Klijanja semena Pančićeve omorike (Picea omorika (Pančić) Purkyně) i antioksidativni enzimi

Pančićeva omorika predstavlja Balkansku endemičnu vrstu smrče i tercijarni relikt. Određen je sastav antioksidativnih enzima tokom klijanja semena omorike u fiziološkim uslovima, a radi boljeg razumevanja mehanizama otpornosti na zagađivače iz spoljašnje sredine.1 Ovo je prva studija aktivnosti enzima katalaze (CAT), superoksid dismutaze (SOD) i peroksidaze (POD) tokom klijanja dve linije semena Pančićeve omorike (A – borealis i S – srpska). Klijanje semena je praćeno tokom 7 dana. Za procenat klijavosti korišćen je proboj radikule za više od 1 milimetar kao kriterijum. Linija A je pokazala veći procenat klijavosti (74%) i veću aktivnost enzima po jedinici sveže mase klijanaca u poređenju sa linijom S (63%). CAT aktivnost kod linije A je povećana po jedinici mase klijanaca i suvih semena, a nakon 7 dana kod neisklijalih semena nije detektovana. SOD aktivnost je ostala na sličnom nivou u obe linije. Aktivnost POD na početku nije detektovana, ali se naglo povećala nakon četvrtog dana da bi sedmog bila 10 U/g sveže mase klijanaca kod linije S i 28 U/g kod A. CAT i SOD su uključeni u očuvanje klijavosti semena i imaju ulogu zaštite od reaktivnih kiseoničnih vrsta tokom skladištenja i klijanja, dok je katalazna aktivnost bitna za procenu klijavosti. Najveću promenu aktivnosti za vreme klijanja pokazala je peroksidaza, čija aktivnost nije detektovana u suvim semenima, a tokom klijanja naglo se povećala i značajna je u kasnijim fazama klijanja

Stability of soybean peroxidase immobilized onto hydrogel micro-beads from tyramine-pectin

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

PREPARATION OF CROSSLINKED TYRAMINE-ALGINATE HYDROGEL USING EDC/NHS WITH SELF-IMMOBILIZED HRP

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

O,O'-diethyl-(S,S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoate dihydrochloride enhances influx of effective NK and NKT cells in murine breast cancer

Background/Aim. O,O'-diethyl-(S,S)-ethylenediamineN,N'-di-2-(3-cyclohexyl)propanoate dihydrochloride (DE-EDCP) has been found to possess promising cytotoxic activity against various tumor cell lines. Also, DE-EDCP reduces tumor progression by several mechanisms such as triggering tumor cell death and inhibition of cell proliferation. The aim of present study was to further evaluate antitumor activity of DE-EDCP by investigating effects on migratory potential of tumor cells and anti-tumor immune response. Methods. Migratory potential of DE-EDCP was evaluated by scratch wound assay. Female BALB/c mice were inoculated with 4T1 breast cancer cells and treatment with DE-EDCP started five days following orthotopic tumor implantation. The frequency and phenotype of tumor-infiltrating natural killer (NK) and natural killer T (NKT) cells were analyzed by flow cytometry. Results. DE-EDCP inhibited migratory potential of highly metastatic 4T1 cells. DE-EDCP facilitated accumulation of CD3+CD49+ NKT cells and CD3-CD49+ NK cells in tumor microenvironment. DE-EDCP treatment led to significant decrement of tumor infiltrating anergic NKT cells expressing cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), killer cell lectin like receptor G1 (KLRG-1) and programmed cell death protein-1 (PD-1). Mice given DE-EDCP had significantly increased percentages of tumoricidal fas ligand (FasL) positive NK cells. Conclusion. DE-EDCP inhibits murine breast cancer progression through direct effects on tumor cells and by facilitating anti-tumor immunity. DE-EDCP enhances accumulation, promotes tumoricidal phenotype and maintenances responsiveness of NK and NKT cells in 4T1 murine breast cancer

OPTIMIZATION OF HORSERADISH PEROXIDASE ENCAPSULATION WITHIN TYRAMINE-ALGINATE FOR PHENOL REMOVAL

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

Characterization of chemically modified pectins as novel material for various applications

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

Optimization of reaction conditions for phenol removal in batch reactor with horseradish peroxidase immobilized within tyramine-alginate micro-beads

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

Covalent immobilization of horseradish peroxidase on novel macroporous poly(GMA-co-EGDMA) for phenol removal

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

STRUCTURAL CHARACTERISATION AND ORIENTATION OF CELL WALL POLYMERS IN MAIZE LEAVES

Cell wall can be considered as a nano-composite in which cellulose, lignin and hemicelluloses are interconnected in a specific manner. Mechanical and physical propreties of plant fibres are dependent on the orientation of constituent polymers (cellulose, hemicellulose, lignin). Fourier transform infrared (FTIR) microscopy was used to examine the orientation of the main plant polymers in transversal and longitudinal direction of the isolated cell wall of the maize leaves. Polarised FTIR measurements indicated an anisotropy, i.e. orientation of the cellulose microfibrils that was more or less parallel to the longitudinal axis of the cell wall. Xylan has parallel orientation with regard to the orientation of cellulose, as well as lignin

IMMOBILIZATION OF TYRAMINE-HRP ONTO TYRAMIDECARBOXYMETHYL CELLULOSE MATRIX FOR WASTEWATER TREATMENT

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