Termofilik mantar Scytalidium thermophilum'un çift aktiviteli katalaz/katekol oksidaz enziminin fenolik maddeler varlığında üretimi ve biyokatalitik özellikleri

TÜBİTAK TBAG01.11.2009Scytalidium thermophilum mantar kompostunda yaşayan ve yenebilir mantar Agaricus bisporus’un üretimini tetikleyen bir termofilik mantardır. S. thermophilum’un çok miktarda melanin üretmesinden hareketle, bu küfte fenol oksidaz enzimlerinin varlığının araştırılması hedeflenmiştir. Sonuç olarak, S. thermophilum hücre-dışı fenol oksidazının bir çift aktiviteli katalaz-fenol oksidaz (KATFO/CATPO) olduğu belirlenmiştir. Bu proje kapsamında S. thermophilum 15 farklı fenolik madde varlığında ve farklı dozlarda büyütülmüş ve KATFO’nun üretimi incelenmiştir. Daha sonra, hem ortam sıvısı hem de saf enzim kullanılarak 15 maddenin oksidasyonu incelenmiştir. Saf enzimle okside olabilen ürünlerden 5 tanesinin oksidasyon ürünleri karakterize edilmiştir. Elde edilen bulgulara göre, KATFO sürekli (konstitütif) ve büyümeye paralel olarak üretilmektedir, ancak fenolikler büyümeyi ve enzim üretimini etkileyebilmektedirler. KATFO katekol, hidrokinon, katekin, kuersetin, klorojenik asit ve kafeik asiti okside edebilmiştir. Bunlardan katekol, hidrokinon, kafeik asit, klorojenik asit ve katekinin C-C ve C-O-C bağları ile polimerleştiği görülmüştür. Özellikle dimer, trimer ve tetramer yapıları belirlenmiş ancak, genel olarak, çoklu ürünlerin varlığı ve polimerleşmenin zamana karşı devam ettiği gözlenmiştir. Katekol, hidrokinon, mirisetin, kaempferol ve kumarik asit artan dozlarda büyümeyi olumsuz etkilemiş, kafeik asit, klorojenik asit, kuersetin, katekin, epikatekin, resorsinol, vanillik asit ve resveratrol varlığında ise büyüme olumsuz etkilenmemiş, hatta %50’ye varan artışlar görülmüştür. Büyümeyi olumlu etkileyen fenolikler varlığında KATFO üretimi ya belirgin bir değişim göstermemiş veya artan dozlarda az ya da çok baskılanmıştır. Toksik etkinin görüldüğü durumlarda genelde KATFO’da olumsuz etkilenmiş, ancak antifungal etki gösteren hidrokinon ve mirisetinde KATFO doza bağlı tetiklenmiştir. Büyümeyi olumlu etkileyen fenoliklerin antioksidan etki gösterdiği düşünülmektedir. Bunların çoğu ve özelinde yapısında orto-difenol bulunduran fenolik asitler ve flavonoidler KATFO tarafından okside olabilmiştir. Diğer yandan, antifungal/toksik etki gösteren maddelerin büyük bir kısmı, mirisetin, kaempferol ve kumarik asit, KATFO tarafından okside olmamıştır. Buna göre, KATFO’nun fenolik maddelerin oksidasyonu ile onların toksik etkilerini azalttığı ve/veya antioksidan etkilerini artırdığı yönünde bir olasılık ortaya çıkmıştır.Scytalidium thermophilum is a thermophilic fungus found in mushroom compost, where it triggers the production of the edible mushroom Agaricus bisporus. Since S. thermophilum produces immense amounts of melanin, phenol oxidase production was analysed. As a result, the extracellular phenol oxidase of S. thermophilum was determined as a bifunctional catalase phenol oxidase (CATPO/KATFO). In the scope of this project S. thermophilum was cultivated in the presence of 15 different phenolic compounds, at different concentrations, to explore CATPO production. Next, the oxidation of these 15 substances was analyzed by using both the supernatant and pure enzyme. The oxidation products of 5 of these compounds was further characterized. According to the findings, CATPO is produced constitutively and in a growth-associated manner. Nonetheless, phenolics could affect the growth and enzyme production. CATPO could oxidize catechol, hydroquinone, catechin, quercetin, chlorogenic acid and caffeic acid. These compounds were mainly polymerized by C-C and C-O-C bonds. Especially dimer, trimer and tetramer structures were determined, however, the presence of multiple products and the progression of polymerization by time was also observed. Catechol, hydroquinone, myricetin, kaempferol and coumaric acid have influenced the growth adversely in an increasing concentration manner, on the other hand under the influence of caffeic acid, chlorogenic acid, quercetin, catechin, epicatechin, recorcinol, vanillic acid and resveratrol the growth was not adversely affected, in fact, upto 50% increase in biomass was detected. Most phenolics that positively affected growth either did not change CATPO production or, more or less, repressed it, in a dose-dependent manner. Under toxic conditions CATPO production was adversely affected, however, hydroquinone and myricetin, which showed antifungal acitivity, enhanced CATPO, in a dose-dependent manner. It is suggested that phenolics that affect growth positively show antioxidant activity. Among these, especially the phenolic acids and flavonoids bearing ortho-diphenols in their structures were oxidized by CATPO and did not inhibit growth. However, most phenolics acting in an antifungal/toxic manner, were not oxidized by CATPO. Accordingly, it is suggested that the oxidation of phenolics by CATPO has an affect either on their detoxification and/or enhancing their antioxidant activity

Production of a novel bifunctional catalase-phenol oxidase of Scytalidium thermophilum in the presence of phenolic compounds

A novel bifunctional catalase with additional phenol oxidase activity (CATPO) is produced by the thermophilic fungus, Scytalidium thermophilum, in a growth-associated manner. In order to study the biological significance of this dual enzyme in relation to phenolic compounds, 14 phenolics were tested for their effect on growth and CATPO production. It was determined that some phenolics exerted a negative effect on growth (catechol, coumaric acid, hydroquinone, kaempferol, myricetin), while others either did not influence growth in a negative manner or enhanced growth by a maximum of 50% increase in biomass (caffeic acid, chlorogenic acid, catechin, gallic acid, resorcinol, vanillic acid). Hydroquinone and myricetin showed an antifungal effect and enhanced CATPO production, while catechol, coumaric acid, and kaempferol decreased CATPO production and showed a toxic effect at higher doses. In general, phenolics acting in an antioxidant manner exhibited a reverse interrelation with CATPO production. These results suggest that the presence of antioxidant phenolic compounds has an effect on enhancing its antioxidant activity; enzyme is therefore no longer required

Frontiers in Vitamin Research: New Antibodies, New Data

Since 2004, the anatomical distribution of vitamins in the monkey brain, studied using immunohistochemical techniques and new tools (specific antisera that discriminate different vitamins reasonably well), has been an ongoing research field. The visualization of immunoreactive structures containing vitamins (folic acid, riboflavin, thiamine, pyridoxal, and vitamin C) has recently been reported in the monkey brain (Macaca fascicularis), all these vitamins showing a restricted or very restricted distribution. Folic acid, thiamine, and riboflavin have only been observed in immunoreactive fibers, vitamin C has only been found in cell bodies (located in the primary somatosensory cortex), and pyridoxal has been found in both fibers and cell bodies. Perikarya containing pyridoxal have been observed in the paraventricular hypothalamic nucleus, the periventricular hypothalamic region, and in the supraoptic nucleus. The fibers containing vitamins are thick, smooth (without varicosities), and are of medium length or long, whereas immunoreactive cell bodies containing vitamins are round or triangular. At present, there are insufficient data to elucidate the roles played by vitamins in the brain, but the anatomical distribution of these compounds in the monkey brain provides a general idea (although imprecise and requiring much more study) about the possible functional implications of these molecules. In this sense, here the possible functional roles played by vitamins are discussed

Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia: The İstanbul Perspective

INTRODUCTION: Alpha thalassemia syndromes are caused by mutations on one or more of the four α-globin genes. Mutations could be either more commonly deletional or non-deletional. As some deletions (3.7 and 4.2) cause α+-thalassemia, some cause (-20.5, MED, THAI, FIL) α0 -thalassemia. The aim of this study was to determine alpha thalassemia mutations in patients with unsolved hypochromic microcytic anemia and to evaluate types of mutations. METHODS: Two hundred six patients with hypochromic microcytic anemia were evaluated for alpha thalassemia. A venous blood sample of 2 mL was drawn from each patient for DNA isolation. The samples were investigated for α-thalassemia mutations by using the Vienna Lab α-Globlin StripAssay TM commercial kit. RESULTS: Fourteen different mutations were determined in 95 (46.1%) patients. The most common mutation was the 3.7 single gene deletion and was found in 37 patients (n=37/95, 39%). Others common mutations were the 20.5 kb double gene deletion (n=20 patients, 21%), MED double gene deletion (n=17 patients, 17.9%), α2 IVS1 (n=10 patients, 10.5%), α2 cd142 Hb Koya Dora (n=6 patients, 6.3%), α2 polyA1 (Saudi type) (n=6 patients, 6.3%), 4.2 single gene deletion (n=4 patients, 4.2%), α1 cd14 (n=2 patients, 2.1%), and -FIL mutation (n=2 patients 2.1%), respectively. Hb Adana, Hb Icaria, α2 init cd and α2 polyA2 (Turkish type) were found in 1% of the patients (n=1). Seven patients (7.4%) had α-thalassemia triplication. In our study, three mutations (Hb Icaria, α1 cd14, α2 init.cd) were determined firstly in Turkey. Seven mutations (-SEA, -THAI, Hb Constant Spring, α2 cd19, α2 cd59, α2 cd125, Hb Paksé) were not determined in this study. DISCUSSION AND CONCLUSION: Alpha thalassemia should be considered in the differential diagnosis of hypochromic microcytic anemia especially in cases without iron deficiency and β-thalassemia carrier state. Genetic testing should be performed for the suspicious cases. We also recommend that a national database with all mutations in Turkey should be created to screen the alpha thalassemia cost-effectively

Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia: The İstanbul Perspective.

Abstract:Objective: Alpha thalassemia syndromes are caused by mutations on one or more of the four α-globin genes. Mutations couldbe either more commonly deletional or non-deletional. As some deletions (3.7 and 4.2) cause α+-thalassemia, some cause(-20.5, MED, THAI, FIL) α0 -thalassemia. The aim of this study was to determine alpha thalassemia mutations in patients withunsolved hypochromic microcytic anemia and to evaluate types of mutations.Material and Methods: Two hundred six patients with hypochromic microcytic anemia were evaluated for alphathalassemia. A venous blood sample of 2 mL was drawn from each patient for DNA isolation. The samples were investigatedfor α-thalassemia mutations by using the Vienna Lab α-Globlin StripAssay TM commercial kit.Results: Fourteen different mutations were determined in 95 (46.1%) patients. The most common mutation was the 3.7single gene deletion and was found in 37 patients (n=37/95, 39%). Others common mutations were the 20.5 kb double genedeletion (n=20 patients, 21%), MED double gene deletion (n=17 patients, 17.9%), α2 IVS1 (n=10 patients, 10.5%), α2 cd142Hb Koya Dora (n=6 patients, 6.3%), α2 polyA1 (Saudi type) (n=6 patients, 6.3%), 4.2 single gene deletion (n=4 patients, 4.2%),α1 cd14 (n=2 patients, 2.1%), and -FIL mutation (n=2 patients 2.1%), respectively. Hb Adana, Hb Icaria, α2 init cd and α2polyA2 (Turkish type) were found in 1% of the patients (n=1). Seven patients (7.4%) had α-thalassemia triplication. In ourstudy, three mutations (Hb Icaria, α1 cd14, α2 init.cd) were determined firstly in Turkey. Seven mutations (-SEA, -THAI, HbConstant Spring, α2 cd19, α2 cd59, α2 cd125, Hb Paksé) were not determined in this study.Conclusion: Alpha thalassemia should be considered in the differential diagnosis of hypochromic microcytic anemiaespecially in cases without iron deficiency and b-thalassemia carrier state. Genetic testing should be performed for thesuspicious cases. We also recommend that a national database with all mutations in Turkey should be created to screen thealpha thalassemia cost-effectively