Effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase in a capillary bed reactor

The effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase were studied in a continuous flow capillary bed reactor. Temperature affects the rates of enzymatic reactions in two ways. Higher temperatures increase the rate of the hydrolysis reaction, but also increase the rate of thermal deactivation of the enzyme. The effect of temperature on the kinetic parameters was studied by performing lactose hydrolysis experiments at 15, 20, 25, 30, and 40 degrees C. The kinetic parameters were observed to follow an Arrhenius-type temperature dependence. Galactose mutarotation has a significant impact on the overall rate of lactose hydrolysis. The temperature dependence of the mutarotation of galactose was effectively modelled by first-order reversible kinetics. The thermal deactivation characteristics of the immobilized enzyme reactor were investigated by performing lactose hydrolysis experiments at 52, 56, 60, and 64 degrees C. The thermal deactivation was modelled effectively as a first order decay process. Based on the estimated thermal deactivation rate constants, at an operating temperature of 40 degrees C, 10% of the enzyme activity would be lost in one year.     

Effects of temperature on lactose hydrolysis by immobilized beta-galactosidase in plug-flow reactor

The hydrolysis of lactose using immobilized beta-galactosidase (from Aspergillus niger) on phenol-formaldehyde resin was studied at temperatures between 8 and 60 degrees C and initial lactose concentrations ranging from 2.5 to 20.0%. A model involving enzyme-galactose complex similar to Michaelis-Menten kinetics with competitive product (galactose) inhibition is suitable to describe the lactose hydrolysis reaction. A small degree of lack of fit between the model and the data was found to be due to the formation of oligosaccharides. Thermal deactivation of lactase follows first-order reaction mechanism. The effect of temperature on the reaction and the deactivation rate constants follows the Arrhenius relationship. The Oligosaccharide formation was not significantly affected by the temperature when the initial lactose concentration was 5%. A design equation for the plug-flow immobilized lactase reactor was developed from the reaction and the deactivation kinetics and was used to find the optimal operating temperature. The optimal temperature was found to be dependent on the operating time but not on the lactose concentration or the conversion. The optimal operating temperature is 60 degrees C when operating time is short but is close to 35 degrees C for a long operating time. A preliminary economic analysis indicates that the optimal operating temperature is 43, 38.5, and 33 degrees C when the operating time is 300 days, 1000 days, and infinity, respectively.     

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.     

A new kinetic model of recombinant beta-galactosidase from Kluyveromyces lactis for both hydrolysis and transgalactosylation reactions

Previous models based on the Michaelis-Menten kinetic equation, that glucose was not used as an acceptor, did not explain our experimental data for lactose conversion by a recombinant beta-galactosidase from Kluyeromyces lactis. In order to create a new kinetic model based on the data, the effects of galactose and glucose on beta-galactosidase activity were investigated. Galactose acted as an inhibitor at low concentrations of galactose and lactose, but did not inhibit the activity of beta-galactosidase at high concentrations of galactose (above 50mM) and lactose (above 100mM). The addition of glucose at concentrations below 50mM resulted in an increased reaction rate. A new model of K. lactis beta-galactosidase for both hydrolysis and transgalactosylation reactions with glucose and lactose as acceptors was proposed. The proposed model was fitted well to the experimental data of the time-course reactions for lactose conversion by K. lactis beta-galactosidase at various concentrations of substrate.     

Kinetic modeling of lactose hydrolysis with an immobilized beta-galactosidase from Kluyveromyces fragilis

The kinetic model of the hydrolysis of lactose with a beta-galactosidase from Kluyveromyces fragilis immobilized on a commercial silica-alumina (KA-3, from Südchemie) has been determined. A wide experimental range of the main variables has been employed: temperature, concentrations of substrate, and products and concentration of enzyme. The runs were performed in a complex buffer with the salt composition of milk. The effect of pH and temperature on the stability and the activity of the enzyme have been studied. The optimum pH for the enzyme activity was, approximately, seven. The immobilized enzyme was more stable than the free one at acidic pH, but more instable at basic pH. The maximum temperature used for the hydrolysis runs performed to select the kinetic model was 40 degrees C, so inactivation of the enzyme during the kinetic runs has been avoided. Agitation, concentration of enzyme in the solid and particle size were selected to ensure that the overall rate was that of the chemical reaction. Eleven kinetic models were proposed to fit experimental data, from first order to more complex ones, such as those taking into account inhibition by one of the compounds involved in the hydrolysis reaction. Applying statistical and physical criteria, a Michaelis-Menten model with a competitive inhibition by galactose has been selected. The model is able to fit the experimental data correctly in the wide experimental range studied. Finally, the model obtained is compared to the one selected in a previous work for the hydrolysis of lactose with the free enzyme.     

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.     

Kinetics of amino acid production from bean dregs by hydrolysis in sub-critical water

Amino acids play an important physiological role in all life-forms and can be recovered from bean dregs waste using sub-critical water hydrolysis. This work deals with the hydrolysis kinetics of bean dregs. Kinetics was conducted in a temperature range of 200–240°C using a 300-ml stainless steel batch reactor. Since the reaction kinetics in sub-critical water is very complicated, a simplified kinetic model to describe the hydrolysis of bean dregs is proposed: a single consecutive reaction. The differential equations resulting from the model were fit to experimental data to obtain kinetic rate constants. By means of the Arrhenius plot, the activation energy as well as the pre-exponential factor was determined. A good agreement between the simplified model and the experimental data was obtained. The kinetic parameters provided useful information for understanding the hydrolysis reaction of bean dregs. The experimental results show that the best hydrolysis technology is: reaction temperature 200°C, reaction time 20 min. Under this condition, the total amino acid yield reaches 52.9%. Based on the results, this method could become an efficient method for bean dregs liquefaction, producing valuable amino acid.     

Optimization of maltodextrin hydrolysis by glucoamylase in a batch reactor

The hydrolysis of maltodextrins (10 DE) by glucoamylase was studied in a batch reactor at temperatures between 40 and 80 degrees C and substrate concentration range from 17 to 300 kg/m(-3). The experimental data were fitted to a model including thermal deactivation of the enzyme. In the model, the reaction rate was correlated with an extended Michaelis-Menten equation including inhibition by product, and the thermal deactivation of glucoamylase was fitted with a first-order reaction. The dependence of rate parameters on temperature was correlated using the Arrhenius equation. The differential equation of the model was integrated and the optimal enzyme demand and temperature were determined for isothermal operation.     

Thermodynamic and kinetic aspects of the hydrolysis of ethyl(R)-2- (benzyloxycarbonylamino)-3-sulfamoylpropionate in the presence of free and immobilized alpha-chymotrypsin

The hydrolysis of ethyl (R)-2-(benzyloxycarbonylamino)-3-sulfamoylpropionate (blocked cysteic acid S-amide) by native and immobilized alpha-chymotrypsin was studied. The experiments were performed using a constant enzyme/substrate ratio of 1:8 and at a temperature of 10-40 degrees C; the immobilized enzyme was bound to a dialdehyde cellulose matrix. A kinetic equation (Eq.10) was found to be applicable which confirms that the mechanism of the enzyme reaction consists of several stages, irrespective of the enzyme state. The temperature dependence of the reaction velocity was investigated and applied using the Arrhenius equation. The constant value thus obtained for the activating energy showed that the active centres retained their character during immobilization. The differences between the velocities of the reaction with immobilized and with native enzyme corresponded to the different number of active centres during the reaction time. Based on these results a kinetic model of the mechanism of the studied reaction is presented which includes an initial balanced stage of the chemosorption type.     

Enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis

The enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis (commercial enzyme Termamyl 300 L Type DX) have been experimentally studied at pH 7.5, within the temperature range of 37-75 degrees C, at initial substrate concentrations of between 0.25 and 2.00 g/L, and enzyme concentrations of between 0.575 x 10(-4) and 13.8 x 10(-4) g/L. To follow the reaction a procedure based on the iodometric method for measuring alpha-amylase activity was used. The kinetics of the enzymatic hydrolysis was fitted to the Michaelis-Menten equation using the integral method, taking into account that the thermal deactivation of the enzyme follows a second-order kinetic. These parameters were fitted to the Arrhenius equation obtaining activation energies of 24.4 and 41.7 kJ/mol and preexponential factors of 734.9 g/L and 1.74 x 10(8) min(-1) for K(M) and k, respectively.     

Kinetics of the alkaline phosphatase catalyzed hydrolysis of disodium p-nitrophenyl phosphate in frozen model systems

The alkaline phosphatase catalyzed hydrolysis of disodium-p-nitrophenyl phosphate was studied in four model systems comprising sucrose, maltodextrin, carboxymethylcellulose (CMC), and CMC-lactose in a temperature range of -28 to 20 degrees C. In the maltodextrin and CMC-lactose model systems, the reaction rate decreased to a very low value as the glass transition temperature was approached. In the CMC and CMC-lactose systems with low initial solute concentration, as a consequence of freeze-concentration, a rate maximum around the initial freezing temperature was observed. The Arrhenius equation described the temperature dependence of the reaction rate both in the liquid and the glassy states in all systems studied, while a slightly curved Arrhenius plot was observed in the "rubbery" state of the CMC and CMC-lactose systems. The WLF equation with system-dependent coefficients described the kinetics in the rubbery state of all the model systems except sucrose, excluding the short temperature range where reaction rate enhancement with decreasing temperature was observed.     

Temperature and pH dependence of mold α-galactosidase

The effect of temperature and pH on kinetic behavior of α-galactosidase of Mortierella vinacea was investigated on the hydrolysis of p-nitrophenyl-α-D -galactopyranoside (PNPG). A very unusual kinetic behavior was observed for the soluble α-galactosidase i.e., substrate inhibition diminished gradually with increasing temperature or near the neutral pH range, and the kinetics approached the ordinary Michaelis-Menten (MM) type. On the other hand, with decreasing temperature or in acidic pH range, substrate inhibition was accelerated. Therefore, Arrhenius plots based on the initial reaction rate did not give straight lines. Furthermore, the slope in the Arrhenius plot changed with substrate concentration, which would make the determination of a characteristic value using conventional methods meaningless. However, the Arrhenius plots of individual kinetic parameters in the rate equation resulted in straight lines in the temperature range 15 to 50°C. From this, the drastic change in kinetic behavior could be explained in connection with the temperature and pH dependence of kinetic parameters in the model. For mold pellets (whole-cell enzyme), however, the influence of temperature and pH was less apparent than that of soluble enzyme because of the limitation in intraparticle diffusion. By using the rate equation that was determined for soluble enzyme and the theoretically derived effectiveness factor, the overall reaction rate for mold pellets at various temperature and pH could be predicted to some extent.     

Kinetic evidence for thermally induced conformational change of butyrylcholinesterase

The effect of temperature on the kinetics of human plasma butyrylcholinesterase-catalyzed reactions was studied. The Arrhenius plot of o-nitrophenylbutyrate hydrolysis presents a break at 21 degrees C. However, nucleophilic competition data indicate that there is no change in the rate-limiting step of the overall reaction. In addition, the temperature dependence of the bimolecular rate constant of enzyme carbamylation shows a break at 18 degrees C. These results argue for the existence of thermally induced conformational active states of the enzyme tetramer. It is suggested that the effects of this transition on kinetics arise at the acylation step.     

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady‐state model for the kinetically controlled synthesis of galacto‐oligosaccharides (GOS) with Aspergillus oryzae β‐galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta‐oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme. Biotechnol. Bioeng. 2011;108: 2270–2279. © 2011 Wiley Periodicals, Inc.     

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.     

Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus

Lactic acid fermentation includes several reactions in association with the microorganism growth. A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus. Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate. During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates. Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose. The inoculum preparation was performed in such a way that healthy young cells were obtained. By using this inoculum, shorter fermentation times with very little lag phase were observed. The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production. A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production. A good fit with a simple equation was obtained. It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9. The Monod kinetic parameters were obtained. The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.     

Reaction kinetics and modeling of the enzyme-catalyzed production of lactosucrose using beta-fructofuranosidase from Arthrobacter sp. K-1

Lactosucrose synthesis from sucrose and lactose was carried out by using beta-fructofuranosidase from Arthrobacter sp. K-1. The transfructosylation mechanism was found to be of an ordered bi-bi type in which sucrose was bound first to the enzyme and lactosucrose was released last. Hydrolysis side-reaction experiments indicated that the reactions were uncompetitively inhibited by glucose and lactose, while no inhibition by fructose was apparent. The overall reaction rates were formulated. The reaction rate constants, equilibrium constant, and dissociation and Michaelis constants were determined at 35 degrees C and 50 degrees C by fitting the experimental concentration changes with the calculated values by a nonlinear least-square method. The average relative derivation for the concentrations was 9.67%. The kinetic parameters were also calculated for 43 degrees C and 60 degrees C by assuming the Arrhenius law, and the course of reaction was predicted. The obtained reaction rate equations well represented the concentration changes during the experiment at all temperatures.     

Kinetics and modeling of temperature effects on batch xanthan gum fermentation

Batch fermentation kinetics of xanthan gum production from glucose by Xanthomonas campestris at temperatures between 22 degrees C and 35 degrees C were studied to evaluate temperature effects on cell growth and xanthan formation. These batch xanthan fermentations were modeled by the logistic equation for cell growth, the Luedeking-Piret equation for xanthan production, and a modified Luedeking-Piret equation for glucose consumption. Temperature dependence of the parameters in this model was evaluated. Growth-associated rate constants increased to a maximum at approximately 30 degrees C and then decreased to zero at approximately 35 degrees C. This temperature effect can be modeled using a square-root model. On the contrary, non-growth-associated rate constants increased with increasing temperature, following the Arrhenius relationship, in the entire temperature range studied. The model developed in this work fits the experimental data very well and can be used in a simulation study. However, due to the empirical nature of the model, the parameter values need to be reevaluated if the model is to be applied to different growth conditions.     

Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides

The aim of this research is to quantify the effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Quantification of these effects is important because temperature and enzyme origin are important process parameters. A kinetic model was used to describe the concentrations in time. The kinetic parameters were determined by using data obtained in batch experiments at various temperatures (20, 30, 40, and 50 degrees C) and by using beta-galactosidases from Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and Kluyveromyces fragilis. The effect of temperature on the kinetic parameters could be described with the Arrhenius equation, except for the inhibition parameter. Slightly higher oligosaccharide yields were found at higher temperatures. However, the influence of the initial lactose concentration was much larger. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Clear differences between the beta-galactosidases were found concerning amount, size, and type of oligosaccharides produced. The beta-galactosidase from B. circulans produced the most abundant amount, the most different, and largest-sized oligosaccharides. The beta-galactosidases from Kluyveromyces spp. produced mainly trisaccharides. The kinetic parameters for the different enzymes were determined and differences were discussed.     

Temperature dependence and mercury inhibition of tonoplast-type H+-ATPase

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H+-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 degrees C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was approximately 14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 degrees C. Lineweaver-Burke analysis of the activities showed that the Km of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg2+. The proton pumping process was more sensitive to the presence of Hg2+. Double-reciprocal analysis of kinetic data indicated that Hg2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H+ pumping and ATP hydrolysis.