Hayvansal kaynaklardan ksantin oksidaz izolasyonu ve saflaştırılması
Başlık çevirisi mevcut değil.
- Tez No: 55897
- Danışmanlar: Y.DOÇ.DR. YÜSEL A. GÜVENİLİR
- Tez Türü: Yüksek Lisans
- Konular: Kimya Mühendisliği, Chemical Engineering
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 1996
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 62
Özet
ÖZET Enzimler bitkisel ve hayvansal dokuların bileşiminde yer alan, canlılarda oluşan kimyasal reaksiyonların vücut ısısında oluşmasını sağlayan protein yapısındaki biyolojik katalizörlerdir. Enzimlerin reaksiyonları katalizleme özelliklerinin belirlenmesinden sonra, endüstride kullanım alanları üzerine yapılan çalışmalar olumlu sonuçlar vermiştir. Hipoksantin ve ksantinin ürik aside yükseltgenmesini katalizleyen ksantin oksidaz enzimi biyokimyada, gıda ve tıp kimyasında kullanılmaktadır. Bu çalışmada inek sütünde ve peynir altı suyunda bulunan ksantin oksidaz enziminin izolasyonu, saflaştırılması deneysel olarak yapılmış ve enzimin aktivitesi farklı süre ve miktarlar için tayin edilmiştir. Ekstraksiyon safhasında tampon çözelti değiştirilerek fosfat bazlı kimyasalların ksantin oksidazı inhibe ettiği gösterilmiştir. Ksantin oksidaz inek sütünden izole edilmiş ve saflaştırma sonunda spesifik aktivitesi 36.96 U/mg olarak tespit edilmiştir. Buna göre saf enzim ham homojenata göre 5310 kez daha saf olarak elde edilmiştir. Saf enzimin pH, sıcaklık ve kararlılık özellikleri araştırılmış, bunun sonucunda optimum pH' sının 8.5, optimum sıcaklığın 50°C olduğu saptanmıştır. Elde edilen enzim pH 7-10 arasında ve 50-60 °C de kararlı özelliğe sahiptir. Sadece ekonomik yönden değil çevre kirlenmesi açısından da değerlendirilmesi zorunlu olan peynir altı suyundan izole edilen ksantin oksidazın spesifik aktivitesi 0.044 U/mg olarak tespit edilmiştir.
Özet (Çeviri)
THE ISOLATION OF XANTHINE OXIDASE FROM COW'S MILK AND CHEESE WHAY AND PURIFICATION OF MILK XANTHINE OXIDASE SUMMARY In this study the isolation of enzymes is discussed, concentrating initially on the principles of the important separation methods employed. Then the operation of these methods is illustrated by considering one specific example, purification of enzymes. Enzymes are catalysts of biochemical origin and are characterized by an extraordinary specifity and reactivity in biological systems. Enzymes are found in all living organisms where they serve to activate and regulate the multitude of chemical reactions essential for the continuing existence of the organism. By the beginning of twentieth century, many enzymes had been found and were recognized as substances associated with living organisms. In 1926, the enzyme urease, was crystallized. Soon after, and almost one hundred years after its discovery, pepsin was also isolated in a crystalline form. The crystallization of the enzymes led to their identification as proteins. Research into the biochemistry of living organisms further demonstrated the role of enzymes, and enzymology became a present day science. The aim of a 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. In addition the preparation should possess the maximum catalytic activity, i.e. there should be no degraded or other inactivated enzyme present, and it should be of the maximum possible purity, i.e. contain no other enzymes or large molecules. A typical purification scheme to isolate a specific enzyme might involve the following steps (all done in aqueous medium near neutral pH, and at low temperature to minimize loss of catalytic activity): 1) Distruption of cells by mechanical or sonic treatment, 2) Differential centrifugation to separate 'soluble' and particulate fractions, x3) Fractionation by differential solubility in solutions of high salt (ammonium sulfate), 4) Ion-exchange chromatographyto separate species of different charge, 5) Adsorption chromatography (i.e., calcium phosphate crystals), 6) Molecular sieve (gel-filtration) chromatography to separate species of different size and, 7) Electrophoresis (again separation by charge differences) on various supports. Analysis for purity can include crystallization, ultracentrifugal analysis, discgel electrophoresis, amino-acid composition (and sequence) determination, and various molecular weight determinations. An ultracentrifuge is capable of generating intense centrifugal fields; in a typical machine a rotor speed of 30x104 times gravity. Under these conditions macromoiecules will have a tendency to sediment, provided that the density of the macromolecule is greater than that of the solution (this generally holds for enzymes in aqueous solution). In gel filtration, the separation between molecules of different sizes is made on the basis of their ability to enter pores within the beads of a beaded gel. The most widely used types of gel are Sephadex (croslinked dextrans) and Bio-Gel P (cross-linked polymers of acrylamide). Small molecules which can enter the pores of beads are retarted as they pass down a column containing the gel; large molecules which are unable to enter the pores pass through the column unimpeded. By varying the size of the pores (which is controlled by the degree of cross-linking in the preparation of the beaded gel) it's possible to change the range of molecular weights which can be fractioned. Sephadex G-100, for example, can fractionate globular proteins in the molecular weight range from 4000 to 15x104. Gel filtration can be carried out on alarge scale but the gels required to fill them are expensive. Ion exchange chromatography depends on the electrostatic attraction between species of opposite charge. Ion exchangers usually consist of modified derivatives of some support material such as cellulose, sephadex, etc., as shown in the examples of DEAE-cellulose and CM-cellulose (Fig.1). During a purification procedure, the enzyme is usually applied to an ion exchanger in a solution of low ionic strength and at a pH where the appropriate interaction will occur (i.e. the enzyme and the ion exchanger have opposite charges). Desorption of the bound species can be brought XI+ CH2CH3 Cellulose-0-CH2-CH2-N Cellulose-O- CH2-C02 N H DEAE-cellulose CM-cellulose (Diethylaminoethyl-cellulose) Carboxymethyl-cellulose possesses a pKa ~ 1 0 possesses a pKa ~ 4 will bind negatively charged species will bind positively and is therefore an anion exchanger charged species and is therefore an cation exchanger. Fig 1 Ion Exchangers Commonly Used in Purification of Enzymes about either by changing the pH, and thus altering the charges, or by increasing the ionic strength of the solution so that the increased concentration of cations or anions will compete with the enzyme for the binding sites on the ion exchanger. Use of a gradient of increasing ionic strength permits the separation of proteins in a mixture on the basis of their ability to bind to the ion exchanger. Ion exchange chromatography can be performed on a large or a small scale. On a large scale it is often convenient to work in a batch wise manner, i.e. adsorb the enzyme by adding the ion exchanger to a solution and then to pour the material into a column for controlled desorption using an ionic strength gradient. On a small scale, both adsorption and desorption are performed in a column. Ion exchange chromatography finds very wide application in present day purification procedures. Electrophoretic separation is based on the differential movement of charged molecules under the influence of an applied potential difference. The rate of movement of a species is governed by the charge it carries and also by its size and shape. In order to minimize convection currents, the buffer (electrolyte) solution is soaked into a support (paper, cellulose powder, starch, or polyacrylamide gels). The position of a protein on the support can be determined by use of a stain such as Coomassie Blue which binds to proteins. Altough the technique is normally performed on a small, analytical scale (a few mg or less), it is possible to use the method on a large preparitive scale. After preparative electrophoresis the separated enzymes can be eluted out of the support or run off the bottom of a column in sequence. Enzymes may increase the rate of a reaction by as much as 1 012 fold. The catalytic activity of a preparation is determined by a suitable assay procedure in which the rate of disappereance of substrate or the rate of appereance of product is determined under defined conditions of substrate concentration, temperature, pH, etc. The international definition of catalytic XIIactivity is a unit (U) and corresponds to a rate of conversion of one micromole (10“6 mole) of substrate to product per minute. The specific activity of an enzyme is measured as U/mg, and this specific activity should rise during purification unit a constant value, indicative of pure protein, is obtained. Xanthine oxidase is widely distributed in mammals, especially in cow1 s milk and calf liver. The enzyme catalyzes the oxidation of hypoxanthine and xanthine to uric acid, but is rather nonspecific enzyme, and a great variety of compounds have been found to serve as substrates (electron donors). The rates of oxidation vary quite markedly among the different types of substrates, with hypoxanthine and xanthine being the best substrates. Molecular oxygen is an electron acceptor for the enzyme, but a large number of dyes, such as methylene blue, 2,6- dichlorophendindiphenol, triphenyltetrazolium chloride, and phenazine methosulfate as well as cytochrome c and ferricyanide can serve as electron acceptors. Milk xanthine oxidase is a molybdoflavoprotein with a molecular weight of 275 000. The metal ions and flavin are bound quite firmly to the protein and are not released by dialysis. Denaturation of the protein by heat or by treatment with acids releases the metal ions and flavin and irreversibly destroys activity. The flavin can be removed by CaCI2 treatment. The isoelectric point of the enzyme is 5.3-5.4 and optimum pH is around 8.3. Many substances can inhibit the activity of the enzyme, including purines, pteridines, and other heterocyclic compounds, which inhibit by competing with the substrate for binding at the active site. Arsenite, cyonide and methanol all appear to inhibit by reacting with molybdenum. Other inhibitors include phosphate, imidazole, sodium and potassium chloride, benzoate, borate, copper, ascorbic acid and dinitrophenol. Substrate hydroxylation occurs at molybdenum center (the reductive half-reaction) whereas 02 reacts at the flavin site (the oxidative half-reaction) and intramolecular electron transfer between the molybdenum center and flavin (mediated by the iron-sulfur centers) is an integral aspect of catalysis. Because of its ease of isolation from a convenient source, the enzyme has become the prototypical moltbdenum hydroxylase, a small but important class of monooxygenases that utilizes water rather than 02 as the source of the oxygen atom incorporated into product. Most of the xanthine oxidase in cow' s milk is closely associated with the milk fat globule membrane (MFGM). The MFGM has a protenaceous surface that interfaces with the milk plasma phase on the exterior and the globule lipids on the interior. XIIIThe objective of this study is to obtain purer xanthine oxidase than produced by prior art by operating under conditions that improve the final product. The study features; - The use of a mild non-ionic detergent, - 20-27% saturation with (NH^SCU, - Maintaining at low temperature to remove more casein and, - The use of multiple chromatographic columns to concentrate the xanthine oxidase and remove non-xanthine oxidase proteins with lower or higher molecular weights. Xanthine oxidase is isolated and purified from raw cow”s milk and cheese whay by a stream line method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Sodium salicylate, ethylenediaminetetraacetate (EDTA), and a 0.2 M phophate buffer are added to fresh milk. After incubation at 40°C-45°C for 105 minutes, the mixture is adjusted to 1% Triton X-100 and allowed to incubate for 15 minutes. The mixture is cooled to 4°C, followed by a 2-step fractionation of the proteins with ammonium sulfate. The crude enzyme is isolated as a red- brown precipitate which is dissolved in 0.1 M Tris/CaCI2 buffer and stored for from 5 days to 3 weeks at -20 °C. Cow milk xanthine oxidase is purified by a 2-step column chromatography (Sephadex G-25). The final stage of purification is accomplished by passing the enzyme preparation through a DEAE-Sephadex A-25 column, by a 2- step application, equlibrated with 0.1 M pyrophosphate buffer. In the first stage of the invention the inhibition affect of phophate buffer to xanthine oxidase activity is determined. In the second stage, milk xsnthine oxidase is purified giving a constant E280/E450 ratio of 1.63. The most sensitive indicator of xanthine oxidase purity is the PFR (E280/E450 ) value. A decrease in the concentration of non-xanthine oxidase protein (at 280 nm) and a simultaneous increase in the concentration of xanthine oxidase (at 450 nm) should give an increasingly lower E280/E450 ratio. Thus, the lower the PFR value, the higher the purity of the preparation. Xanthine oxidase is purified 5310 fold from the starting material. The specific activity of the pure enzyme is 36.96 U/mg. A variety of stability and activity profiles are determined for the purified cow" s milk xanthine oxidase. These include optimum pH, optimum temperature, a pH stability profile and a temperature stability profile. The pH optimum is around 8.5 and the temperature optimum is around 50 °C. The enzyme is stable at pH 7-10 and 50-60 °C. Analyses show that milk xanthine oxidase contains 35.45 ppm Fe and 8.147 Mo. In the final stage of the invention xanthine oxidase is isolated from cheese whay giving a specific activity of 0.044 U/mg. XIV
Benzer Tezler
- Bazı hayvansal kaynaklardan sülfatlanmış glikozaminoglikan eldesi ve işlem parametrelerinin optimizasyonu
Extraction of sulphated glycosaminoglycan from animal sources and optimization of the process parameters
ÇİSE KARAMAN
Yüksek Lisans
Türkçe
2022
Gıda MühendisliğiEge ÜniversitesiGıda Mühendisliği Ana Bilim Dalı
DOÇ. DR. SEHER KUMCUOĞLU
- Çevresel ve hayvansal kaynaklardan izole edilen hareketli aeromonas türlerinde bazı patojenite kriterlerinin araştırılması
The research of some pathogenicity criterions in motile aeromonas species isolated from environmental and animal sources
SEDA ERDOĞAN
- Hayvansal atıklardan elde edilen proteinlerin verimlerinin ve jelleşme özelliklerinin araştırılması
Investigation of gelation properties and efficiencies of proteins obtained from animal-based wastes
AYŞEGÜL GÜNDEM
Yüksek Lisans
Türkçe
2021
Gıda MühendisliğiUşak ÜniversitesiGıda Mühendisliği Ana Bilim Dalı
DR. ÖĞR. ÜYESİ ÖZGÜR TARHAN
- Keçiboynuzu ekstraktının hyaluronik asit üretiminde karbon kaynağı olarak değerlendirilmesi ve üretim şartlarının optimizasyonu
Evaluation of carob extract as a carbon source in hyaluronic acid production and optimization of production conditions
ALİ ÖZCAN
Yüksek Lisans
Türkçe
2020
Gıda MühendisliğiAkdeniz ÜniversitesiGıda Mühendisliği Ana Bilim Dalı
DOÇ. DR. İRFAN TURHAN
- Tavuk derisinden ohmik sistem yardımıyla jelatin eldesi ve geleneksel yöntemle karşılaştırılması
Obtaining gelatin from chicken skin with the help of ohmic system and comparison with the traditional method
ÇİĞDEM IŞIK
Yüksek Lisans
Türkçe
2023
Gıda MühendisliğiBursa Teknik ÜniversitesiGıda Mühendisliği Ana Bilim Dalı
DOÇ. DR. FURKAN TÜRKER SARICAOĞLU