Improvement of chloroperoxidase stability by covalent immobilization on chitosan membranes

Chloroperoxidase (CPO) from Caldariomyces fumago was optimally covalently immobilized on chitosan membranes pretreated with 0.8 M glutaraldehyde at pH 3.5 to give 3.18 mg CPO g⁻¹ support. Using monochlorodimedone (MCD) as assay substrate, the immobilized-CPO retained 40% activity at 50°C after 40 min whereas free CPO retained only 0.02%. The residual activity for immobilized-CPO was 99 and 58% compared with 68 and 43% for free CPO in the presence of 1.5 M urea and 300 μM H₂O₂, respectively, after 20 h.     

Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose

Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD(+). In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD(+)-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.     

Improvement of activity and stability of chloroperoxidase by chemical modification

Background  

Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes.     

Control of acetyl-CoA carboxylase by covalent modification

Summary In this review, various experiments which establish the occurrence of covalent modification mechanisms, both in vivo and in vitro, in the control of acetyl-CoA carboxylase have been presented. It is interesting to note that phosphorylation of the carboxylase results in disaggregation of the active species. These studies indicate that aggregation and disaggregation of the enzyme are involved in the control of carboxylase activity. Our covalent modification mechanism and the allosteric control mechanism share a common ground in that both mechanisms affect the equilibrium between protomers and polymers of the enzyme. However, it is clear that the allosteric control mechanism cannot functon alone under normal physiological conditions. Covalent modification of the carboxylase is prerequiste for efficient functioning of the allosteric mechanism.There are many aspects of the regulation of acetyl-CoA carboxylase which require further clarification. However, it is now established that short-term control of acetyl-CoA carboxylase involves the covalent modification mechanism.This research was supported by a grant from National Institutes of Health (AM 12865).This is Journal Paper No. 7701 from Purdue Agriculture Experiment Station.     

Desensitization by covalent modification of the chemoreceptor of Escherichia coli

Chemoreceptors in Escherichia coli were studied in situ in chemotactic mutants, deficient in the ability to modify the receptors, by using membrane vesicles prepared from the mutants. The affinity of the receptors for the ligands is related to the level of modification of the receptors. Unmodified serine receptor had a dissociation constant of 0.8 microM, while modified receptor had a dissociation constant that was at least 100-times higher. The results are discussed in relation to the two-state model of the chemoreceptor.     

Reversible covalent modification of DNA

The reaction of bromomethylbenzoyl esters of choline and dimethylaminoethanol with DNA and model compounds led predominantly to phosphotriester formation. In model compounds the phosphotriester formation was verified by uv spectrometry. The bromomethylbenzoyl cationic esters reacted with DNA at room temperature at neutral pH values. The amount of the reagent chromophores was assessed semiquantitatively by spectrophotometry. The maximum binding appeared to be stoichiometric, i.e., one residue per phosphorus. The binding of one mole of reagent per phosphorus was confirmed by electron spectroscopic measurements of the phosphorus atom electron emission of maximally modified DNA. The modified DNA showed altered CD spectra indicating that the reagent chromophores are arranged in an orderly fashion affording a strong (Δ_? > 4), positive, apparently extrinsic CD band at ~240 nm; a double helical array is proposed. The introduced chromophores were readily removed by heat treatment or by treatment with nucleophiles at neutral pH values at moderate temperatures (<37 °C); no measurable fraction of the DNA became dialyzable. A decrease in viscosity accompanied the reversal, indicative of some chain breaking. The modified DNA's show higher T_m values than the native DNA and some display a higher and some a lower degree of cooperativity in their melting curves. No chemically detectable amounts of base alkylation, depurination, or depyrimidination were found when dialyzates of treated DNA and hydrolyzed samples of modified DNA were examined. However, presumptive evidence for some base alkylation by these novel alkylating agents was found utilizing Salmonella typhimurium tester strains sensitive to reversion by alkylation. No comparable binding of benzoylcholine, a nonalkylating analogue, by DNA was seen under conditions utilized here.     

Reversible inactivation and superactivation by covalent modification of thermolysin

Diethylpyrocarbonate (DEP) in the pH range 6.1 – 7.5 inactivates thermolysin by ethoxyformylation. Restoration of activity by hydroxylamine at pH 6.2 correlates with the regeneration of a single histidyl residue. Exposure of the enzyme to DEP together with the reversible inhibitor β-phenylpropionyl-L-phenylalanine, or acylation with the mixed anhydride of β-phenylpropionyl-L-phenylalanine and ethoxyformic acid or with β-phenylpropionyl-L-phenylalanyl-imidazole increases the activity by an order of magnitude toward both peptide and ester substrates. Treatment with NH₂OH restores the catalytic properties of this superactive derivative to that of the native enzyme while modification with DEP destroys activity completely.     

Functional stabilization of invertase by covalent modification with pectin

Pectin was attached to ethylenediamine-activated carbohydrate moieties of invertase using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent. The modified enzyme retained 57% of the original activity and contained 2.7 mol polymer per mol holoenzyme. Its optimum temperature was increased by 8°C and its thermostability by 7.3°C. The half-life at 65°C was increased from 5 min to 2 days. The enzyme stability was enhanced by 33% at pH 2.0, and also by 27% at pH 12.0. The conjugate retained about 96% of its initial activity after 3 h incubation in 6 M urea.     

Energy expenditure in the control of biochemical systems by covalent modification

Regulation by reversible, covalent modification of proteins requires a continuous expenditure of energy, even in a steady-state situation. The cost of this energy drain is evaluated for the case of an effector controlling the modifying enzyme and an effector controlling the demodifying enzyme and for the case of dual control in which an effector activates one of these enzymes and inhibits the other. Energy consumption is determined when the converter enzymes are functioning in the first-order and zero-order domains. The profile of energy expenditure versus fractional protein modification at steady state varies both as a function of the mechanism of control of the converter enzymes and of the kinetic domain in which they operate. This theory allows one to predict the strategies that would minimize energy costs. Dual control appears to provide maximum sensitivity with minimal energy expenditure. The analysis is applied to two experimental systems. Comparison of ATP turnover rates with rates for individual modification enzymes in living systems shows that a significant fraction of the total energy expenditure of an organism is required for the large number of reactions which involve covalent modification of proteins. It is concluded that there will be selection pressure for energy-efficient control of covalent regulation.     

Effects of cellulase on the modification of cellulose

Multicomponent cellulases, purified endoglucanases and cellobiohydrolases were assayed and shown to modify pure natural cellulose (softwood pulp). Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme, and the treatment conditions. The reactivity of cellulose for some dissolving and derivatization processes may be improved by enzymatic hydrolysis. Endoglucanases decreased the average degrees of polymerization (DP) and improved the alkaline solubility of cellulose most efficiently. The variation in the supramolecular structure estimated from the infrared spectra of the cellulose samples was found to be correlated with the reactivity and might represent wide variations in conformation caused by the breakdown of the hydrogen bonds.     

Chemical modification of L-asparaginase with a comb-shaped copolymer of polyethylene glycol derivative and maleic anhydride.

L-Asparaginase from Escherichia coli, an anti-tumor enzyme, was chemically modified with two types of maleic anhydride copolymers with a comb-shaped form, the one composed of polyoxyethylene allyl methyl diether with the molecular weight of 13,000 (activated PM13) and the other of polyoxyethylene 2-methyl-2-propenyl methyl diether with 100,000 (activated PM100). The modified asparaginases (PM13- and PM100-asparaginases) exhibited the complete loss of immunoreactivity towards anti-asparaginase serum. The enzymic activity of PM100-asparaginase without immunoreactivity was well retained by 85% of non-modified one, while that of PM13-asparaginase was retained 46%. These results were discussed in relation to the chemical structure of modifying reagents including chain shaped-polyethylene glycol derivatives.     

Regulation of nitrogenase activity by covalent modification in Chromatium vinosum

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH ₄ ⁺ . Activity of the Fe protin component of nitrogenase required both Mn²⁺ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.Abbreviation HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid     

Impairment by covalent modification of the ability of Phaseolus vulgaris phytohemagglutinin to stimulate cultured human lymphocytes: a comparison of different forms of covalent modification

Phytohemagglutinin (PHA) isolated from Phaseolus vulgaris has been modified by treatment with various chemical reagents and the modified proteins have been tested for their ability to stimulate peripheral lymphocytes from two healthy human donors, in vitro. Reaction of PHA with citraconic anhydride, S-methyl isothiourea, or 2-hydroxy-5-nitrobenzyl bromide produced derivatives which retained the ability to stimulate lymphocytes, at low concentrations. Acylation of the lectin with acetic anhydride or masking of the carboxyl side chains by reaction with glycinamide-carbodiimide impaired stimulation. When PHA was treated with N-bromosuccinimide or with tetranitromethane, the derivatives were ineffective as lymphocyte stimulants. Chemical modifications affected, in some cases, the quaternary structure of the lectin. Glycinamide-, homoarginine-, and nitro-PHA were tetramers whereas acetyl-, citraconyl-, and N-bromosuccinimide-treated lectin were dimers. Antinative lectin antiserum cross-reacted with all the modified proteins, except in the case of the N-bromosuccinimide derivative. The results show that, in the human lymphocyte transformation assay, the mitogenic property of PHA may depend on intact aspartic, glutamic, and tyrosine residues whereas lysine residues do not appear to be essential.     

Inhibition of staphylococcal alpha-toxin by covalent modification of an arginine residue

The effects of 1,2-cyclohexanedione and phenylglyoxal on staphylococcal alpha-toxin were studied. Modification of one arginine residue in alpha-toxin was sufficient to render the toxin nonhemolytic with no conformational change. Modified alpha-toxin did not protect cells from hemolysis by native alpha-toxin. An arginine residue is therefore at or near the binding site of alpha-toxin. Trypsin digestion of modified alpha-toxin generated a 20 kDa fragment which was isolated using a boric acid gel column. Upon regeneration, this 20 kDa fragment was not recognized by a population of antibodies which prevented alpha-toxin binding. The fragment was recognized by antibodies directed against post-binding events. However, the antibinding antibodies recognized the intact modified toxin. This leads us to conclude that antibinding determinants are not found directly in the binding site or are conformationally masked.     

Metabolic oscillations in biochemical systems controlled by covalent enzyme modification

We analyze the conditions under which sustained oscillations develop in a biochemical system regulated autocatalytically by reversible, covalent enzyme modification. The analysis applies, for example, to the situation where adenylate cyclase (or guanylate cyclase) is activated through phosphorylation by a cAMP (or cGMP)-dependent protein kinase. The model then provides a non-allosteric mechanism for the periodic generation of cAMP or cGMP pulses. For certain parameter values close to those that produce oscillations, the system is excitable since it can amplify in a pulsatory manner suprathreshold perturbations. The results on excitable and oscillatory behavior are discussed in relation with the mechanism of cAMP relay and oscillation in the slime mold Dictyostelium discoideum.     

Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability

To obtain regenerable magnetic nanoparticles, triethoxy(3-isocyanatopropyl)silane and iminodiacetic acid (IZ) were used as the starting material and immobilized on Fe₃O₄ nanoparticles. Copper ions (Cu²⁺ ions) were loaded on the Fe-IZ nanoparticles and used for cellulase immobilization. The support was characterized by spectroscopic methods (FTIR, NMR) and thermogravimetric analysis, transmission electron microscopy, scanning electron microscope, X-ray diffraction, energy dispersive X-ray analysis, and vibrating sample magnetometer techniques. As a result of experiments, the amount of protein bound to immobilized cellulase (Fe-IZ-Cu-E) and cellulase activity was found to be 33.1 mg/g and 154 U/g at pH 5, 50°C, for 3 h. The results indicated that the free cellulase had kept only 50% of its activity after 2 h, while the Fe-IZ-Cu-E was observed to be around 77%, at 60°C. It was found that the immobilized cellulase maintained 93% of its initial catalytic activity after its sixth use. Furthermore, the Fe-IZ-Cu-E retained about 75% of its initial activity after 28 days of storage. To reuse the support material (Fe-IZ-Cu), it was regenerated by thorough washing with ammonia or imidazole.