Substrate-inhibition

The dehaloperoxidase-hemoglobin (DHP) from the terebellid polychaete Amphitrite ornata is a multifunctional hemoprotein that catalyzes the oxidation of a wide variety of substrates, including halo-/nitro-phenols, haloindoles, and pyrroles, via peroxidase and/or peroxygenase mechanisms. To probe whether substrate substituent effects can modulate enzyme activity in DHP, we investigated its reactivity against a panel of o-guaiacol substrates given their presence (from native/halogenated and non-native/anthropogenic sources) in the benthic environment that A. ornata inhabits. Using biochemical assays supported by spectroscopic, spectrometric, and structural studies, DHP was found to catalyze the H2O2-dependent oxidative dehalogenation of 4-haloguaiacols (F, Cl, Br) to 2-methoxybenzoquinone (2-MeOBQ). 18O-labeling studies confirmed that O-atom incorporation was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The 2-MeOBQ product furthe...

Oligopeptidase B (OPB; EC 3.4.21.83) from 2 Gram-negative bacteria, Stenotrophomonas maltophilia (Stm) and Serratia marcescens (Sem), and the Gram-positive bacterium Rhodococcus erythropolis (Re) were cloned and characterized to clarify their activities and substrate specificities using peptidyl-MCA substrates containing Arg or Lys. The cloned enzymes, Stm, Sem and ReOPBs, in addition to Escherichia coli OPB (EcOPB) were expressed using a pET expression system. Although the Stm and SemOPBs share 45% sequence identity to each other and up to 60% identity with respect to their catalytic domains, their activities towards MCA substrates were quite different. StmOPB is approximately 100-500 times more active than SemOPB and 3-30 times more active than EcOPB. The activity of ReOPB is comparable to that of StmOPB and it shares 40% and 36% identity to StmOPB and SemOPB, respectively. Some features of Stm, Re and EcOPBs are similar to those of previously cloned OPBs, which were also strongly inhibited by substrates, but SemOPB differs from all other OPBs in that it is not inhibited by substrates; even substrates containing double arginine at 35 µM did not inhibit SemOPB. On the other hand, the same substrates at only 5 µM inhibited the activity of the Stm, Re, and EcOPB. This phenomenon was not observed with substrates containing single or double lysine.

Fatty-acyl-glutamate (FA-Glu), a surfactin variant has been successfully produced using a genetically modified strain of Bacillus subtilis grown on glucose. However, yields with soybean hulls (SBH) replacing glucose were lower. This work was undertaken to reduce the yield loss when using SBH as the carbon source and to evaluate two other soy by-products, namely fiber and skim from aqueous oil extraction as alternative carbon and nitrogen sources. Fermentation of soybean hulls, fibers and skim at various concentrations produced lower FA-Glu titers compared to S-7 medium. Neither increasing their amount nor supplementing with glucose increased the FA-Glu titer, suggesting the presence of an inhibitor in these feedstocks. By using a mixture of polysaccharidedegrading enzymes, over 65% of SBH solids were converted to soluble carbohydrates. FA-Glu titers obtained from SBH hydrolysates containing residual hull solids were still low; however, with the removal of the solids, cell growth improved and FA-Glu yield was 60% higher than with glucose. Thus, this low-cost material can be converted to a substrate for production of FA-Glu biosurfactant. Unmodified fiber and skim components of aqueous oil extraction were not beneficial.

The oxidation of D-sorbitol and D-dulcitol by potassium permanganate in alkaline medium showed substrate inhibition for both substrates due to the formation of 1:1 manganese-sugar alcohol complex which resists oxidation. Consequently, the reaction was inverse first order with respect to sugar alcohol for both substrates, first order each with respect to [KMnO4], [OH-] and independent of ionic strength for both substrates investigated. Activation parameters were obtained and a plausible mechanism was proposed from kinetic and spectroscopic studies.

Ruthenium(III) catalyzed oxidation of propane-1,3-diol by potassium periodate was studied in aqueous perchloric acid medium. Orders of reaction with respect to concentrations of oxidant, substrate, acid and catalyst were determined. First order in oxidant and catalyst concentrations, and inverse fractional order in acid medium were observed. In addition, substrate inhibition (i.e. a decrease in reaction rate with an increase in substrate concentration) was observed. Effect of addition of salt and solvent was studied. Based on the studies of temperature variation, Arrhenius parameters were calculated. Plausible mechanism was also proposed based on observed kinetics.

We analyse the steady-state operation of a continuous flow bioreactor in which the biochemical reaction is governed by noncompetitive substrate inhibition (Andrews kinetics). A generalized reactor model is used in which the well-stirred bioreactor and the idealized membrane bioreactor are special cases. As generic properties of systems subject to substrate inhibition have been obtained by other authors, we discuss reaction engineering features specific to Andrews kinetics.

The dehaloperoxidase-hemoglobin (DHP) from the terebellid polychaete Amphitrite ornata is a multifunctional hemoprotein that catalyzes the oxidation of a wide variety of substrates, including halo-/nitro-phenols, haloindoles, and pyrroles, via peroxidase and/or peroxygenase mechanisms. To probe whether substrate substituent effects can modulate enzyme activity in DHP, we investigated its reactivity against a panel of o-guaiacol substrates given their presence (from native/halogenated and non-native/anthropogenic sources) in the benthic environment that A. ornata inhabits. Using biochemical assays supported by spectroscopic, spectrometric, and structural studies, DHP was found to catalyze the H2O2-dependent oxidative dehalogenation of 4-haloguaiacols (F, Cl, Br) to 2-methoxybenzoquinone (2-MeOBQ). 18O-labeling studies confirmed that O-atom incorporation was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The 2-MeOBQ product furthe...

The kinetics of oxidation of Fe(CN)z-by periodate in acetate buffers follow the rate law (i): dfFe(CN)z-]/dt = k,&Fe(CN);f-1 (i) in large excess of periodate concentrations, where k obs = 0.20 + 2.60 X 10-3/[Periodate]. Copper(U) but not Fe(U) ions catalyse the reaction. The inhibiting effect of periodate is believed to be due to the formation of a Cu(III)-periodate complex which is less reactive than a pen'odate free Cu(III) species. The rate of the reaction is greatly lowered by EDTA and in its presence the kinetics obey the rate law (ii): d[Fe(CN)z-]/2dt = (a + b[Na'] + k,fH'])-[Fe(CN)z-J[Periodate/ (ii) where a = 0.22 M-' set-', b = 0.66 Mm2 set-' and k3 = 5.5 X 104 Mb2 set-' at 25 'C. Mechanisms consistent with kinetics in absence and in presence of EDTA have been proposed.

The kinetic studies of N-chlorosaccharin (NCSA) oxidation of propan-1,3-diol and butan-1,4-diol have been reported in presence of phophotungstic acid and in aqueous acetic acid medium. The reactions follow first-order in NCSA and one to zero order with respect to substrate and phosphotungstic acid. Increase in the concentration of added perchloric acid increases the rate of oxidation. A negative effect on the oxidation rate is observed for solvent whereas the ionic strength does not influence the rate of reaction. Addition of the reaction product, saccharin, exhibited retarding effect. Various activation parameters have been evaluated. The products of the reactions were identified as the corresponding aldehydes. A suitable scheme of mechanism consistent with the experimental results has been proposed.

CAREY, LEIAH MARIE. Probing the Structure-Function Relationship of a Multifunctional Enzyme using Crystallographic Diffraction Methods. (Under the direction of Dr. Reza Ghiladi.) The marine annelid Amphitrite ornata possesses the ability to chemically detoxify deleterious aromatic compounds prevalent in its environment, which is afforded by its coelomic hemoglobin, Dehaloperoxidase (DHP). In addition to its oxygen transport function, DHP also possesses peroxidase, peroxygenase, oxidase and oxygenase enzymatic functions, resulting in 5 activities that coexist at a single heme reactive center. Structurally, DHP possesses the canonical -helical globin fold associated with oxygen transport, yet lacks the structural homology with archetypical monofunctional examples of which it shares enzymatic reactivity, such as horseradish peroxidase, AaeAPO, cytochrome c oxidase and cytochrome

Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron...

The N-methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N-methylation by nicotinamide N-methyltransferase (NNMT) and its effect upon 4-phenylpyridine toxicity in vitro. Human recombinant NNMT possessed 4-phenylpyridine N-methyltransferase activity, with a specific activity of 1.7 ± 0.03 nmol MPP+ produced/h/mg NNMT. Although the K for 4-phenylpyridine was similar to that reported for nicotinamide, its k of 9.3 × 10 ± 2 × 10 s and specificity constant, k/K, of 0.8 ± 0.8 s M were less than 0.15% of the respective values for nicotinamide, demonstrating that 4-phenylpyridine is a poor substrate for NNMT. At low (<2.5 mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a K of 34 ± 8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a K of 4 ± 1 mM. In silico molecular ...

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.

Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. The absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional "hole" into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members: up to 32 times increased substrate inhibition and 17 times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to those of the native enzyme and the most proficient haloalkane dehalogenase reported to date (k cat = 57 s −1 with 1,2-dibromoethane at 37°C and pH 8.6). Crystallographic analysis and molecular dynamics simulations confirmed the successful introduction of a structurally defined and functional transport tunnel. Our study demonstrates that, whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels, and we anticipate that this engineering strategy will facilitate the creation of a wide range of useful biocatalysts.

of the distal histidine is due to the stability of the final bound state rather than an intrinsic bond strength difference. The implications of increased dynamics on the distal side of the pocket after ligand binding will be discussed in terms of ligand migration inside GlbN and related globins.

The simultaneous effects of temperature and pH on the biokinetic properties of thiocyanate biodegradation under mixed-culture, autotrophic conditions were investigated using response surface analysis (RSA) combined with biokinetic modeling. A partial cubic model, based on substrate inhibition biokinetics, was constructed for each kinetic coefficient in Andrew model (i.e., maximum specific growth rate (m m), saturation coefficient (K S), and substrate inhibition coefficient (K SI)). Each model proved statistically reliable to approximate the responses of the kinetic coefficients to temperature and pH changes (r 2 > 0.8, p < 0.05). The response surface plots demonstrated that the biokinetic coefficients change with respect to temperature and pH significantly and in different ways. The model response surfaces were substantially different to each other, indicating distinct correlations between the independent (temperature and pH) and dependent (model response) variables in the models. Based on the estimated response surface models, temperature was shown to have significant effects on all biokinetic coefficients tested. A dominant influence of temperature on m m response was observed while the interdependence of temperature and pH was apparent in the K S and K SI models. Specific growth rate (m) versus substrate (i.e., thiocyanate) concentration plots simulating using the obtained response surface models confirmed the significant effects of temperature and pH on the microbial growth rate and therefore on the thiocyanate degradation rate. Overall, the response surface models able to describe the biokinetic effects of temperature and pH on thiocyanate biodegradation within the explored region (20e30 C and pH 6.0e9.0) were successfully constructed and validated, providing fundamental information for better process control in thiocyanate treatment.

We analyse the steady-state operation of a continuous flow bioreactor in which the biochemical reaction is governed by noncompetitive substrate inhibition (Andrews kinetics). A generalized reactor model is used in which the well-stirred bioreactor and the idealized membrane bioreactor are special cases. As generic properties of systems subject to substrate inhibition have been obtained by other authors, we discuss reaction engineering features specific to Andrews kinetics.

determining the stability and rigidity of proteins, shifting the pKa values of buried ionizable residues, and modulating dynamical processes such as folding, catalysis, and proton transfers. Detecting these internal water molecules is sometimes obscured in x-ray crystallography due to positional disorder. We have developed a spectrokinetic assay that accurately detects the presence of a non-coordinated water molecule in the distal heme pocket of myoglobin and in a series of distal pocket mutants, including many where this water molecule is positionally disordered. We also have shown that this water plays a major role in determining the observed bimolecular recombination rate constant. We show that 1) this water molecule modulates the ligand binding dynamics of a series of H64, L29 and V68 mutants; 2) it plays the major role in the observed pH dependence of the CO recombination kinetics between pH 4 and 7 with the protonation of the distal histidine acting as a switch to change water occupancy; and 3) it may also modulate ligand binding dynamics in isolated hemoglobin chains, with the occupancy being larger in the alpha chains. Accurately measuring water occupancy in heme proteins answers crucial questions about water in apolar or slightly polar protein cavities and clarifies the role internal water molecules play in modulating protein function.

Tyrosinemia type 1 (TT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Neurocognitive deficiencies have been described in TT1 patients, that have, among others, been related to changes in plasma large neutral amino acids (LNAA) that could result in changes in brain LNAA and neurotransmitter concentrations. Therefore, this project aimed to investigate plasma and brain LNAA, brain neurotransmitter concentrations and behavior in C57 Bl/6 fumarylacetoacetate hydrolase deficient (FAH−/−) mice treated with 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and/or diet and wild-type mice. Plasma and brain tyrosine concentrations were clearly increased in all NTBC treated animals, even with diet (p &lt; 0.001). Plasma and brain phenylalanine concentrations tended to be lower in all FAH−/− mice. Other brain LNAA, were often slightly lower in NTBC treated FAH−/− mice. Brain neurotransmitter concentrations were usually within a normal range, a...

The N-methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N-methylation by nicotinamide N-methyltransferase (NNMT) and its effect upon 4-phenylpyridine toxicity in vitro. Human recombinant NNMT possessed 4-phenylpyridine N-methyltransferase activity, with a specific activity of 1.7 ± 0.03 nmol MPP+ produced/h/mg NNMT. Although the K for 4-phenylpyridine was similar to that reported for nicotinamide, its k of 9.3 × 10 ± 2 × 10 s and specificity constant, k/K, of 0.8 ± 0.8 s M were less than 0.15% of the respective values for nicotinamide, demonstrating that 4-phenylpyridine is a poor substrate for NNMT. At low (&lt;2.5 mM) substrate concentration, 4-phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a K of 34 ± 8 mM. At high (&gt;2.5 mM) substrate concentration, enzyme activity followed substrate inhibition kinetics, with a K of 4 ± 1 mM. In silico molecular ...

Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. The absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional "hole" into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members: up to 32 times increased substrate inhibition and 17 times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to those of the native enzyme and the most proficient haloalkane dehalogenase reported to date (k cat = 57 s −1 with 1,2-dibromoethane at 37°C and pH 8.6). Crystallographic analysis and molecular dynamics simulations confirmed the successful introduction of a structurally defined and functional transport tunnel. Our study demonstrates that, whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels, and we anticipate that this engineering strategy will facilitate the creation of a wide range of useful biocatalysts.

of the distal histidine is due to the stability of the final bound state rather than an intrinsic bond strength difference. The implications of increased dynamics on the distal side of the pocket after ligand binding will be discussed in terms of ligand migration inside GlbN and related globins.

Using Resonance Raman spectroscopy and activity assays we have determined that there are potent inhibitors of DHP activity and these inhibitors play a major role in the biology of DHP. Resonance Raman spectroscopy can detect substrate or inhibitor binding by monitoring shifts in the core size marker modes, n 2 and n 3 , and the vinyl substituent mode, n C¼C , of the heme. Para-halogenated phenols bind internally, displacing the sixth position water molecule of the iron (in agreement with X-ray crystallography) resulting in a 5-coordinate high spin heme. Tri-halogenated phenols bind externally, forcing the distal histidine into

Highlights  BMP of citrus waste (peel, pulp and rotten fruit) was 354-398 L•kgVS-1.  Grinding the citrus waste did not improve BMP but slowed the kinetics.  IC50 of limonene was 423 mg•kg-1 in batch anaerobic digestion of cellulose.  Inhibition of anaerobic digestion process by limonene was reversible.  IC50 value increased to 669 mg•kg-1 for a second load of limonene, indicating that some biomass adaptation may occur.

The SHARON process allows partial nitrification of wastewaters with high ammonium content and, when coupled with the Anammox process, is a more sustainable alternative for N-removal than a conventional nitrification-denitrification. A mathematical model describing a continuously aerated SHARON reactor is presented. Haldane kinetics is used in both steps of the nitrification, since ammonium-oxidizing and nitrite-oxidizing organisms are inhibited by their own substrates. Special attention is given to pH because it is a key factor, implementing an algorithm for its calculation. A preliminary evaluation of the model using synthetic feed is presented.

obtained with respect to each the diol and hydrogen ion. The oxidation of [1,1,2,2- 2 H4]ethanediol exhibited primary kinetic isotope effect ( kH/kD = 6.61 at 298 K). The temperature dependence of the kinetic isotope effect suggested the symmetrical transition state in the rate-determining step. The rate constants of oxidation of four vicinal diols show excellent correlation with Taft’s Σ σ * values with negative reaction constant, ρ * . The rate of oxidation of ethanediol has been determined in nineteen different solvents. An analysis of the solvent effect indicates the importance of the cation-solvating power of the solvents. A suitable mechanism has been postulated involving the formation of chromate ester in a pre-equilibrium.

Kinetics of oxidation of four vicinal, four non-vicinal diols and two of their monoethers by quinolinium bromochromate (QBC) have been studied in dimethyl sulphoxide (DMSO). The main product of oxidation is the corresponding hydroxycarbonyl compound. The reaction is first order each in QBC and the diols. The reaction is catalysed by hydrogen ions. The hydrogen ion dependence is taking the form : k obs = a + b[H+]. The oxidation of [1,1 ,2,2-2H4] ethanediol exhibits a substantial primary kinetic isotope effect (kH/kD = 5.83 at 298 K). The reaction has been studied in nineteen different organic solvents and the solvent effect has been analysed using Taft's and Swain's multiparametric equations. The temperature dependence of the kinetic isotope effect indicates the presence of a symmetrical transition state in the rate-determining step. A suitable mechanism has been proposed .

The oxidation of D-sorbitol and D-dulcitol by potassium permanganate in alkaline medium showed substrate inhibition for both substrates due to the formation of 1:1 manganese-sugar alcohol complex which resists oxidation. Consequently, the reaction was inverse first order with respect to sugar alcohol for both substrates, first order each with respect to [KMnO4], [OH-] and independent of ionic strength for both substrates investigated. Activation parameters were obtained and a plausible mechanism was proposed from kinetic and spectroscopic studies.

Oxidation of alcohols has industrial importance as it yields several useful products.Toxic and costly metal ions like Os(VIII), Cr(VI), and Ru in different oxidation states are widely used for the oxidation of a variety of organic compounds. We are reporting herein the oxidation of the industrially useful primary alcohols, 2-Chloroethanol, 2-Butoxyethanol and 2-Phenoxyethanol using Ammonium metavanadate in acidic medium. Relatively less toxic and cheaper transition metal ions of the first series are effectively used as homogeneous catalysts for the oxidation of the alcohols to the corresponding aldehydes. 2- Chloroethanol is used as a precursor for ethylene oxide and is useful in the manufacture of crop protection chemicals, and pharmaceuticals.2-Butoxyethanol finds use in the making of paints, varnishes and industrial and household cleaners.

The oxidation of propane-1,3-diol by potassium permanganate in aqueous solution was investigated at λmax 525 nm. The rate of the reaction was found to increase with increase in [KMnO4] and [Propane-1,3-diol]. The reaction showed first order dependence each in [KMnO4] and [Propane-1,3-diol] and independent on the ionic strength of the solution. The activation parameters were evaluated from Arrhenius and Erying's equations and the values of〖∆H〗 #(kJ mol-1), 〖∆S〗^#(kJK-1 mol-1) and〖∆G〗^#(kJmol-1) were 24.98,-0.22 and 90.50 respectively. Negative entropy of activation revealed an association mechanism and an ordered transition state for the reaction. Stoichiometric study showed 1:1 consumption of KMnO4 and propane-1,3-diol. Spectroscopic studies and FTIR analysis revealed the product of the reaction to be 3-hydroxyl-propanal. A suitable reaction mechanism was proposed for the reaction investigated.

In the oxidation of D sorbitol and D mannitol by potassium periodate in alkaline media, sub strate inhibition was observed with both substrates, i.e., a decrease in the rate of the reaction was observed with an increase in the concentration of substrate. The substrate inhibition was attributed to the formation of stable complex between the substrate and periodate. The reactions were found to be first order in case of periodate and a positive fractional order with hydroxide ions. Arrhenius parameters were calculated for the oxidation of sorbitol and mannitol by potassium periodate in alkali media.

The comparative kinetics and mechanism of oxidation of maltose and lactose by copper (II) and hexacyanoferrate (III) ion has been studied in alkaline medium. The rate of reaction was monitored spectrophotometrically under a wide range of experimental conditions. For both oxidants, the reaction was found to be zero and first order in oxidant and sugar concentration respectively. The rate of reaction shows no significant dependence on the ionic strength of the medium. The values of ΔG # revealed that both oxidants follow the same mechanistic pathway. The mechanism involves the formation of enediol intermediate. From rate equation, the rate of reaction was found to be proportional to k[sugar] [OH -],where k is the rate constants for the reaction. Arrhenius activation energy and other thermodynamic parameters were also evaluated and reported.

of the distal histidine is due to the stability of the final bound state rather than an intrinsic bond strength difference. The implications of increased dynamics on the distal side of the pocket after ligand binding will be discussed in terms of ligand migration inside GlbN and related globins.

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediate...

The oxidation of propane-1,3-diol by potassium permanganate in aqueous solution was investigated at λmax 525 nm. The rate of the reaction was found to increase with increase in [KMnO4] and [Propane-1,3-diol]. The reaction showed first order dependence each in [KMnO4] and [Propane-1,3-diol] and independent in the ionic strength of the solution. The values of 〖∆H〗^#(kJ mol-1), 〖∆S〗^# (kJK-1mol-1) and〖 ∆G〗^#(kJmol-1) were 24.98, -0.22 and 90.50 respectively. Negative entropy of activation revealed an ordered transition state for the reaction. Spectroscopic studies and FTIR analysis revealed the product of the reaction to be 3-hydroxyl-propanal.

The oxidation of propane-1, 3-diol by potassium permanganate in aqueous solution was investigated at λmax 525 nm. The rate of the reaction was found to increase with increase in [KMnO4] and [Propane-1, 3-diol]. The reaction showed first order dependence each in [KMnO4] and [Propane-1, 3-diol] and independent in the ionic strength of the solution. The values of ∆ # (kJ mol-1), ∆ # (kJK-1 mol-1) ∆ # (kJ mol-1) were 24.98,-0.22 and 90.50 respectively. Negative activation of entropy revealed an ordered transition state for the reaction. Spectroscopic studies and FTIR analysis revealed the product of the reaction to be 3-hydroxy-propanone.

The oxidation of D-sorbitol and D-dulcitol by potassium permanganate in alkaline medium showed substrate inhibition for both substrates due to the formation of 1:1 manganese-sugar alcohol complex which resists oxidation. Consequently, the reaction was inverse first order with respect to sugar alcohol for both substrates, first order each with respect to [KMnO4], [OH-] and independent of ionic strength for both substrates investigated. Activation parameters were obtained and a plausible mechanism was proposed from kinetic and spectroscopic studies.

A structured model is presented giving a mathematical description of batch cultures ofAlcaligenes eutrophus strain H 16 under chemolithoantotrophic growth conditions. A mass-spectrometer with a membrane inlet system was used to measure and control the concentrations of the dissolved gaseous substrates H2, 02 and CO 2. Growth and storage of PHB (Poly-/~-hydroxybutyric acid) H + are described as a function of the limiting substrate S (N 4), the residual biomass R, and the product P (PHB), Model parameters were evaluated by regression analysis followed by linearization. The differential equation model was solved numerically and compared with experimental data.

Polarographic oxygen electrodes, a mass spectrometer with a membrane inlet system, and a redox electrode were used to measure dissolved H2, 02, and CO 2 continuously during chemolithoautotrophic cultivation of Alcaligenes eutrophus H 16. A mass spectrometer is a versatile instrument for measuring dissolved gases. Its dynamic characteristics are comparable to conventional sterilizable oxygen electrodes. A redox electrode combined with an oxygen electrode is a simple but somewhat limited sensor to measure dissolved H 2.

Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutropbus HI6 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly-13-hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate proportional to the specific growth rates of total biomass. After complete consumption of ammonium, the increase of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass (cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutropbus, strain H 16, high concentrations of dissolved oxygen strongly influence growth as well as PHB synthesis.

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediate...

The biochemical route is identified to be one of the simplest and cheapest means by which valuable chemicals are being synthesized. Microorganisms play a vital role in carrying out these processes. However, the kinetic studies relevant to this process is scarce. Most often inhibition effects due to either cells or substrates or products affect the performance of such processes. This paper deals with the study of various model equations for substrate inhibition kinetics. An attempt has been made to study the applicability of various model equations for substrate inhibited systems by fitting their experimental data and evaluating various parameters including the standard deviations in each case. Finally, a new model has been brought out which gives the best fit for almost all the systems.

A novel copolymer system has been synthesized using methyl hydroquinone diacetate (MHQDA), 1,4 naphthalene dicarboxylic acid (1,4 NDCA), and polybutylene terephthalate (PBT) using the melt polymerization technique. The optimum kinetic parameters pertinent to this system are reported in this study. A simple second-order reaction sequence summarizes catalyzed and uncatalyzed reactions between MHQDA, 1,4 NDCA, and PBT. The kinetic parameters for the copolymerization reaction were determined using a new second-order model. This technique was used to compute the moles of acetic acid generated in the polymerization process. The model was compared to that of the experimentally determined data. Close comparison (percentage error of less than 5%) was obtained between the experimental and theoretical data. The kinetic data support block copolyester formation. The thermal data also supports formation of block polymers.

A novel copolymer system has been synthesized using methyl hydroquinone diacetate (MHQDA), 1,4 naphthalene dicarboxylic acid (1,4 NDCA), and polybutylene terephthalate (PBT) using the melt polymerization technique. The optimum kinetic parameters pertinent to this system are reported in this study. A simple second-order reaction sequence summarizes catalyzed and uncatalyzed reactions between MHQDA, 1,4 NDCA, and PBT. The kinetic parameters for the copolymerization reaction were determined using a new second-order model. This technique was used to compute the moles of acetic acid generated in the polymerization process. The model was compared to that of the experimentally determined data. Close comparison (percentage error of less than 5%) was obtained between the experimental and theoretical data. The kinetic data support block copolyester formation. The thermal data also supports formation of block polymers.

Oxidation of alcohols has industrial importance as it yields several useful products.Toxic and costly metal ions like Os(VIII), Cr(VI), and Ru in different oxidation states are widely used for the oxidation of a variety of organic compounds. We are reporting herein the oxidation of the industrially useful primary alcohols, 2-Chloroethanol, 2-Butoxyethanol and 2-Phenoxyethanol using Ammonium metavanadate in acidic medium. Relatively less toxic and cheaper transition metal ions of the first series are effectively used as homogeneous catalysts for the oxidation of the alcohols to the corresponding aldehydes. 2- Chloroethanol is used as a precursor for ethylene oxide and is useful in the manufacture of crop protection chemicals, and pharmaceuticals.2-Butoxyethanol finds use in the making of paints, varnishes and industrial and household cleaners.