ABSTRACT
In this research, protease enzyme was purified and characterized from milk of Euphorbia amygdaloides. (NH4)2SO4 fractionation and CM-cellulose ion exchange chromatography methods were used for purification of the enzyme. The optimum pH value was determined to be 5, and the optimum temperature was determined to be 60 degrees C. The V(max) and K(M) values at optimum pH and 25 degrees C were calculated by means of Linewearver-Burk graphs as 0.27 mg/L min(-1) and 16 mM, respectively. The purification degree was controlled by using SDS-PAGE and molecular weight was found to be 26 kD. The molecular weight of the enzyme was determined as 54 kD by gel filtration chromatography. These results show that the enzyme has two subunits. In the study, it was also researched whether purified and characterized protease can be collapsed to milk. It was determined that protease enzyme can collapse milk and it can be used to produce cheese.
Subject(s)
Cheese , Euphorbia/enzymology , Latex/chemistry , Peptide Hydrolases/isolation & purification , Caseins/metabolism , Catalysis , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , TemperatureABSTRACT
Four isozymes of alkaline phosphatase (AP) were purified from Elephas trogontherii (Steppe elephant) from different locations in the bone (outer and inner peripheral, cytosolic, and integral) using Sephadex G-200 gel filtration and TEAE-cellulose anion-exchange chromatography. The specimen was obtained from Erzurum Museum and its age was approx. 0.3-0.5 million years old. No fungi or bacteria were present in the bone sample. The enzyme activity was determined by using p-nitrophenylphosphate as a substrate. SDS-PAGE of all the isozymes gave a single band at the same location. The molecular mass of the four isozymes as determined by using gel filtration was about 60 kDa. Optimum pHs for the four isozymes were between 8-8.5. The optimum temperatures of the isozymes were: outer peripheral, 37.5 degrees C, cytosolic, 37.5 degrees C, inner peripheral, 35 degrees C and integral, 40 degrees C. The values of V(max) and K(m), as well different optimum temperatures indicated that isozymes were structurally different.
Subject(s)
Alkaline Phosphatase/isolation & purification , Alkaline Phosphatase/metabolism , Bone and Bones/enzymology , Cytosol/enzymology , Elephants/metabolism , Alkaline Phosphatase/classification , Animals , Anion Exchange Resins/chemistry , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Isoenzymes , Kinetics , Molecular Weight , Nitrophenols/metabolism , Organophosphorus Compounds/metabolism , Substrate Specificity , TemperatureABSTRACT
In this research, acid phosphatase was purified and characterized from approximately 3000-year-old human bones from archeological excavations. Using anion exchange chromatography, two isoenzymes, TrACP and TsACP, were isolated from the bone. TrACP and TsACP were eluted separately, with a concentration gradient, from a CM-sepharose column. The resulting TrACP was further purified on a cellulose phosphate column. The activity was determined by using pNPP as substrate. Additionally, protein was determined by the Bradford and Coomassie Brilliant Blue method. The optimum pHs of TsACP and TrACP were 6 and 5, respectively. The optimum temperatures were 0 and 10 degrees C, respectively. Molecular weights were measured by gel filtration chromatography. The isoenzyme purity was checked with SDS-PAGE. Finally, the effects of sodium molybdate and tartrate on isoenzyme activity were determined.
