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Research paper thumbnail of An amperometric sensor employing glucose oxidase immobilized on nylon membranes with different pore diameter and grafted with different monomers

Journal of Molecular Catalysis B-enzymatic, Sep 1, 2002

Glucose oxidase (GOD) was immobilized on nylon membranes having three different pore diameters an... more Glucose oxidase (GOD) was immobilized on nylon membranes having three different pore diameters and chemically grafted with glycidyl methacrylate (GMA) or butyl methacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GA) were used as spacer and coupling agent, respectively. The biochemical and electrochemical behaviour of the membranes has been studied as a function of pH, temperature and glucose concentration with reference to the grafted monomer and the membrane pore diameter. The behaviour of the soluble GOD has also been studied in order to see the modification induced by the immobilization process on the enzyme activity. It was found that the values of the biosensor sensitivity, maximum saturation current and electrochemical affinity increase with the membrane pore diameter, indipendently of the nature of the graft monomer. Opposite behaviour was found relatively to the extension of the linear response ranges and the average response times. With reference to the parameters increasing with the pore diameter it was found that membranes grafted with GMA had higher values than those of the membranes grafted with BMA. The contrary occurred to the values of the parameters decreasing with the increase of the pore diameter. Biochemical and electrochemical results have been discussed in terms of the different limitations to the diffusion of substrate and reaction products across the catalytic membrane introduced by the different pore diameters and by the different hydrophobicity of the graft monomers.

Research paper thumbnail of A DNA Oligonucleotide−Hemin Complex Cleaves t-Butyl Hydroperoxide through a Homolytic Mechanism

Inorganic Chemistry, 2001

Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been emplo... more Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex (∼35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of ∼250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of e5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from-scission of the primary tert-butyloxyl radical product.

Research paper thumbnail of The Peroxidase Activity of a Hemin−DNA Oligonucleotide Complex:  Free Radical Damage to Specific Guanine Bases of the DNA

Journal of the American Chemical Society, 2001

A specific DNA oligonucleotide-hemin complex (PS2.M-hemin complex) that exhibits DNAenhanced pero... more A specific DNA oligonucleotide-hemin complex (PS2.M-hemin complex) that exhibits DNAenhanced peroxidative activity was studied by EPR and UV-visible spectroscopy and by chemical probing analysis. EPR data obtained from low-temperature experiments on the PS2.M-hemin complex showed both a low-field g ∼6 and a high-field g ∼2 signal. These EPR signals are typical of high-spin ferric heme with axial symmetry as judged by the EPR spectrum of six-coordinate heme iron in acidic Fe(III)-myoglobin. This similarity is consistent with the presence of two axial ligands to the heme iron within the PS2.M-hemin complex, one of which is a water molecule. Optical analyses of the acid-base transition for the hemin complex yielded a pK a value for the water ligand of 8.70 (0.03 (mean (SD). Low-temperature EPR analysis coupled with parallel spin-trapping investigations following the reaction of the PS2.M-hemin complex and hydrogen peroxide (H 2 O 2) indicated the formation of a carbon-centered radical, most likely on the PS2.M oligonucleotide. Chemical probing analysis identified specific guanine bases within the PS2.M sequence that underwent oxidative damage upon reaction with H 2 O 2. These and other experimental findings support the hypothesis that the interaction of specific guanines of PS2.M with the bound hemin cofactor might contribute to the superior peroxidative activity of the PS2.M-hemin complex.

Research paper thumbnail of A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites

Chemistry & Biology, 1999

Background: An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was ... more Background: An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporphyrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-enhanced peroxidative activity. PS2.M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guanine-quaduplex. In investigating the specific catalytic features of PS2.M we tested the peroxidative properties of its RNA version (rPS2.M) as well as that of an unrelated DNA guanine-quadruplex, OXY4. Results: The hemin-binding affinity of rPS2.M was found to be 30-fold weaker than that of PS2.M. The UV-visible spectra and kinetics of enzymatic peroxidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially greater than those of heme proteins such as catalase and Fe(III)-myoglobin. Kinetic analysis suggested that PS2.M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M) had a distinct absorption spectrum and only a minor peroxidase activity above the background level. Conclusions: The results indicated that it is possible for RNA and DNA of the same sequence to fold to form comparable cofactor-binding sites, and to show comparable catalytic behavior. The results further suggest that only a subset of cofactor-binding sites formed within folded nucleic acids might be able to function as active sites, by providing the appropriate chemical environments for catalysis.

Research paper thumbnail of DNA and RNA enzymes with peroxidase activity — An investigation into the mechanism of action

Canadian Journal of Chemistry, 2006

A DNA–hemin complex (PS2.M–hemin), and its RNA counterpart (rPS2.M–hemin), have previously been r... more A DNA–hemin complex (PS2.M–hemin), and its RNA counterpart (rPS2.M–hemin), have previously been reported, in the presence of nitrogenous buffers such as HEPES, to show enhanced peroxidative activity relative to both uncomplexed hemin and a control DNA–hemin complex (Chem. Biol. 5, 505, 1998). A kinetic analysis of these two hemin-utilizing nucleic acid enzymes provides key insights into the mechanisms for their catalyzed peroxidation reactions. First, control experiments indicate that charge on the added detergent, required for solubility reasons, has little effect on the efficiency of the nucleic-acid-catalyzed reactions. Second, the key functional impact of the two nucleic acid frameworks, either DNA or RNA, appears to be a reduction in the acidity of a water molecule coordinated to the iron atom of the hemin that is bound to the ribozyme and DNAzyme scaffolds. This effect could result from a polar environment and possibly hydrogen bond(s) at the axial position of the hemin, along...

Research paper thumbnail of Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: The penicillin G acylase as enzyme model

Biotechnology and Bioengineering, 2002

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, ... more A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzymebinding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3°C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed.

Research paper thumbnail of A Heme–Peptide Metalloenzyme Mimetic with Natural Peroxidase‐Like Activity

Chemistry – A European Journal, 2011

Mimicking enzymes with alternative molecules represents an important objective in synthetic biolo... more Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity. Herein, we describe the spectroscopic and functional characterization of Fe(III)-mimochrome VI, a 3.5 kDa synthetic heme-protein model, which displays a peroxidase-like catalytic activity. By the use of hydrogen peroxide, Fe(III)-mimochrome VI efficiently catalyzes the oxidation of several substrates, with a typical Michaelis-Menten mechanism and with several multiple turnovers. The catalytic efficiency of Fe(III)-mimochrome VI in the oxidation of 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and guaiacol (k(cat)/K(m)=4417 and 870 mM(-1) s(-1), respectively) is comparable to that of native horseradish peroxidase (HRP, k(cat)/K(m)=5125 and 500 mM(-1) s(-1), respectively). Fe(III)-mimochrome VI also converts phenol to 4- and 2-nitrophenol in the presence of NO(2) (-) and H(2) O(2) in high yields. These results demonstrate that small synthetic peptides can impart high enzyme activities to metal cofactors, and anticipate the possibility of constructing new biocatalysts tailored to specific functions.

Research paper thumbnail of DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex

Chemistry & Biology, 1998

A new class of catalytic activity for nucleic acids is reported. The aptamer-hemin complexes desc... more A new class of catalytic activity for nucleic acids is reported. The aptamer-hemin complexes described are novel DNA enzymes and their study will help elucidate the structural and functional requirements of peroxidase enzymes in general and the ways that a nucleic acid 'apoenzyme' might work to enhance the intrinsic peroxidatic ability of hemin. These aptamerhemin complexes could be regarded as prototypes for redox-catalyzing ribozymes in a primordial 'RNA world'.

Research paper thumbnail of Enzyme Reaction Engineering: Effect of Methanol on the Synthesis of Antibiotics Catalyzed by Immobilized Penicillin G Acylase under Isothermal and Non-Isothermal Conditions

Biotechnology progress (Print), Oct 4, 2002

Research paper thumbnail of An amperometric sensor employing glucose oxidase immobilized on nylon membranes with different pore diameter and grafted with different monomers

Journal of Molecular Catalysis B-enzymatic, Sep 1, 2002

Glucose oxidase (GOD) was immobilized on nylon membranes having three different pore diameters an... more Glucose oxidase (GOD) was immobilized on nylon membranes having three different pore diameters and chemically grafted with glycidyl methacrylate (GMA) or butyl methacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GA) were used as spacer and coupling agent, respectively. The biochemical and electrochemical behaviour of the membranes has been studied as a function of pH, temperature and glucose concentration with reference to the grafted monomer and the membrane pore diameter. The behaviour of the soluble GOD has also been studied in order to see the modification induced by the immobilization process on the enzyme activity. It was found that the values of the biosensor sensitivity, maximum saturation current and electrochemical affinity increase with the membrane pore diameter, indipendently of the nature of the graft monomer. Opposite behaviour was found relatively to the extension of the linear response ranges and the average response times. With reference to the parameters increasing with the pore diameter it was found that membranes grafted with GMA had higher values than those of the membranes grafted with BMA. The contrary occurred to the values of the parameters decreasing with the increase of the pore diameter. Biochemical and electrochemical results have been discussed in terms of the different limitations to the diffusion of substrate and reaction products across the catalytic membrane introduced by the different pore diameters and by the different hydrophobicity of the graft monomers.

Research paper thumbnail of A DNA Oligonucleotide−Hemin Complex Cleaves t-Butyl Hydroperoxide through a Homolytic Mechanism

Inorganic Chemistry, 2001

Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been emplo... more Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex (∼35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of ∼250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of e5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from-scission of the primary tert-butyloxyl radical product.

Research paper thumbnail of The Peroxidase Activity of a Hemin−DNA Oligonucleotide Complex:  Free Radical Damage to Specific Guanine Bases of the DNA

Journal of the American Chemical Society, 2001

A specific DNA oligonucleotide-hemin complex (PS2.M-hemin complex) that exhibits DNAenhanced pero... more A specific DNA oligonucleotide-hemin complex (PS2.M-hemin complex) that exhibits DNAenhanced peroxidative activity was studied by EPR and UV-visible spectroscopy and by chemical probing analysis. EPR data obtained from low-temperature experiments on the PS2.M-hemin complex showed both a low-field g ∼6 and a high-field g ∼2 signal. These EPR signals are typical of high-spin ferric heme with axial symmetry as judged by the EPR spectrum of six-coordinate heme iron in acidic Fe(III)-myoglobin. This similarity is consistent with the presence of two axial ligands to the heme iron within the PS2.M-hemin complex, one of which is a water molecule. Optical analyses of the acid-base transition for the hemin complex yielded a pK a value for the water ligand of 8.70 (0.03 (mean (SD). Low-temperature EPR analysis coupled with parallel spin-trapping investigations following the reaction of the PS2.M-hemin complex and hydrogen peroxide (H 2 O 2) indicated the formation of a carbon-centered radical, most likely on the PS2.M oligonucleotide. Chemical probing analysis identified specific guanine bases within the PS2.M sequence that underwent oxidative damage upon reaction with H 2 O 2. These and other experimental findings support the hypothesis that the interaction of specific guanines of PS2.M with the bound hemin cofactor might contribute to the superior peroxidative activity of the PS2.M-hemin complex.

Research paper thumbnail of A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites

Chemistry & Biology, 1999

Background: An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was ... more Background: An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporphyrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-enhanced peroxidative activity. PS2.M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guanine-quaduplex. In investigating the specific catalytic features of PS2.M we tested the peroxidative properties of its RNA version (rPS2.M) as well as that of an unrelated DNA guanine-quadruplex, OXY4. Results: The hemin-binding affinity of rPS2.M was found to be 30-fold weaker than that of PS2.M. The UV-visible spectra and kinetics of enzymatic peroxidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially greater than those of heme proteins such as catalase and Fe(III)-myoglobin. Kinetic analysis suggested that PS2.M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M) had a distinct absorption spectrum and only a minor peroxidase activity above the background level. Conclusions: The results indicated that it is possible for RNA and DNA of the same sequence to fold to form comparable cofactor-binding sites, and to show comparable catalytic behavior. The results further suggest that only a subset of cofactor-binding sites formed within folded nucleic acids might be able to function as active sites, by providing the appropriate chemical environments for catalysis.

Research paper thumbnail of DNA and RNA enzymes with peroxidase activity — An investigation into the mechanism of action

Canadian Journal of Chemistry, 2006

A DNA–hemin complex (PS2.M–hemin), and its RNA counterpart (rPS2.M–hemin), have previously been r... more A DNA–hemin complex (PS2.M–hemin), and its RNA counterpart (rPS2.M–hemin), have previously been reported, in the presence of nitrogenous buffers such as HEPES, to show enhanced peroxidative activity relative to both uncomplexed hemin and a control DNA–hemin complex (Chem. Biol. 5, 505, 1998). A kinetic analysis of these two hemin-utilizing nucleic acid enzymes provides key insights into the mechanisms for their catalyzed peroxidation reactions. First, control experiments indicate that charge on the added detergent, required for solubility reasons, has little effect on the efficiency of the nucleic-acid-catalyzed reactions. Second, the key functional impact of the two nucleic acid frameworks, either DNA or RNA, appears to be a reduction in the acidity of a water molecule coordinated to the iron atom of the hemin that is bound to the ribozyme and DNAzyme scaffolds. This effect could result from a polar environment and possibly hydrogen bond(s) at the axial position of the hemin, along...

Research paper thumbnail of Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: The penicillin G acylase as enzyme model

Biotechnology and Bioengineering, 2002

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, ... more A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzymebinding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3°C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed.

Research paper thumbnail of A Heme–Peptide Metalloenzyme Mimetic with Natural Peroxidase‐Like Activity

Chemistry – A European Journal, 2011

Mimicking enzymes with alternative molecules represents an important objective in synthetic biolo... more Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity. Herein, we describe the spectroscopic and functional characterization of Fe(III)-mimochrome VI, a 3.5 kDa synthetic heme-protein model, which displays a peroxidase-like catalytic activity. By the use of hydrogen peroxide, Fe(III)-mimochrome VI efficiently catalyzes the oxidation of several substrates, with a typical Michaelis-Menten mechanism and with several multiple turnovers. The catalytic efficiency of Fe(III)-mimochrome VI in the oxidation of 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and guaiacol (k(cat)/K(m)=4417 and 870 mM(-1) s(-1), respectively) is comparable to that of native horseradish peroxidase (HRP, k(cat)/K(m)=5125 and 500 mM(-1) s(-1), respectively). Fe(III)-mimochrome VI also converts phenol to 4- and 2-nitrophenol in the presence of NO(2) (-) and H(2) O(2) in high yields. These results demonstrate that small synthetic peptides can impart high enzyme activities to metal cofactors, and anticipate the possibility of constructing new biocatalysts tailored to specific functions.

Research paper thumbnail of DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex

Chemistry & Biology, 1998

A new class of catalytic activity for nucleic acids is reported. The aptamer-hemin complexes desc... more A new class of catalytic activity for nucleic acids is reported. The aptamer-hemin complexes described are novel DNA enzymes and their study will help elucidate the structural and functional requirements of peroxidase enzymes in general and the ways that a nucleic acid 'apoenzyme' might work to enhance the intrinsic peroxidatic ability of hemin. These aptamerhemin complexes could be regarded as prototypes for redox-catalyzing ribozymes in a primordial 'RNA world'.

Research paper thumbnail of Enzyme Reaction Engineering: Effect of Methanol on the Synthesis of Antibiotics Catalyzed by Immobilized Penicillin G Acylase under Isothermal and Non-Isothermal Conditions

Biotechnology progress (Print), Oct 4, 2002