Domates filizinden asit fosfatazın kısmi saflaştırılması ve özelliklerinin incelenmesi
Partial purification and characterization of tomato seedling acid phosphatase
- Tez No: 101130
- Danışmanlar: PROF.DR. NURAN DEVECİ
- Tez Türü: Yüksek Lisans
- Konular: Kimya Mühendisliği, Chemical Engineering
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2000
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 85
Özet
DOMATES FİLİZİNDEN ASİT FOSFATAZ ENZİMİNİN KISMI SAFLAŞTIRILMASI VE ÖZELLİKLERİNİN İNCELENMESİ ÖZET Enzimler ortam sıcaklığında hayatın devam etmesini sağlayan çok özel moleküllerdir. Protein yapısındadırlar ve diğer bütün proteinlerde olduğu gibi birbirlerine peptid bağları ile bağlanmış uzun aminoasit zincirleri içerirler. Bütün yaşayan hücrelerde mevcutturlar ve bulundukları yerde meydana gelen metabolik prosesleri kontrol etmek gibi çok önemli işlevleri vardır. Enzimler biyokatalizdirler. Proseste harcanmadan, mevcut olmadıkları taktirde çok yavaş ilerleyecek olan kimyasal reaksiyonları hızlandırırlar. Asit Fosfataz (ortofosforik monoester fosfohidrolaz; EC 3.1.3.2) asidik şartlar altında fosfomonoesterlerin hidrolizini katalizleyen, doğada çok yaygın olan fakat az miktarlarda elde edilebilen bir enzimdir.Asit fosfataz ismi bu enzimin asidik aralık içindeki optimum pH larda yüksek aktivite göstermesinden ileri gelmektedir. Bu çalışmanın amacı domates Sizinden Asit Fosfataz enziminin izolasyonu, saflaştırılması ve bazı özelliklerinin incelenmesi olarak belirlenmiştir. Enzim domates filizlerinden ilk etapta tampon çözelti kullanılarak ve homojenizatörde parçalanarak ekstrakte edilmiştir. Saflaştırma işlemleri olarak, santrifüjleme, amonyum sülfat ile doyurma, diyaliz, DEAE Sellüloz ve DEAE Sepharose iyon değiştirme kolon kromotogrofısi uygulanmıştır. Çalışmada iki, dört ve altı haftalık domates filizlerinin spesifik aktiviteleri karşılaşılmıştır. Buna göre iki haftalık ham homojenatin spesifik aktivitesi 0.330 U/mg, %60 doyurma sonrası spesifik aktivitesi 0.532 U/mg dır. Doyurma sonrası iki kat saflaşma elde edilmiştir. Dört haftalık domates filizinden elde edilen ham homojenatin spesifik aktivitesi 0.289 U/mg, %60 doyurma sonrası 0.632 U/mg ve DEAE selüloz iyon değiştirme kolon kromotogrofısi sonrası spesifik aktivitesi 6.98 U/mg dır. Doyurma sonrası iki, kolon sonrası 24 kat saflaşma elde edilmiştir. Altı haftalık domates filizleri ile yapılan çalışmada ise ham homojenatin spesifik aktivitesi 0.06 U/mg, %60 doyurma sonrası spesifik aktivitesi 0.182 U/mg dır. 3 kat saflaşma olduğu tespit edilmiştir. Çalışmada aynı zamanda farklı substratlarla da çalışılmış, iki ve dört haftalık domates filizlerinden elde edilmiş olan enzim çözeltisinin optimum pH ve optimum sıcaklık ile pH ve sıcaklık kararlılığı incelenmiştir. e-- VII
Özet (Çeviri)
PARTIAL PURIFICATION AND CHARACTERIZATION OF TOMATO SEEDLING ACID PHOSPHATASE SUMMARY Enzymes are catalysts. Each reaction taking place in the cell is catalyzed by its own particular enzyme so that there are large numbers of enzymes. Enzymes are bought and sold by activity rather than by weight. The aim of purification procedure should be to isolate a given enzyme with the maximum possible yield, based on the percentage recovered activity compared with the total activity in the original extract. The preparation should possess the maximum catalytic activity. There is no need to use more highly purified, or modified, enzyme for industrial applications, it would be necessary for the particular process like clinical analyses. As work done on the enzyme, it becomes more expensive. Acid phosphatase or ortophosphoric monoester phosphohydrolase that catalyze the hydrolysis of a large number of orthophosphoric monoester compounds within the acid range of pH, is widely distributed in nature and has been studied in various plants and animals. Acid Phosphatases mostly catalyze the following reaction at an optimum pH below 6.0: Orthophosphoric monoester +H2O ? alcohol+H3P04 Acid phospahatase activity was observed in 1954 in preparations of a wheat germ lipase. The enzyme has been purified and its properties has described by at least three different laboratories. It was purified 3000-fold and its activity on a variety of phosphate esters was measured. The biological role of plant and animal acid phosphatase remains largely unknown. Acid phosphatases are ubiquitous in nature having already been identified in numerous organisms and tissues, perhaps one of the most concentrated sources being human prostate gland. In general, acid posphatases occur in very small quantities, are unstable in diluted solutions and are subject to surface denaturation in pure state. These properties, and their tendencies to occur in multiple forms, often make the isolation of acid phosphatase difficult. Studies on pure acid phosphatases have so far focused mainly on those of clinical significance from animal sources. Unlike the phosphatases of animal origin, those from plant sources have so far been examined only to a limited extend and some of the reported results are incompatible with each other. Acid phosphatase is found in various organs in human body, e.g. the prostate, gland, liver and spleen and in blood cells. When tissue cells are damaged, acid phosphatase is transferred to the plasma. Therefore, the determination of Acid phosphatase in body fluids is a valuable tool in the diagnosis of diseases of the organs where is normally found. VIIIPerhaps because of the difficulty of obtaining the pure enzyme, the function of the nonspecific acid phosphatases is poorly defined. Among possible functions of Acid phosphatases might be the role of providing organic phosphate for metabolic excretory and some secretory purposes. Acid phosphatases may also have a role in the mammalian reproductive processes. Many roles have been postulated for these enzymes in plant, including a role in the release of inorganic phosphate from organic phosphate in the environment. Phosphorous not only plays a vital role in energy transfer and in metabolic regulation but is also an important constituent of macromolecules such as phospholipids, proteins and nucleic acids. In plants, Acid Phosphatase activity in seeds often increases greatly during germination, indicating possible role in phosphate mobilization. The purpose of this study was the partial isolation and purification of acid phosphates from one of the plant sources tomato seedlings and to determine its characteristics. In this study, the purification scheme to isolate the enzyme involves the following steps: 1. Extraction: Tomato seedlings were disrupted by a homogenizator and the enzyme was extracted by means of 0.1 mM sodium acetate buffer (pH 5.5, 0.1 M NaCl, %0.1 TritonX-100) 2. Centrifugation: Done at 20000-22000 rpm. 3. Saturation: Relatively %40, %60 and % 80 ammonium sulfate saturation. 4. Dialysis: Used a dialysis membrane via sodium acetate buffer. 5. Ion-exchange chromatography: DEAE Celluose and DEAE Sephadex. In this study, the relationship between tomato seedlings' growth rate and enzyme activity was established in the first place. Above-mentioned procedure was applied partially to two, four, six -week tomato seedlings. The results obtained by using 4- nitrophenylphosphate as substrate are mentioned below:. In the study of two-week tomato seedlings, the specific activity of crude extract is 0.330 Unit/mg, the specific activity of the sample after %60 ammonium sulfate fraction is 0.532 Unit/mg then the purification fold is 2.. In the study of four-week tomato seedlings, the specific activity of crude extract is 0.289 Unit/mg, the specific activity of the sample after %60 ammonium sulfate fraction is 0.62 Unit/mg then the purification fold is 2. After DEAE Cellulose ion- exchange chromatography, the specific activity increased up to 6.98 Unit/mg, and the purification fold is almost 24.. In the study of six- week tomato seedlings, the specific activity of crude extract is 0.06 Unit/mg, the specific activity of the sample after %60 ammonium sulfate fraction is 0.182 Unit/mg then the purification fold is 3. As a result, existence of acid phosphatase enzyme is the highest in two-week tomato seedlings and the lowest in six-week tomato seedlings. Specific activity of four-week tomato seedlings' seems quite near to that of two-week's results. After the activity tests of crude extracts 1 5 days later, it was seen that four and six -week tomato seedlings were lost most of their activities while that of two-week tomato seedlings was almost the same. But two-week tomato seedlings samples after chromatography lost their activities 15 days later. It shows that the dilute enzyme samples are not stable. IXAfter ammonium sulfate procedures, it is concluded that %40 and %60 fractionation is enough for the purification because after %80 fractionation specific activity decreases significantly. In the study, three different substrates were used, Paranitrophenylphosphate, bis- paranitrophenylphosphate and 4-nitrophenylphosphate. The highest activity is obtained, respectively, from PNPP, 4NPP and bis-PNPP. In literature, PNPP is known as second appropriate substrate after pyrophosphates. Plant acid phosphatases are classified into two distinct categories based on their relative substrate specificities. One category represents specialized acid phosphatases, having clear, but not absolute, substrate selectivity and a distinct metabolic function. The second type is so-called“non-specific”Acid Phosphatases that exhibit a lesser degree of substrate selectivity. The activity of these non-specific acid phospahatses can be induced by various developmental processes including seed germination, flowering, senescence and fruit ripening. In another study with the two-week tomato seedlings, after the general purification procedures, sample was applied to DEAE Sephadex ion-exchanger in a solution of low ionic strength and at a pH and for the activity testes PNPP was used as the substrate. At the end of the procedure, only 3 -fold purification could be reached. The specific activity of the crude extract is 4.3 Unit/mg, and the specific activity of the sample after DEAE Sephadex chromatography is 13 U/mg. Some parameters of the partially purified Acid Phosphatase enzyme obtained from two-week and four-week tomato seedlings, which were mentioned below, were determined. Two-week tomato seedlings;. The highest activity was obtained at 30 °C and pH 5.60. The enzyme was stable between 30-50 °C and pH 5-6.8. Four-week tomato seedlings;. The highest activity was obtained at 40 °C and pH 5.63.. The enzyme was stable between 30-60 °C and pH 5.9-7.07. The activity of acid phosphatase depends on the type of the seeds and the growing conditions. Two-week tomato seedlings were grown from different kinds of seeds and in different environmental conditions. After two weeks, it was observed that the heights and the forms were quite different from each other besides their activities and proteins. Besides there is a study with the ripen tomatoes, tomato seedlings were not tested before. As a result of this study, it could be concluded that considering the specific activities despite the little amount of crude extract, tomato seedlings consist of significant amount of acid phosphatase enzyme. This study may be carried on further purification by a more appropriate procedure and tomato seedlings might become an economic source of acid phosphatase enzyme.
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