Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: IV. Effects of temperature

A lipase from Aspergillus niger, immobilized by adsorption on microporous polypropylene hollow fibers, was used to effect the hydrolysis of the glycerides of melted butterfat at pH. 7.0 at 40, 50, 55, and 60 degrees C. Mcllvane buffer was pumped upward through the lumen, and melted butterfat was pumped upward through the shell side of a hollow fiber reactor. Nonlinear regression methods were employed to determine the kinetic parameters of models based on combinations of three nested rate expressions for the hydrolysis reaction with three nested rate expressions for thermal deactivation of the enzyme. A rate expression containing four lumped parameters is sufficient to model the release of free fatty acids as a function of reactor space time and time elapsed after immobilization. Nonlinear regression methods were also employed in global fits of the data to rate expressions containing an explicit dependence on temperature. For the reaction conditions used in this research, a 14-parameter rate expression is necessary to accurately model the overall release of free fatty acids as a continuous function of the absolute temperature, initial substrate concentrations, reactor space time, and time elapsed after immobilization of the lipase.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part II. Uniresponse kinetic studies

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the hydrolysis of the glycerides of melted butterfat at 40 degrees C and pH 7.0. Mcllvane buffer was pumped through the lumen and melted butterfat was pumped courrently through the shell side of a shell-and-tube reactor. Nonlinear regression methods were employed to determine the kinetic parameters of three nested rate expressions derived from a Ping Pong Bi Bi enzymatic mechanism coupled with three nested rate expressions for the thermal deactivation of the enzyme. For the reaction conditions used in this research, a four-parameter rate expression (which includes a two-parameter deactivation rate expression and a two-parameter hydrolysis rate expression) is sufficient to model the overall release of free fatty acids from the triglycerides of butterfat as a function of space time and time elapsed after immobilization. At a space time of 3.7 h immediately after immobilization of lipase, 50% of the fatty acid residues esterified in the sn-1,3 positions of the triglycerides can be released in the hollow-fiber reactor.     

Use of novel immobilized beta-galactosidase reactor to hydrolyze the lactose constituent of skim milk

beta-galactosidase from Aspergillus oryzae immobilized in an axial-annular flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Nonlinear regression methods were employed to determine the kinetic parameters of four rate expressions derived from a proposed enzymatic mechanism. Data taken at three different temperatures (30 degrees C, 40 degrees C, and 50 degrees C) were fit via nonlinear regression methods assuming an Arrhenius temperature model for each of the parameters. For the reaction conditions used in this research, a three-parameter rate expression which includes the separate competitive inhibition effects of alpha- and beta-galactose (and the associated mutarotation reaction) is sufficient to model the hydrolysis of lactose in skim milk. The effects of temperature on the individual kinetic parameters are small. The most significant effect appears in the term for inhibition by the beta anomer of galactose (E(A) = 10.3 kcal/mol). At 40 degrees C and a space time of 10 min, 70% of the lactose present in skim milk can be hydrolyzed with the axial-annular flow reactor. This reactor can be used to hydrolyze the lactose in skim milk without the problems observed with other reactor configurations, namely, plugging due to particulates, microbial contamination, and large pressure drop.     

Hydrolysis of menhaden oil by a Candida cylindracea lipase immobilized in a hollow-fiber reactor

A lipase from Candida cylindracea immobilized by adsorption on microporous polypropylene fibers was used to selectively hydrolyze the saturated and monounsaturated fatty acid residues of menhaden oil at 40 degrees C and pH 7.0. At a space time of 3.5 h, the shell and tube reactor containing these hollow fibers gives a fractional release of each of the saturated and monounsaturated fatty acid residues (i.e., C14, C16, C16:1, C18:1) of ca. 88% of the corresponding possible asymptotic value. The corresponding coproduct glycerides retained over 90% of the initial residues of both eicosapentaenoic (EPA; C20:5) and docosahexaenoic (DHA; C22:6) acids. The half-life of the immobilized lipase was 170 h when the reactor was operated at the indicated (optimum) conditions. Rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for the lipase catalyzed reaction. Both uni- and multiresponse nonlinear regression methods were employed to determine the kinetic parameters associated with these rate expressions. The best statistical fit of the uniresponse data was obtained for a rate expression, which is formally equivalent to a general Michaelis-Menten mechanism. After reparameterization, this rate expression reduced to a pseudo-first-order model. For the multiresponse analysis, a model that employed a normal distribution of the ratio of Vmax/Km with respect to the chain length of the fatty acid residues provided the best statistical fit of the experimental data.     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: II. Multiresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow-fiber reactor

The hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow-fiber reactor was studied at 30 degrees C. To assess the selectivity of this immobilized enzyme, the effluent concentrations of five different free fatty acids were measured using high-performance liquid chromatography (HPLC). Several rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for this lipase-catalyzed reaction. A multiresponse nonlinear regression method was employed to determine the kinetic parameters associated with these rate expressions. Quasi-optimum operating conditions corresponded to 30 degrees C and a buffer pH value of 7.0. Under these conditions, the concentration of free linoleic acid (C18:2) (the fatty acid of primary interest) in the effluent oil stream for a fluid residence time of 6 h was approximately 0.5 M.     

Metabolic modeling of polyhydroxybutyrate biosynthesis

A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium.     

Glycosylation of Aromatic Amines I: Characterization of Reaction Products and Kinetic Scheme

The reactions of aliphatic and aromatic amines with reducing sugars are important in both drug stability and synthesis. The formation of glycosylamines in solution, the first step in the Maillard reaction, does not typically cause browning but results in decreased potency and is hence significant from the aspect of drug instability. The purpose of this research was to present (1) unreported ionic equilibria of model reactant (kynurenine), (2) the analytical methods used to characterize and measure reaction products, (3) the kinetic scheme used to measure reaction rates and (4) relevant properties of various reducing sugars that impact the reaction rate in solution. The methods used to identify the reversible formation of two products from the reaction of kynurenine and monosaccharides included LC mass spectrometry, UV spectroscopy, and 1-D and 2-D ¹H–¹H COSY NMR spectroscopy. Kinetics was studied using a stability-indicating HPLC method. The results indicated the formation of α and β glycosylamines by a pseudo first-order reversible reaction scheme in the pH range of 1–6. The forward reaction was a function of initial glucose concentration but not the reverse reaction. It was concluded that the reaction kinetics and equilibrium concentrations of the glycosylamines were pH-dependent and also a function of the acyclic content of the reacting glucose isomer.     

Study of the reaction of grafting acrylamide onto xanthan gum

The present study aimed to study the reaction conditions of grafting of acrylamide on xanthan gum. It was analyzed the influence of reaction conditions, mainly type of initiator activation, initiator concentration and initiator/acrylamide ratio, on graft parameters and copolymer properties. Potassium persulfate was employed as an initiator and heating or N,N,N',N'-tetramethylethylenediamine was used to activate the initiator. Reaction time and initiator concentration were varied and final values for grafting percentage and grafting efficiency were the same for both methods, whereas speed in reaching these values differs from one technique to another. We found that reaction time was inversely proportional to intrinsic viscosity, likely due to main chain degradation promoted by potassium persulfate (KPS); furthermore, the increasing in the KPS concentration lowers grafting percentage, acrylamide conversion and chain degradation, possibly as a result of O(2) formation at high KPS concentrations.     

Hydrolysis of lactose in skim milk by immobilized beta-galactosidase in a spiral flow reactor

beta-galactosidase from Aspergillus Oryzae immobilized in a spiral flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Residence time distribution measurements were used to assess the amount of longitudinal dispersion occurring as a consequence of the spiral flow pattern and the semiporous nature of the polymeric material used to construct the spiral. It was possible to model the flow conditions as tubular flow with a Peclet number that was a linear function of the reactor space time. Nonlinear regression methods were used to determine the kinetic parameters of three proposed enzymatic rate expressions. The best fit of the data was obtained using a rate expression containing separate terms for competitive inhibition of the reaction by both the a and beta anomers of galactose. This kinetic model also incorporates the kinetics of the mutarotation between these forms. At 30 degrees C and a space time of 7 minutes, 80% of the lactose present in skim milk can be converted to glucose and galactose.     

Tubular bioreactor models that include Onsager-Curie scalar cross-phenomena to describe stress-dependent rates of cell proliferation

The theory of heterogeneous catalysis in chemical reactors is employed to simulate laminar flow through tubes at large mass transfer Peclet numbers in which anchorage-dependent cells (i) adhere to a protein coating on the inner surface at r = R_wall, (ii) receive nutrients and oxygen from an aqueous medium via transverse diffusion toward the active wall, and (iii) proliferate in the presence of viscous shear at the cell/aqueous-medium interface. This process is modeled as convective diffusion in cylindrical coordinates with chemical reaction at the boundary, where chemical reaction describes the rate of nutrient consumption. The formalism of irreversible thermodynamics is employed to describe an unusual coupling between viscous shear, or velocity gradients at the cell/aqueous-medium interface, and rates of nutrient consumption. Linear transport laws in chemically reactive systems that obey Curie's theorem predict the existence of cross-phenomena between fluxes (i.e., scalar reaction rates) and driving forces (i.e., 2nd-rank velocity gradient tensor) whose tensorial ranks differ by an even integer—in this case, two. This methodology for stress-dependent chemical reactions yields an additional zeroth-order contribution, via the magnitude of the velocity gradient tensor, to heterogeneous kinetic rate expressions because nutrient consumption and cell proliferation are stress-sensitive. Computer simulations of nutrient consumption suggest that bioreactor designs should consider stress-sensitive reactions when the shear-rate-based Damköhler number (i.e., defined for the first time in this study as the stress-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the tube wall) is greater than 10–20% of the stress-free Damköhler number. Models of bioreactor performance are presented for simple 1st-order, simple 2nd-order, and complex chemical kinetic rate expressions, where the latter considers adsorption/desorption equilibria via the Fowler–Guggenheim modification of the Langmuir isotherm for cell–protein docking on active sites, accompanied by cell–cell attraction. Stress sensitivity is magnified in physically realistic cell-based tubular bioreactors with complex stress-free kinetic rate expressions relative to simulations with simple 1st- and 2nd-order kinetics.     

Lipase-catalyzed esterification of conjugated linoleic acid with sorbitol: a kinetic study

The kinetics of esterification of conjugated linoleic acid (CLA) with sorbitol in acetone was investigated. An immobilized lipase from Candida antarctica (Chirazyme L-2) was used as the biocatalyst. A 2(2) x 3 factorial design was employed to find an experimental region in which one obtains a high rate of formation of the diester product. Best results were obtained at 10 degrees C using a CLA to sorbitol molar ratio of 5 and a biocatalyst loading of 150 mg/mL of acetone. Under these conditions, in 72 h one obtains a nearly quantitative yield (ca. 98%) of the diester of sorbitol with CLA. To minimize formation of products with degrees of esterification greater than two, the reaction should be carried out at 10 degrees C. A kinetic model developed using the King-Altman method was employed to fit the data. Use of the steady-state approximation for the monoester and an assumption that the concentration of sorbitol was constant and equal to its solubility limit permit one to minimize the number of parameters necessary to model the reaction network. Nonlinear regression analysis based on either two or three parameters provides very good fits of the multiresponse data in the presence or absence of triesters, respectively.     

General methods for analysis of sequential "n-step" kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding

Helicase-catalyzed DNA unwinding is often studied using "all or none" assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using "n-step" sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the "kinetic step size", m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using "n-step" sequential mechanisms has previously been limited by an inability to float the number of "unwinding steps", n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, f(ss)(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain f(ss)(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation.     

Kinetics of lactose hydrolysis by beta-galactosidase of Kluyveromyces lactis immobilized on cotton fabric

A mathematic model for describing the Michaelis-Menten-type reaction kinetics with product competitive inhibition and side-reaction is proposed. A multiresponse nonlinear simulation program was employed to determine the coefficients of a four-parameter rate expression. The rate expression was compared with the conventional Michaelis-Menten reaction rate models with and without product inhibition. Experimental data were obtained using beta-galactosidase of Kluyveromyces lactis immobilized on cotton fabric in a batch system at a temperature of 37 degrees C and at various initial concentrations of dissolved lactose ranging from 3-12.5% (w/v). The reaction is followed by concentration changes with time in the tank. Samples were obtained after the outlet stream of the packed bed reactor is mixed in a well-stirred tank. High-performance liquid chromatography (HPLC) was applied to monitor the concentrations of all the sugars (reactants as well as products). The four-parameter rate model is featured with a term to describe the formation of trisaccharides, a side-reaction of the enzymatic hydrolysis. The proposed model simulates the process of lactose hydrolysis and the formation of glucose and galactose, giving better accuracy compared with the previous models.     

Lipoxygenase-another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid.

In this study, we examined the ability of human term placental lipoxygenase (HTPLO) to catalyze glutathione (GSH) conjugate formation from ethacrynic acid (EA) in the presence of linoleic acid (LA) and GSH. HTPLO purified by affinity chromatography was used in all the experiments. The results indicate that the process of EA-SG is enzymatic in nature. The reaction shows dependence on pH, the enzyme, and the concentration of GSH, LA, and EA. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.3 mM LA, 0.2 mM EA, 2.0 mM GSH, and approximately 300 microg HTPLO in the reaction medium buffered at pH 9.0. Under the experimental conditions employed, the reaction exhibited K(m) values of 1.1 mM, 200 microM, and 130 microM for GSH, LA, and EA, respectively. The estimated specific activity of HTPLO-catalyzed EA-GS formation was approximately 4.4 +/- 0.4 micromol/min/mg protein. This rate is more than twofold greater than the rate noted for the reaction mediated by the purified human term placental glutathione transferase. Under physiologically relevant conditions (20 microM LA, 2.0 mM GSH, at pH 7.4), HTPLO produced EA-SG at 56% of the maximal rate noted under optimal assay conditions. Nordihydroguaiaretic acid, the classical inhibitor of different lipoxygenases, significantly blocked the reaction. It is proposed that free radicals are involved in the process of EA-SG formation by HTPLO. The evidence gathered in this in vitro study suggests for the first time that lipoxygenase present in the human term placenta is capable of EA-SG formation.     

Open tubular heterogeneous enzyme reactors: preparation and kinetic behavior

Tubes with immobilized enzymes on the inner wall, called open tubular heterogeneous enzyme reactors, were prepared by binding enzymes either directly to the tube inside surface or to a layer of a porous matrix attached to the inner wall. Kinetic studies of the hydrolysis of N-benzoyl-L -arginine ethylester as a model reaction indicated that the reaction was kinetically controlled in reactors with surface bound trypsin and the kinetic parameters were evaluated by conventional methods. On the other hand, substrate diffusion in both the porous matrix and the bulk substrate solution strongly affected the rate of reaction in porous layer trypsin reactors. The highest overall rates of reaction were obtained when the reaction was bulk diffusion controlled and the measured rates were in agreement with those calculated from expressions derived from heat transfer theory. The design of reactors for the limiting cases of kinetic and bulk diffusion controlled reaction as well as a method for the determination of substrate diffusivity are outlined.     

Myonemal contraction of Spirostomum. III. The thermal dependence of contraction, relaxation and excitation-contraction coupling.

A microphotometric technique that displays rapid length changes of Spirostomum has been used to follow the variation with temperature of these kinetic parameters of myonemal contraction: contraction rate, relaxation rate and stimulus duration at threshold. In each case the exponential form of the relationship indicated that the gross rate constant might be equated with the limiting rate constant, k, of a driving chemical reaction, and from standard expressions of chemical kinetics the change in activation free energy appropriate to this reaction has been computed.     

Reactions of d-glyceraldehyde 3-phosphate dehydrogenase facilitated by oxidized nicotinamide–adenine dinucleotide

Transient kinetic methods have been used to study the influence of NAD(+) on the rate of elementary processes of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. In the pH range 5-8 NAD(+) is bound to the enzyme during the following elementary processes of the mechanism: phosphorolysis of the acyl-enzyme, its formation from 1,3-diphosphoglycerate and the enzyme and the formation and breakdown of the glyceraldehyde 3-phosphate-enzyme complex. The rates of these four elementary processes only equal or exceed the turnover rate of the enzyme when NAD(+) is bound and are as much as 10(4) times the rates in the absence of NAD(+). Autocatalysis of the reductive dephosphorylation of 1,3-diphosphoglycerate occurs when glyceraldehyde 3-phosphate release is rate determining because NAD(+) is a reaction product. An important feature of the enzyme mechanism is that the negative-free-energy change of a chemical reaction, acyl-enzyme formation, is linked in a simple way to the positive-free-energy change of a dissociation reaction, NAD(+) release.     

Allosteric interactions in phosphorylase B

An allosteric model of phosphorylase B is proposed based on the following assumptions. The enzyme consists of two sub-units and undergoes a concerted transition from the inactive T to the active R state. The binding of substrates, phosphate, and glycogen is regarded as exclusive, but the binding of the activator AMP is nonexclusive. The enzyme model is of the K, V type, i. e., the activator AMP is important, not only for the T-R transition and the substrates binding, but also for the formation of the active site. Therefore, it displays a big influence on the maximal reaction rate. Calculations based on this model lead to an equation containing 5 constants, which can be easily computed from kinetic data. All kinetic measurements fit the expressions derived from the model. Independent methods for the measurement of all the constants involved were developed. They are based on the study of binding of phosphorylase with the substrates and the activator. These measurements are in satisfactory agreement with the data obtained from enzyme kinetics.     

The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution in chloroplasts.

The rate of reaction between superoxide anion (O2) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O2. A kinetic spectrophotometric method utilizing competition between p-benzoquinone and tiron for O2 was employed. In this system, the known rate of reduction of p-benzoquinone was compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5x10-8 M-minus1-s-minus1. Ascorbate reduced O2 to hydrogen peroxide with a rate constant of 10-8 M-minus1-s-minus1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O2 compared to the rate of O2 formation is most enzymatic systems. Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O2 since O2 was consumed in the reaction and tiron did not reduce the P700 cation radical or other components of Photosystem I under anaerobic conditions.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.