Effect of Chemical Modification of Lipase on the Regulation of Its Lipolytic Activity in Reversed Micelles

Hydrophilized and hydrophobized forms of the lipase from Mucor miehei were obtained by its chemical modification with cellobiose and N-succinimidyl palmitate with a modification degree of 4 in both cases. A comparative analysis of the regulation of the catalytic activities of the native and modified lipases was carried out in the system of reversed micelles of OT aerosol (AOT) in isooctane. The level of catalytic activity of all the lipase preparations in the micellar medium was found to be higher than that in aqueous solution. The chemical modification of lipase did not result in a change in the regulation of the oligomeric composition of the enzyme controlled by the degree of micelle hydration Ω₀ (micelle size). The k _cat dependences on Ω₀ for each lipase preparation exhibit two maxima, corresponding to the functioning of lipase monomers and tetramers. The changes in the hydrophilic-lipophilic balance of the lipase surface significantly affect the character of the regulation of enzyme activity due to changes in the surfactant concentration (the number of micelles). The lipase hydrophobization results in a decrease in the enzyme activation effect with an increase in the AOT concentration in comparison with the native lipase. The lipase hydrophilization dramatically decreases the activity of lipase tetramer when the AOT concentration is increased. The catalytic activity of the monomer of hydrophilized lipase is practically independent of the AOT concentration. Kinetic data indicate a mixed type of activation of both oligomeric forms of the native and the hydrophobized lipase by AOT molecules and the noncompetitive type of the activation and AOT inhibition of the monomer and the tetramer of the hydrophilized lipase, respectively.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Flow dynamics of immobilized enzyme reactors

Summary Enzymic conversion of glucose to fructose was carried out in a packed bed and in a fluidized bed reactor. The flow dynamics of these two flow systems, loaded with two different types of immobilized loaded with two different types of immobilized glucose isomerase particles, were studied. The theoretical RTD curve calculated from the axial dispersed plug flow model equation was matched to the experimental RTD curve by an optimization technique. The effect of fluid velocity on the extent of liquid dispersion was established. Theoretical predictions on the conversion of glucose to fructose were calculated using three mathematical models, namely, a plug flow model, a continuous stirred tank reactor (CSTR) model and an axial dispersed plug flow model. The experimental results showed that the axial dispersed plug flow model was superior in predicting the performance of both the packed bed and fluidized bed reactor.Abbreviations C Dimensionless concentration - D Dispersion coefficient [cm²/sec] - d _p Mean particle diameter [cm] - E Enzyme concentration [mol/gm] - F Fructose concentration [mol/cm³] - F _e Equilibrium fructose concentration [mol/cm³] - G Glucose concentration [mol/cm³] - G _e Equílibrium glucose concentration [mol/cm³] - G _o Initial glucose concentration [mol/cm³] - Reduced glucose concentration [mol/cm³] - K Equilibrium constant - K _mf Forward reaction rate constant [mol/cm³] - K _mr Reserve reaction rate constant [mol/cm³] - K _m Rate constant [mol/cm³] - L Total length of the reactor bed [cm] - l Length [cm] - Q Flow rate [cm³/s] - r Rate of reaction based on volume of substrate - u Superficial liquid velocity [cm/s] - v Interstitial liquid velocity [cm/s] - V Reactor bed volume [cm³] - V _mf Forward reaction rate constant [mol/s·g enzyme] - V _mr Reserve reaction rate constant [mol/s·g enzyme] - z Dimensionless distance along the reactor - Density [g/cm²]     

Modeling the cucumber fermentation: Growth ofLactobacillus plantarum

Summary Specific growth rate models of product-inhibited cell growth exist but are rarely applied to fermentations beyond ethanol and large-scale antibiotic production. The present paper summarizes experimental data and the development of a model for growth of the commercially important bacterium,Lactobacillus plantarum, in cucumber juice. The model provides an excellent correlation of data for the influence on bacterial growth rate of NaCl, protons (H⁺), and the neutral, inhibitory forms of acetic acid and the fermentation product, lactic acid. The effects of each of the variables are first modeled separately using established functional forms and then combined in the final model formulation.Nomenclature [C] inhibitory component concentration, mM - [C]_max concentration of the inhibitory component where the specific growth rate is zero, mM, determined by model fitting - [H⁺] hydrogen ion concentration, mM - [HLa] undissociated lactic acid concentration, mM - [La^–] dissociated lactic acid concentration, mM - [La_t] total lactic acid ([HLa]+[La^–]) concentration, mM - [HAc] undissociated acetic acid concentration, mM - [Ac^–] dissociated acetic acid concentration, mM - [Ac_t] total acetic acid ([HAc]+[Ac^–]) concentration, mM - [NaCl] sodium chloride concentration, %, w/v - specific growth rate, h^–1 - _max maximum specific growth rate, h^–1 - ₀ specific growth rate, h^–1, at 0 concentration of additive - K _ij inhibition coefficient - , ,K _m coefficients determined by model fitting Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other products that may be suitable.     

Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt

Zusammenfassung Es wurden analysenreine Proben der Romanowsky-Farbstoffe Eosin Y, Erythrosin B und Tetrachlorfluoreszein hergestellt.Im DC der Farbstoffproben konnten keine Verunreinigungen nachgewiesen werden. Die Absorptionsspektren der Farbstoffdianionen in wäßriger alkalischer Lösung und der Farbstoffsäuren in 95%igem Ethanol wurden bei sehr kleinen Farbstoffkonzentrationen gemessen und der molare Extinktionskoeffizient der längstwelligen Absorptionsbande der monomeren Farbstoffspezies bestimmt (Tabelle 1). Die Extinktionskoeffizienten können zur Standardisierung von Farbstoffproben verwendet werden. Die Absorptionsspektren von Eosin Y hängen in wäßriger Lösung von der Farbstoffkonzentration ab. Aus der Konzentrationsabhängigkeit wurden mit einem neuen, sehr empfindlichen Verfahren zwei Assoziationsgleichgewichte ermittelt. Bereits in sehr verdünnter Lösung bilden sich Dimere, bei erhöhter Konzentration Tetramere, Die Dissoziationskonstante der DimerenD in MonomereM beträgt bei pH=12, 293K:K ₂₁=2,9 × 10^–5 M; der TetramerenQ in DimereD:K ₄₂=2,4 × 10^–3 M. Aus den gemessenen Spektren von Eosinlösungen verschiedener Konzentration, pH=12, und den GleichgewichtskonstantenK ₂₁,K ₄₂ haben wir die Spektren der reinen Monomeren, Dimeren und Tetrameren bestimmt.M hat eine langwellige Absorptionsbande: , _M =1,03 x 10₅ M_-1 cm_-1;D eine Bande: , _D =1,74 x 10₅ M_-1 cm_-1;Q zwei Banden: , , _Q1=1,65 x 10⁵, _Q2=1,96 x 10⁵ M^-1 cm^-1. Das Absorptionsspektrum der Dimeren wird quantenmechanisch interpretiert.

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Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, Erythrosin B, tetrachlorofluorescein, Spectroscopic characterization of pure dyes, association of Eosin Y

Summary Analytically pure smaples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependend on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimersD in monomersM at 293 K, pH=12, isK ₂₁=2,9×10^–5 M; of the tetramersQ in dimersDK ₄₂=2,4×10^–3 M. From the experimental spectra of eosin solutions at various concentrations, pH=12, and the equilibrium constantsK ₂₁,K ₄₂ the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, , _M =1,03 x 10₅ M_-1 cm_-1;D also one absorption band, , _D =1,74 x 10₅ M_-1 cm_-1;Q two absorption bands, , , _Q1=1,65 x 10⁵, _Q2=1,96 x 10⁵ M^-1 cm^-1. The absorption spectrum of the dimers is discussed by quantum mechanics.

     

Simulation of glucose isomerase reactor: optimum operating temperature

A design equation for immobilized glucose isomerase (IGI) packed bed reactor is developed assuming enzyme deactivation and substrate protection. The developed equation is used to simulate the performance of the reactor at various temperatures (50–80 °C). Enzyme deactivation is significant at high temperature. Substrate protection showed to have significant effect in reducing enzyme deactivation and increasing the enzyme half-life. Factors affecting the optimum operating temperature are discussed. The optimum operating temperature is greatly influenced by the operating period and to a lesser extent with both initial glucose concentration and glucose conversion.Two modes of reactor operation are tested i.e., constant feed flow rate and constant conversion. Reactor operating at constant conversion is more productive than reactor operating at constant flow rate if the working temperature is higher than the optimum temperature. Although at lower temperatures than the optimum, the two modes of operation give the same result.List of Symbols a residual enzyme activity - E [mg/l] concentration of active enzyme - E _a [kJ/mole] activation energy - E ₀ [mg/l] initial concentration of active enzyme - k [Specific] kinetic parameter - k _d [h^–1] first order thermal deactivation rate constant - k _e equilibrium constant - k _m [mole/l] apparent Michaelis constant - k _p [mole/l] Michaelis constant for product - k _s [mole/l] Michaelis constant for substrate - k ₀ [Specific] pre-exponential factor - Q [1/h] volumetric flow rate - ¯Q [1/h] average volumetric flow rate - R [kJ/mol·k] ideal gas constant - s [mole/l] apparent substrate concentration - s [mole/l] substrate concentration - s _e [mole/l] substrate concentration at equilibrium - s ₀ [mole/l] substrate concentration at reactor inlet - p [mole/l] product concentration - p _e [mole/l] product concentration at equilibrium - P _r [mole fructose/l·h] reactor productivity - T [k] temperature - t [h] time - t _p [h] operating time - V [l] reactor volume - v [mole/l·h] reaction rate - v [mole/l] reaction rate under enzyme deactivation and substrate protection - v _m [mole/l·h] maximum apparent reaction rate - v _p [mole/l·h] maximum reaction rate for product - v _s [mole/l·h] maximum reaction rate for substrate - x substrate fractional conversion - x _e substrate fractional conversion at equilibrium Greek Symbols effectiveness factor - mean effectiveness factor - substrate protection factor - [h] residence time - [h] average residence time - ₀ [h] initial residence time     

A mathematical model for solid state fermentation in tray bioreactors

A simple mathematical model for the interaction of mass transport with biochemical reaction in solid state fermentations (SSF) in static tray type bioreactors under isothermal conditions has been developed. The analysis has enabled scientific explanations to a number of practical observations, through the concept of critical substrate bed thickness. The model will be most useful in the prediction of the concentration gradients as also in efficient design of these bioreactors.List of Symbols C g/cm³ Oxygen concentration in the bed - C _g g/cm³ Atmospheric oxygen concentration - C ^* Dimensionless oxygen concentration, C/C _g - D _e cm²/h Effective diffusivity - H cm Bed thickness or height - H _c cm Critical bed thickness or height - H _m cm Maximum height of zone of zero oxygen concentration - p _i mg/(g · h) Productivity (Eq. 13) - R g/(cm³ · h) Biochemical reaction rate - t h Fermentation time - t ^* Dimensionless time, D _e t/H² - X mg/cm³ Biomass concentration - X _max mg/cm³ Maximum biomass concentration - y Dimensionless thickness or height, (y = z/H) - y cm Thickness of zone of zero oxygen concentration (Eq. 12) - Y Yield coefficient - z cm Bed thickness or height along tray axis - Bed void fraction - _max h^–1 Specific growth rate - Thiele modulus      

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Characteristics of Lipase-Catalyzed Glycerolysis of Triglyceride in AOT-Isooctane Reversed Micelles

Chromobacterium viscosum lipase, solubilized in microemulsion droplets of glycerol containing small amounts of water and stabilized by a surfactant, could catalyze the glycerolysis of triolein. Kinetic analysis of the lipase-catalyzed reaction was possible in the reversed micellar system. Among surfactants and organic solvents tested, bis(2-ethylhexyl)sodiumsulfosuccinate (AOT) and isooctane were respectively most effective, for the glycerolysis of triolein in reversed micelles. Temperature effects, pH profile, K_m,app, and V_max,app were determined. Among various chemical compounds, Fe³⁺, Cu²⁺, and Hg²⁺ inhibited the lipase-catalyzed glycerolysis severely. However, the glycerolysis activity was partially restorable by adding histidine or glycine to the system containing these metal ions. The glycerolysis activity was dependent on water content and maximum activity was obtained at an R value of 1.21. Higher stability of the lipase was obtained in the reversed micellar system.     

Purification,characterization and thermostability of lipase from a thermophilic Bacillus sp. J33

A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The K_m and V_max for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 M min^-1 ml^-1 respectively. The immobilized enzyme was stable for 12 h at 60°C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70°C for 150 min in the presence of additives.     

One-dimensional model of the acid hydrolysis of cellulosic substrates in a solid-liquid cyclone reactor

The use of rotating flow in an annulus is investigated as a means of enhancing the yield of glucose and xylose in the acid hydrolysis of cellulosic slurries. A one-dimensional model of such a cyclone reactor is developed for flow cases, co-current and counter-current flow. For the case of 250°C, 1% w/w acid, the one-dimensional model indicates an increase in the maximum glucose yield from 48.1% in a plug flow reactor to 69.3% in a co-current cyclone reactor, and up to 81.0% in a countercurrent cyclone reactor. The corresponding xylose yields are 91.6% for co-current operation and 97.7% for countercurrent operation. In the co-current case the maximum glucose and xylose yields do not occur at the same location in the reactor; however, in the countercurrent case they do. Although product yields are dramatically improved over those obtained in a plug flow reactor, the product concentrations are lower than would typically be obtained in a plug flow reactor.List of Symbols A cm² cross sectional area perpendicular to radial flow - A _c cm² cross sectional area of slurry inlet - A _c cm² cross sectional area of steam inlet - A _w cm² cross sectional area of water inlet - C _c concentration of cellulose as potential glucose (grams of potential glucose/cm³ of total stream) - C _c ^* grams cellulose/cm³ of solids concentration of cellulose as potential glucose - C _ginitial ^* grams glulose/cm³ of solids concentration of cellulose entering reactor - C _g grams glucose/cm³ of total stream concentration of glucose - C _g ^* grams glucose/cm³ of liquid stream concentration of glucose - C _cinitial ^* grams cellulose/cm³ of liquid concentration of glucose entering reactor - C _xn concentration of xylan as potential xylose (grams of potential xylose/cm³ of total stream) - C _xs grams xyclose/cm³ of total stream concentration of nylose - d _f dilution factor - dr cm radial increment - g cm/s² gravitational acceleration - g ^* centrifugal acceleration proportionality constant - h cm height of cyclone reactor - j cm/s flux - K constant in general equation for vortex flow, Eq. (4.9) - k ₁ 1/s kinetic rate constant of cellulose hydrolysis - k _a 1/s kinetic rate constant of xylan hydrolysis - k ₂ 1/s kinetic rate constant of glucose decomposition - k _2a 1/s kinetic rate constant of xylose decomposition - m vortex exponent - M _steam g/s mass rate of steam addition at outer radius - M _water g/s mass rate of cold water addition at outer radius - n cm³/s empirically determined settling parameter - Q cm³/s net volumetric flow in outward radial direction - Q _tot cm³/s total volumetric flow through reactor - q _c cm³/s volumetric flow of slurry feed - q _s cm³/s volumetric flow of stream feed - q _water cm³/s volumetric flow of cold water feed - r cm radial position - r _c 1/s rate of cellulose hydrolysis - r _g 1/s rate of glucose decomposition - r _i cm inner radius - r _o cm outer radius - r _xn 1/s rate of xylan hydrolysis - r _xs 1/s rate of xylose decomposition - s _mom cm g/s² inlet steam momentum - T _bulk s bulk residence time in reactor - T °C reactor temperature - v _c cm³/g specific volume of slurry feed - v _s cm³/g specific volume of steam - v _w cm³/g specific volume of water - V _f cm/s velocity of liquid as a function of radius - V _i cm/s inlet velocity - V _s cm/s velocity of solids as a function of radius - V _steam cm/s inlet steam velocity to cyclone - V cm/s terminal settling velocity - V _q cm/s tangential velocity - w _mom cm g/s² water inlet momentum - Y grams product out/grams reactant in yield of product - solids volumetric fraction - _f solids volumetric fraction in slurry feed - _i initial solids volumetric fraction of slurry - Pi     

Simulation of a multicolumn recirculated packed bed reactor (MRPBR) for penicillin acylase

Enzyme reactors for the industrial hydrolysis of penicillin are analyzed in terms of biocatalyst stability to pH. A multicolumn system with packed beds placed in parallel and operating under recirculating conditions is proposed as an adequate reactor for this process. The system is studied both experimentally and with the aid of a simulation program.List of Symbols A transversal area (cm²) - C _A ammonia concentration in the reaction mixture (M) - C ₁ concentration of KH₂PO₄ in buffer (M) - C ₂ concentration of K₂HPO₄ in buffer (M) - d _p biocatalyst diameter (cm) - E enzyme or biocatalyst concentration (gcat l^–1) - K _APA APA non competitive inhibition constant (M) - K _IS excess substrate inhibition constant (M) - Km constant Michaelis-Menten (M) - K _PAA PAA competitive inhibition constant (M) - Q recirculation flow rate (cm³ min^–1) - Q _T recirculation flow rate per column (cm³ min^–1) - Re Reynolds number - S _E substrate concentration entering the neutralization tank (M) - S ₀ initial substrate concentration (M) - S _T substrate concentration in neutralization tank (M) - t time (min) - v _i initial reactor rate (mol min^–1 gcat^–1) - V _s superficial velocity (cm seg^–1) - V _T volume of neutralization tank (cm³) - X _E substrate conversion entering tank - X _T substrate conversion in neutralization tank - X conversion - Z reactor length (cm) - z axial position in reactor (cm) - z ^* non-dimensional axial position in reactor - biocatalyst's density (gcat cm^–3) - p pressure drop in the packed-bed reactor     

Packed-bed immobilized enzyme reactors for complex processes

Utilization of enzymic reactors for biotechnological-biomedical applications is currently developing at a sustained pace.Our present study concentrates on development of procedures for describing the performance of devices where enzyme-catalyzed reactions between two substrates take place, and for the rational design and optimization of the reactors considered. Within this context, an analytical model was developed for immobilized enzyme packed-bed reactors; it takes into account internal diffusion limitations for the cosubstrates, and hydrodynamic backmixing effects. In order to overcome the complex mathematical problems involved, the compartmental analysis approach was employed.Using this model, performance was simulated for various configurations of the enzymic unit, i.e. from a continuously operated stirred tank reactor (CSTR) to an essentially plug flow type. In addition, an experimental method is described for quantitatively assessing the backmixing effects prevailing in the reactor.The procedures established also provide the ground for further developments, particularly for systems where, in parallel to the enzymic reaction, additional processes (e. g. complexation) take place.List of Symbols C _j,i mM Concentration of substrate j in the pores of stage - iD _j cm²/s Internal (pore) diffusion coefficient of substrate j; defined in Eq. (7) - D _e cm²/s Axial dispersion diffusion coefficient - D _j, cm²/s cm²/s Bulk diffusion coefficient for substrate j - E mM Enzyme concentration inside the catalytic pores - J _j,immol/s/cm² Net flux of substrate j taking place from the bulk of stage i into the corresponding pores; defined in Eq. (6) - K _m,1, K _m,2 mM Michaelis-Menten constants for cosubstrates 1 and 2, respectively - k s ^–1 Catalytic constant - k _s cm/s Catalytic constant - n Total number of elementary stages in the reactor - Q cm³/s Volumetric flow rate throught the reactor - r cm Radius of the pore - R _j,i mM/s Reaction rate of substrate j in stage i, in terms of volumetric units - S cm² Internal surface of a pore - S _j,0 mM Concentration of substrate j in the reactor feed - S _j,i–1, S _j,i mM Concentration of substrate j in the bulk phase leaving stages i — 1 and i, respectivley - V _i cm³ Total volume of stage i (bulk phase + pore phase + inert solid carrier) - V cm³ Total volume of the reactor - V _m ^* mmol/s/cm² Maximal reaction rate in terms of surface units; defined in Eq. (8) - V _m mM/s Maximal reaction rate in terms of volumetric units; defined in Eq. (8) - V _p cm³ Volume of one pore - y cm Axial coordinate of the pores - y ₀ cm Depth of the pores - Z cm Axial coordinate of the reactor - Z ₀ cm Length of the reactor - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - Ratio between the radius of the enzyme molecule and the radius of the pore (dimensionless) - V₁ Dimensionless parameter; defined in Eq. (21) - v₂ Dimensionless parameter; defined in Eq. (21) - Q Volumetric packing density of catalytic particles (dimensionless) - Ø Porosity of the catalytic particles (dimensionless) - Ø Dimensionless concentration of substrate j in pores of stage i; defined in Eq. (16) - _j,i-1,_j,i Dimensionless concentration of substrate j in the bulk phase of stage i; defined in Eq. (18) - Dimensionless position; defined in Eq. (16) - ² s² Variance; defined in Eq. (33) - Mean residence time in the reactor; defined in Eq. (33)     

A simple phenomenological thermodynamic model for protein partitioning in reversed micellar systems

A simple thermodynamic model is developed for the partitioning of proteins between a bulk aqueous solution and a reversed micellar organic phase by assuming that a pseudo-chemical equilibrium is established when proteins in solution interact with a non-integral number of empty micelles to form the protein-micelle complex. From the equilibrium constant for this reaction, which is related to both the chemical and electrical free energy changes associated with the transfer of the proteins between the two phases, a simple expression is derived for the partition coefficient as a function of pH and surfactant concentration. Assumptions include a linear variation in protein net charge with pH, and a linear decrease in protein-micelle complex size with increasing protein charge. Results on the solubilization of ribonuclease-a and concanavalin-a in Aerosol-OT/isooctane organic solvents were well-correlated by the model equation, and the estimated parameters were of the expected order of magnitude as estimated based on the known physical properties of the system components.List of Symbols F C/mol Faraday's constant - G J/mol standard free energy change on solubilization - G J/mol standard free energy change in the absence of charge effects - K partition coefficient - K _eq (mol/m³)^–n equilibrium constant for pseudo-reaction (1) - M micelle - N _ag empty micelle aggregation number - n number of empty micelles required to form protein/micelle complex - n ₀ number of empty micelles required at zero net protein charge - P protein - PM protein/micelle complex - pI protein isoelectric point - R J/mol K gas law constant - S surfactant - z protein charge - slope of protein titration curve - change in micelle size, n, per unit change in charge - V electrostatic potential difference     

Comparative study on hydrolysis of triolein in reversed micellar system catalyzed by different types of lipases

Hydrolysis of triolein in AOT/isooctane reversed micelles by an sn-1,3-regioselective and a non-selective lipase were studied. Kinetics of the multistep reaction: decomposition of tri-, di- and monoacylglycerols and production of fatty acid were investigated separately. All the reactions was found to obey the Michaelis-Menten model and the apparent parameters (Michaelis-constants (K_m) and maximal reaction rates (V_max)) were determined both for non-selective and regioselective preparations.     

Separation process of binary protein solution by ultracentrifuge

Separation process of a binary protein solution by ultracentrifuge with an angle rotor was discussed by considering the calculated distribution of concentration in an ultracentrifugal tube. The weight fraction of the desired protein and the recovery index after the ultracentrifugation were calculated from the distribution of the concentration. When the weight fraction after the ultracentrifugation is given, the optimal ultracentrifugal time was determined so as to maximize the recovery index.List of Symbols c _B kg/cm³ concentration of Bovine serum albumin - c _L kg/cm³ concentration of Lysozyme - D cm²/s diffusion coefficient - d cm diameter of ultracentrifugal tube - R dimensionless collecting range - r ^* dimensionless radial coordinate - r ₁ cm minimum radius of ultracentrifugal tube - r ₂ cm maximum radius of ultracentrifugal tube - s s sedimentation constant - t s ultracentrifugal time - X _L weight fraction of Lysozyme - X _LO initial weight fraction of Lysozyme - Y _L recovery index of Lysozyme - inclination of ultracentrifugal tube - s^–1 angular velocity of rotation     

Substrate-induced stability of the lipase from candida cylindracea in reversed micelles

Summary Activity of lipase (candida cylindracea) in reversed micelles was found to be sustained over extended periods of time in the presence of amphiphilic substrates. Esterification of palmitic or oleic acid and octanol was studied to characterize the lipase activity in AOT/isooctane reversed micelles. Complete conversion was possible even in the presence of stoichiometric excess of water. In the absence of acyl substrates, the enzyme lost all its activity within a few hours in reversed micelles. Thermal effects on the enzyme activity were studied, and the enzyme stability in reversed micelles was compared to that in a bulk organic solvent.     

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ₀ (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C₁₂E₄), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C₁₂E₄ addition. The stabilizing effect of C₁₂E₄ resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C₁₂E₄ may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.     

Two stable electron-conformational states of photoactivated reaction center and their observation in primary donor recovery kinetics

We investigated the peculiarity of primary donor recovery kinetics in the reaction centers from the purple bacteriaRb. Sphaeroides at low levels of their cw photoactivation. A pronounced biphasity of the relaxation kinetics was found for the total light activating intensity <5×10¹² quanta·cm^-2·s^-1. The effect was attributed to strong dependence of an electron transfer rate constant for the reactionP ⁺Q _infA ^pup- Q_B P ⁺Q_AQ _infB ^sup- upon the RC conformational state controlled by the light. We showed the existence of two different electron-conformational states for the photoexcited RC. The first reveals itself at low intensity of cw photoactivation while the second becomes actual under the intensity >5×10¹² quanta · cm^-2 · s^-1.     

The relationship between the redox state of Q A and photosynthesis in leaves at various carbon-dioxide,oxygen and light regimes

The response of chlorophyll fluorescence elicited by a low-fluence-rate modulated measuring beam to actinic light and to superimposed 1-s pulses from a high-fluence-rate light source was used to measure the redox state of the primary acceptor Q _A of photosystem II in leaves which were photosynthesizing under steady-state conditions. The leaves were exposed to various O₂ and CO₂ concentrations and to different energy fluence rates of actinic light to assess the relationship between rates of photosynthesis and the redox state of Q _A. Both at low and high fluence rates, the redox state of Q _A was little altered when the CO₂ concentration was reduced from saturation to about 600 l·l^-1 although photosynthesis was decreased particularly at high fluence rates. Upon further reduction in CO₂ content the amount of reduced Q _A increased appreciably even at low fluence rates where light limited CO₂ reduction. Both in the presence and in the absence of CO₂, a more reduced Q _A was observed when the O₂ concentration was below 2%. Q _A was almost fully reduced when leaves were exposed to high fluence rates under nitrogen. Even at low fluence rates, Q _A was more reduced in shade leaves of Asarum europaeum and Fagus sylvatica than in leaves of Helianthus annuus and Fagus sylvatica grown under high light. Also, in shade leaves the redox state of Q _A changed more during a transition from air containing 350 l·l^-1 CO₂ to CO₂-free air than in sun leaves. The results are discussed with respect to the energy status and the CO₂-fixation rate of the leaves.Abbreviations and symbols L 1,2 first and second actinic light beam - Q _A primary acceptor of photosystem II - q _Q Q-quenching