Studies on isocitrate lyase isolated from Lupinus cotyledons

1979 ◽  
Vol 57 (9) ◽  
pp. 1131-1137 ◽  
Author(s):  
P. Vanni ◽  
M. T. Vincenzini ◽  
F. M. Nerozzi ◽  
S. P. Sinha
Keyword(s):  
Enzyme Activity ◽  
Solid State ◽  
Specific Activity ◽  
Isocitrate Lyase ◽  
Spectral Characteristics ◽  
Random Orientation ◽  
Ph Optimum ◽  
Helix Conformation ◽  
Α Helix ◽  
Fluorescence Peak

Isocitrate lyase (threo-Ds-isocitrate glyoxylate-lyase, EC 4.1.3.1) was isolated from cotyledons of Lupinus seedlings, purified 100-fold with respect to its initial specific activity and characterized (Km, pH optimum, Mg2+ requirement, sulfhydryl inhibitors, and synthase activity). The final purified preparation consisted of two homogeneous protein bands clearly separated by electrophoresis on polyacrylamide gel and chromatography on Sephadex G 200.Reducing agents are necessary for the maintenance of enzyme activity. The most effective reducing agent studied was 1,4-dithioerythreitol. The effect of several metabolites (oxalate, malonate, phosphoenolpyruvate, succinate, malate, tartrate, gluconate-6-phosphate, sorbose, sorbitol, and inositol) on the activity of purified preparations was tested. Oxalate proved to be the strongest inhibitor, seconded closely by phosphoenolpyruvate. The spectral characteristics of the purified enzyme are as follows: ultraviolet peak at 280 nm and fluorescence peak at 340 nm. The solid state infrared spectrum of the enzyme (lyophilized) showed that the enzyme was mostly in the α-helix conformation with very slight random orientation.

UDP-Glucose: Flavonol 7-O-glucosyltransferase Activity in Flower Extracts of Chrysanthemum segetum

1997 ◽  
Vol 52 (3-4) ◽  
pp. 153-158 ◽  
Author(s):  
K. Stich ◽  
H. Halbwirth ◽  
F. Wurst ◽  
G. Forkmann
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Temperature Stability ◽  
Inhibitory Effect ◽  
Hydroxyl Group ◽  
Ph Optimum ◽  
Yellow Colour ◽  
Commercial Strain ◽  
Strong Inhibitory Effect ◽  
Chrysanthemum Segetum

Abstract The yellow colour of Chrysanthemum segetum petals is due to the presence of the 7-O-glucosides of quercetin and particularly gossypetin (8-hydroxyquercetin). In petal extracts of C. segetum an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5'-diphosphoglucose (UDPG) to the 7-hydroxyl group of flavonols with gossypetin and quercetin as the best substrates. Besides flavonols flavanones and flavones were found to be glucosylated in the 7-position. The pH-optimum of the reaction highly depended on the substrate used. With quercetin as substrate, maximal enzyme activity occurred at a pH of 8.25 and a temperature of 25 °C, but 7-O-glucosylation also proceeded at low temperatures. Studies on temperature stability revealed, that there was no influence on the glucosylation reaction up to 40 °C. Higher temperatures led to a loss of enzyme activity. Using gossypetin as a substrate a similar course of temperature stability was observed. Addition of Mg2+, Ca2+ and KCN slightly stimulated 7-O-glucosylation, whereas Co2+, Cu2+, Fe2+, Hg2+, p-hydroxymercuribenzoate and N-ethylmaleimide showed a strong inhibitory effect. Additional enzymatic studies were performed with the commercial strain " Stern des Orients" where gossypetin 7-O-glucoside is restricted to the inner parts of the petals. For enzyme extracts from both parts of the petals gossypetin was found to be the most attractive substrate. In comparison to quercetin (133.4 μkat / kg protein) an about three times higher specific activity of the 7-O-glucosyltransferase(s) was determined with gossypetin (382.1 μkat/ kg protein) as substrate, indicating that hydroxylation of quercetin in 8-position to gossypetin precedes 7-O-glucosylation.

scholarly journals Biochemical and cytochemical evidence for ATPase activity in basal bodies isolated from oviduct.

1977 ◽  
Vol 74 (2) ◽  
pp. 547-560 ◽  
Author(s):  
R G Anderson
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Basal Body ◽  
Divalent Cation ◽  
Specific Activity ◽  
Basal Bodies ◽  
Ph Optimum ◽  
Outer Portion ◽  
Chicken Oviduct ◽  
Basal Foot

Biochemical and cytochemical techniques were used to determine whether oviduct basal bodies have ATPase activity. All studies were carried out on basal bodies isolated and purified from the chicken oviduct. These preparations contained structurally intact basal bodies with basal feet, rootlet, and alar sheet accessory structures. Whereas the specific activity of the basal body ATPase in 2 mM Ca++ or 2 mM Mg++, 1 mM ATP, pH 8.0, averaged 0.04 mumol Pi/min per mg protein, higher concentrations of either cation inhibited the enzyme activity. Furthermore, the pH optimum for this reaction was pH 8.5. In comparison, the ATPase activity in cilia purified and measured under conditions identical to those for determining the basal body ATPase activity averaged 0.07 mumol Pi/min per mg protein. However, the activity increased at higher concentrations of divalent cation, and the pH optimum was pH 10.0. By cytochemical procedures for localizing ATPase activity, ATP-dependent reaction product in isolated basal bodies was found to be confined to: (a) the cross-striations of the rootlet; (b) the outer portion of the basal foot; (c) the alar sheets; and (d) the triplet microtubules. It is concluded that basal bodiesve an intrinsic ATPase activity that, by a variety of criteria, can be distinguished from the ATPase activity found in cilia.

3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Spruce ( Picea abies). Properties and Regulation

1990 ◽  
Vol 45 (9-10) ◽  
pp. 973-979 ◽  
Author(s):  
Meinrad Boll ◽  
Angelika Kardinal
Keyword(s):  
Enzyme Activity ◽  
Picea Abies ◽  
Cell Suspension ◽  
Specific Activity ◽  
Reductase Activity ◽  
Cell Suspension Cultures ◽  
Callus Cultures ◽  
Suspension Cultures ◽  
Phase Cell ◽  
Ph Optimum

Abstract HM GCoA reductase was identified in seedlings, callus cultures, cell suspension cultures and in needles of spruce ( Picea abies) (L.) (Karst). Activity was found in both the 18 K pellet and in the 105 K pellet with different ratios between the two fractions from the various sources. The enzyme has a pH-optimum of 7.9 and an absolute requirement for NADPH . The presence of a thiol reagent such as dithiothreitol is required for activity. Km for HM G CoA is 20 -25 μM. Detergents have differential effects on the activity. In seedlings, enzyme activity was considerably higher in the hypocotyls than in the cotyledons. Enzyme activity was high in dark-grown and low in light-grown seedlings. When the light conditions were reversed, levels of activity adapted to the respective new conditions (increase or decline of specific activity). Aerobic incubations of seedlings, callus cultures or needles in medium containing a carbon source, resulted in a large (up to 20-fold) transient increase of HMGCoA reductase activity. Transfer of stationary phase cell suspension cultures into new medium caused a similarly large increase of activity. A number of carbohydrates induced the enzyme, glucose, fructose and sucrose being most effective. The increase of activity was prevented by cycloheximide. All changes of activity were much more pronounced in the 18 K pellet HMG CoA reductase

scholarly journals Optium Conditions for the Production of Neutral Protease from local strain Aspergillus niger var cabonarius

2008 ◽  
Vol 5 (4) ◽  
pp. 472-481
Author(s):  
Baghdad Science Journal
Keyword(s):  
Enzyme Activity ◽  
Solid State ◽  
Aspergillus Niger ◽  
Wheat Bran ◽  
Specific Activity ◽  
Local Strain ◽  
Neutral Protease ◽  
Optimum Conditions ◽  
Solid Materials ◽  
Fermentation System

The optimum conditions for the production of neutral protease from local strain Aspergillus niger var carbonarius by solid – state fermentation system (Wheat bran) moisted with 0.2 M phosphate buffer (PH7.0) . the hydration ratio was 1:5 (V:W) . the concentration of inoculum was 1×106 spores per 10 gram of solid materials , initial P H 6.5 and 96 hours of incubation period at 30? C .the enzyme activity was 1300 unit / ml and specific activity was 1550 unit / mg protein .

On the role of octopine dehydrogenase in cephalopod mantle muscle metabolism

1976 ◽  
Vol 54 (6) ◽  
pp. 871-878 ◽  
Author(s):  
Jeremy H. A. Fields ◽  
John Baldwin ◽  
Peter W. Hochachka
Keyword(s):  
Enzyme Activity ◽  
Muscle Activity ◽  
Specific Activity ◽  
Ph Dependence ◽  
Muscle Metabolism ◽  
Ph Optimum ◽  
Wet Weight ◽  
Octopine Dehydrogenase

Octopine dehydrogenases from the mantle muscle of the squid, Symplectoteuthis oualaniensis, and of the octopus, Octopus ornatus, were kinetically characterized and compared. In the squid, the specific activity of the enzyme was about 110 μmol product formed per minute per gram wet weight; in the octopus that value was over 600. Both enzymes show similar pH dependence; in the direction of octopine formation the pH optimum was about 6.5, whereas in the direction of octopine oxidation it was about 8.5. The affinities for NADH, arginine, and pyruvate were similar (Km values were about 0.04 mM, 7 mM, and 2 mM respectively). Increasing the concentration of either arginine or pyruvate increased the affinity for the cosubstrate (pyruvate or arginine), this mechanism being a means of regulating the enzyme activity in vivo. In the direction of octopine oxidation, the octopus enzyme showed a much higher affinity for octopine (Km = 0.8 mM) than did the squid enzyme (Km = 4.4 mM), suggesting that it may be better geared for reconverting octopine to arginine and pyruvate after anaerobic bursts of muscle activity.

scholarly journals The metabolism of cyclopentanol by Pseudomonas N.C.I.B. 9872

1972 ◽  
Vol 129 (3) ◽  
pp. 595-603 ◽  
Author(s):  
Martin Griffin ◽  
Peter W. Trudgill
Keyword(s):  
Specific Activity ◽  
Isocitrate Lyase ◽  
Deae Cellulose ◽  
Sole Source ◽  
Similar Rate ◽  
High Yield ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Nadph Oxidation ◽  
Cellulose Column

1. Pseudomonas N.C.I.B. 9872 grown on cyclopentanol as carbon source oxidized it at a rate of 228μl of O2/h per mg dry wt. and the overall consumption of 5.9μmol of O2/μmol of substrate. Cyclopentanone was oxidized at a similar rate with the overall consumption of 5.2μmol of O2μmol of substrate. Cells grown with sodium acetate as sole source of carbon were incapable of significant immediate oxidation of these two substrates. 2. Disrupted cells catalysed the oxidation of cyclopentanol to cyclopentanone by the action of an NAD+-linked dehydrogenase with an alkaline pH optimum. 3. A cyclopentanolinduced cyclopentanone oxygenase (specific activity 0.11μmol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1μmol of NADPH and 0.9μmol of O2 in the presence of 1μmol of cyclopentanone. NADPH oxidation did not occur under anaerobic conditions. The only detectable reaction product with 100000g supernatant was 5-hydroxyvalerate. 4. Extracts of cyclopentanol-grown cells contained a lactone hydrolase (specific activity 7.0μmol hydrolysed/min per mg of protein) that converted 5-valerolactone into 5-hydroxyvalerate. 5. Cyclopentanone oxygenase fractions obtained from a DEAE-cellulose column were almost devoid of 5-valerolactone hydrolase and catalysed the formation of 5-valerolactone in high yield from cyclopentanone in the presence of NADPH. 6. Incubation of 5-hydroxyvalerate with the 100000g supernatant, NAD+ and NADP+ under aerobic conditions resulted in the consumption of O2 and the conversion of 5-hydroxyvalerate into glutarate. 7. The high activity of isocitrate lyase in cyclopentanol-grown cells suggests that the further oxidation of glutarate proceeds through as yet uncharacterized reactions to acetyl-CoA. 8. The reaction sequence for the oxidation of cyclopentanol by Pseudomonas N.C.I.B. 9872 is: cyclopentanol → cyclopentanone → 5-valerolactone → 5-hydroxyvalerate → glutarate → → acetyl-CoA.

scholarly journals Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

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.

Poly(β-L-malate) hydrolase from Plasmodia of Physarum polycephalum

1995 ◽  
Vol 41 (13) ◽  
pp. 192-199 ◽  
Author(s):  
Christian Korherr ◽  
Michael Roth ◽  
Eggehard Holler
Keyword(s):  
Hydrophobic Interaction ◽  
Malic Acid ◽  
Physarum Polycephalum ◽  
Culture Medium ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Reduced Form ◽  
Serine Esterase ◽  
Hydroxybutyric Acid ◽  
Ph Optimum

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.

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