Enzymatic decolorization of anthraquinone dyes from wastewaters

Osnovni predmet istraţivanja u okviru ove doktorske disertacije je mogućnost primene peroksidaze iz rena, komercijalnog i neprečišćenog preparata, u obezbojavanju sintetičkih, antrahinonskih boja. Strukturno, ova doktorska disertacija se moţe podeliti u četiri dela. Prvi deo odnosi se na ispitivanje mogućnosti obezbojavanja AV 109 boje slobodnim enzimom (komercijalnim, neprečišćenim iz ekstrakta). Slobodni enzimski preparati su okarakterisani sa aspekta primene u reakciji obezbojavanja AV 109 boje. UtvrĎeno je optimalno vreme kontakta, koncentracije enzima, koncentracije boje, koncentracije vodonik-peroksida, pH i temperature. Kinetička ispitivanja u početnom periodu reakcije pokazala su najbolje slaganje sa ping pong bi-bi dvosupstratnim modelom koji uzima u obzir inhibiciju bojom i supstratom. Navedeni kinetički model model je korišćen za modelovanje dobijenih eksperimentalnih podataka u cilju odreĎivanja kinetičkih konstanti..

Enzimsko obezbojavanje antrahinonskih boja iz otpadnih voda

Osnovni predmet istraţivanja u okviru ove doktorske disertacije je mogućnost primene peroksidaze iz rena, komercijalnog i neprečišćenog preparata, u obezbojavanju sintetičkih, antrahinonskih boja. Strukturno, ova doktorska disertacija se moţe podeliti u četiri dela. Prvi deo odnosi se na ispitivanje mogućnosti obezbojavanja AV 109 boje slobodnim enzimom (komercijalnim, neprečišćenim iz ekstrakta). Slobodni enzimski preparati su okarakterisani sa aspekta primene u reakciji obezbojavanja AV 109 boje. UtvrĎeno je optimalno vreme kontakta, koncentracije enzima, koncentracije boje, koncentracije vodonik-peroksida, pH i temperature. Kinetička ispitivanja u početnom periodu reakcije pokazala su najbolje slaganje sa ping pong bi-bi dvosupstratnim modelom koji uzima u obzir inhibiciju bojom i supstratom. Navedeni kinetički model model je korišćen za modelovanje dobijenih eksperimentalnih podataka u cilju odreĎivanja kinetičkih konstanti..

Biotehnološki postupak uklanjanja sintetičkih boja iz otpadnih voda imobilisanom peroksidazom iz rena

Opšte je poznata činjenica da postoji veliki ekološki problem zagađenja životne sredine otpadnim vodama što zahteva kolektivne napore da bi se sprečila nadolazeća ekološka katastrofa. U ovom radu je razvijen postupak utemeljen na principima biotehnologije, tačnije peroksidaza iz rena je primenjena u tretmanu sintetičkih boja koje su neizostavna komponenta otpadnih voda. Novi biotehnološki postupak je razmatran sa aspekta ekološki prihvatljive tehnike imobilizacije korišćenjem oksidovanog pektina kao agensa za aktiviranje Purolite® A109 nosača koji je biokompatibilan, sa ciljem dobijanja stabilne i efikasne imobilisane peroksidaze iz rena za primenu u tretmanu sintetičkih boja iz otpadnih voda. Za aktivaciju makroporozne polistirenske jonoizmenjivačke smole, Purolite® A109 korišćen je oksidovani pektin (1% w/v). Specifična aktivnost peroksidaze imobilisane na pektinom modifikovan Purolite® A109 iznosila je 37,58 ± 2,21 IU/mg. Imobilisana peroksidaza se pokazala kao veoma efikasna u biorazgradnji antrahinonske boje, 92,03 ± 7,32 % AV109 boje je uklonjeno nakon 30 minuta tretmana. Pored toga, imobilisani enzim je značajno doprineo kvalitetu tretirane obojene otpadne vode potvrđene testovima hemijske potrošnje kiseonika (HPK) i ukupnog organskog ugljenika (UOU). Operativna stabilnost je potvrđena nakon 10 ciklusa primene sa 45 % zadržane aktivnosti

Sinteza 4,6-dimetil-3-cijano-2-piridona katalizovana aminokiselinama

In this work the synthesis of 4,6-dimethyl-3-cyano-2-pyridone catalyzed by amino acids was studied. In the first part of the work, the optimization was carried out by varying the initial concentration of glycine, the molar ratio of the reactants (acetylacetone and cyanoacetamide) and the reaction time. It has been found that it is necessary to heat the reaction mixture and that the optimum concentration of the amino acid is 0.2 mmol cm-3. It was also found that the optimal concentration of cyanoacetamide is 3.0 mmol cm-3 (molar ratio cyanoacetamide: acetyl acetone = 3:1), and that the reaction time is 24 h. In the second part of the work, the influence of the structure of 19amino acids on the yield of 4,6- dimethyl-3-cyano-2-pyridone was investigated, using the optimal reaction conditions. It was found that the highest yield was achieved with arginine (almost 90%) and than with histidine (about 85%). With the most of other amino acid the yield was 50-60%. .U okviru rada prikazana je sinteza 4,6-dimetil-3-cijano-2-piridona katalizovana aminokiselinama. U prvom delu izvršena je optimizacija reakcije sinteze piridona varirajući početnu koncentraciju glicina, molski odnos reaktanata (acetilacetona i cijanoacetamida) i reakciono vreme. U drugom delu rada ispitan je uticaj strukture još 18 aminokiselina na prinos 4,6-dimetil-3-cijano-2-piridona. Najveći prinosi ostvareni su sa argininom (90%) i histidinom (85%) kao katalizatorima.

Optimization and kinetic study of anthraquinone dye removal from colored wastewater using soybean seed as a source of peroxidase

As water contamination emerges as a serious threat to the environment, ventures for cleaner and sustainable solutions are continuously being developed. The present study investigates the ability of crude peroxidase extract from soybean seeds to degrade the anthraquinone dye Acid Violet 109. The influence of the essential parameters pH, dye concentration, hydrogen peroxide dosage, and temperature were inspected. The enzyme had 81.9 % biodegradation at pH 4 in 30 min with 0.1 U peroxidase, 40 mg/l dye concentration, and 1 mM hydrogen peroxide. Considering that substrate concentration can cause reaction inhibition, a kinetic study was performed. Kinetic data fitting using bisubstrate kinetics with a substrate inhibition model revealed the high inhibitory effect of the dye, which was confirmed by the inhibition constant, 7.123.10(-5) mM. Alongside the inhibition constant values, the Ping-Pong Bi-Bi model gave the maximum rates 15.788 and 14.321 mM/min for hydrogen peroxide and dye inhibition, respectively

New life of waste material: immobilized horseradish peroxidase for degradation of antraquinone dye

Various strategies have been developed for the removal of synthetic dyes from the wastewater of the textile industry due to their stability as well as their negative effect on the environment. Enzymatic wastewater treatments as environmentally safe processes have many benefits. An enzyme suitable for use in the treatment of wastewater from the textile industry is horseradish peroxidase (HRP) because it can degrade different types of dyes. Immobilized enzymes have many advantages over free enzymes in solution such as higher stability and the opportunity to be removed easily from the reaction mixtures. Consequently, immobilized enzymes can be used more than once. Many different materials are used as a carrier for enzyme immobilization, so if residual material is used as a carrier, waste valorization will be achieved. In this study immobilized commercial HRP was used for decolorization of anthraquinone dye Acid green 40 (AG 40). Waste material – potato peels were used as a carrier for the immobilization of HRP. Potato peels were oxidized with sodium periodate to introduce aldehyde groups that can react with the primary amino groups of the enzyme to form Schiff bases, resulting in covalently immobilized HRP. The success of the oxidation was verified by the 2,4-dinitrophenylhydrazine (DNPH) test, and subsequently, the oxidized potato peels were characterized by SEM and FTIR. After oxidation, the influence of the mass of the immobilization carrier on enzyme activity and immobilization efficiency was investigated. The optimal conditions of pH, hydrogen peroxide concentration, concentration of AG 40, and enzyme activity were determined for decolorization of AG 40 with immobilized HRP. Adsorption of AG 40 on the carrier was also determined. In addition, the operational stability of the enzyme was determined. The results obtained show that oxidized potato peels are a promising material that can be used as a carrier for the immobilization of HRP

Dekolorizacija antrahinonskih boja peroksidazom iz rena imobilisanom na kaolin

Antrahinonske boje su veoma značajne za tekstilnu industriju i, odmah nakon azo boja, imaju najveću primenu. Nažalost, kao i azo boje, i antrahinonske boje predstavljaju ozbiljan problem zagađenja životne sredine. Većina njih je toksična, mutagena, karcinogena i rezistentna na degradaciju. Osnovni cilj ovog rada je razvoj efikasnog, ekološki prihvatljivog imobilisanog biokatalizatora za primenu u procesima dekolorizacije antrahinonskih boja. Ispitivanje je fokusirano na primeni kaolina, i to njegovog aktiviranog oblika, kao nosača za imobilizaciju peroksidaze iz rena. Peroksidaza je imobilisana adsorpcijom i ispitan je uticaj početne koncentracije enzima na masu vezanog enzima kao i na aktivnost dobijenog imobilisanog preparata. Koncentracija enzima varirana je u opsegu 0,5 - 3 mg cm-3 pri čemu su dobijeni visoki prinosi imobilizacije, a fitovanje dobijenih eksperimentalnih podataka Lengmirovom adsorpcionom izotermom (R2=0,911) ukazuje na to da se na površini nosača formira monosloj. Dobijeni biokatalizatori su ispitani u reakciji dekolorizacije dve antrahinonske boje (C.I.Acid violet 109 i C.I.Acid blue 225). Ostvareni rezultati ukazuju na veliki potencijal pripremljenih biokatalizatora. Biokatalizator dobijen pri početnoj koncentraciji enzima 1,5 mg cm-3 već nakon 30 min ukloni preko 90 % C.I.Acid blue boje. Dobijeni biokatalizatori su takođe pokazali dobru operativnu stabilnost i mogućnost ponovne upotrebe

Removal of Synthetic Dyes from Wastewaters Using Horseradish Peroxidase

Horseradish peroxidase is a heme-containing plant enzyme (HRP; EC 1.11.1.7) with a wide range of biotechnological and medical applications. In combination with hydrogen peroxide (H2O2) as an oxidant HRP can be used in the oxidation of various substrates (e.g. considered as toxic, muthagenic, recalcitrant and carcinogenic compounds with harmful impact on human health and ecosystems and main textile industry problem when it comes to environment pollution with wastewaters. Traditional methods are inefficient, and in recent years, with a view to preserving the environment, the scientific community turns to the application of enzymes as the main catalysts in the removal of synthetic dyes. Based on the data available in the literature, HRP proved to be an enzyme with a high potential for the application of synthetic dyes removal by the mechanism of polymerization or ring opening. In this chapter well-studied enzyme is described with the focus on its promising use in colored wastewater treatment. C. I. Acid Violet 109 (AV 109) was used as a model dye in order to examine the feasibility of HRP application in removal of anthraquinone class of synthetic dyes. The reaction conditions were optimized in terms of dye, enzyme and H2O2 concentration as well as temperature and pH influence. It was established that the dye removal shows good agreement with two substrate ping-pong mechanism with substrate inhibition. The industrial application of this enzyme requires the economic feasibility. Accordingly, an expensive commercial preparation HRP was replaced with partially purified enzyme isolated from horseradish root which showed high efficiency in model dye removal. The next step towards the industrial application of this enzyme was the immobilization of various techniques (adsorption, covalent immobilization, cross-linked aggregates) to various carriers such as kaolin and the synthetic carrier Purolite® A109. Improvement in storage and operational stability after immobilization was confirmed and immobilized preparations were applied in different bioreactor configurations, batch and packed bed. HRP catalyzed removal of synthetic dyes appeared to be promising technique dye to high efficiency in dye removal and phytotoxicity and acute toxicity decrease, measured by Mung bean seeds and A. salina assays

Production of wheat gluten hydrolysates with improved functional properties: Optimization of operating parameters by statistical design

Native wheat gluten is a deluxe bread improver and may be utilized as a functional protein additive in multifarious non-bakery foodstuffs due to its desirable structure-enhancing properties. Also its utilization would be economically interesting, but lack of some desirable functional properties limited their expanding utilization in foodstuff formulations. This study was designed to examine the relationship between process parameters and functional properties of the obtained hydrolysates using Box-Benken experimental design and response surface methodology (RSM). The hydro-lysate showing the highest improvement of solubility and foaming ability was further separated by sequential ultrafiltration to obtain molecular weight distribution profile. The progress of wheat gluten hydrolysis was followed by monitoring the degree of hydrolysis (DH) using the pH-stat method and functional properties were measured by our methods already adopted. The effects of process parameters (pH, T, [S], [E]/[S] ration) and their interactions were investigated by the means of the four-factor Box-Behnken experimental design with 29 experimental points (5 central points). Experiments were carried out in triplicates and expressed as means with SD. The effects of different parameters under the significance level of p < 0.05 were examined using one-way analysis of variance (ANOVA) and Student t-test. The coefficients of the response function and their statistical significance were evaluated by the response surface regression analysis, using the Design software. Non-significant terms (p ≥ 0.05) were deleted from the second order polynomial and a new polynomial has been recalculated. The Fisher test (F-value) was used to determine whether the second-order model was adequate to describe the obtained data while the goodness of fit of the model was evaluated by the determination coefficient (R2). The obtained results showed that the second-order models developed for DH, solubility and foaming properties of gluten hydrolysates were significant (p < 0.05) with a high value of coefficients of determination (0.944-0.981). The statistical analysis showed that each variable had a significant effect on degree of hydrolysis and the functional properties of tested system. Almost the linear increase in DH was observed with the rise in temperature at the highest substrate concentration, while on the other hand increasing the concentration of the substrate leads to a decrease in DH. In terms of foaming properties results showed that foam capacity range are in the range of 24.2-80.3%, depending on the independent variables that were tested. Results are relevant to the protein ingredient industry because of the economic importance of novel glu-ten-based functional products and can provide useful information for the design an efficient enzymatic process for their production in high yield and with improved functionality

Kontinualni sistem za obezbojavanje otpadnih voda. Primena umrežene peroksidaze iz poljoprivrednog otpada u uklanjanju boje

Agro-industrial waste has gained special attention as an abundatly available natural carbon source, which can be used for value-added products such as generating power, biofuel, biogas and biofertilizers production. Further application of agro-industrial waste may be as a substrate for solid state fermentation for production of antibiotics, enzymes, and phytochemicals. Isolation of enzymes from waste material as crude extracts contributes to the concept of sustainability and lowers their cost significantly. The enzyme of interest in this study was peroxidase isolated from soybean hull. Furthermore, the crude peroxidase from soybean hull was crosslinked by oxidized pectin onto the inner walls of PTFE microtubular reactor. The influence of the crosslinker concentration on the enzyme activity and crosslinking efficiency was evaluated, together with the effect of the reagents flow rate on the enzyme activity and crosslinking efficiency. Two different microtubular diameters were examined for the crude peroxidase crosslinking: 0.5 and 0.8 mm. After the immobilization, the possibility of anthraquinone dye removal by crosslinked peroxidase in the microtubular reactor was examined. The reusability of the immobilized enzyme was also evaluated in this study.Poljoprivredni otpad predstavlja lako dostupan prirodni izvor ugljenika, koji se može koristiti za dobijanje proizvoda sa dodatom vrednošću. Poljoprivredni otpad se može koristiti kao sirovina za proizvodnju električne energije, biogorivo, biogas i proizvodnju biođubriva. Dalja primena poljoprivrednog otpada može biti kao supstrat za fermentaciju u čvrstom stanju (eng. solid state fermentation), za proizvodnju antibiotika, enzima i fitohemikalija. Izolovanje enzima iz otpadnog materijala kao sirovi ekstrakt doprinosi konceptu održivosti i značajno smanjuje cenu enzima. Enzim od interesa u ovom radu je peroksidaza izolovana iz sojinih ljuspica. Sirova peroksidaza iz sojinih ljuspica umrežena je oksidovanim pektinom na unutrašnje zidove PTFE cevnog mikroreaktor, i ispitivan je uticaj koncentracije umreživača na aktivnost enzima i efikasnost umrežavanja, kao i uticaj protoka reagensa na aktivnost enzima i efikasnost umrežavanja. Nakon toga, vršeno je ispitivanje uticaja prečnika cevnog mikroreaktora na efikasnost umrežavanja i aktivnost peroksidaze: 0,5 i 0,8 mm. Nakon imobilizacije ispitana je mogućnost uklanjanja antrahinonske boje umreženom peroksidazom u cevnom mikroreaktoru. U ovom radu je takođe je ispitivana mogućnost ponovne upotrebe imobilisanog enzima