Purification of lactoperoxidase from bovine milk and investigation of the kinetic properties.

Lactoperoxidase (LPO) was purified from bovine milk using Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. During the purification steps, the activity of enzyme was measured using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diamonium salt (ABTS) as a chromogenic substrate at pH 6. Optimum pH and optimum temperature values for LPO were determined for ABTS, p-phenylendiamine, catechol, epinephrine, and pyrogallol as substrates, and then Km and Vmax values for the same substrate were obtained by means of Lineweaver-Burk graphics. The purification degree of the enzyme was controlled by SDS-PAGE and Rz (A412/A280) values. Km values, at optimum pH and 20 degrees C, were 0.197 mM, 0.063 mM, 0.64 mM, 25.2 mM, and 63.95 mM for p-phenylendiamine, ABTS, epinephrine, pyrogallol, and catechol, respectively. Vmax values, at optimum pH and 20 degrees C, were 3.5x10(-5) EU/mL, 4.0x10(-5) EU/mL, 5.8x10(-4) EU/mL, 8.4x10(-4) EU/mL, and 1.01x10(-3) EU/mL for the same substrates, respectively. p-Phenylendiamine was first found as a new substrate for LPO.     

Effects of the water-miscible organic solvents on lactoperoxidase purified from creek-water buffalo milk

Water buffalo lactoperoxidase (WBLPO) was purified with Amberlite CG-50 (NH₄ ⁺ form) resin, CM-Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel-filtration chromatography from skimmed buffalo milk. The purity of the WBLPO was shown with SDS-PAGE. The R_z(A ₄₁₂/A ₂₈₀) value for the WBLPO was 0.9. The optimum pH for the WBLPO was at 6.0. The K _m value at optimum pH and 25°C was 0.13 mM. The V _max value at optimum pH and 25°C was 5.3 mol/min per ml. The K _i values for methanol, ethanol, dimethyl sulfoxide (DMSO), acetonitrile, isopropanol, tetrahydrofuran (THF), N,N"-dimethylformamide (DMF), and ethylene glycol were 1.087, 0.364, 0.302, 0.459, 0.330, 0.126, 0.093, and 2.125 M, respectively. All the solvents showed competitive inhibition. The I ₅₀ values of methanol, ethanol, dimethyl sulfoxide, acetonitrile, isopropanol, tetrahydrofuran, N,N"-dimethylformamide, and ethylene glycol were 2.910, 0.942, 0.537, 1.320, 0.875, 0.470, 0.405, and 3.920 M, respectively. Ethylene glycol, methanol, acetonitrile, and ethanol have been found to be very promising solvents for performing biocatalytic reactions with LPO in organic media.     

Purification of bovine milk lactoperoxidase and investigation of antibacterial properties at different thiocyanate mediated

Bovine lactoperoxidase (LPO) was purified with amberlite CG 50 H+ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography from skim milk. The activity of lactoperoxidase was measured by using 2.2-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH 6.0. Purification degree for the purified enzyme was controlled with SDS-PAGE and Rz value (A412/A280). Rz value for the purified LPO was 0.8. Km value at pH 6.0 at 20 degrees C for the LPO was 0.20 mM. Vmax value was 7.87 micromol/ml min at pH 6.0 at 20 degrees C. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate--100 mM H2O2 medium for some pathogenic bacteria, such as Aeromonas hydrophila ATCC 7966, Micrococcus luteus LA 2971, Mycobacterium smegmatis RUT, Bacillus subtilis IMG 22, Pseudomonas pyocyanea, Bacillus subtilis var. niger ATCC 10, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 15753, Bacillus brevis FMC3, Klebsiella pneumoniae FMC 5, Corynebacterium xerosis UC 9165, Bacillus cereus EU, Bacillus megaterium NRS, Yersinia enterocolytica, Listeria monocytogenes scoot A, Bacillus megaterium EU, Bacillus megaterium DSM32, Klebsiella oxytocica, Staphylococcus aerogenes, Streptococcus faecalis, Mycobacterium smegmatis CCM 2067 and compared with well known antibacterial substances such as penicilline, ampicilline, amoxicillin-clavulanate and ceftriaxon. The LPO--100 mM thiocyanate--100 mM H2O2 system was purposed as an effective agent against many of the diseases causing organisms in human and animals.     

Thiocyanate mediated antifungal and antibacterial property of goat milk lactoperoxidase

Goat milk lactoperoxidase was purified using CM-Sephadex-C-50 ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein was found to have high antifungal and antibacterial activity in a thiocyanate-H2O2 medium. The results are relevant enough to suggest the exploitation of LP-thiocyanate-H2O2 system as an effective agent against many of the disease causing organisms in plants and animals.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

Purification of NAD malic enzyme from potato and investigation of some physical and kinetic properties

A procedure is described for purification of NAD malic enzyme (EC 1.1.1.39) to near homogeneity from potato tuber mitochondria. The purified enzyme is active with either NAD or NADP, and functions with either Mg²⁺ or Mn²⁺. V_app is greatest when the enzyme is assayed with Mg²⁺ and NAD. When Mn²⁺ replaces Mg²⁺ the V_app of the NAD-linked reaction decreases but the K_m values for all substrates drop substantially. When NADP is used in place of NAD, the V_app of the Mg²⁺-linked reaction decreases and the K_m values for most substrates increase. The pH optimum of the enzyme depends on the metal ion and cofactor used and varies between 6.4 and 6.8. At pH 6.8, with saturating levels of Mg²⁺ and NAD, the turnover number of the enzyme is 37,000 min^?1. The shape of the pH profile indicates the involvement of two to three protons in the activation of the enzyme, whereas only one proton is involved in the inactivation process. The molecular weight of the enzyme in the presence of 5 mm dithiothreitol and 2 mm MgCl₂ is 490,000 as determined by gel filtration. A lower molecular weight form of the enzyme predominates in gel filtration at lower levels of dithiothreitol and in native gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme reveals two main bands with molecular weights of 61,000 and 58,000, suggesting that the subunit stoichiometry of the high-molecular-weight form may be α₄β₄. However, given the possibility that the smaller subunit may be a proteolytic artifact, the enzyme may prove to be an octamer of identical subunits.     

Purification and properties of buffalo serum albumin

Based on a detailed study of the solubility of serum albumin, a procedure for its purification by selective ammonium sulphate precipitation has been developed. Using buffalo serum, first extraneous proteins were precipitated by making the serum 2.26 M saturated with ammonium sulphate at pH 7.0 and then albumin was precipitated from the supernatant at 1.9 M ammonium sulphate concentration at pH 4.2. The overall yield of serum albumin thus isolated was about 55% with a purity of 97%. The protein preparation gave a single nearly symmetrical peak on Sephadex G-100 column and virtually a single band on polyacrylamide gel electrophoresis in the presence and absence of SDS. Buffalo serum albumin has a molecular weight of 69,000 Da. The hydrodynamic properties such as Stoke's radius (3.70 nm), diffusion coefficient (6.03 X 10(-7) cm2/s) and frictional ratio (1.36) obtained by analytical gel chromatography, bilirubin binding characteristics and its interaction with anti-bovine serum albumin antiserum suggest that buffalo serum albumin is very similar to BSA in its molecular properties.     

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.     

Purification of glucose 6-phosphate dehydrogenase from chicken erythrocytes. investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from chicken erythrocytes, and some characteristics of the enzyme were investigated. The purification procedure was composed of three steps: hemolysate preparation, ammonium sulfate precipitation, and 2',5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 20.862 EU/mg proteins, was purified with a yield of 54.68% and 9,150-fold. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, Ki values and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADH, and NADPH.     

Purification and kinetic properties of galactokinase from human placenta

Galactokinase from human placenta was purified about 350-fold using DEAE-Sephadex-A-50 chromatography followed by Sephadex-G-200 and CM-Sephadex-C-50 filtration. The final steps of purification involved electrofocusing and ammonium sulfate precipitation. In analytical disc electrophoresis the purified enzyme moved as a single protein band.     

Purification and kinetic properties of sialidase from Clostridium perfringens

Clostridium perfringens sialidase was isolated from a culture medium of bacterial cells by ammonium sulfate precipitation (42-85%), followed by purification through Sephadex G-75 gel chromatography, DEAE A-50 anion exchange chromatography, FPLC medium pressure anion exchange chromatography and finally FPLC medium pressure isochromatofocussing. From 9 l culture medium 1.17 mg sialidase was isolated with a specific activity of 295 U/mg. The enzyme appeared to be homogeneous by analytical polyacrylamide gel electrophoresis. The molecular mass was measured to be 66 kDa. Km values ranging from 0.6 to 1.6mM were determined for several oligosaccharides as substrates. The enzyme catalyzed transglycosylation reactions with methanol as a nucleophilic reagent competitive with water. In the enzymatic hydrolysis of the (3'-methoxyphenyl)glycoside of alpha-N-acetylneuraminic acid, increase of methanol concentration had no effect on the release of 3-methoxyphenol. This finding suggests that the formation of the enzyme-glycon intermediate is the rate-determining step for this substrate.     

Purification and kinetic properties of polynucleotide kinase from rat testes

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.     

An immobilized two-enzyme system for the activation of the lactoperoxidase antibacterial system in milk

Lactoperoxidase catalyzes the oxidation of thiocyanate by hydrogen peroxide and an intermediary product is formed with antibacterial properties. The components of this system, with the exception of hydrogen peroxide, are present in milk. H₂O₂ may be introduced by means of enzymatic, generation and thus make the system complete. A two-enzyme system consisting of β–galactosidase and glucose oxidase has been developed for this purpose. The coupled enzyme reaction is shown to work with high efficiency at the neutral pH of milk although the enzymes as such, particularly lactases suitable for immobilization, have optimal activities at much lower pH values. The results indicate that the lactoperoxidase system may in this way be employed to inactivate bacteria present in milk.     

Purification and some properties of buffalo spleen cathepsin B

Purification of cathepsin B from buffalo-spleen, a hitherto unstudied system has been achieved by a simple procedure developed by incorporating suitable modifications in the existing methods for isolation of the enzyme from other sources. The purified enzyme has a molecular weight of 25 KDa and its Stokes radius was found to be 2·24 nm. Effects of several reducing agents, urea and thiol-protease inhibitors such as leupeptin and antipain, have been studied and the data unequivocally support the contention that the buffalo-enzyme is similar to cathepsin B from other tissues with respect to these properties.     

Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties

In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.     

Purification and kinetic properties of chalcone-flavanone isomerase from soya bean.

The chalcone-flavanone isomerase from soya bean seed has been purified 8300-fold. A molecular weight of 15,600 plus or minus 1000 has been determined for the enzyme. Effects of iodoacetamide and sodium tetrathionate on the enzyme, and pH dependence of the catalytic step, indicate that a sulphydryl group is not involved in the reaction mechanism and the catalytic group is probably an imidazole side chain in the basic form. The kinetics of the isomerisation of isoliquiritigenin to liquiritigenin have been examined and show that at pH 7.6 the reaction is reversible with an equilibrium constant of 37 in favour of flavanone. A number of flavonoid compounds competitively inhibit the reaction, suggesting a possible regulatory role for this enzyme in flavonoid biosynthesis.     

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Purification of glucose 6-phosphate dehydrogenase from Buffalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from buffalo (Bubalus bubalis) erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: hemolysate preparation and 2('),5(')-ADP-Sepharose 4B affinity gel chromatography. Thanks to the two consecutive procedures, the enzyme, having a specific activity of 69.7EU/mg proteins, was purified 650-fold with a yield of 31%. Optimal pH, stable pH, optimal temperature, molecular weight, and K(M) and V(max) values for NADP(+) and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition, K(i) values and the type of inhibition were determined by means of Lineweaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADPH, and NADH.