Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

The extracellular enzyme dextransucrase was produced from Leuconostoc mesenteriodes NRRL B512F and purified by ultracentrifugation and cross-flow ultrafiltration for use in the biosynthesis of the macromolecule dextran by ion exchange chromatographic reaction—separation techniques. The two-stage purification process yielded over 90% pure dextransucrase with overall enzyme recovery of over 60%. A second stage of centrifugation was required to achieve complete cell removal. The purified enzyme contained 1–2 g l⁻¹ of solute ions, which affected the operation of the chromatographic system. Gel filtration removed over 93% of the remaining ions but resulted in high activity losses. Two-phase separation with polyethylene glycol (PEG) and purification by ion exchange chromatography were less successful in desalting the enzyme. PEG precipitation was successful in concentrating the enzyme, but the ions remained predominantly with the enzyme portion of the two phases. The purified enzyme was found to be unstable during storage.Use of the enzyme in chromatographic reactor—separators for the production of dextran resulted in over 33% more high molecular weight dextran (the desired product) and a useful pure fructose byproduct being obtained than for a conventional reactor. Sodium and potassium ions in the enzyme hampered continuous operation by displacing calcium ions from the resin and thus reducing the separation efficiency of the system. Partial regeneration of the resin with calcium nitrate rather than complete enzyme desalting, which was very expensive and resulted in high activity losses, helped overcome this effect.     

Kiwifruit Actinidin: A Proper New Collagenase for Isolation of Cells from Different Tissues

Actinidin is a cysteine protease abundant in Kiwifruit. This enzyme is known as a meat-tenderizing protease. In this project, actinidin was purified from kiwifruit by salt precipitation and ion exchange chromatography. Collagenolytic effect of the purified enzyme was tested in four different buffer systems. Thereafter, the enzyme was used for isolation and culture of cells from three different tissues: endothelial cells from human umbilical vein, hepatocytes from rat liver, and thymic epithelial cells from rat thymus. Our results revealed that actinidin can hydrolyze collagen types I and II at neutral and alkaline buffers. Furthermore, actinidin compared with type II or IV collagenase isolated intact human umbilical vein endothelial cells, hepatocytes, and thymic epithelial cells with viability more than 90%. These results address a novel and valuable collagenase, which can be used efficiently for hydrolysis of collagen and isolation of different cell populations from various solid tissues.     

Determination of the big head carp myofibrillar (Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography

A method for the determination of adenosine triphosphatase (ATPase) activity of myofibrils of big head carp by using ion chromatography was introduced. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and orthophosphate (Pi) were separated completely. Recoveries for ATP, ADP and Pi were 98+/-5%, 97+/-4% and 98+/-5%, respectively. Pi liberated from ATP during reaction was monitored by ion chromatography using the suggested method. This method was applicable to the determination of myofibrils ATPase activity for quick quality evaluation of surimi.     

Synthesis of 5'-methylthio coformycins: specific inhibitors for malarial adenosine deaminase

Transition state theory suggests that enzymatic rate acceleration (kcat/knon) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Because transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2'-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5'-methylthioadenosine. In contrast, neither human adenosine deaminase (HsADA) nor the bovine enzyme (BtADA) can deaminate 5'-methylthioadenosine. 5'-Methylthiocoformycin and 5'-methylthio-2'-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum, and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to be toxic to humans, and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors described here have potential for development as antimalarials without inhibition of host ADA.     

Affinity Chromatography of Ribitol Dehydrogenase,a Possible New Zinc Enzyme from Mycobacterium Butyricum

Abstract

An effective purification procedure of ribitol dehydrogenase (RDH), a possible new zinc enzyme from Mycobacterium butyricum is described. The procedure took advantage of different chromatographic methods in which the most significant were two affinity chromatography steps. One of them was the immobilized metal ion affinity chromatography (IMAC), with the use of iminodiacetate-Sepharose 6B (IDA-Sepharose 6B) chelating Zn²⁺ ions (IDA-Zn) as an affinity sorbent. The enzyme was eluted with a decreasing pH gradient from 7 to 4. The other step was a biospecific affinity chromatography, where the enzyme retained on 5′ AMP-Sepharose 6B was eluted with 10 mM adenosine 5′-monophosphate (AMP). RDH was purified 174-fold with 10.2% of recovery, and the final preparation was homogenous in polyacrylamide gel electrophoresis.     

Fe(III)-ion-catalysed non-enzymatic transformation of adenosine diphosphate into adenosine triphosphate part II. Evidence of catalytic nature of Fe ions

Non-enzymatic phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) in aqueous solutions using acetyl phosphate has been studied extensively. The reaction proceeds in the presence of Fe(III) ions. Normally, the yield of ATP never exceeds the amount of Fe ions but, when a sufficient amount of Mg(II) ions is added to the reaction system, the yield of ATP increases beyond the amount of Fe ions. This is because the Fe(III) ion is tightly bound to not only ADP but also ATP, the phosphorylation product. When, however, the Fe(III) ion is liberated from ATP by exchanging with an Mg(II) ion, it regains catalytic activity and contributes to the surplus production of ATP. The Fe(II) ion with either a single molecule of 1,10-phenanthroline or 2,2′-bipyridyl exhibits a remarkably high catalytic activity. This suggests that the Fe(II) aquo ion should also be an excellent catalyst.     

Zinc deficient bovine erythrocyte superoxide dismutase has low specific activity.

Zinc deficient bovine superoxide dismutase (Cu2E2SOD (E = empty)) was prepared and purified by high performance liquid chromatography (HPLC). Each peak was characterized as to protein, copper content and specific activity. The Cu2E2SOD peak fractionated by HPLC has a low specific activity at pH 7.8 (about 10% of the native enzyme (Cu2Zn2SOD)). With the addition of zinc ions, the specific activity of Cu2E2SOD was quantitatively restored to that of the native enzyme. This behavior implies that the zinc ion is very important for the appearance of enzyme activity.     

Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus

The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.     

Properties of chicken liver phosphofructokinase-2.

Phosphofructokinase-2 was purified to homogeneity from chicken livers by homogenization, polyethylene glycol fractionation and column chromatography on DEAE-Sephadex A-50 and Blue-Sepharose 4B. Some properties of the enzyme were as follows: (i) The saturation curve of the enzyme for fructose 6-phosphate showed hyperbolic and the Km of fructose 6-phosphate was affected by inorganic phosphate while Vmax was not; (ii) the binding of ATP to the enzyme was of negative cooperativity with a Hill coefficient of 0.56; (iii) the activity of the enzyme was completely lost in the presence of EDTA. The enzyme was activated by Mg2+ at low concentrations, but inhibited by Mg2+ at high concentrations; (iv) the enzyme was stable below 30 degrees C and easily lost its activity when the temperature was above 40 degrees C; (v) the activity of the enzyme was stable at the range of pH 7-9, increased at pH 9.0-9.5 and decreased when pH was over 9.5; (vi) the enzyme was sensitive to trypsin and ATP protected the enzyme against the proteolysis of trypsin.     

Expression,Purification, and Characterization of a Recombinant Methionine Adenosyltransferase pDS16 in Pichia pastoris

Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using l-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93 %, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ～5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 μmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg²⁺ and was slightly stimulated by the monovalent cation K⁺. It showed an improved thermostability, about 50 % of the enzyme activity was retained even after preincubation at 50 °C for 2 h.     

In Vitro Inhibition Effect of Some Dihydroxy Coumarin Compounds on Purified Human Serum Paraoxonase 1 (PON1)

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76?U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K _i of 0.0080?±?0.256 and 0.0003?±?0.018?mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K _i of 0.0010?±?0.173?mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC₅₀ of 0.012, 0.022 and 0.003?mM values, respectively. IC₅₀ value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178?mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.     

Effects of acute gamma-irradiation on extracellular adenine nucleotide hydrolysis in developing rat brain

Cell membrane is highly sensitive to irradiation which, acting directly or indirectly, may disturb functions of constitutive proteins including membrane enzymes. Plasma membrane surface-located enzyme chain of ecto-nucleotide triphospho diphosphohydrolases (NTPDases) and 5′-nucleotidase are involved in termination of cell purinergic signalization by hydrolyzing extracellular, excitatory adenosine triphosphate (ATP), as well as nucleotide di-, and mono-phosphate (ADP and AMP) to neuroprotective adenosine. Extracellular ATP, ADP, and AMP hydrolyzes were examined in purified synaptic plasma membranes after whole-body acute irradiation. All measurements were done 24 h after irradiation of developing (15-, 30-day-old) and adult (90-day-old) rats with low (50 cGy) and high (2 Gy) dose of gamma-rays. Both, high and low doses inhibited nucleotide hydrolyses in 15-day-old rats; in 30-day-old rats low dose of radiation inhibited ADP and AMP hydrolyses while high dose inhibited only ATP hydrolyse. In adult rats high dose induced no effects, while low dose stimulated nucleotides hydrolyses. According to obtained results it was concluded that ecto-nucleotidases of young rats are more sensitive to irradiation, since even low dose induces inhibition of ecto-nucleotidases activities. Ionizing radiation, by decreasing brain nucleotide hydrolyses in developing rats, induces accumulation of ATP and decreases production of adenosine in synaptic cleft which could be neurocytotoxic. On the contrary, in adult rats low dose of radiation stimulates NTPDase and 5′-nucleotidase activity and protective adenosine production which indicates protective and adaptive mechanisms developed in adult brain neuronal cells. The article is published in the original.     

ATP synthesis in the disk membranes of rod outer segments of bovine retina

ATP is synthesized on the disk membrane isolated from rod outer segments of the bovine retina. Together with a slow component which accounted for a constant rate of about 22 nmol ATP/min/mg of protein and which was due to the adenylate kinase activity, a fast component with a maximal activity of about 58 nmol ATP/min/mg of protein was measured at physiological calcium concentrations. This fast activity disappeared in the presence of the Ca(2+) ionophore A23187, was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting that this ATP synthesis is due to the reversal functioning of the Ca(2+)-ATPase previously found on the disk membranes.     

Preparative purification of adenosine deaminase from human erythrocytes by affinity chromatography

The purification of adenosine deaminase from human erythrocytes is reported. By means of classical procedures and by using affinity chromatography as the last step, the enzyme is purified 760,000-fold with a yield of 32%. The affinity resin is composed of purine riboside (nebularine) linked to Sepharose CL6B. Since the compound has no leaving group at the C-6 position the affinity gel is stable and the chromatography can be repeated several times (up to fifteen times in eight months). Purine riboside was chosen because its potency as a reversible inhibitor of adenosine deaminase is greater than that of inosine (a low-affinity inhibitor), but lower than that of erythro-9-(2-hydroxy-3-nonyl)adenine (a high-affinity inhibitor).     

Fischer–Tropsch Chemistry at Room Temperature?

The unique catalytic activity of vanadium nitrogenase suggests a new direction for the direct production of biofuels from CO with either synthetic catalysts or nitrogenase-containing bacteria. The reduction of CO by V?nitrogenase to light hydrocarbons shows striking similarities to the established Fischer-Tropsch process; however, the enzyme does not use H(2) directly for this reaction. ADP=adenosine diphosphate, ATP= adenosine triphosphate.     

Rapid purification of detergent-solubilized bovine hormone-sensitive lipase by high performance hydrophobic interaction chromatography

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.     

Microelectrode Sensing of Adenosine/Adenosine‐5′‐triphosphate with Fast‐Scan Cyclic Voltammetry

Adenosine and adenosine‐5′‐triphosphate (ATP) are important extracellular signaling molecules. Here, we studied adenosine and ATP using fast‐scan cyclic voltammetry at carbon‐fiber microelectrodes. Although ATP and adenosine have similar oxidation potentials, ATP oxidation current was highly dependent on buffer pH and divalent cation concentrations but adenosine current was not. Therefore, they can be distinguished by adding a divalent cation chelator or calibrating electrodes at different pH values. The enzymatic degradation of adenosine by adenosine deaminase was monitored in a mixture of adenosine and ATP in presence of EDTA (ethylenediaminetetraacetate). This sensing method is promising for enzyme kinetics or in vitro studies.     

Simultaneous determination of thymidylate and thymidine diphosphate by capillary electrophoresis as a rapid monitoring tool for thymidine kinase and thymidylate kinase activities

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of thymidylate (TMP) and thymidine 5'-diphosphate (TDP) in enzyme assays without using radioactive-labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. The separation of micromolar TMP and TDP from millimolar adenosine 5'-triphosphate (ATP) was performed at 25 degrees C using sodium tetraborate as the background electrolyte. Under the optimal condition, a good separation with high efficiency was achieved in 6 min. Several parameters affecting the separation were studied, including the pH of electrolyte, the applied voltage, and acetonitrile-salt sample stacking. The fronting of the ATP peak resulting from the interference of magnesium ion in the enzyme assay buffer was suppressed by the addition of sodium ethylenediaminetetraacetate to the sample solution. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of TMP and TDP were 2.6 and 3.8 microM, respectively. Application of the proposed method for simultaneous determination of TMP and TDP in enzyme assays was demonstrated by the activity assays of thymidine kinase and thymidylate kinase from white spot syndrome virus. This is a sensitive, nonradioactive method for thymidine kinase and thymidylate kinase assays.     

Towards an integrated device that utilizes adherent cells in a micro-free-flow electrophoresis chip to achieve separation and biosensing

We immobilized adherent human embryonic kidney (HEK) cells—which are able to trace adenosine triphosphate (ATP) —inside a microfluidic free-flow electrophoresis (μFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 μM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.     

Characterization of calcium and phospholipid dependent protein kinase in isolated rat adipocytes

Calcium and phospholipid-dependent protein kinase (protein kinase C) from isolated rat adipocytes has been partially purified using DEAE-Sepharose CL-6B and characterized. The enzyme was shown to have similar properties as the kinase isolated from brain or spleen. When histone was used as substrate, an equal amount of cAMP-dependent and calcium and phospholipid-dependent kinase activity was detected from the DEAE Sepharose CL-6B fractions. The major part of protein kinase C (72%) was isolated from the soluble adipocyte fraction. Of the membranous fractions, the plasma membrane exhibited the highest specific activity. The protein kinase preparations bound [3H]-phorbol-12,13-dibutyrate (PDBU) with high affinity (Kd = 2 nM) and the number of PDBU binding sites per cell was calculated to 63 000.