Kaldirik (trachystemon orientalis) bitkisinden polifenol oksidaz enziminin kısmi saflaştırılması ve karakterizasyonu

06.03.2018 tarihli ve 30352 sayılı Resmi Gazetede yayımlanan “Yükseköğretim Kanunu İle Bazı Kanun Ve Kanun Hükmünde Kararnamelerde Değişiklik Yapılması Hakkında Kanun” ile 18.06.2018 tarihli “Lisansüstü Tezlerin Elektronik Ortamda Toplanması, Düzenlenmesi ve Erişime Açılmasına İlişkin Yönerge” gereğince tam metin erişime açılmıştır.Bu çalışmada, Trachystemon orientalis (Kaldirik) bitkisinden ekstrakte edilen Polifenol oksidaz (PPO) enzimi jel filtrasyon kromatografisiyle kısmi olarak saflaştırılmıştır. Yapılan karakterizasyon çalışmalarında ham enzim ekstraktı kullanılmıştır. Enzim monofenolaz aktivitesi göstermemekle birlikte difenolaz ve trifenolaz aktivitesine sahiptir. Katekol, 4-metil katekol, kafeik asit ve pirogallol substratlarına karşı aktivite gösteren enzimin kafeik asit substratına karşı etkinliği en fazladır. Enzimin optimum pH ve sıcaklık değerlerinin substrata göre farklandığı görülmüştür. Optimum pH değeri katekol ve pirogallol için 7,5; 4-metil katekol için 5,0; kafeik asit için ise 5,5'dur. Optimum sıcaklık değerleri ise katekol için 10C,4-metil katekol için 5°C, kafeik asit için 20C ve pirogallol için 30C'dir. Pekçok PPO enzimi üzerinde şiddetli inhibisyon etkisi yaratan kalay ve kurşun metallerine ve sodyum azid inhibitörüne karşı kaldirik PPO enziminin dayanıklı olduğu tespit edilmiştir. Asidik, bazik ve nötral aminoasitlerle gerçekleştirilen inhibisyon denemelerinde, denenen tüm aminoasitlerin enzimi inhibe ettiği ve en yüksek inhibisyon etkisini asidik aminoasitlerin yarattığı belirlenmiştir. Ayrıca yapılan termal inaktivasyon ve depolanma kararlılığı çalışmaları da enzimin diğer bitki kaynaklı PPO enzimlerine göre daha dayanıklı olduğunu göstermiştir.In this study, the Polyphenol oxidase enzyme which was extracted from Trachystemon orientalis (Borage) was partially purified by using gel filtration column. The crude extract was used for the characterization studies of the enzyme. Whereas PPO enzyme did not show monophenolase activity, it had diphenolase and triphenolase activity. PPO enzyme that had activity toward catechol, 4-methylcatechol, cafeic acid and pyrogallol has the greatest activity toward cafeic acid. The enzyme showed different optimum pH and temperature values for different substrates. The optimum pH was at 7,5; 5,0; 5,5 for catechol and pyrogallol, 4-methyl catechol, cafeic acid, respectively. The enzyme had an optimum temperature at 10C for catechol, 5C for 4-methyl catechol, 20C for cafeic acid and 30C for pyrogallol. We detected that Trachystemon orientalis PPO was stable against tin and lead metals and sodium azide which could cause heavy inhibition effect on other PPOs. Inhibition assays with acidic, basic and neutral aminoacids showed that all tried aminoacids had inhibitory effect on enzyme and acidic aminoacids were the most effective ones. We also found that Trachystemon orientalis PPO is more stable than other herbal PPOs depending on heat inactivation and storage stability studies

A novel serine protease from strawberry (Fragaria ananassa): Purification and biochemical characterization

In this study, a protease enzyme was purified from strawberry by using Sepharose-4B-L-tyrosine-p-amino benzoic acid affinity chromatography. The molecular weight of pure protease was determined 65.8 kDa by SDS-PAGE. The single band observed on the gel showed that the enzyme had a single polypeptide chain and was successfully purified. Purification of the protease by the chromatographic method resulted in a 395.6-fold increase in specific activity (3600 U/mg). Optimum pH and temperature for the enzyme were 6 and 40 degrees C, respectively. The protease was stable at a wide temperature range of 40 to 70 degrees C and a pH range of 3.0 to 9.0. Co2+ ions stimulated protease activity very strongly. Cu2+, Hg2+, Cd2+ and Mn2+ ions significantly inhibited protease activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin among all the natural substrates tested. The specificity of the enzyme towards synthetic substrates was also investigated and it was concluded that it has broad substrate specificity. The obtained results indicated that this purified protease was highly-likely a serine protease and its activity was significantly affected by the presence of metal ions. (C) 2018 Published by Elsevier B.V

Purification of polyphenol oxidase from borage (Trachystemon orientalis L.) by using three-phase partitioning and investigation of kinetic properties

In this study a Polyphenol oxidase from borage plant was purified with 3.59-fold enrichment in the specific activity and 68.75% recovery of the total activity by using three-phase partitioning purification technique for the first time. Its molecular weight was found around 80 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature values of the enzyme for the used four substrates ranged between the pH 5.0-7.5 and 5-30 degrees C. The kcat/K-m values showed that the enzyme has the greatest reactivity toward caffeic acid among the substrates used. Ascorbic acid, L-cysteine and sodium metabisulfite markedly inhibited borage polyphenol oxidase activity. (C) 2016 Elsevier B.V. All rights reserved

Partial Purification and Characterization of Polyphenol Oxidase from the Wild Edible Mushroom Lepiota Procera Using Three-Phase Partitioning

Polyphenol oxidase (PPO) from the wild edible mushroom Lepiota procera is partially purified and biochemically characterized using three-phase partitioning (TPP), which is an easily applied and effective method. This method includes ammonium sulfate saturation at different concentrations, t-butanol addition (1:1; 1:5), and adjustment of pH. Optimum purification parameters with 20% ammonium sulfate saturation and 1:1 t-butanol conditions led to the highest activity at bottom phase with 8.4 fold purification. Sodium dodecyl sulfate- poly-acrylamide gel electrophoresis (SDS-PAGE) analysis showed the molecular weight of the purified enzyme to be 35 kDa. The partially purified PPO presented a high level of activity with L-DOPA (Michaelis-Menten constant, Km - 0.12 mM), followed by caffeic acid (0.27 mM) and 4-methylcatechol (0.46 mM) and it is classified as a catecholase type of PPO. The enzyme had peak activity at a temperature of 40 degrees C and a pH value of 7.0. These results demonstrate a new enzyme source and easy purification method, useful for various industrial applications

Synthesis, characterization, aggregation, fluorescence and antioxidant properties of bearing (4-(methylthio)phenylthio) tetra substituted phthalocyanines

A new substituted phthalonitrile derivative, 4-(4-(methylthio)phenylthio) phthalonitrile was prepared by nucleophilic displacement reaction between 4-(methylthio)phenylthio and 4-nitrophthalonitrile. Tetra phthalocyanines [M: 2H(2), Zn(II)(3), Co(II)(4)] with four peripheral 4-(methylthio)phenylthio groups were synthesized by cyclotetramerization. They were characterized by elemental analysis, FTIR, H-1 NMR, C-13 NMR, MALDI-TOF, UV-vis, Fluorescence, AFM, and spectral data. Their aggregation properties were examined in THF by UV-vis and Fluorescence spectrophotometers. The antioxidant properties of newly synthesized metal-free and metallophthalocyanines were also investigated by using three different antioxidant methods: free radical scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ion chelating ability and reducing power activity. The highest DPPH activity was found from cobalt phthalocyanines (4) compound respectively. All tested phthalocyanines compounds had fairly ferrous ion chelating activity and showed very good reducing power. They had even better reducing power than the standards antioxidant compounds such as ascorbic acid and BHT. Thus, Tetra phthalocyanines [M: 2H (2), Zn(II)(3), Co(II)(4)] with four peripheral 4-(methylthio)phenylthio groups could be used as potential antioxidant compounds. (C) 2017 Elsevier B.V. All rights reserved

Formation, characterization, aggregation, fluorescence and antioxidant properties of novel tetrasubstituted metal-free and metallophthalocyanines bearing (4-(methylthio)phenoxy) moieties

The synthesis and characterization of peripherally tetra 4-(methylthio)phenoxy substituted metal-free(2), Zn(II) (3) and Co(II) (4) phthalocyanine derivatives were reported. These newly synthesized phthalocyanine derivatives showed the enhanced solubility in organic solvents and they were characterized by a combination of elemental analysis, FTIR, H-1 NMR, C-13 NMR, UV-vis and MALDI-TOF/MS spectral data. Their aggregation properties were investigated in THE by UV-vis and fluorescence. These metal-free and metallophthalocyanine compounds were also evaluated for their total antioxidant abilities by using three different antioxidant methods such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, ferrous ion chelating and reducing power activity. All tested compounds showed radical scavenging activity. The highest radical scavenging activity was found from cobalt phthalocyanine (4) compound respectively. IC50 values of the compounds and standards (BHT and Trolox) were also determined. The results showed that the compound 4 had the highest antioxidant activity among all tested compounds including standards. The tested phthalocyanine compounds had ferrous ion chelating activity. In addition, they showed very high reducing power. All tested compounds had higher reducing power than the standards such as ascorbic acid and BHT. The present study shows that the synthesized tetra phthalocyanine [M: 2H(2), Zn(II)(3), Co(lI)(4)] with four peripheral 4-(methylthio) phenoxy compounds have the effective antioxidant properties that can be used as antioxidant agents. (C) 2017 Elsevier B.V. All rights reserved

alpha- or beta-Substituted functional phthalocyanines bearing thiophen-3-ylmethanol substituents: synthesis, characterization, aggregation behavior and antioxidant activity

The tetra - or -thiophene substituted metal and metal-free phthalocyanines (Pcs) M[Pc(-OCH(2)Thiopen)(4)] and M[Pc(-OCH(2)Thiopen)(4)] {(-ThMet-MPc), (-ThMet-MPc) [ThMet: Thiophene methoxy], M=Zn(II), Co(II) and, 2H} were synthesized from the corresponding 3'-(thiophen-3-ylmethoxy)phthalonitrile or 4'-(thiophen-3-ylmethoxy)phthalonitrile (ThMePN). The structural characterization, spectral, and antioxidant properties of a series of new Pcs were also presented. Both - and -substituted Pc complexes increased solubility in polar solvents, such as THF, DMF, and DMSO. FT-IR, H-1-NMR, C-13-NMR, UV-vis, MALDI-TOF/MS spectral, and elemental analysis data were used to characterize the compounds. The aggregation behaviors of 3-8 were also investigated at different concentrations in THF. Antioxidant test methods, ,-diphenyl--picrylhydrazyl radical scavenging activity, metal chelating activity, and reducing power, were used to determine the antioxidant activities. 6 showed very good ferrous ion chelating activity of 81 +/- 1%. 6, 5, 4, and 3 showed better reducing power than trolox, ascorbic, acid and butylated hydroxytoluene, commercially used antioxidants

Synthesis, characterization, antioxidant and antibacterial properties of non-peripherally and peripherally tetra-substituted phthalocyanines

.This study focuses on the synthesis, spectral, antioxidant and antibacterial properties of the metal-free, zinc and cobalt phthalocyanines (3–8) bearing 4-methoxy-phenoxy substituents on the nonperipheral [(1(4), 8(11), 15(18), 22(25)] or peripheral [2(3), 9(10), 16(17), 23(24)] positions. The new synthesized complexes 7 and 8 have been characterized by elemental analysis, FT-IR, MALDI-MS, and UV-vis spectral data. The antioxidant activities of all tested compounds were investigated by applying three different antioxidant methods such as radical scavenging ability of 1,1-diphenyl-2-picrylhydrazyl, chelating ability to ferrous ions and reducing power activity methods. In addition, the antibacterial activities of the compounds were screened by disc diffusion method against one gram-negative and four gram-positive bacteria. The tested phthalocyanine compounds showed very good antioxidant activity and promising antibacterial properties

Antioxidant properties of water-soluble phthalocyanines containing quinoline 5-sulfonic acid groups

In the present study, the antioxidant properties of peripherally and nonperipherally water-soluble tetra (quinoline 5-sulfonic acid) substituted metal-free (1, 2), Zn(II) (3, 4), Co(II) (5, 6), and Mn(III)Cl (7, 8) phthalocyanine (Pc) derivatives were reported. In order to determine the antioxidant properties of the Pc compounds, three different commonly known antioxidant methods were used: DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) radical scavenging, ferrous ion chelating, and reducing power assays. Compound 6 showed the best DPPH activity among the other tested compounds except for the standards. The ferrous ion chelating IC(50)value of compound 7 was nearly as good as that of the EDTA standard. The reducing power activity results indicated that the tested Pc compounds were as sensitive as standard reducing agents such as butylated hydroxytoluene and ascorbic acid. Overall, all tested compounds showed good antioxidant properties and it may be suggested that they could be used as new positive controls for reducing power assays in the future works