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At least 703 records · Page 39Linked to original sources
PubMed1988

Induction of alcohol dehydrogenase by ethanol in Drosophila melanogaster.

When Drosophila melanogaster larvae were fed a defined fat-free, low sucrose medium, alcohol dehydrogenase (ADH) was increased to a higher activity with a moderate, nontoxic level of ethanol (2.5% vol/vol) within 5 h. Ethanol-stimulated increases in ADH activity and cross-reacting material in late third-instar larvae were paralleled by increases in the larval ADH mRNA as indicated by dot blot analysis. Northern blot observations indicated that both adult and larval ADH messages were increased by dietary ethanol. The increased levels of the ADH mRNA transcribed from the proximal and distal promoters of ethanol-fed larvae argue that the induction is a consequence of elevated levels of mRNA, not a result of changes in enzyme stability or synthesis. To determine whether the induction is of nutritional significance to larvae, the rate of flux from ethanol to lipid was estimated in control larvae and larvae that were pre-fed ethanol. Flux changes occurred; the rate of incorporation of [14C]ethanol into body lipid showed a strong association with larval ADH activity. Because the induced increase in larval ADH activity did not extend into the adult stage and attempts to stimulate ADH activity by exposing adults to ethanol were unsuccessful, the modulation of ADH activity by dietary ethanol may be a mechanism by which larvae utilize environmental ethanol as a resource, especially when free sugar levels are low. In addition, ADH in larvae is postulated to perform a second, nonethanol function that expedites the conversion of sugars to lipid when habitats are low in fats, low in ethanol and high in sugars.

Age Factors↗
PubMed1999

Enzymatic synthesis of alpha-butylglucoside lactate: a new alpha-hydroxy acid derivative.

An alpha-hydroxy acid derivative, alpha-butylglucoside lactate, was successfully prepared by enzymatic transesterification of alpha-butylglucoside with a lactate alkyl ester in a non-aqueous medium using immobilized lipase as biocatalyst. Ester synthesis in organic solvent was optimized. Solvent choice was made on the basis of substrate solubility and enzyme stability in the medium. A solvent-free reaction using butyllactate as lactate donor led to the highest yields. In the presence of 0.5M alphabutylglucoside and 100 g/L Novozym(R), a 67 % yield could be obtained within 40 h at 50 degrees C. However, the presence of butanol by-product limited the reaction to a maximum that could not be exceeded in closed systems. The elimination of the alcohol under reduced pressure resulted in the complete equilibrium shift of the transesterification reaction in favor of synthesis; below 15 mbars, more than 95% of 0.5M alpha-butylglucoside could be converted within 30 h. Moreover, simultaneous evaporation of water allowed hydrolysis of butyllactate to be eliminated. Consequently, a very high alpha-butylglucoside lactate concentration (170 g/) could be obtained in a single batch reaction. A single purification procedure, consisting of butyllactate extraction with hexane, enabled the product to be obtained at a purity above 95% (w/w). 1H and 13C NMR analysis later demonstrated that lactic acid was exclusively grafted onto the primary hydroxyl group of alphabutylglucoside.

Bioreactors↗
PubMed1984

Isolation and characterization of revertants from four different classes of aryl hydrocarbon hydroxylase-deficient hepa-1 mutants.

Revertants were selected from aryl hydrocarbon hydroxylase (AHH)-deficient recessive mutants belonging to three complementation groups and from a dominant mutant of the Hepa-1 cell line. The recessive mutants had low spontaneous reversion frequencies (less than 4 X 10(-7] that were increased by mutagenesis. The majority of these revertants also had reacquired only partial AHH activity. Revertants of group A mutants were identical to the wild type with respect to both in vivo and in vitro enzyme stability and the Km for the substrate, benzo [alpha]pyrene, and therefore failed to provide evidence that gene A is the AHH structural gene. Group B and group C mutants are defective in the functioning of the Ah receptor required for AHH induction. Revertants of these groups were normal with respect to in vivo temperature sensitivity for AHH induction and for the 50% effective dose for the inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and thus provided no evidence that the B and C genes code for components of the receptor. Two rare group C revertants possessed AHH activity in the absence of induction. The phenotype of one of these was shown to be recessive to the wild type. Spontaneous revertants of the dominant mutant occurred at a frequency 300-fold greater than those of the recessive mutants, and this frequency was not increased by mutagenesis. These revertants all displayed complete restoration of AHH activity to wild type levels. These observations and the results from cell hybridization studies suggest that the dominant revertants arose by a high frequency event leading to functional elimination of the dominant mutation.

Animals↗
PubMed2001

Tyrosine residue 300 is important for activity and stability of branching enzyme from Escherichia coli.

Branching enzyme belongs to the alpha-amylase family, which includes enzymes that catalyze hydrolysis or transglycosylation at alpha-(1,4)- or alpha-(1,6)-glucosidic linkages. In the alpha-amylase family, four highly conserved regions are proposed to make up the active site. From amino acid sequence analysis a tyrosine residue is completely conserved in the alpha-amylase family. In Escherichia coli branching enzyme, this residue (Y300) is located prior to the conserved region 1. Site-directed mutagenesis of the Y300 residue in E. coli branching enzyme was used in order to study its possible function in branching enzymes. Replacement of Y300 with Ala, Asp, Leu, Ser, and Trp resulted in mutant enzymes with less than 1% of wild-type activity. A Y300F substitution retained 25% of wild-type activity. Kinetic analysis of Y300F showed no effect on the Km value. The heat stability of Y300F was analyzed, and this was lowered significantly compared to that of the wild-type enzyme. Y300F also showed lower relative activity at elevated temperatures compared to wild-type. Thus, these results show that Tyr residue 300 in E. coli branching enzyme is important for activity and thermostability of the enzyme.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1993

Stability and activity of immobilized hydrolytic enzymes in two-liquid-phase systems: acid phosphatase, beta-glucosidase, and beta-fructofuranosidase entrapped in poly(2-hydroxyethyl methacrylate) matrices.

Enzyme storage stability and hydrolysis yield were measured in experiments carried out with three model hydrolytic enzymes: acid phosphatase (EC 3.1.3.2), beta-glucosidase (EC 3.2.1.4), and beta-fructofuranosidase (EC 3.2.1.26) entrapped in hydrogels of poly(2-hydroxyethyl methacrylate). Runs were performed at 30 degrees C, under intensive stirring (500 rev min-1), in 50% v/v biphasic media prepared with buffer and organic solvents, whose log P value varied from 0.68 to 8.8. Storage stability was also monitored in the pure solvents. The small average particle size (125-210 microns) and the intensive stirring eliminate hindrances of intra- and interphase mass transfer resistances. The hydrophilic matrix protects the enzymes against thermal and chemical deactivation, thus allowing good production per unit weight of biocatalyst. In biphasic media, storage stability, with the exception of acid phosphatase, was not dependent on solvent polarity. On the contrary, a significant trend was observed when the enzymes were stored in neat organic solvents.

Acid Phosphatase↗
PubMed1990

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: mechanism of O2-inactivation of the enzyme and its stability in the aerobe.

O2-inactivation of pyruvate:NADP+ oxidoreductase from mitochondria of Euglena gracilis was studied in vitro, and a mechanism which consists of two sequential stages was proposed. Initially, the enzyme is inactivated by the direct action of O2 in a process obeying second-order kinetics. Although the catalytic activity for pyruvate oxidation is lost by this initial inactivation, NADPH oxidation with artificial electron acceptors still occurs. Subsequently, a secondary, O2-independent inactivation occurs, rendering the enzyme completely inactive. Pyruvate stimulates the O2-inactivation while CoA and NADP+ protect the enzyme from O2. The O2-inactivation is accelerated by reduction of the enzyme with pyruvate and CoA. Reactivation of the O2-inactivated enzyme was studied in Ar by incubation with Fe2+ in the presence of some other reducing reagent such as dithiothreitol. The evidence obtained indicates that the partially inactivated enzyme, which retains catalytic activity for NADPH oxidation, can be reactivated, but the completely inactivated enzyme is not. When Euglena cells were exposed to 100% O2 the enzyme in the cells was inactivated by O2, but the rate was quite slow compared with that observed in vitro. The enzyme inactivated by O2 in the cells was almost completely reactivated in vitro by incubation with Fe2+ and other reducing reagents in Ar, suggesting that the secondary, O2-independent inactivation does not occur in situ. When the cells were returned to air, reactivation of the O2-inactivated enzyme in the cells began immediately. The enzyme, kept in isolated, intact mitochondria, was stable in air; however, the enzyme was inactivated by O2 when the mitochondria were incubated with a high concentration of pyruvate.

Animals↗
PubMed2004

Effect of genetic circular permutation near the active site on the activity and stability of an enzyme inhibitor.

We report here the effect of circular permutation on the structure and function of a model protein tendamistat, a 74 amino acid competitive inhibitor of porcine pancreatic alpha-amylase. The activity and stability of wild type and two permuted tendamistat variants were characterized by measurement of alpha-amylase kinetic and thermodynamic binding parameters and their thermodynamics of unfolding. Our results show that large variations in structure and function can occur upon circularly permuting tendamistat near its active site that are not obvious, a priori, from the structure of the native protein and we propose a structural thermodynamic explanation of the experimental observations.

Animals↗
PubMed1998

Activity losses among T4 lysozyme variants after adsorption to colloidal silica.

Maintaining a specific molecular conformation is essential for the proper functioning of an enzyme. A substantial loss of catalytic activity can occur from the displacement caused by even a single amino acid substitution. Activity may also be lost as an enzyme undergoes a conformational change during adsorption. In this study, we investigated the effect of thermostability on the activities of three T4 lysozyme variants after adsorption to 9 nm colloidal silica particles. Less-stable T4 lysozyme variants lost more activity after adsorption than did more stable variants, apparently because they experienced more extensive structural alteration.

Adsorption↗
PubMed1999

Reversible denaturation of carbonic anhydrase provides a method for its adsorptive immobilization.

Palmityl-substituted sepharose 4B has been used for adsorptive immobilization of heat-denatured carbonic anhydrase. The native form of this enzyme does not show any affinity for binding to this hydrophobic support. However, through the process of denaturation-renaturation performed by heating and subsequent cooling of an enzyme solution in the presence of the matrix, it was possible to obtain a catalytically active immobilized preparation, which was used successfully in continuous catalytic transformations. It is suggested that this simple procedure may provide a convenient method of immobilization for proteins, which are not normally adsorbed on hydrophobic supports.

Adsorption↗
PubMed2000

Continuous enzymatic esterification of glycerol with (poly)unsaturated fatty acids in a packed-bed reactor.

Enzymatic synthesis of mono-, di-, and triacyglycerols from (poly)unsaturated fatty acids (linoleic, oleic, and conjugated linoleic acids) has been studied as a solvent-free reaction in a packed-bed reactor containing an immobilized lipase from Mucor miehei. The extents of the esterification reactions of interest are primarily determined by the molar ratio of glycerol to fatty acid because the presence of excess glycerol as a immiscible phase is responsible for reducing the activity of the water produced by the esterification reactions. For molar ratios of fatty acid to glycerol of less than 1.5, the percentage of the fatty acid esterified decreases quasi-linearly with an increase in this molar ratio. By appropriate manipulation of the fluid-residence time, one can control the relative proportions of the various acylglycerols in the effluent stream. At the outlet of the reactor, one observes excellent spontaneous separation of the glycerol and acylglycerol/fatty acid phases. At 50 degrees C and a fluid residence time of 1 hour, as much as 90% of the fatty acid can be esterified when the molar ratio of fatty acid to glycerol is 0.33 or less.

Bioreactors↗
PubMed2000

Immobilization of alliinase on porous aluminum oxide.

Membrane filters prepared from porous aluminum oxide (Anopore) were investigated for their potential use as a durable support for enzymes. Alliinase (EC 4.4.1.4) was chosen as a model enzyme for immobilization experiments. To allow for smooth fixation, the enzyme was immobilized indirectly by sugar-lectin binding. Monomolecular layers of the lectin concanavalin A and alliinase were applied by self-assembling processes. As an anchor for these layers, the sugar, mannan, was covalently coupled to the membrane surface. This procedure exhibits several advantages: (i) enzyme immobilization can be carried out under smooth conditions; (ii) immobilization needs little time; and (iii) protein layers may be renewed.

Absorption↗
PubMed2000

Highly efficient immobilization of glycosylated enzymes into polyurethane foams.

Glycosylated enzymes, including aminoacylase from Aspergillus melleus, chloroperoxidase from Caldariomyces fumago, and phytase from Aspergillus ficuum, were covalently immobilized into polyurethane foams with very high enzyme loadings of up to 0.2 g protein per gram dry foam. The immobilization efficiency (retained activity) ranged from 100% at a low loading to 60% at high loadings. In contrast to many other immobilization methods no leaching of the enzyme from the support took place under the reaction conditions. In short, a universal method for the immobilization of enzymes from fungal sources was developed, affording a highly active, stable, and reusable biocatalyst.

6-Phytase↗
PubMed2003

In vitro studies of the influence of ELF electromagnetic fields on the activity of soluble and insoluble peroxidase.

The influence of an extremely low frequency (ELF) magnetic field (50 Hz and 1 mT, EMF) on the activity of a soluble and insoluble horseradish peroxidase (E.C. 1.11.17) has been studied as a function of time. Insoluble derivatives were obtained by enzyme entrapment into two different gelatin membranes or by covalent attachment of the enzyme on two nylon membranes, differently preactivated. Results have shown that the field affects the inactivation rate of the soluble enzyme, while no effects are observed with insoluble derivatives. Since in vivo enzymes are immobilised into the biomembrane bilayer or entrapped into the cytoplasmic mixture, one might speculate that our experimental conditions do not reflect the catalytic activity of the enzymes in vivo.

Benzothiazoles↗
PubMedDate not supplied

Continuous-flow bioluminescent determination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate.

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25 degrees C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The ATP content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.

Adenosine Triphosphate↗
PubMedDate not supplied

The effect of detergents on firefly luciferase reactions.

The reaction rate of ATP-limited firefly luciferase-catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several-fold with a sharply defined optimum concentration of detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non-ionic and zwitterionic detergents increase reaction rate over a broad range of detergent concentrations. Enzyme stability during the reaction is not affected by non-ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates of some chemiluminescent reactions, have little effect on firefly luciferase activity. Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents are present.

Adenosine Triphosphate↗