Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

scholarly journals (335) Purification and Characterization of ADP-glucose Pyrophosphorylase from Apple Leaves

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1083C-1083
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Purification And Characterization ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations ◽  
Dual Functions

Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.

scholarly journals Optimization, Purification, and Starch Stain Wash Application of Two Newα-Amylases Extracted from Leaves and Stems ofPergularia tomentosa

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Imen Lahmar ◽  
Hanen El Abed ◽  
Bassem Khemakhem ◽  
Hafedh Belghith ◽  
Ferjani Ben Abdallah ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Amylase Activity ◽  
Detergent Industry ◽  
Sepharose Column ◽  
Stems And Leaves ◽  
Commercial Detergents ◽  
Starch Substrate

A continuous research is attempted to fulfil the highest industrial demands of natural amylases presenting special properties. Newα-amylases extracted from stems and leaves ofPergularia tomentosa, which is widespread and growing spontaneously in Tunisia, were studied by the means of their activities optimization and purification. Some similarities were recorded for the two identified enzymes: (i) the highest amylase activity showed a promoted thermal stability at 50°C; (ii) the starch substrate at 1% enhanced the enzyme activity; (iii) the twoα-amylases seem to be calcium-independent; (iv) Zn2+, Cu2+, and Ag2+were considered as important inhibitors of the enzyme activity. Following the increased gradient of elution on Mono Q-Sepharose column, an increase in the specific activity of 11.82-fold and 10.92-fold was recorded, respectively, for leaves and stems with the presence of different peaks on the purification profiles.Pergulariaamylases activities were stable and compatible with the tested commercial detergents. The combination of plant amylase and detergent allowed us to enhance the wash performance with an increase of 35.24 and 42.56%, respectively, for stems and leaves amylases. Characterized amylases were reported to have a promoted potential for their implication notably in detergent industry as well as biotechnological sector.

scholarly journals Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus

1972 ◽  
Vol 128 (2) ◽  
pp. 243-252 ◽  
Author(s):  
J. S. Heller ◽  
C. L. Villemez
Keyword(s):  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Soluble Enzyme ◽  
Phaseolus Aureus ◽  
Original Activity ◽  
Solubilized Enzyme ◽  
High Concentrations

A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-α-d-mannose was used as a substrate was a β-(1→4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent Km is 55–62μm, and a disproportionate increase in rate is observed at high concentrations. GDP-α-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent Ki of 6.2μm. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0°C or in 24h at −20°C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at −20°C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.

scholarly journals The biosynthesis of granulose by Clostridium pasteurianum

1974 ◽  
Vol 144 (3) ◽  
pp. 503-511 ◽  
Author(s):  
R L Robson ◽  
R M Robson ◽  
J G Morris
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Partial Purification ◽  
Clostridium Pasteurianum ◽  
Saturation Curve ◽  
Wild Type ◽  
Competitive Inhibitors ◽  
Fine Control ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like α-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose–α-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5′-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (Ki=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5′-phosphate were competitive inhibitors, ADP being most effective (Ki about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.

scholarly journals Immobilization of Soybean Lipoxygenase on Nanoporous Rice Husk Silica by Adsorption: Retention of Enzyme Function and Catalytic Potential

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 291
Author(s):  
Putheary Ngin ◽  
Kyoungwon Cho ◽  
Oksoo Han
Keyword(s):  
Thermal Stability ◽  
Rice Husk ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Silica Particles ◽  
Soybean Lipoxygenase ◽  
Desorption Kinetics ◽  
Rice Husk Silica ◽  
Kinetics Of ◽  
Thermal Stability Of

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (kcat/Km) and the Michaelis–Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.

scholarly journals Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate

1987 ◽  
Vol 246 (2) ◽  
pp. 263-269 ◽  
Author(s):  
P J Casey ◽  
J M Lowenstein
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
Rat Skeletal Muscle ◽  
Adenylosuccinate Lyase ◽  
Apparent Homogeneity ◽  
High Concentrations

Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at −70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.

scholarly journals Study of thermal stability of ascospores Aspergillus (Neosartorya) fischeri depending on the concentration of soluble solids in apple juice

2019 ◽  
Vol 81 (3) ◽  
pp. 91-98
Author(s):  
V. V. Kondratenko ◽  
M. V. Trishkaneva ◽  
M. T. Levshenko ◽  
T. A. Pozdnyakova ◽  
A. Y. Kolokolova
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Apple Juice ◽  
Thermal Inactivation ◽  
Soluble Solids ◽  
Stability Parameters ◽  
Mold Fungus ◽  
Rate Of Increase ◽  
Thermal Stability Parameters ◽  
Thermal Stability Of

Kinetic studies of the thermal inactivation of test crop spores are necessary to develop optimal heat treatment regimes for fruit juices. The purpose of the work is to study the dynamics of changes in the thermal stability parameters DT and z depending on changes in the soluble solids content in canned fruit products using the example of certain types of apple juice products with a pH of 3.80. The regularity of thermal inactivation of ascospores of the mesophilic mold Aspergillus fischeri in concentrated apple juice (JAC) with a soluble dry matter (RSV) content of 70%, in restored apple juice with RSV – 11.2%, and in restored apple juice with pulp with RSV – 16% was studied. The parameters of thermal stability were determined by the capillary method at temperatures of 80, 85, 90, and 95 °C. It was experimentally established that the heat resistance of A. fischeri spores in clarified apple juice was DT 95 °С = 0.16 min, and the parameter value z = 6.76 °C, in apple juice with pulp parameters: DT 95 °C = 0.24 min, z – 7.12 °C, in YaKS – DT 95 °C = 0.39 min, and z – 7.8 °C. The dynamics of thermal stability parameters D and z of A. fischeri mold fungus spores (test cultures) versus RSV concentration of juice products was established. The research results showed that with an increase in the concentration of RSV, the thermal stability of spores increases exponentially. The rate of increase in thermal stability decreases with increasing concentration of RSV. Since the concentration of RSV affects the rheological properties of the product (viscosity), this leads to a change in the kinetics of heating in products with convection heat transfer. Therefore, an increase in the concentration of RSV should inevitably lead not only to an increase in the thermal stability of spores of microorganisms, but also to a shift in the region of optimal modes of heat treatment of products toward an increase in the thermal load to ensure regulatory requirements for microbiological safety.

scholarly journals Thermostable Adenosine 5’-Monophosphate Phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies

2020 ◽  
Author(s):  
Sarah Kamel ◽  
Miriam Walczak ◽  
Felix Kaspar ◽  
Sarah Westarp ◽  
Peter Neubauer ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Inclusion Bodies ◽  
Specific Activity ◽  
High Thermal Stability ◽  
Active Protein ◽  
E Coli ◽  
Thermococcus Kodakarensis ◽  
Active Inclusion Bodies ◽  
Catalytically Active

Abstract Catalytically active inclusion bodies (CatIBs) produced in E. coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5'-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of solubilized protein induces aggregation of active protein which phosphorolysis all natural 5’-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2−6 folds higher specific activity compared to the heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.

Effect of the hydrophilicity of the polymer matrix on immobilized α-chymotrypsin

1984 ◽  
Vol 49 (6) ◽  
pp. 1552-1556
Author(s):  
Minoru Kumakura ◽  
Isso Kaetsu
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Pore Size ◽  
Polymer Matrix ◽  
Low Temperatures ◽  
Radiation Polymerization ◽  
Thermal Stability Of

α-Chymotrypsin was immobilized by radiation polymerization at low temperatures and the effect of the hydrophilicity of the polymer matrix on the enzyme activity and thermal stability was studied. The activity and thermal stability of immobilized chymotrypsin increased with the increasing hydrophilicity of the polymer matrix or monomer. The thermal stability was affected by the form and pore size of the polymer matrix; chymotrypsin immobilized on a soft-gel polymer matrix exhibited an enhanced thermal stability.

Decomposition of hydrogen peroxide on cerium dioxide-nickel oxide two-component catalysts and the effect of ionizing radiation on them

1984 ◽  
Vol 49 (1) ◽  
pp. 14-24 ◽  
Author(s):  
Viliam Múčka
Keyword(s):  
Heat Treatment ◽  
Hydrogen Peroxide ◽  
Ionizing Radiation ◽  
Nickel Oxide ◽  
Cerium Dioxide ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Ion Pairs ◽  
Composition Region ◽  
Two Component

Some physical and catalytic properties of cerium dioxide-nickel oxide two-component catalysts have been studied over the entire composition region, employing the decomposition of hydrogen peroxide in aqueous solution as a model catalytic process. The two oxides have been found to affect each other, particularly for NiO contents of 9.1 and 96.7 mol%; the mutual influencing, the nature of which in the conditions applied remains unaffected by heat treatment of the sample or by its exposition to ionizing radiation, is manifested by the nonmonotonic dependences of the oxidation power and of the specific activity of the catalysts on their composition. This can be interpreted in terms of the concept of bivalent catalytic centres, assuming that for nickel oxide the centres consist of Ni2+-Ni3+ ion pairs, for cerium dioxide they consist of Ce3+-Ce4+ ion pairs, and that in the region of the mutual influencing , Ni2+-Ce4+ ion pairs play a major role. Within the scope of this concept, the increase in the oxidation power of all the catalysts in question and a simultaneously decrease in the specific activity of the pure nickeloxide exposed to ionizing radiation can be explained in terms of the ionization effect.

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