Enhanced stability of β-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran

In order to enhance the stability of β-galactosidase, we conjugated the enzyme with dextran T-10 (M_r approx. 10 000). The conjugate contained 9–10 mol dextran/mol protein (β-galactosidase, M_r 68 000), and the specific activity retained after conjugation was 90 ± 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated β-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when β-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated β-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50°C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

Conjugation of α-amylase with dextran for enhanced stability: Process details, kinetics and structural analysis

The influence of enzyme polysaccharide interaction on enzyme stability and activity was elucidated by covalently binding dextran to a model enzyme, α-amylase. The conjugation process was optimized with respect to concentration of oxidizing agent, pH of enzyme solution, ratio of dextran to enzyme concentration, temperature and time of conjugate formation, and was found to affect the stability of α-amylase. α-Amylase conjugated under optimized conditions showed 5% loss of activity but with enhanced thermal and pH stability. Lower inactivation rate constant of conjugated α-amylase within the temperature range of 60-80°C implied its better stability. Activation energy for denaturation of α-amylase increased by 8.81kJ/mol on conjugation with dextran. Analysis of secondary structure of α-amylase after covalent binding with dextran showed helix to turn conversion without loss of functional properties of α-amylase. Covalent bonding was found to be mandatory for the formation of conjugate.     

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.     

Covalent attachment of epoxide hydrolase to dextran

Epoxide hydrolase (EC 3.3.2.3) purified from rat liver microsomes has been immobilized by covalent linking to dextran activated by imidazolyl carbamate groups, under mild conditions. K^app_m values of free and dextran bound epoxide hydrolase toward benzo(a)pyrene-4,5-oxide were 0.5 and 0.35 μM respectively, while V^app_max was lowered from 300 to 120 nmol min^?1mg^?1protein. The activity lost upon coupling could not be restored by digestion of the support by dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) treatment. This fact, along with the similarity of the activation energy values for both native and bound epoxide hydrolase, indicated that steric hindrance effects due to the polymer support played only a minor role in this loss of activity. Evidences of changes in the conformation of epoxide hydrolase were obtained by a comparative study of u.v. circular dichroism and tryptophan fluorescence emission spectra of the native and dextran bound enzymes. On the other hand, the enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation.     

Preparation and characterization of soluble conjugates of defined molecular sizes prepared by linkage of pepsin and carboxypeptidase A to dextrans

Soluble conjugates of pepsin and carboxypeptidase A were prepared by covalent linkage of the enzymes to an amino derivative of dextran. By fractionating the dextran derivatives before and after enzyme coupling, three conjugates, with median Stokes radii between 4.0 and 11.7 nm and with a range of 25% of the median, were prepared from each enzyme. The pepsin and carboxypeptidase A conjugates contained about 35% and 3% protein, respectively. Both types had specific activities close to those of the native enzymes and were stable at -20 degrees C. The pH-activity curve was unaffected by linkage of either enzyme to dextran. The stabilities at 30 degrees C of pepsin at pH 6-7 and carboxypeptidase A at pH 3.5-9.0 were increased by linkage to dextran. No significant amount of unbound enzyme was released from either type of conjugate in skim milk. The molecular sizes, deduced from the intrinsic viscosities and the diffusion coefficients of all conjugates, were close enough to the Stokes radii to indicate that the molecules were approximately spherical. Physical measurements also indicated that the molecules were dextranlike and highly hydrated.     

Stabilisation and immobilisation of penicillin amidase

Penicillin amidase was coupled to a periodate-oxidised dextran by reductive alkylation in the presence of sodium cyanoborohydride. A loss of activity (25%) was observed but the conjugate enzyme dextran was more thermostable than the native enzyme. Native and dextran-conjugated penicillin amidase were immobilised on amino activated silica (Promaxon, Spherosil, Aerosil) by a classical method using glutaraldehyde for the native enzyme and reductive alkylation for the modified enzyme. Good relative activity of the enzymes was obtained after insolubilisation. Immobilisation of both native and modified enzymes resulted in the thermostabilisation of the penicillin amidase.     

Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.     

Studied on ribonucleoside-diphosphate reductase in permeable animal cells. I. Reversible permeabilization of mouse L cells with dextran sulfate

The treatment of mouse L cells with sodium dextran sulfate 500 for 20 min at 4 °C rendered them selectively permeable to small molecules. The degree of permeabilization was reproducible and depended on dextran sulfate concentration: as the dextran sulfate concentration increased, the cells became more permeable to the dye erythrosin B, lost the ability to incorporate [³H]thymidine into DNA, and acquired the capacity for nucleotide-dependent incorporation of [³H]dTTP into DNA and for K⁺- and ATP-dependent incorporation of [³H]lysine into protein. The ability to engage in macromolecular synthesis indicated that the permeabilized cells had retained a considerable amount of integrity, and this was confirmed by the observation that the cells could regain viability after an appropriate repair treatment. Permeabilization was rapidly reversed by incubating the cells in suspension culture medium plus 10% calf serum or in a defined medium composed of NaCl, NaH₂PO₄, glucose, glutamine, and CaCl₂, calcium ion being the most critical component. Using this minimally disruptive permeabilization procedure, it was possible to assay the enzyme ribonucleoside-diphosphate reductase in situ. The enzymatic conversion of CDP and GDP to the corresponding deoxyribonucleotides was studied; enzyme activity was almost entirely intracellular and responded to the allosteric activators and inhibitors that are known to modulate the activity of ribonucleotide reductase in vitro.     

Design,and facile synthesis of 1,3 diaryl-3-(arylamino)propan-1-one derivatives as the potential alpha-amylase inhibitors and antioxidants

Diabetes is the most prevalent metabolic disorder causing a high rate of mortality and morbidity. Recently alpha-amylase is reported to be good drug design target for the treatment of diabetes mellitus. We have designed 116 molecules based on aza-Michael adduct of trans-chalcone as 1,3 diaryl-3-(arylamino)propan-1-ones which were studied by molecular docking and among them best six derivatives were synthesized easily via aza-Michael addition on trans-chalcone using KOH as a catalyst and evaluated for alpha-amylase inhibition along with antioxidant activity. It was observed that all compounds have alpha-amylase inhibitory activity but at different extents. The molecule 3e is the most potent alpha-amylase inhibitor of this series. 3a is the second most potent compound, whereas only one molecule 3d has shown antioxidant activity.     

Poly(N-acryloyl-4- and -5-aminosalicylic acids). 3. Uses as their titanium complexes for the insolubilisation of enzymes

The titanous and titanic complexes of the water-insoluble poly(N-acryloyl-4- and -5-aminosalicylic acids) have been prepared by several methods, and alpha-amylase, glucoamylase, and polygalacturonase (pectinase) have been coupled to the various preparations. The products from alpha-amylase and glucoamylase were enzymically active, but the alpha-amylase was washed off after only one use. With glucoamylase, the derivative withstood extensive washing and could be used continuously in a column. Particular advantages of the glucoamylase preparation were that maximal coupling of the enzyme was achieved in one hour and that a very high specific activity towards a macromolecular substrate was achieved. The polygalacturonase derivative was inactive, possibly because the polysalicylic acid acts as an inhibitor of the enzyme.     

Influence of heat treatment on rabbit liver ferritin

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

Hydrolysis of amylopectin by the alpha-amylase of B. subtilis

The mixture of oligosaccharides obtained after initial hydrolysis of waxy-maize amylopectin by the alpha-amylase from Bacillus subtilis was fractionated on a Sepharose CL 6B column. The molecular-weight-distribution curves indicated that intermediate products of defined molecular weights were formed as the reaction proceeded towards smaller dextrins. The results can be explained in terms of the cluster model for the fine structure of the amylopectin molecules, assuming that the intermediate products represent one or more unit clusters or cluster residues. Simultaneously with the formation of the intermediate products of molecular weights ⩾30,000, small dextrins were also produced, most probably representing the residues from outer chains in the macromolecule. Thus, alpha-amylase does not hydrolyse amylopectin molecules in a random manner, but, in the initial stages, preferentially attacks the glucosidic bonds between the unit clusters, which in turn are of defined sizes.     

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.     

Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors

Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the V_max value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s⁻¹ (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s⁻¹ (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s⁻¹, and the apparent K_m value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent K_m value of 1.8 units·ml⁻¹ in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.     

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).     

Influence of rheopolyglucin and dextran dialdehyde on physicochemical properties and thermostability of human hemoglobin

A study was made of the influence of rheopolyglucin and dextran dialdehyde derived therefrom on the structural characteristics and thermostability of human hemoglobin. The effects of solution pH, incubation time and temperature, and the degree of dextran oxidation on the conjugation between human hemoglobin and dextran dialdehyde were assessed. Formation of the hemoglobin-dextran dialdehyde complex resulted in shielding of the protein chromophore groups by the polysaccharide and transition of a part of hemoprotein molecules from a low-spin (HbO₂) to a high-spin (Hb and MetHb) state. It was found that the temperature of denaturation transition for the native protein and hemoglobin in the presence of rheopolyglucin was 60°C versus 80°C for the hemoprotein-dextran dialdehyde conjugate. Presumably, the latter is determined by the enhancement of hydrophobic interactions within the protein globule caused by dextran dialdehyde and the ability of the surface-bound carbohydrate components to prevent the association of hemoglobin molecules.     

Lysosomal nature of hormonally induced enzymes in wheat aleurone cells

The subcellular distribution of the enzymes alpha-amylase, protease and ribonuclease in wheat aleurone layers after treatment with gibberellic acid was determined by differential centrifugation. Of the alpha-amylase 56% was precipitable from cell homogenates, indicating that it is a particulate enzyme. Similar results were recorded with protease. Particulate alpha-amylase showed distinct structural latency, and membrane-rupturing mechanical or chemical treatments were required to release the enzyme in an active form; the results were completely analogous to results with lysosomal enzymes found in animal tissues. The identification of the hormonally induced enzymes as lysosomal suggests that the hormonal mechanism may be more closely associated with extracellular enzyme synthesis rather than with nucleic acid metabolism.     

Maltose adaptive mutant of heterotrophic marine bacterium, Alteromonas espejiana Bal-31: changes in the growth and the induction of extracellular alpha-amylase

Growth of the heterotrophic marine bacterium, Alteromonas espejiana Bal-31 was inhibited in the presence of sucrose, maltose and even glucose, but not with starch. Extracellular alpha-amylase was induced with a lag phase of 2 h in the presence of starch. In contrast, cell growth of the S2a mutant was not affected by the addition of maltose, and starch was ineffective in the induction of extracellular alpha-amylase in this mutant. Activity of extracellular alpha-amylase was induced from the S2a mutant with a 4-h lag phase in the presence of maltose, and the high level of enzyme activity was maintained for at least 24 h. Activity of alpha-amylase induced by both wild type starch and S2a mutant maltose cultures were mainly observed in extracellular locations. This activity could be stopped by tetracycline treatment, indicating that enzyme induction was dependant on gene expression and not on enzyme protein secretory mechanisms. Our results showed that the mutation in S2a changed the growth and the modulation of the specific alpha-amylase in response to carbon nutrients.     

Membrane-bound and soluble extracellular alpha-amylase from Bacillus subtilis.

Extracellular alpha-amylase was purified to homogeneity from a Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67,000. Its NH2-terminal amino acid sequence is Leu-Thr-Ala-Pro-Ser-Ile-Lys. A membrane-derived alpha-amylase was solubilizing from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-alpha-amylase-protein A-Sepharose column. Membrane-derived alpha-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulfate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform:methanol. The extracted phospholipid was predominately phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound alpha-amylase was latent in membrane vesicles. Release of membrane-bound alpha-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and diisopropyl fluorophosphate and was stimulated by Ca2+ and Mg2+. The amount of membrane-bound alpha-amylase was related to the level of secretion.     

Enhanced intracellular stability of dextran-horse radish peroxidase conjugate: an approach to enzyme replacement therapy.

Horse radish peroxidase (HRP), a mannose-containing glycoprotein was covalently modified by conjugation with dextran. The rapid uptake of HRP by the liver is markedly inhibited by mannan. The uptake of dextran-HRP conjugate by the liver, though lower compared to that of the free enzyme, is also partially inhibited by mannan. Liposomes were therefore used as carriers for delivering the free and the modified HRP to the liver. The dextran-HRP conjugate showed greater stability intracellularly as compared to the free enzyme. The enhanced stability of enzymes upon their extensive glycosylation with nondegradable sugar polymers would be of importance in extending the catalytic life of therapeutically active enzymes and thereby improve their therapeutic potential for the treatment of certain enzyme deficiency disorders.