Lipase-catalyzed hydrazinolysis of phenyl benzoate: Kinetic modeling approach

Immobilized lipase-catalyzed synthesis of benzoic acid hydrazide from hydrazine and phenyl benzoate is reported in this work. A series of immobilized lipases such as Candida antarctica lipase B, Mucor miehei lipase and Thermomyces lanuginosus lipase were screened to establish that C. antarctica lipase B was the best lipase for hydrazinolysis. When phenyl benzoate (0.01 mol) and hydrazine (0.02 mol) in toluene (15 ml) were reacted with C. antarctica lipase B (Novozym 435) at 50 °C, 95% of phenyl benzoate was converted to benzoic acid hydrazide after 2 h. The effects of various parameters such as speed of agitation, concentration of the substrates, temperature, enzyme concentration, and reusability of the enzyme were studied to deduce kinetics and mechanism of the reaction. A mechanism based on an ordered bi–bi dead end complex with hydrazine was found to fit the data. Systematic deactivation studies indicated that the enzyme was deactivated due to the hydrazine and phenol, enzyme deactivation obeys first-order series model. The kinetic parameters deduced from these models were used to simulate the lipase activity. There was a very good agreement between the simulated and experimental values.     

Kinetics and optimization of lipase-catalyzed synthesis of rose fragrance 2-phenylethyl acetate through transesterification

Lipase from Candida rugosa was immobilized on a polyvinylidene fluoride membrane for synthesis of rose flavor ester, 2-phenylethyl acetate. Response surface methodology (RSM) was employed for kinetic modeling of process and prediction the yield. The RSM was used in practice for determining the kinetic models by fitting the initial rate dates based on the equations of ping-pong bi–bi and order bi–bi model. The maximum reaction rate and kinetic constants were matched with the order bi–bi model. The specificity constant of the immobilized lipase was 10-folds higher than the free form indicated the enzyme–substrate affinity, and catalytic ability was enhanced after immobilization. Moreover, the effects of reaction parameters on the yield were evaluated by RSM using a Box–Behnken experimental design. Based on a ridge max analysis, the maximum conversion was 95.33 ± 2.57% at 38.78 h, 35.85 °C, and substrate mole ratio of 3.65:1. Furthermore, the order bi–bi kinetic model was simulated successfully in a batch reaction. A good prediction existed between the RSM results and integrated equation was found.     

Encapsulation in a sol–gel matrix of lipase from Aspergillus niger obtained by bioconversion of a novel agricultural residue

Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol–gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t _1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis–Menten equation. The K _m values indicated that the encapsulation process reduced enzyme–substrate affinity and the V _max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.     

pH and thermal stability studies of chitinase from Trichoderma harzianum: A thermodynamic consideration

The combined effect of pH and temperature on chitinase was investigated using response surface methodology. A central composite design for two variables was employed. The optimal pH and temperature for the least degree of deactivation were found out to be 5.4 and 24°C respectively. The deactivation rate constants and the half life of chitinase were estimated at different pH and temperature combinations. At the optimal pH of 5.4, the rate of the deactivation was found to be the least. Thermodynamic parameters, viz., ΔH^*, ΔS^*, ΔG^* and activation energy of thermal deactivation of chitinase were calculated in the temperature range from 50°C to 60°C.     

Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli

The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS–PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.     

Non-linear kinetic modelling of reversible bioconversions: Application to the transaminase catalyzed synthesis of chiral amino-alcohols

This work describes the establishment of a full kinetic model, including values of apparent kinetic parameters, for the whole cell E. coli mediated synthesis of the chiral amino-alcohol (2S,3R)-2-amino-1,3,4-butanetriol (ABT), using (S)-(−)-α-methylbenzylamine (MBA) as amino donor. The whole cell biocatalyst expressed the CV2025 ω-transaminase from Chromobacterium violaceum. Establishment of the most suitable reaction mechanism and determination of the complete forward and reverse kinetic parameter values for the reversible bioconversion where obtained using a hybrid methodology. This combined traditional initial rate experiments to identify a solution in the vicinity of the global minimum, with nonlinear regression methods to determine the exact location of the solution. The systematic procedure included selection and statistical evaluation of different kinetic models that best described the measured reaction rates and which ultimately provided new insights into the reaction mechanism; in particular the possible formation of a dead end complex between the amino donor and the cofactor enzyme complex. The hybrid methodology was combined with a microscale experimental platform, to significantly reduce both the number of experiments required as well as the time and material required for full kinetic parameter estimation. The equilibrium constant was determined to be 849, and the forward and reverse rate constants were found to be 97 and 13 min⁻¹, respectively, which greatly favoured the asymmetric synthesis of chiral ABT. Using the established kinetic model, the asymmetric synthesis of ABT was simulated, and excellent agreement was found between the experimental and predicted data over a range of reaction conditions. A sensitivity analysis combined with various simulations suggested the crucial bottleneck of the reaction was the second half reaction of the ping pong bi–bi mechanism, in part due to the low Michaelis constant of substrate l-erythrulose (ERY). The toxicity of MBA towards the transaminase was identified as another major bottleneck. The kinetic model was useful to give early insights into the most appropriate bioconversion conditions, which can improve the rate and yield of ABT formation, as well as minimizing the toxicity and inhibition effects of the substrates and products. The systematic methodology developed here is considered to be generic and useful in regard to speeding up bioconversion process design and optimization.     

Allosteric inhibition by phosphoenolpyruvate of glucose-6-phosphate dehydrogenase from bacteria and its taxonomic importance

Purified glucose-6-phosphate dehydrogenase from Zymomonas mobilis was examined with respect to inhibition by phosphoenolpyruvate, ADP and ATP. Its molecular weight was 260,000 and the kinetics of substrate conversion indicated a random bi bi mechanism. This enzyme and the dehydrogenases from Z. anaerobia, Azotobacter chroococcum, A. vinelandii, and “Corynebacterium” autotrophicum strain 19/-/x were found to be allosterically inhibited by phosphoenolpyruvate, while those from several coryneform bacteria and from Escherichia coli or Pseudomonas fluorescens were not.     

Effect of ionic liquids alkyl chain length on horseradish peroxidase thermal inactivation kinetics and activity recovery after inactivation

The effects of different alkyl chain lengths of ionic liquids 1-ethyl-, 1-butyl- and 1-hexyl-3-methylimidazolium chloride, on the catalytic activity, thermal stability and deactivation kinetics of horseradish peroxidase were studied in the temperature range of 45–85 °C. The presence of 1-ethyl- and 1-butyl-ionic liquids up to 25 % (w/v) did not affect significantly the enzyme activity at 25 °C, whereas the addition of 1-hexyl-solvent resulted in lower activity of enzyme. Typical biphasic deactivation profiles were obtained and adequately fitted by a bi-exponential equation. When increasing ionic liquids concentration up to 25 % (w/v), the second phase of deactivation became more prominent, till leading to apparent first-order kinetics. Occurrence of activity regain, following thermal deactivation was found, reaching up 60–80 % of the initial activity, especially in 1-hexyl-3-methylimidazolium chloride. Activity regain was particularly noticeable in the first phase of deactivation. Temperature sensitivity of the Soret band maxima indicated that the enzyme prepared in buffer or 1-hexyl-3-methylimidazolium chloride had similar conformational changes in the haem region, but no correlations were found with activity decrease.     

Mathematical modelling of the dehydrogenase catalyzed hexanol oxidation with coenzyme regeneration by NADH oxidase

The hexanol oxidation catalyzed by alcohol dehydrogenase from baker's yeast (YADH) has been investigated with two different forms of the biocatalyst: the isolated YADH as well as the YADH in the permeabilized whole cells. It was found that in this reaction, equilibrium is shifted to the reduction side. Hence, to increase the conversion it was necessary to regenerate NAD⁺. For that purpose, enzyme NADH oxidase isolated from Lactobacillus brevis was used. All biocatalysts were kinetically characterized. The overall reaction rate was described by the mathematical model which consisted of kinetics and balance equations. Due to the deactivation of NADH oxidase, only 50–58% hexanol was converted to hexanal in the batch reactor where the hexanol oxidation was catalyzed by isolated YADH. In the case of permeabilized baker's yeast cells, no enzyme deactivation occurred and 100% hexanol conversion in the hexanoic acid was detected.     

Optimization of immobilization conditions of Thermomyces lanuginosus lipase on styrene–divinylbenzene copolymer using response surface methodology

Microbial lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa) was immobilized by covalent binding on a novel microporous styrene–divinylbenzene polyglutaraldehyde copolymer (STY–DVB–PGA). The response surface methodology (RSM) was used to optimize the conditions for the maximum activity and to understand the significance and interaction of the factors affecting the specific activity of immobilized lipase. The central composite design was employed to evaluate the effects of enzyme concentration (4–16%, v/v), pH (6.0–8.0), buffer concentration (20–100 mM) and immobilization time (8–40 h) on the specific activity. The results indicated that enzyme concentration, pH and buffer concentration were the significant factors on the specific activity of immobilized lipase and quadratic polynomial equation was obtained for specific activity. The predicted specific activity was 8.78 μmol p-NP/mg enzyme min under the optimal conditions and the subsequent verification experiment with the specific activity of 8.41 μmol p-NP/mg enzyme min confirmed the validity of the predicted model. The lipase loading capacity was obtained as 5.71 mg/g support at the optimum conditions. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation (12%) occurred after being used repeatedly for 10 consecutive batches with each of 24 h. The effect of methanol and tert-butanol on the specific activity of immobilized lipase was investigated. The immobilized lipase was almost stable in tert-butanol (92%) whereas it lost most of its activity in methanol (80%) after 15 min incubation.     

Kinetics of Pyruvate Decarboxylase Deactivation by Benzaldehyde

Based on experimental data, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0–200 mM) in MOPS buffer (2.5 M) has been developed. An initial lag period prior to deactivation was found to occur. With first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration, a reaction time deactivation constant of 2.64×10^?3 h^?1 and a benzaldehyde deactivation coefficient of 1.98×10^?4 mM^?1 h^?1 were determined for benzaldehyde concentrations up to 200 mM. The PDC deactivation kinetic equations established in this study are an essential component in an overall model being developed to describe the enzymatic biotransformation of benzaldehyde and pyruvate to produce the pharmaceutical intermediate (R)-phenylacetylcarbinol (R-PAC).     

Simulation of terrestrial carbon equilibrium state by using a detachable carbon cycle scheme

Determining the equilibrium state of terrestrial carbon is a prerequisite for scientific analysis on the carbon cycle. However, the mechanism through which the carbon cycle reaches the equilibrium state remains unclear. Moreover, the carbon cycle in most of the short–term field experiments rarely reaches the equilibrium state. In this study, a detachable carbon cycle (DCC) model was proposed to simulate the equilibrium state of each carbon pool. The model was established based on a pool–and–flux scheme and contained 14 carbon pools, or carbon flow processes, each process could be detached from the main model and evaluated as an independent component. The environmental scalar algorithms of the Integrated Terrestrial Ecosystem Carbon budget model (InTEC) and Community Atmosphere Biosphere Land Exchange (CABLE) were incorporated in the DCC model. Four situations were compared using the two environmental scalar algorithms and model structure (9 vs. 14 carbon pools). Furthermore, the size and turnover time of each carbon pool were analyzed at the equilibrium state. A sensitivity analysis was then conducted to investigate the responses of carbon density and equilibrium time to 12 key parameters of the model. Results indicated that the combination of the CABLE environmental scalar algorithm and 14 pools exhibited improved performance on carbon storage simulation than that of the other combinations, and the effect of the environmental scalar algorithm was considerably larger than that of the carbon pool number. Sensitivity analysis indicated that the carbon density of grassland and cropland was more vulnerable and sensitive to key parameters of the model than that of the other biomes. This study elucidates influencing factors and underlying control mechanisms in the carbon accumulation, and provides a framework for quantitative analysis of each component of the carbon cycle.     

Glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes. Kinetic mechanism and competitive substrate inhibition by glyceraldehyde 3-phosphate

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was isolated from human erythrocyte ghosts by a simple procedure utilizing ammonium sulfate precipitation and affinity chromatography on NAD⁺-Sepharose 4B. The purified enzyme had a specific activity of 98 units/mg protein. The kinetic mechanism of GAPD was studied by product and deadend inhibition using NADH, α-glycerophosphate, nitrate, and 2,3-diphosphoglycerate. The results indicated that the human erythrocyte GAPD-catalyzed reaction follows an ordered ter bi mechanism characterized by the sequential addition of NAD⁺, glyceraldehyde 3-phosphate (GAP), and phosphate to the enzyme and the sequential release of 1,3-diphosphoglycerate and NADH from the enzyme. This contrasts with the mechanism (rapid equilibrium random ter bi) proposed by Oguchi (1970, J. Biochem. (Tokyo)68, 427–439) who based his conclusion on the initial rate data alone. Since the Michaelis-Menten kinetics were not applicable to this enzyme because of the competitive substrate inhibition by GAP, we devised a new kinetic approach for determining the parameters of the GAPD-catalyzed reaction. Results of this study indicate that the GAPD-catalyzed reaction is regulated by both ATP and GAP. We propose that GAP acts as an “amplifier” for the feedback inhibition effect of ATP. We discuss the effect this may have played in causing controversy over the regulatory role of this enzyme in glycolysis.     

Estimating in vivo passive forces of the index finger muscles: Exploring model parameters

We compared predicted passive finger joint torques from a biomechanical model that includes the exponential passive muscle force–length relationship documented in the literature with finger joint torques estimated from measures in ten adult volunteers. The estimated finger joint torques were calculated from measured right index fingertip force, joint postures, and anthropometry across 18 finger and wrist postures with the forearm muscles relaxed. The biomechanical model predicting passive finger joint torques included three extrinsic and three intrinsic finger muscles. The values for the predicted passive joint torques were much larger than the values calculated from the fingertip force and posture measures with an average RMS error of 7.6 N cm. Sensitivity analysis indicated that the predicted joint torques were most sensitive to passive force–length model parameters compared to anthropometric and postural parameters. Using Monte Carlo simulation, we determined a new set of values for the passive force–length model parameters that reduced the differences between the joint torques calculated from the two methods to an average RMS value of 0.5 N cm, a 94% average improvement of error from the torques predicted using the existing data. These new parameter values did vary across individuals; however, using an average set for the parameter values across subjects still reduced the average RMS difference to 0.8 N cm. These new parameters may improve dynamic modeling of the finger during sub-maximal force activities and are based on in vivo data rather than traditional in vitro data.     

Kinetic modelling of the production of methyl oleate by Celite® supported lipase sol–gels

This study illustrates the benefits of Celite^® supported lipase sol–gels for the transesterification of triolein to produce methyl oleate. A ping–pong bi–bi kinetic model was developed and validated taking into account the inhibition effects of methanol and glycerol as well as the effect of temperature. Although initial reaction rate models are useful for predicting the kinetics in the absence of products, a kinetic model beyond the initial conditions that considers glycerol inhibition is important. The model developed was consistent with the experimental data (R² = 0.95) predicting an increase in methyl oleate production with increasing methanol concentration up to an optimal range of 1.3 M to 2.0 M depending on the temperature. In general, increasing the temperature increased the initial reaction rate for the immobilized lipase over the temperature range of 40–60 °C. Based on the kinetic constants, the maximum velocity of the reverse reaction is about 25% slower than that of the forward reaction and glycerol inhibition has a more significant effect on the reaction kinetics than methanol inhibition. The model developed would be useful for understanding the effects of methanol and glycerol inhibition as well as temperature on the production of methyl oleate using lipase-mediated enzymatic transesterification.     

Enhanced biocatalytic activity of immobilized <Emphasis Type="Italic">Pseudomonas cepacia</Emphasis> lipase under sonicated condition

The present work reports the use of biocatalyst and ultrasound for greener synthesis of cinnamyl propionate. The lipase Pseudomonas cepacia was immobilized on a copolymer of hydroxypropyl methyl cellulose and polyvinyl alcohol. This biocatalyst was used for ultrasound-assisted synthesis of cinnamyl propionate with the detailed optimization of various reaction parameters. Besides this, protocol was extended to synthesize various industrially important propionate esters. In addition to this, different enzyme-kinetic parameters such as r _max and K _{m(vinyl propionate),} K _{m(cinnamyl alcohol)} and K _{i(cinnamyl alcohol)} were studied which presented ordered bi–bi mechanism with an inhibition by cinnamyl alcohol. The developed biocatalyst demonstrated enhancement in catalytic activity and recyclability up to five recycles. Moreover, the biocatalyst was tested to investigate the effects of sonication via various characterization techniques such as scanning electron microscopy, thermogravimetry, and water content analysis.     

Physically correlated muscle activation for a human head and neck computational model

A computational 50th percentile male head and neck complex model was correlated to physical experimental data. The computational model utilizes 15 muscle pairs represented by the Hill Muscle Model with the complete head/neck system modeled using MADYMO?. The model was used for analysis and optimization of activation and deactivation of muscle activity in flexion and extension. Sensitivity analysis performed using the model shows that, of the multiple parameters within the Hill Model, activation level and timing prove to have the greatest effect on the system kinematics. In addition, the rate by which an activation level is changed becomes an important factor in the simulation. With the use of numerical optimization techniques, a pattern was determined for the applied activation/deactivation rates and timing of flexors and extensors during flexion and extension of the head. The numerical optimization result correlated to within 9% of measured value during the initial flexion of the head. The optimized activation model reflected an activation onset 90 ms after the start of the impulse load, which agrees with published reaction times of muscles. Activation and deactivation rates for the extensors were found to be 1.7 and 0.29%, respectively. While the onset of activation of the flexor muscles occurred before rebound, it was found that muscles, at near the mid-plane, were triggered by the optimized model to abate the flexion. Rates of activation and deactivation of the flexors were found to be 0.9 and 0.3%, respectively. Both the extensors as well as the flexors were found to activate only up to 70% before deactivating. Therefore, it was evident from this study that using the Hill Muscle Model with the activation parameter modeled as binary, 0 or 100%, may lead to inaccurate simulation results.     

Deactivation studies of immobilized glucose oxidase

Studies have been performed in a tubular flow reactor to characterize the deactivation of immobilized glucose oxidase. The effects of oxygen concentration in the range of 0.09 to 0.467mM and hydrogen peroxide concentrations in the range of 0.1 to 10mM were studied. A simple mathematical model assuming first-order reaction and deactivation was found to describe the deactivation behavior adequately. The deactivation rate constant was found to increase with increasing levels of feed oxygen. Hydrogen peroxide was found to deactivate the enzyme severely and the deactivation rate constants were higher than those for oxygen deactivation. The influence of external and internal diffusion effects on the deactivation rate constant were examined. Although diffusional restrictions were negligible for oxygen transfer to the pellet, they were significant for transfer of hydrogen peroxide to the bulk stream. Increasing deactivation rates. Severe internal diffusion limitations were observed for the glucose oxidase system. However, for particle sizes in the range of 500 to 2000 μm, no effect on the rate of deactivation of the enzyme was observed.     

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: I. uniresponse kinetics

A calf pregastric esterase immobilized in a hollow-fiber reactor was employed to hydrolyze milkfat, thereby producing a lipolyzed butteroil. The reaction kinetics can be modeled by a two-parameter model of the general Michaelis-Menten form based on a ping-pong bi-bi mechanism; the rate of enzyme deactivation can be modeled as a first-order reaction. The initial concentration of accessible glyceride bonds, [G](O), was estimated by complete saponification of the substrate butteroil as 2400 mM. An extra sum of squares test indicated that not only the parameters of the kinetic generalized Michaelis-Menten model, but also the deactivation-rate constant varied significantly with pH. The optimum pH, for lypolysis is near 6.0 at a temperature of 40 degrees C because at this pH the rate of deactivation of the esterase is minimized.     

Determination of internal diffusion limitation and its macroscopic kinetics of the transesterification of CPB alcohol catalyzed by immobilized lipase in organic media

Optically active cyanohydrin esters such as (S)-α-cyano-3-phenoxybenzyl (CPB) acetate can be obtained with enzymatic transesterification of racemic cyanohydrins in organic media. The influence of internal diffusion limitation on transesterification of CPB alcohol with vinyl acetate catalyzed by immobilized lipase was studied. Internal diffusion limitation could not be ignored since the immobilized lipase granule was not small enough. In order to express the effect of internal diffusion limitation quantitatively, a method was proposed which calculates the effective diffusion coefficient (D_e) first and then calculates apparent Thiele modulus (Φ) and finally obtains internal diffusion effectiveness factor, η value (0.55). D_e was calculated to be 3.1 × 10⁻¹⁰ m² s⁻¹ by supposing that the immobilized lipase was a sphere granule and the diffusion only existed in one dimension. Φ was calculated to be 2.2 by presuming that transport of substrate through the catalyst could be described by Fick's law. Using King–Altman method, a kinetic model of the transesterification of CPB alcohol with vinyl acetate in organic media in the presence of internal diffusion limitation was proposed based on ping-pong bi–bi mechanism. The relative error of the model was 11.18%. A practicable method to evaluate η value and relatively simple model for such kind of reaction system was proposed in this paper.