potential of all four samples after exposure in SIF followed a similar pattern. Predmet ovog rada je ispitivanje promena IgG vezivanja nemodifikovanih alergena polena A. vulgaris i tri tipa alergoida malih molekulskih masa dobijenih tretmanom sa kalijum-cijanatom, i anhidridima ćilibarne i meleinske posle izlaganja uslovima gastrointestinalnog trakta (saliva, želudac, tanko crevo i kisela sredina želudačnog soka) i optimizacija domaćeg ELISA testa za određivanje IgG vezivanja alergena i alergoida polena pelina.UELISA testu je za pločicu kuplovan ekstrakt polena pelina i korišćeni su serumi 12 pacijenata alergičnih na pelin. Izlaganje salivi u trajanju od 2 minuta ne utiče na IgG vezujuće osobine alergena i alergoida. Izlaganje kiseloj sredini želudačnog soka značajno ne utiče na IgG vezujuće osobine alergena i alergoida polena pelina. Posle izlaganja simuliranom želudačnom soku alergena i derivata u trajanju od 1 sata, procenat IgG vezujućih epitopa u nemodifikovanom uzorku se smanjuje na polovinu početne vrednosti i na oko 30 % kod sva tri derivata. Jedino se kod karbamil-derivata % IgG vezivanja dodatno smanjuje sa produžavanjem izlaganja SGF-u.Promene u IgG vezujućem potencijalu sva 4 uzorka posle izlaganja simuliranim uslovima tankog creva prate sličan obrazac.

The physiological role of fig latex is to protect the plant from pathogens. Latex is a rich source of proteases, predominantly ficin. Fig latex also contains collagenolytic protease and chitinolytic enzymes. Our aim was to investigate changes in protein composition, enzyme and antifungal activities of fig latex during fruit ripening.Comparison of latex samples in different time periods showed a uniform increase of protein concentration in chronological order. The content of collagenolytic protease did not differ significantly in the latex samples, while the content of ficin decreased. Ficin-specific activity towards casein was the highest at the beginning of fruit development (about 80 U mg(-1)). Specific milk clotting activity increased as well as the abundance of casein band in the clots. Specific chitinolytic activity at the beginning of flowering was 6.5 times higher than the activity in the period when fruits are ripe. Antifungal activity is the most extensive in spring.Ficin forms with different casein specificities are present in different proportions during fruit ripening, which is of importance for applications in the dairy industry. The protection mechanism against insects and fungi, which relies on chitinolytic activity, is the most important in the early phases of flowering and is replaced with other strategies over time.

New square-planar Pd(II) and Pt(II) complexes with 8-quinolinecarboxaldehyde selenosemicarbazone have been synthesized and characterized by use of elemental analysis, molar conductivity measurements, and IR and NMR spectroscopy. The cytotoxic activity of the ligand, new Pt(II) and Pd(II) compounds, and previously synthesized Pd(II), Pt(II), Cd(II), and Ni(II) complexes with the analogous ligand, 2-quinolinecarboxaldehyde selenosemicarbazone, was tested against two human cancer cell lines: lung carcinoma (H460) and glioma (U251). The potential of these compounds to induce perturbations of the H460 cell cycle was also evaluated. These substances had an excellent radical-scavenging effect against ABTS radical cations. The best antimicrobial activity, among two yeasts and eight bacterial strains tested, was against Bacillus cereus. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3683]

A novel collagenolytic serine protease was identified and then purified (along with ficin) to apparent homogeneity from the latex of fig (Ficus carica, var. Brown Turkey) by two step chromatographic procedure using gel and covalent chromatography. The enzyme is a monomeric protein of molecular mass of 41 +/- 9 kDa as estimated by analytical gel filtration chromatography. It is an acidic protein with a pI value of approximately 5 and optimal activity at pH 8.0-8.5 and temperature 60 degrees C. The enzymatic activity was strongly inhibited by PMSF and Pefabloc SC, indicating that the enzyme is a serine protease. The enzyme showed specificity towards gelatin and collagen (215 GDU/mg and 24.8 CDU/mg, respectively) and non-specific protease activity (0.18 U/mg against casein). The enzyme was stable and retained full activity over a broad range of pH and temperature. The fig latex collagenolytic protease is potentially useful as a non-microbial enzyme with collagenolytic activity for various applications in the fields of biochemistry, biotechnology and medicine. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

Objective: Human BPIFA2 (parotid secretory protein) is a ubiquitous soluble salivary protein, which belongs to the PLUNC family of proteins. Having sequence similarity to bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein, PLUNC proteins are probably involved in local antibacterial response at mucosal sites, such as oral cavity. The aim of the study was to isolate and characterize human BPIFA2. Design: In this paper, we report one-step affinity chromatography method for BPIFA2 purification from whole human saliva. The isolated BPIFA2 was identified by trypsin mass fingerprinting and characterized by electrophoretic methods. Antibacterial activity of BPIFA2 against model microorganism Pseudomonas aeruginosa was shown in minimum inhibitory concentration and time kill study assays. Results: The protein showed microheterogeneity, both in molecular weight and pI value. BPIFA2 inhibited the growth of P. aeruginosa in microgram concentration range determined by minimum inhibitory concentration assay. In the time kill study, 32 mu g/mL BPIFA2 showed clear bactericidal activity and did not cause any aggregation of bacteria. Conclusion: Affinity chromatography is well suited for isolation of functional BPIFA2 with a potent bactericidal activity against P. aeruginosa. (C) 2013 Elsevier Ltd. All rights reserved.

Actinidin, an abundant cysteine protease from kiwifruit, is a specific biomarker of isolated allergy to kiwifruit. This study evaluates the IgE-binding properties of biologically active and thermally inactivated actinidin. Employing two different activity assays (caseinolytic assay and zymogram with gelatin) we showed that actinidin obtained from kiwifruit extract under native conditions represents a mixture of inactive and active enzyme. The structural integrity of actinidin was confirmed by SDS-PAGE. Edman degradation, mass fingerprint and Western blot with polyclonal antibodies. Although it was capable of inducing positive skin prick test reactions, we failed to detect IgE reactivity of active actinidin in Western blot with patient sera. Thermally inactivated actinidin exhibited IgE reactivity both in vivo and in vitro, indicating that heat processed kiwifruit products may induce clinical reactivity. These findings imply that apart from the allergenic epitopes on its surface, actinidin also contains hidden epitopes inside the protein which become accessible to IgE upon thermal treatment. (C) 2011 Elsevier Ltd. All rights reserved.

P gt Background House dust mites (HDM) are well-known as a source of indoor aeroallergens and for causing allergic airway diseases. Some proteolytic HDM allergens are known to activate respiratory epithelial cells to produce pro-inflammatory mediators, while there is limited knowledge regarding such activity among non-proteolytic HDM allergens. Objective To investigate whether Der p 2, a major non-proteolytic allergen of Dermatophagoides pteronyssinus, activates respiratory epithelial cells to produce mediators involved in asthma pathogenesis and to elucidate the mechanism of such activation. Methods The human bronchial epithelial cell line BEAS-2B, normal human bronchial epithelial (NHBE) cells and the alveolar epithelial cell line A549 were exposed to recombinant Der p 2. Following exposure, we analysed a panel of soluble mediators and cell adhesion receptors involved in asthma pathogenesis by promoting recruitment, survival and binding of inflammatory cells. The involvement of nuclear factor (NF)-kappa B and mitogen-activated protein kinases (MAPKs) was studied using specific inhibitors. Results Der p 2 activated bronchial BEAS-2B and NHBE cells, but not alveolar A549 cells. In BEAS-2B cells Der p 2 induced dose-dependent up-regulation in both mRNA level and protein secretion of granulocyte-macrophage colony-stimulating factor, IL-6, IL-8, monocyte-chemotactic protein-1 and macrophage inflammatory protein-3 alpha. Secretion as well as surface expression of intercellular adhesion molecule (ICAM)-1 was also up-regulated, which was associated with increased adhesion of monocytes to the epithelial cells. The release of cytokines and chemokines was regulated by NF-kappa B and MAPK activation in different ways, while expression of ICAM-1 was solely dependent on NF-kappa B activation. Conclusion These results show that Der p 2 activates respiratory epithelial cells, indicating that this non-proteolytic allergen, in addition to its immunogenic properties, can aggravate respiratory airway disease by adjuvant-like activation of the lung epithelium.

The use of enzymes may improve the functional properties of various food ingredients. The aim of this study was to examine the effects of proteolytic contaminants in phenol oxidases on beta-lactoglobulin (BLG). In the presence of Trametes versicolor laccase and Agaricus bisporus tyrosinase, both variants of BLG (A and B) underwent removal of a peptide from the N-terminus. The truncated forms were more susceptible to digestion by pepsin. The truncation of BLG resulted from contaminating proteases and not due to the action of phenol oxidases. The removal of N-terminal peptides proceeded quickly, while the rest of the globular protein remained resistant to proteolysis for up to 3 h. In the case of the application of enzymes in food bioprocessing, it may be important to carefully monitor the effects of contaminating proteases in enzyme preparations used. (C) 2009 Elsevier Ltd. All rights reserved.

A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study here theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use. (c) 2007 Elsevier B.V. All rights reserved.

Background: It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low-pH conditions. Objective: The purpose of this study was to investigate the effect of hydrogel forming polysaccharide-rich fruit matrixes on in vivo gastric and in vitro pepsic digestion of fruit allergens. Methods: Fruit extract proteins (kiwi, banana, apple and cherry) and a purified major kiwi allergen Act c 2 were digested with simulated gastric fluid in accordance with the US Pharmacopeia. In vivo experiments on kiwi fruit digestion were performed on four healthy non-atopic volunteers by examining the gastric content 1 h after ingestion of kiwi fruit. The Act c 2 and kiwi proteins were detected in immunoblots using monoclonal anti-Act c 2 antibodies and rabbit polyclonal antisera. Results: Crude fruit extracts were resistant to digestion by pepsin when compared with commonly prepared extracts. In the gastric content of all volunteers, following kiwi fruit ingestion and immunoblotting, intact Act c 2 was detected with anti-Act c 2 monoclonal antibodies, while kiwi proteins of higher molecular weights were detected using rabbit polyclonal antisera. Addition of apple fruit pectin (1.5% and 3%) to the purified kiwi allergen was able to protect it from pepsin digestion in vitro. Conclusion: The matrix effect in pectin-rich fruits can influence the digestibility of food proteins and thereby the process of allergic sensitization in atopic individuals.