Natural polymers: suitable carriers for enzyme immobilization

Enzyme immobilization onto support carriers has the potential to overcome some of the limitations of soluble enzymes in practical applications. Various materials have been used as carriers, such as inorganic matrices, as well as natural and synthetic polymers. Production of carriers from natural biopolymers and their derivatives has been the focus of research worldwide, and a summary of their applications for enzyme immobilization is presented in this paper. Enzymes, or cells as an enzyme source, are entrapped inside a three-dimensional polymeric network, called a hydrogel, that is able to retain large amounts of water. This network can 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 physico-chemical characteristics. The most frequently used biopolymers as carriers for immobilization include alginate, cellulose, chitosan, collagen, xylan, pectin, and others

Immobilization of soybean peroxidase (Glycine max) onto macroporous glycidyl-methacrylate and chemically modified pectin

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

Immobilization of soybean peroxidase (Glycine max) onto macroporous glycidyl-methacrylate and chemically modified pectin

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

Immobilization of soybean peroxidase (Glycine max) onto macroporous glycidyl-methacrylate and chemically modified pectin

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

Characterization of colored maize seed fractions using fluorescence spectroscopy and multivariate analysis

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

Antioxidant activity estimation of inner and outer seed fractions of the legumes Vigna radiata L. and Glycine max L.

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

Antioxidant activity and fluorescence of colored maize (Zea Mays L.) seeds under various temperature conditions

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

Characterization of colored maize seed fractions using fluorescence spectroscopy and multivariate analysis

Application of fluorescence spectroscopy combined with chemometrics algorithms provides rapid and non-destructive screening method in seed quality estimation, widely used in the agricultural industry and crop breeding. Fluorescence spectroscopy is a technique capable of detecting differs fluorophores among various colored maize seed cultivars and through different seed fractions. In the present study, we used the Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) algorithm to analyse the excitation-emission matrices (EEMs) 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 330 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 component 1 (C1) varied among the samples. On the other hand, the position and shape were similar for component 2 (C2). C1 could be used as a marker for the discrimination of colored seeds and their fractions. The observed variations in C1 between the analysed seeds may be due to the presence of their individual fluorophores, assigned to anthocyanins, proteins, and phenolics. In conclusion, the MCR-ALS analysis of the seed emission spectra has a great potential for the rapid and non-expensive characterization of various cultivars of colored seeds

CHARACTERISATION OF MUNG BEAN (VIGNA RADIATA L.) SEEDS USING FLUORESCENCE SPECTROSCOPY AND MULTIVARIATE ANALYIS

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

Characterization of Mung bean (Vigna radiata L.) seeds: antioxidant activity, chlorophyll and carotenoid content

Mung bean (Vigna radiata L.) is a leguminous plant with high nutritional value, traditionally known as a functional food. Legume seeds are a rich source of proteins, vitamins, minerals, and essential amino acids but also contain bioactive components and polyphenols which possess a high antioxidant capacity. Pigments content (chlorophyll a and b, carotenoids) was determined as good parameter for estimation of seed quality and an indicator of tolerance to different types of stress. The antioxidant activity of the seeds was determined using DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay. The concentration of chlorophile a and b and carotenoids were determined by a spectrophotometric method. Obtained results indicate a higher content of chlorophyll a than chlorophyll b, 0.352 μg/ml and 0.220 μg/ml respectively, while total carotenoids content was 0.108 μg/ml and DPPH radical scavenging activity was 54.52% ± 1.77. The advancement in this research lies in collecting information about bioactive compounds, such as chlorophylls and carotenoids, that are useful in improving the functional and antioxidant properties of quality seeds used in daily diet