Yaşar Demir

Identification of protease from Euphorbia amygdaloides latex and its use in cheese production.

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.

Activation of electron deficient cycloheptatrienes by tricarbonyliron complexation

Abstract (1,6-Dimethoxycarbonyl-cycloheptatriene)Fe(CO)3, unlike its uncoordinated counterpart, readily reacts with TCNE to give the (3 + 2) adduct, and with NPTD to give a mixture of the (3 + 2), (4 + 2) and (5 + 2) adducts.

Carbonic Anhydrase from Vicia Canencens Leaves

Carbonic anhydrases (CA: Carbonate hydrolase; E, C,4.2.1.1) from leaves Vicia canencens were purified and were characterized. The purification level of enzyme was 76-fold. The optimum temperature of this enzyme was 70 degrees C. pH optimum was 9.2. Each of the enzyme molecules was a decamer having MW of 262,000 and the subunit MW was 26,400.

THE ACTIVITIES OF CARBONIC ANHYDRASE AND ALKALINE PHOSPHATASE IN ANCIENT HUMAN BONES. PURIFICATION AND CHARACTERIZATION OF OUTER PERIPHERAL, CYTOSOLIC, INNER PERIPHERAL, AND INTEGRAL CA

In the present study, bone carbonic anhydrase was isolated from ancient human bones and its characteristic features were determined. For this purpose, the skull bone of about 3000 years age was used. The purification was performed in four steps. Four different isoenzymes of CA, including outer peripheral, inner peripheral, integral, and cytosolic were purified and characterized. Affinity chromatography using Sepharose-4B-L-tyrosyn sulfanilamide as a support material was used in its purification. Two different methods were used for enzymatic activity determination: a) hydratase, and b) esterase methods. Bradford and Coomassie Brillant Blue methods were used for protein determination. Optimal pH, temperature, and molecular weight determinations were performed by conventional methods. The purification degree and the subunits, if present, were determined by SDS-PAGE. The effects of some chemicals on the enzyme were also investigated. The most cardinal finding was that the enzymatic activity has been found in antique human bone, showing some other enzymatic activity. That the alkaline phosphatase activity has been determined in the same sample supports the finding of carbonic anhydrase.

A Different Structural Feature for Carbonic Anhydrases in Human Erythrocytes

This study presents a different structural feature for carbonic anhydrase in human erythrocytes. Carbonic anhydrase isozymes (CA-I and CA-II) were purified from an erythrocyte pool of 20 healthy subjects. For purification, Sepharose-4B-L-tyrosine-sulfanilamide affinity column was used. Resnets from 3-10% discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band for CA-I and two distinct bands for CA-II. The molecular weights of the two bands were similar. One peak for CA-I and two peaks for CA-II were obtained in gel filtration. The enzymatic activities of the bands in question were also of different value. Native electrophoresis showed two bands for CA-I, and it showed three bands for CA-II. It can be concluded that CA-I is a polymer composed of a single promoter and CA-II has three different polymers composed of two distinct promoters, suggesting a new structural feature of human erythrocyte carbonic anhydrase isozymes.

Carbonic anhydrases from leaves and roots of Daucus carota

Abstract Carbonic anhydrases (CAs) (carbonate hydrolase; EC 4.2.1.1) from leaves and roots of mature Daucus carota cv. nantes were purified and characterized. The purification levels were 75- and 73-fold in leaves and roots, respectively. The optimum temperature for the enzymes from both sources was 75°. pH optima were 8.5 and 9.0 in leaves and in roots, respectively. Each enzyme molecule was a hexamer having M r 137 800 and the subunit M r was 22 800. A dimer CA which has a M r of 45 700 was also detected. The amount of this dimer was higher in leaves than in roots.

Production of pectin lyase from Geobacillus pallidus p26, purification, characterization and fruit juice application

Abstract A bacterial strain was isolated from Pasinler hot spring, Erzurum, Turkey. The purified thermophilic isolate was identified as Geobacillus pallidus P26 and used to produce extracellular pectin lyase (EC 4.2.2.10). Pectin lyase enzyme was purified 34 fold by using DEAE-cellulose anion exchange column chromatography and characterized. Molecular weight of the enzyme was determined as 56 kDa by using Sephadex G-100 gel filtration chromatography. Purification of enzyme was verified by SDS-PAGE. The pH- and temperature optima of enzyme were determined (pH 9.0 and 60°C, respectively). Pectin lyase was mostly stable at 50 oC for 24 hours. Its’ activity decreased to 50 % for 24 h at 60°C. KM and Vmax were calculated as 24.8 mg/mL and 2.28 μmol/L min, respectively. Purified pectin lyase was inhibited by Fe3+, Zn2+, Cu2+, Ca2+, Co2+ and Hg2+ but not by Mg2+. The purified pectin lyase enzyme was used for getting fruits juices. It was found that yields of fruits juices increased when they were compared with control.

CYTOSOLIC AND NON-CYTOSOLIC CARBONIC ANHYDRASE ENZYMES FROM BOVINE LEUCOCYTES

In this study, carbonic anhydrase (CA) enzyme has been purified and separately characterized according to bound form in 4 steps as outer peripheral, cytosolic, inner peripheral, and integral from bovine leukocyte. Affinity chromatography has also been used for purification of the enzyme in four steps. CA has been found for each step. Measurment of enzyme activity has been done by CO2 hydratase activity and esterase activity methods. Optimum pH and optimum temperature have been defined for each step of purified enzyme. The behaviors of CA with specific inhibitors, such as KSCN and NaN3 have been investigated. In each step, molecular weight and purity have been determined by gel filtration and SDS-PAGE electrophoresis. In addition, enzyme KM and Vmax values have been determined with the method of Lineweaver-Burk.

CARBONIC ANHYDRASE FROM BOVINE BONE

In this research, carbonic anhydrase enzyme, which was taken from the bones of an animal, was purified and characterized for the first time. For this, the bones of a young cow were used. The purification treatment was completed in three steps. Three different isoenzymes, such as peripheral, cystolic, and integral from the bone-cell cytozolic isoenzyme were purified and characterized. In purification of the three isoenzymes, the technique of affinity chromatography, which utilized Sepharose-4B-L-Tyrosine-Sulphanylamide, was used. In measuring the activities of enzymes, two different methods were applied. These are the esterase methods that utilize hydratase and p-nitrophenylacetate as substrate. The measurement of proteins was done with the methods of Bradford and Coomassie Brillant Blue. The optimum pH and temperature of each enzyme were measured and molecular weights were measured by gel-filtration. Its purity was examined by SDS-PAGE (3-10% alternating) electrophoresis and the inferior unit was defined. The inhibition effects of some chemicals were tested for each of the three isoenzymes.

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-l-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their Km and Vmax values for the same substrate by Lineweaver–Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The Km values at optimum pH and 20°C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective Vmax values at optimum pH and 20°C were 0.875 μmol/L min, 0.186 μmol/L min, 0.214 μmol/L min and 0.253 μmol/L min with the same substrate. The Ki and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.