Sarımsak filizinden asit fosfatazın kısmi saflaştırılması ve kinetik özelliklerinin incelenmesi

Abstract

Enzimler bütün yaşayan hücrelerde bulunan ve metobolik prosesleri kontrol eden, protein yapısındaki katalizörlerdir. Proseste harcanmadan, çok yavaş ilerleyen kimyasal reaksiyonları hızlandırırlar. Asit Fosfataz enzimi doğada çok yaygın fakat düşük miktarlarda bulunan bir enzimdir. Çeşitli fosfataz esterlerini katalizleyen pH optimumu 6 olan bir enzimdir. Çalışmada, sarımsak filizlerinden asit fosfatazm izolasyonu ve kısmi saflaştırılması hedeflenmiş ve enzimin bazı kinetik özellikleri saptanmıştır. Saflaştırmada, p-nitrofenilfosfat (pNPP) ve p-nitrofenol (pNP) kullanılarak yapılan deneysel çalışmalar sonucunda başlangıca göre sırasıyla 20 ve 17 kat saflaşma sağlanmıştır. Çalışma kapsamında substrat konsantrasyonu, pH, sıcaklık, enzim konsantrasyonu, aktivasyon enerjisi ve stabilite profilleri incelenmiştir. Optimum izolasyon pH ve sıcaklık değerleri 6 ve 50 °C olarak tespit edilmiştir. Enzimin aktivasyon enerjisi ise 10 ile 20 kcal. olarak belirlenmiştir.Kinetik sabitlerinden biri olan Michealis Menten sabitleri de Vm - Km olmak üzere sırasıyla 100 mM/sn -21.27 mM (pNPP) ve 20 mM/sn - 8.33 (pNP) hesaplanmıştır.

Today's biotechnological methods are capable to supply a multitude of enzyme variations. To develop economically and ecologically competitive bioconversion processes one has to gather as much information as possible as early stage of process development to determine further investigations.The application of enzymes in organic/inorganic synthesis has become increasingly important in the last decade. Like any other catalyst, an enzyme brings the reaction catalyzed to its equilibrium position more quicly than would occur otherwise; an enzyme cannot bring about a reaction with an unfavorablechange in free energy unless that reaction can be coupled to one whose free energy change is more favorable. This situation is common in biological systems, but the true role of enzymes involved should not be mistaken. Asid phosphatases are enzymes that catalse the hydrolysis of variety of phosphatase esters and appear to exhibit pH optima below 6. Phosphate esters are widely distributed in any organism. While many metabolic intermediates are activated through the transfer of phosphate groups it is equally important that phosphate esters can also be rapidly broken down. The hydrolitic removal of phosphate groups from phosphoesters is catalyzed by phosphatases. Depending on the pH at which such phosphatases have optimal activity, we distinguish between acidic phosphatases and alkaline phosphatases. Asid phosphatase has been widely used for medicine, pharmacy, the preparation of food additives, the analitical area or the synthesis of organic compounds. Asid phosphatase that catalyze the hidrolysis of large number of orthophosphoric plants and animals. Monoester compounds, is widely distributed in nature and has been studied in various in plants, asid phophatase's activity in seeds often increases greatly during germination. Sunflower seed asid phosphatase consists of two polypeptide chains, each of which similarin size to the one that is present in wheat germ enzyme. Garlic asid phosphatase is a metalloprotein as a monomer with a molecular weight 56000 - 58000. The aim of the presented experiments is partial isolation and purification of acide phophatase from three week garlic seedlings and to determine its characteristics and kinetic parameters. In this study two different substrates were used in the beginning. Para nitro phenyl phosphate and para nitro phenol. The highest activity is obtained from pNPP. So kinetic parameters are controlled by using pNPP. VIII The study steps ; -Extraction and homogenization : Three week garlic seedlings were distrupted by 0,1 mM Sodium Acetate buffer. (0,1 M NaCl, pH 5,5,%0,1 TritonX-100 ) -Centrifiguration : Solution is centrifugated at 20000-22000 rpm. -Three Step Amonium Sulfate Fraction : %40, %60,%80 Amonium Sulfate Fraction -Dialysis :Used a dialysis membrane (24 hours, Sodium Acetate buffer ) Garlic seedling was homogenated with 0.1 M acetate buffer. After homogenating supernatant was filtrated with paper filters. Filtrated superaated was cooled to 4°C, followed by a three step fractination of proteins with ammonium sulfate. After ammonium sulfate fractions, enzyme solution was dialysed to decreased ammonium sulfate concentration. Every step is controlled by measuring protein and activity with spectrophotometer at 280 nm and 405 nm respectively. -Asid phosphatase was purified 17.40 (pNPP) and 14.40 (pNP) fold from the starting material. -After dialysis, asid phosphatase was purified 20.70 (pNPP) and 17.96 (pNP) fold. -The highest activity is obtained at 60 ° C. -The pH optimum is 6. -The enzyme is stable at pH 4- 6 and 40 - 60 ° C. -Activation energy of enzyme is between 10-20 kcal.