Fikocijanin iz mikroalge Spiruline: prečišćavanje i vezivanje odabranih (poli)fenola

Stabilization of the vivid colors of phycobiliproteins of micro/macroalgae is a prerequisite for their greater use in the food industry. Phycocyanin (C-PC) was purified from cyanobacteria Spirulina (Arthrospira) Pacifica raw extract by ammonium sulfate protein precipitation and anion ion-exchange chromatography. Purity was confirmed electrophoretically (SDS-PAGE). The binding of ten selected bioactive polyphenols to CPC (including quercetin, coenzyme Q10, gallic acid, vanillic acid, and resveratrol) was examined by standard spectroscopic methods. Quercetin is shown to have the strongest binding (Ka~3x105 M-1), with stabilization of the secondary protein structure under physiological conditions.Stabilizacija živopisnih boja fikobilinskih proteina mikro/makroalgi preduslov je za njihovo veće korišćenje u industriji hrane. Fikocijanin (C-PC) je prečišćen iz sirovog ekstrakta cijanobakterije Spirulina (Arthrospira) Pacifica, taloženjem proteina amonijumsulfatom i anjonskom jonoizmenjivačkom hromatografijom. Čistoća je potvrđena elektroforetski (SDS-PAGE). Vezivanje deset odabranih bioaktivnih polifenola za C-PC (uključujući kvarcetin, koenzim Q10, galnu kiselinu, vanilinsku kiselinu i resveratrol) ispitano je standardnim spektroskopskim metodama. Kvarcetin je pokazao najjače vezivanje (Ka~3x105 M-1), uz stabilizaciju sekundarne strukture proteina pod fiziološkim uslovima

C-Phycocyanin from cyanobacteria Artrhorspira platensis: Binding of selected food-derived ligands

To minimize the impact of artificial food colouring (e.g., in drinks) on health, chemical dyes are increasingly replaced by natural ones. C-phycocyanin (C-PC), hexameric light-harvesting phycobiliprotein from cyanobacteria Artrhorspira platensis, has been proposed as an alternative. The intensive blue colour of C-PC arises from phycocyanobilin (PCB), the covalently attached tetrapyrrole chromophores. The presence of PCB chromophores gives C-PC a broad range of bioactive effects (antioxidant, anticancer, and immunomodulatory ones), substantially increasing their potential for applications in the food industry. However, C-PC issensitive to temperature, and its colour significantly diminishes by thermal treatment, limiting its use in the food industry. Hence, improving C-PC stability is the major challenge for successful application in food and beverage colouring. It is well known that binding small, high-affinity ligands significantly improve protein stability. Therefore, selecting food-derived ligands (such as vitamins, polyphenols, sugars, etc.) with the ability to bind C-PC firmly could be a promising strategy to increase the C-PC stability and preserve its colour, which should increase its application potential in the food industry. The main aim of this study is to characterize the binding of selected food-derived ligands (including quercetin, coenzyme Q10, gallic acid, vanillic acid, vanillin, resveratrol, glucose, fructose, sucrose, vitamin K, menthol, and dihydrolipoic acid) to C-PC by standard spectroscopic methods (UV/VIS absorption spectroscopy, spectrofluorimetry, and CD spectroscopy). Quercetin has the strongest binding affinity to C-PC (Ka~3.7x105 M-1 ), and its effects on C-PC structure and stability have been further investigated. CD spectroscopy revealed that quercetin induces stabilization of the protein secondary structure under simulated physiological conditions, while the conformation of the PCB chromophore is altered upon quercetin binding. Furthermore, quercetin binding increases the thermal stability of C-PC. Overall, our study revealed the ability of high-affinity, food-derived ligands to increase the stability of C-PC, which may enhance its application potential in the food industry

Prečišćavanje i strukturna karakterizacija R-fikocijanina

Purple R-phycocyanin is a protein from red algae with the potential for application in the food industry (colorant) and wastewater treatment (binding of heavy metals). Analytical grade R-phycocyanin was purified from the buffered extract of dried Nori flakes (Porphyra spp.) by combining ammonium sulfate precipitation, hydroxyapatite, and DEAE-Sepharose column chromatography. The multimeric protein had absorption maxima characteristic for phycoerythrobilin (at 580 nm) and phycocyanobilin (at 640 nm) chromophores, high α- helical content, and melting temperature of 52°C. The secondary R-PC structure was stable under a wide range of pH values (3–9). R-phycocyanin immobilized in calcium alginate beads showed increased thermal stability and preserved antioxidant activity.Ljubičasti R-fikocijanin je protein crvenih algi sa mogućnostima primene u industriji hrane (kolorant) i za tretman otpadnih voda (vezuje teške metale). R-fikocijanin analitičke čistoće je izolovan iz puferisanog ekstrakta osušenih Nori algi (Porphyra spp.), kombinacijom taloženja amonijum-sulfatom, hidroksiapatitne i hromatografije na DEAE-Sepharose koloni. Multimerni protein imao je apsorpcione maksimume karakteristične za fikoeritrobilinsku (na 580 nm) i fikocijanobilinsku (na 640 nm) hromoforu, visok sadržaj α-zavojnica i temperaturu topljenja od 52°C. Sekundarna struktura proteina bila je stabilna u širokom rasponu pH vrednosti (3–9). R-fikocijanin imobilisan u kuglice kalcijum-alginata pokazao je povećanu toplotnu stabilnost i očuvana antioksidativna svojstva

Prečišćavanje i strukturna karakterizacija R-fikocijanina

Purple R-phycocyanin is a protein from red algae with the potential for application in the food industry (colorant) and wastewater treatment (binding of heavy metals). Analytical grade R-phycocyanin was purified from the buffered extract of dried Nori flakes (Porphyra spp.) by combining ammonium sulfate precipitation, hydroxyapatite, and DEAE-Sepharose column chromatography. The multimeric protein had absorption maxima characteristic for phycoerythrobilin (at 580 nm) and phycocyanobilin (at 640 nm) chromophores, high α- helical content, and melting temperature of 52°C. The secondary R-PC structure was stable under a wide range of pH values (3–9). R-phycocyanin immobilized in calcium alginate beads showed increased thermal stability and preserved antioxidant activity.Ljubičasti R-fikocijanin je protein crvenih algi sa mogućnostima primene u industriji hrane (kolorant) i za tretman otpadnih voda (vezuje teške metale). R-fikocijanin analitičke čistoće je izolovan iz puferisanog ekstrakta osušenih Nori algi (Porphyra spp.), kombinacijom taloženja amonijum-sulfatom, hidroksiapatitne i hromatografije na DEAE-Sepharose koloni. Multimerni protein imao je apsorpcione maksimume karakteristične za fikoeritrobilinsku (na 580 nm) i fikocijanobilinsku (na 640 nm) hromoforu, visok sadržaj α-zavojnica i temperaturu topljenja od 52°C. Sekundarna struktura proteina bila je stabilna u širokom rasponu pH vrednosti (3–9). R-fikocijanin imobilisan u kuglice kalcijum-alginata pokazao je povećanu toplotnu stabilnost i očuvana antioksidativna svojstva

A study of biological active molecules immobilised in non aqueous media: microemulsions - organogels

This study was made in the field of industrial biotechnology and its purpose was the development and optimization of systems which would be used for biocatalysis. Enzymes -and specifically lipases entrapped in microemulsions- have been used for achieving ester synthesis. Series of experiments have been done on structure and kinetic studies to solve several problems that occur, such as catalyst recovery and system stability, in order to be able to move further to using these systems in high added value products production. In order to overcome the problems that occur on catalyst recovery due to the presence of surfactant molecules in conventional microemulsions, surfactantless ternary solutions were studied. Structural studies have indicated that these are systems with defined water structures and propanol-reach interface between water and the organic solvent (hexane). When lipase from Rhizomucor miehei and lipase from Candida antarctica were entraped in the surfactantless microemulsions, they both retained their catalytic activity and showed very good stability. Remarkable is also the possibility of using these systems for the esterification of phenolic acids (anti-oxidants). Moreover, gels were prepared based on lecithin, AOT or surfactantless microemulsions, using natural polymers such as agar, gelatine or cellulose derivatives (HPMC). These organogels are stable in non polar organic solvents and consist a suitable matrix for lipase immobilization. They can effectively be used for enzymatic synthesis of fatty or phenolic acid esters. The stability of the lipases immobilized in these media is very good and appears to be better than their stability in relative microemulsions. Furthermore, the reusability of the catalyst was successful. The kinetic study revealed the ping pong bi bi mechanism with inhibition by alcoholic substrate. The external organic solvent can successfully be replaced by environmentally friendly supercritical CO2, without affecting systems efficiency. Finally, it has been proved that these organogels can successfully been used for transdermal drug delivery.Η εργασία αυτή εντάσσεται στο πεδίο της βιομηχανικής βιοτεχνολογίας και σκοπός της ήταν η ανάπτυξη και βελτιστοποίηση συστημάτων που θα χρησι-μοποιηθούν για την πραγματοποίηση βιοκαταλύσεων. Χρησιμοποιήθηκαν ένζυμα -και συγκεκριμένα λιπάσες εγκλωβισμένες σε μικρογαλακτώματα- για τη σύνθεση εστέρων. Με σειρές πειραμάτων τόσο πάνω σε δομικές μελέτες όσο και σε κινητικές μελέτες προσπαθήσαμε να επιλύσουμε τα διάφορα προβλήματα που προκύπτουν, όπως την ανάκτηση του καταλύτη και τη σταθερότητα του συστήματος, ώστε να μπορέσουμε να προχωρήσουμε στην παραγωγή προϊόντων υψηλής προστιθέμενης αξίας. Με σκοπό να ξεπεραστούν τα προβλήματα που προκύπτουν κατά την ανάκτηση του καταλύτη λόγω της παρουσίας επιφανειοενεργού στα μικρογαλακτώματα, μελετήθηκαν τριαδικά συστήματα χωρίς επιφανειοενεργό. Οι δομικές μελέτες έδειξαν ότι πρόκειται για συστήματα μικρογαλακτωμάτων στα οποία οι υδατικές μικροδομές περιβάλλονται από μια μεσεπιφάνεια πλούσια σε αλκοόλη, η οποία τις διαχωρίζει από τον εξωτερικό διαλύτη (εξάνιο). Οι λιπάσες από Rhizomucor miehei και Candida antarctica που εγκλωβίστηκαν στα μικρογαλακτώματα χωρίς επιφανειοενεργό διατηρούν την καταλυτική τους δραστικότητα, επιδεικνύουν πολύ καλή σταθερότητα, ενώ αξιοσημείωτη είναι και η δυνατότητα εφαρμογής αυτών των συστημάτων στην επίτευξη βιοκαταλυόμενων αντιδράσεων σύνθεσης εστέρων ορισμένων φαινολικών οξέων (αντι-οξειδωτικά). Επίσης σχηματίστηκαν πηκτώματα (γέλες) με βάση μικρογαλακτώματα χωρίς επιφανειοενεργό ή λεκιθίνης ή ΑΟΤ, με φυσικά πολυμερή όπως το άγαρ, η ζελατίνη και παράγωγα κυτταρίνης (HPMC). Τα συστήματα αυτά είναι σταθερά σε μη πολικούς οργανικούς διαλύτες και αποτελούν κατάλληλο φορέα για την ακινητοποίηση λιπασών και την ενζυμική σύνθεση εστέρων τόσο λιπαρών όσο και φαινολικών οξέων με αλειφατικές αλκοόλες. Η σταθερότητα των λιπασών που ακινητοποιήθηκαν στα συστήματα αυτά είναι πολύ καλή και μάλιστα εμφανίζεται αυξημένη σε σύγκριση με τις ίδιες λιπάσες εγκλωβισμένες σε μικρογαλακτώματα. Επίσης, η επαναχρησιμοποίηση του καταλύτη ήταν επιτυχής. Η κινητική μελέτη έδειξε ότι στα συστήματα αυτά ακολουθείται ο μηχανισμός ping pong bi bi με παρεμπόδιση από το αλκοολικό υπόστρωμα. Ο εξωτερικός οργανικός διαλύτης μπορεί να αντικατασταθεί επιτυχώς από το φιλικό προς το περιβάλλον υπερκρίσιμο CO2, χωρίς να επηρεαστεί η αποδοτικότητα του συστήματος. Τέλος, αποδείχτηκε ότι τα πηκτώματα αυτά μπορούν να χρησιμοποιηθούν επιτυχώς για τη διαδερμική χορήγηση φαρμακευτικών ουσιών

Oxidation Catalysis by Enzymes in Microemulsions

Microemulsions are regarded as “the ultimate enzyme microreactors” for liquid oxidations. Their structure, composed of water nanodroplets dispersed in a non-polar medium, provides several benefits for their use as media for enzymatic transformations. They have the ability to overcome the solubility limitations of hydrophobic substrates, enhance the enzymatic activity (superactivity phenomenon) and stability, while providing an interface for surface-active enzymes. Of particular interest is the use of such systems to study biotransformations catalyzed by oxidative enzymes. Nanodispersed biocatalytic media are perfect hosts for liquid oxidation reactions catalyzed by many enzymes such as heme peroxidases, phenoloxidases, cholesterol oxidase, and dehydrogenases. The system’s composition and structural properties are important for better understanding of nanodispersion-biocatalyst interactions

Molecular Catalysis

The present work reports on the use of a hybrid blend of biopolymers as a matrix for lipase immobilization. (Hydroxypropyl)methyl cellulose (HPMC) and Chitosan (CS) were combined in order to formulate a film on which Mucor miehei lipase was immobilized. The biocatalyst was studied upon the model reaction of propyl laurate synthesis. The system was examined in terms of its capability to provide an appropriate environment where lipase will maintain its activity. The ratio of the polymers used was examined and HPMC:CS=2:1 proved to form the most promising matrix. Increasing the amount of the immobilized enzyme appears to improve the reaction yield indicating, however, mass transfer limitations. Apparent activation energy was calculated and energy input showed that ultra-sonication accelerated the initial rate of the reaction. Different reaction solvents were tested with isooctane being the most effective. The enzyme-containing film showed a remarkable reusability, since it can be used for up to 35 times without loss of activity. Finally, Atomic Force Microscopy (AFM) was performed to observe the morphology of the most promising films. The HPMC/CS film exhibits a nanostructure without a unique characteristic length and a roughness of 42.8 nm while the presence of enzyme smoothens the film as the roughness decreases to 5.5 nm

(Hydroxypropyl)methyl Cellulose-Chitosan Film as a Matrix for Lipase Immobilization. Part ΙΙ: Structural Studies

International audienceThe present work reports on the structural study of a film made of a hybrid blend of biopolymers used as an enzyme carrier. A cellulose derivative (HPMC) and chitosan (CS) were combined in order to formulate a film on which Mucor miehei lipase was immobilized. The film was successfully used as a biocatalyst; however, little is known about the structure of the system. Therefore, small-angle X-ray scattering, Fourier transform infrared spectroscopy (FTIR), optical microscopy, and scanning electron microscopy (SEM), as well as microindentation measurements, were used to shed light on the structure of the promising biocatalyst. Among the results, intermolecular hydrogen bonds were observed between the amide groups of the two polymers and the lipase. The presence of the enzyme does not seem to affect the mechanical properties of the matrix. The used film after 35 cycles of reaction seemed to be fatigued and had lost part of its humidity, explaining the reduction of the enzyme activity

Immobilization and activity of Rhizomucor miehei lipase. Effect of the matrix properties prepared from nonionic fluorinated surfactants

International audienc

Exploring and strengthening the potential of R-phycocyanin from Nori flakes as a food colourant

This study aimed to purify, characterise and stabilise the natural food colourant, R-phycocyanin (R-PC), from the red algae Porphyra spp. (Nori). We purified R-PC from dried Nori flakes with a high purity ratio (A618/A280 ≥ 3.4) in native form (α-helix content 53%). SAXS measurements revealed that R-PC is trimeric ((αβ)3) in solution. The thermal denaturation of α-helix revealed one transition (Tm at 52 ◦C), while the pH stability study showed R-PC is stable in the pH range 4–8. The thermal treatment of R-PC at 60 °C has detrimental and irreversible effects on RPC colour and antioxidant capacity (22 % of residual capacity). However, immobilisation of R-PC within calcium alginate beads completely preserves R-PC colour and mainly retains its antioxidant ability (78 % of residual capacity). Results give new insights into the stability of R-PC and preservation of its purple colour and bioactivity by encapsulation in calcium alginate beads