Modelling thermal stability and activity of free and immobilized enzymes as a novel tool for enzyme reactor design.

In this work, a novel method is proposed to establish the most suitable operational temperature for an enzyme reactor. The method was based on mathematical modelling of the thermal stability and activity of the enzyme and was developed using thermodynamic concepts and experimental data from free and immobilized inulinases (2,1-beta-D fructan frutanohydrolase, EC 3.2.1.7) from Kluyveromyces marxianus, which were used as examples. The model was, therefore, designed to predict the enzyme activity with respect to the temperature and time course of the enzymatic process, as well as its half-life, in a broad temperature range. The knowledge and information provided by the model could be used to design the operational temperature conditions, leading to higher enzyme activities, while preserving acceptable stability levels, which represent the link between higher productivity and lower process costs. For the inulinase used in this study, the optimum temperature conditions leading to higher enzyme activities were shown to be 63 degrees C and 57.5 degrees C for the free and immobilized inulinases, respectively. However, according to the novel method of approach used here, the more appropriate operating temperatures would be 52 degrees C for free and 42 degrees C for immobilized inulinases, showing that the working temperature is not necessarily the same as the maximum reaction rate temperature, but preferably a lower temperature where the enzyme is much more stable.

Enhancement of availability of cloricromene at brain level by a lipophilic prodrug.

The pharmacokinetics of a lipophilic alkylamino acid (LAA) prodrug of cloricromene (AD6), name CLOR-C4, was studied in rat plasma and brain. In particular, we observed that the intraperitoneal administration of CLOR-C4 to rats was able to provide a slight but statistically significant higher concentration of the active drug metabolite (cloricromene acid) in the brain compared with the parent drug administered by the same way. The correlation between pharmacokinetic data and calculated partition (LogP) and brain distribution coefficients (LogBB) supported the hypothesis that the amphiphilic nature of the LAA promoiety could be responsible for a better penetration into the brain, more than the simple increase of lipophilicity gained with respect to the parent drug.

Optimization of extracellular lipase production by Geotrichum sp. using factorial design.

Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.

Continuous cephalosporin C purification: dynamic modelling and parameter validation.

A mathematical kinetic model for the adsorption and desorption of cephalosporin C on Amberlite XAD-2 resin is proposed. The model can represent Langmuir, Freundlich or linear isotherms at equilibrium. The intrinsic kinetic parameters and adsorption isotherms as well as physical parameters such as the effective diffusivity and the external mass transfer coefficient were obtained at different temperatures and ethanol concentrations. An unfavourable cephalosporin C adsorption occurred when ethanol was present in the solution. It has been shown that at 25 degrees C the ethanol, at concentrations from 1.5% to 2.5%, decreases the cephalosporin C adsorption. However, this behaviour was not observed at 10 degrees C. The kinetic model fitted the experimental data well under different conditions. The model was validated in a continuous process of cephalosporin C purification using the same resin. The model with the validated parameters is able to predict the behaviour of the reactor system. The continuous process is composed of two stirred tank reactors with adsorber recycle. The adsorption occurs in the first stage, and elution of the product takes place in the second stage with ethanol as eluent. The dynamic behaviour of the process was described using the following parameters: hydraulic residence time for the first (theta(h1)) and second stage (theta(h2)), solid residence time (theta(s)), initial concentration of CPC (C(0)), inlet ethanol concentration (C(ET0)) and kinetics parameters.

Optimization of inulinase production by Kluyveromyces marxianus using factorial design.

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2(5-1)) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K2HPO4), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K2HPO4 had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K2HPO4. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.

Studies on lipase-affinity adsorption using response-surface analysis.

Lipases are widely distributed enzymes that can be obtained from animals, plants and micro-organisms. Coupling lipases with a wide range of substrates allows the opportunity for synthesis of optically pure pharmaceutical preparations, flavour compounds and other food additives. Affinity chromatography owes its power as a purification method to specific biological interactions. Response-surface analysis was chosen to study column efficiency. This method allows the understanding of interactions between variables with advantages over conventional methods, which involve changing one variable while fixing others at certain levels. The aim of this work was to study the influence of the ratio bed height/column diameter (L/D) and superficial velocity (V) on the column efficiency. The experimental design involved the two variables, L/D (2-10) and v (1-2 cm/min), at five levels. Lipase was obtained from Geotrichum sp. culture in a complex medium composed of 5% corn-steep liquor, 0.5% NH(4)NO(3) and 1% olive oil at 30 degrees C, with 1VVM (air volume/medium volume per min) aeration and 400 rev./min agitation. Maximum lipase activity was 19 units/ml after almost 9 h of fermentation. This lipase could potentially be used in esterification reactions to increase the content of gamma-linolenic acid and to produce bioaromas for food industries. The adsorption assays were carried out in a fixed-bed column with an affinity adsorbent, which was obtained by reaction of a gel with oleic acid as ligand. Breakthrough curves were obtained for all experiments. It has been shown that the lower the values of both L/D and v, the higher the column efficiency (maximum 65.43%). Also, it was observed from the response surface that the efficiency reached a minimum at an L/D of around 8.

Kinetics of ethanol fermentation with high biomass concentration considering the effect of temperature.

A model of ethanol fermentation considering the effect of temperature was developed and validated. Experiments were performed in a temperature range from 28 to 40 degrees C in continuous mode with total cell recycling using a tangential microfiltration system. The developed model considered substrate, product and biomass inhibition, as well as an active cell phase (viable) and an inactive (dead) phase. The kinetic parameters were described as functions of temperature.

Simulated dynamics and control of an extractive alcoholic fermentation.

In this study, we investigated the dynamics of a computer simulation of a continuous alcoholic fermentation process combined with a flash column under vacuum. The alcohol was partially extracted in order to maintain its concentration at about 40 kg/m3 in the fermentor. The mathematical model of the fermentation was developed for industrial conditions and considers the effect of the temperature on the kinetic parameters. The performance of the dynamic matrix control algorithm, single input single output and multiple input multiple output, for the control of the extractive process was studied. The concepts of factorial design were used in a simulation study to determine the best control structures for the process.