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At least 1,063 records · Page 59Linked to original sources
PubMed1987

Tinkering with enzymes: what are we learning?

It is now possible, by site-directed mutagenesis of the gene, to change any amino acid residue in a protein to any other. In enzymology, application of this technique is leading to exciting new insights both into the mechanism of catalysis by particular enzymes, and into the basis of catalysis itself. The precise and often delicate changes that are being made in and near the active sites of enzymes are illuminating the interdependent roles of catalytic groups, and are allowing the first steps to be taken toward the rational alteration of enzyme specificity and reactivity.

Amino Acids↗
PubMed2006

Trigonopsis variabilis D-amino acid oxidase: control of protein quality and opportunities for biocatalysis through production in Escherichia coli.

Trigonopsis variabilis D-amino acid oxidase accounts for 35% of Escherichia coli protein when added D-methionine suppresses the toxic activity of the recombinant product. Permeabilized E. coli cells are reusable and stabilized enzyme preparations. The purified oxidase lacks the microheterogeneity of the natural enzyme. Oriented immobilization of a chimeric oxidase maintains 80% of the original activity in microparticle-bound enzymes.

Alanine↗
PubMed1988

Biotransformation of Dehydroabietic, Abietic, and Isopimaric Acids by Mortierella isabellina Immobilized in Polyurethane Foam.

Polyurethane foam supports immobilization of Mortierella isabellina ATCC 38063, a zygomycete that hydroxylates and thereby detoxifies resin acids found in certain pulp mill effluents. The fungus becomes entrapped as it threads growing mycelia through the open foam matrix. The tenacity of binding and the amount of biomass bound depend on initial spore numbers and nutrient concentration. Optimal dehydroabietic acid transformation occurs with early-stationary-phase foam-bound mycelia suspended in buffer at pH 6.5 to 8.5 with aeration >0.1 liter liter min and near a temperature maximum of 33 degrees C. Neither a greater number of similarly loaded foam cubes nor a greater surface area for similar amounts of biomass increased transformation rates. Compared to free mycelial cultures, foam presence retards growth and decreases the rate of precursor transformation but does not alter the nature of the metabolites formed from dehydroabietic acid, abietic acid, or isopimaric acid. Diffusional barriers or binding of substrates in an inaccessible manner may cause the observed inhibitions. Overall, foam immobilization stabilizes enzymic activity and is a facile laboratory process with scaleup potential.

Journal Article↗
PubMed1991

Effect of the lactoperoxidase system on Listeria monocytogenes behavior in raw milk at refrigeration temperatures.

Activity of raw milk lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system on four Listeria monocytogenes strains at refrigeration temperatures after addition of 0.25 mM sodium thiocyanate and 0.25 mM hydrogen peroxide was studied. The LP system exhibited a bactericidal activity against L. monocytogenes at 4 and 8 degrees C; the activity was dependent on temperature, length of incubation, and strain of L. monocytogenes tested. D values in activated-LP system milk for the four strains tested ranged from 4.1 to 11.2 days at 4 degrees C and from 4.4 to 9.7 days at 8 degrees C. The lactoperoxidase level in raw milk declined during a 7-day incubation, the decrease being more pronounced at 8 degrees C than at 4 degrees C and in control milk than in activated-LP system milk. The thiocyanate concentration decreased considerably in activated-LP system milk at both temperatures during the first 8 h of incubation. LP system activation was shown to be a feasible procedure for controlling development of L. monocytogenes in raw milk at refrigeration temperatures.

Animals↗
PubMed1998

Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant alpha-amylase.

Alpha-Amylase from the antarctic psychrophile Altermonas haloplanktis is synthesized at 0 +/- 2 degrees C by the wild strain. This heat-labile alpha-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18 degrees C.

Escherichia coli↗
PubMed1975

Metalloprotease from Bacillus thuringiensis.

Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2. The addition of Ca24 was required for enzyme stabilization...

Bacillus thuringiensis↗
PubMed1983

Glucose-6-phosphatase: is activity regulated by phosphorylation-dephosphorylation?

Incubation of rat liver microsomes with ATP and Mg2+ in the absence or presence of an exogenous protein kinase showed no changes in the activity of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). These observations confirm the recent findings of the Burchells and colleagues and refute on methodological grounds the earlier conclusions of Begley and Craft implicating regulation of this enzyme by protein phosphorylation-dephosphorylation. In other studies, the time-dependent inactivation of microsomal glucose-6-phosphatase by incubation with deoxycholate was used to obtain the inactive enzyme which in the presence of a protein kinase, ATP, and Mg2+ could not be restored to its original level. A number of substrates and competitive inhibitors of glucose-6-phosphatase, most notably vanadate which is the most potent inhibitor of the enzyme identified, stabilized this enzyme against its time-dependent inactivation in the presence of detergent as effectively as did fluoride and molybdate which are also effective competitive inhibitors of glucose-6-phosphatase. An alternative explanation to the involvement of a phosphoprotein phosphatase, as discussed by the Burchells, in the time-dependent inactivation of glucose-6-phosphatase is thus suggested.

Adenosine Triphosphate↗
PubMed1988

A new bilirubin-degrading enzyme from orange peels.

A novel enzyme activity that catalyzes the degradation of unconjugated bilirubin (Bu) has been demonstrated in extracts of the peels of edible oranges. Unlike the few known bilirubin-oxidizing enzymes, the orange enzyme does not produce biliverdin as a product, does not seem to require oxygen, and has a unique absorption spectrum of its products. Even at the crude stage, the enzyme has a specific activity that is 10 and 20 times higher, respectively, than those reported for the crude or partially purified Bu-degrading enzymes from mushrooms and rat liver. The enzyme has a pH optimum near 7.5 and a Km value of 50-100 microM for Bu. The enzyme is remarkably stable, retaining over 50% activity after prolonged digestion with proteinase K or heating at 100 degrees C. Similar treatments inactivated the bilirubin oxidase from Myrothecium verrucaria MT-1. The enzyme is poorly soluble in water but can be partially solubilized with cholic acid, with a doubling in specific activity.

Bilirubin↗
PubMed2002

The role of dynamics in enzyme activity.

Although protein function is thought to depend on flexibility, precisely how the dynamics of the molecule and its environment contribute to catalytic mechanisms is unclear. We review experimental and computational work relating to enzyme dynamics and function, including the role of solvent. The evidence suggests that fast motions on the 100 ps timescale, and any motions coupled to these, are not required for enzyme function. Proteins where the function is electron transfer, proton tunneling, or ligand binding may have different dynamical dependencies from those for enzymes, and enzymes with large turnover numbers may have different dynamical dependencies from those that turn over more slowly. The timescale differences between the fastest anharmonic fluctuations and the barrier-crossing rate point to the need to develop methods to resolve the range of motions present in enzymes on different time- and lengthscales.

Catalysis↗
PubMed2001

High-Pressure Biotechnology in Medicine and Pharmaceutical Science.

High-pressure (HP) biotechnology is an emerging technique initially applied for food processing and more recently in pharmaceutical and medical sciences. Pressure can stabilize enzymes and modulate both their activity and specificity. HP engineering of proteins may be used for enzyme-catalyzed synthesis of fine chemicals, pharmaceuticals, and production of modified proteins of medical or pharmaceutical interest. HP inactivation of biological agents is expected to be applicable to sterilization of fragile biopharmaceuticals, or medical compounds. The enhanced immunogenicity of some pressure-killed bacteria and viruses could be applied for making new vaccines. Finally, storage at subzero temperatures without freezing is another potential application of HP for cells, animal tissues, blood cells, organs for transplant, and so forth.

Journal Article↗
PubMed2002

Chronic haemolytic anaemia and glucose-6 phosphate dehydrogenase deficiency. Case report and review of the literature.

Deficiency in glucose-6-phosphate dehydrogenase (G6PD) is the most common enzymopathy, and more than 125 different mutations causing G6PD deficiency have been identified. Chronic haemolytic anaemia (CHA) associated with G6PD deficiency is rare, but there is a cluster of mutations causing CHA between amino acids 361-428 which are encoded by exon 10 of the G6PD gene. This region is involved in the dimer formation of the active G6PD enzyme and therefore plays an important role for enzyme stability and activity. Here, we report a 17-year-old patient with CHA, who carries a rare G --> A mutation at nucleotide 1160 which causes an R387H amino acid substitution. We review the reports of the seven previously described patients with this mutation, concluding that G6PD deficiency should be considered as a rare differential diagnosis of chronic haemolytic, non-spherocytic anaemia.

Adolescent↗
PubMed1976

[Glutathione reductase deficiency with membrane defect in hereditary hemolytic anemia].

Glutathione reductase activity and phospholipid metabolism in red cell membranes were determined in a family with hereditary hemolytic anemia. A marked decrease of glutathione reductase activity and stability of reduced glutathione was found in combination with enhanced phospholipid-phosphate metabolism and decreased activity of the membrane-stabilizing enzyme lysolecithin-acyltransferase. In all cases splenectomy beneficially influenced the hemolytic process. Family studies revealed a dominant-autosomal genetic transmission.

Adolescent↗
PubMed1971

Detection of intravascular coagulation.

A method is described for the measurement of soluble thrombin-altered fibrinogen (circulating fibrin) in human plasma. This method is dependent upon the enzymatic incorporation of glycine ethyl ester-(14)C (GEE-(14)C) into circulating fibrin by the action of the fibrin-stabilizing enzyme, factor XIII. The mean incorporation of GEE-(14)C into the fibrinogen of normal human plasma controls was 167 +/-47 dpm/mg fibrinogen. The addition of 0.03 NIH U/ml of thrombin to normal human plasma resulted in a two to threefold increase in the incorporation of GEE-(14)C into the fibrinogen. The addition of plasmin split products of fibrinogen to normal plasma did not increase the incorporation of GEE-(14)C unless these products were also exposed to thrombin. The addition of plasmin split products of a fibrin clot resulted in only minimal increase in the incorporation of GEE-(14)C (57 dpm/mg fibrinogen) at 37.5% concentration. The method was therefore sensitive to thrombin alterations of fibrinogen but insensitive to plasmin alterations of fibrinogen and fibrin.Clinically, the method was found to provide useful information for the diagnosis and treatment of disseminated intravascular coagulation in two patients with meningococcemia, two patients with Rocky Mountain spotted fever, and three patients in whom therapeutic abortions were induced by the injection of hypertonic saline.

Abortion, Therapeutic↗
PubMed2002

Estrogen receptors alpha and beta have similar activities in multiple endothelial cell pathways.

The presence of both estrogen receptor alpha (ERalpha) and ERbeta in vascular cells has greatly increased the complexity of potential estrogen regulatory pathways in the cardiovascular system. Here, human umbilical vein endothelial cells were engineered using adenovirus vectors to express either ERalpha or ERbeta. The activities of ERalpha and ERbeta were compared in three distinct gene regulatory pathways, including inhibition of IL-1beta induction of E-selectin expression, inhibition of basal endothelin-1 production, and the ability to induce two matrix-stabilizing enzymes: tissue transglutaminase and a novel member of the lysyl oxidase family. Both ERs were active on these end points, although ERbeta was typically less efficacious than ERalpha. As no class of gene regulation could differentiate ERalpha from ERbeta activity, we characterized a novel steroid (7alpha-thiophenyl-E2) that bound with similar affinities to ERalpha and ERbeta, but functioned as an ERalpha agonist and ERbeta antagonist for all of these endothelial responses. This pattern of receptor subtype-selective activity was not unique to endothelial cells, but was also seen in metabolically active HepG2 cells, suggesting potential in vivo utility. The panel of endothelial responses coupled with a selective modulator should provide a means to characterize the roles of ERalpha and ERbeta in endothelial cells in vivo.

Amino Acid Oxidoreductases↗
PubMed1975

Cyclic 3', 5'-adenosine monophosphate levels in separated bone cells.

It has been shown that both parathyroid hormone (PTH) and calcitonin (CT) increase the concentration of cyclic 3',5'-adenosine monophosphate (cyclic AMP) in skeletal tissue. Since these two hormones have opposing actions on bone resorption, it has been postulated that the cell types upon which the hormones act in skeletal tissue may be different. We have previously shown that osteoblasts and osteocytes contain adenylate cyclase responsive to both PTH and CT whereas the enzyme prepared from periosteum and marrow cells did not respond to either. To further examine the postulate, the effect of these hormones on the concentration of cyclic AMP was determined. Bovine PTH (5U/ml equal to 4.2 times 10-7M) significantly increased the concentration of cyclic AMP/mug DNA in periosteum, osteoblasts, and osteocytes but not in marrow cells. Porcine CT (0.5 U/ml equal to 8.0 mug/ml approximately equal to 3.0 times 10-6M) significantly increased the concentration of cyclic AMP/mug DNA in periosteum, osteoblasts, osteocytes, and marrow cells. Adenylate cyclase activity prepared from periosteum and marrow cells was re-examined using EGTA and DMSO to improve enzyme stability and activity. Wth these additions PTH (5 U/70 mul) increased activity of the preparation from periosteum, and CT (0.5 U/70 mul) increased activity from marrow cells and periosteum. These studies provide evidence that: 1) periosteum, osteoblasts, and osteocytes respond directly to both PTH and CT and 2) marrow cells respond to CT and not PTH.

Animals↗
PubMed2006

Thermal inactivation of immobilized lipase in 1-alcohols.

The thermal inactivation of immobilized lipase from Candida antarctica in 1-alcohols with four to eight carbons at various temperatures could be well expressed using a model in which heterogeneity in the susceptibility of the enzyme to the inactivation was assumed. It was found that the enthalpy-entropy compensation held for inactivation in 1-alcohols.

Alcohols↗