Biomedical subjects

H H Beeftink

Publications and source records attributed to H H Beeftink.

7 recordsLinked to original sources

PubMed2002

Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin.

Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary.

Adsorption↗

PubMed2001

Modeling of the enzymatic kinetic synthesis of cephalexin--influence of substrate concentration and temperature.

During enzymatic kinetic synthesis of cephalexin, an activated phenylglycine derivative (phenylglycine amide or phenylglycine methyl ester) is coupled to the nucleus 7-aminodeacetoxycephalosporanic acid (7-ADCA). Simultaneously, hydrolysis of phenylglycine amide and hydrolysis of cephalexin take place. This results in a temporary high-product concentration that is subsequently consumed by the enzyme. To optimize productivity, it is necessary to develop models that predict the course of the reaction. Such models are known from literature but these are only applicable for a limited range of experimental conditions. In this article a model is presented that is valid for a wide range of substrate concentrations (0-490 mM for phenylglycine amide and 0-300 mM for 7-ADCA) and temperatures (273-298 K). The model was built in a systematic way with parameters that were, for an important part, calculated from independent experiments. With the constants used in the model not only the synthesis reaction but also phenylglycine amide hydrolysis and cephalexin hydrolysis could be described accurately. In contrast to the models described in literature, only a limited number (five) of constants was required to describe the reaction at a certain temperature. For the temperature dependency of the constants, the Arrhenius equation was applied, with the constants at 293 K as references. Again, independent experiments were used, which resulted in a model with high statistic reliability for the entire temperature range. Low temperatures were found beneficial for the process because more cephalexin and less phenylglycine is formed. The model was used to optimize the reaction conditions using criteria such as the yield on 7-ADCA or on activated phenylglycine. Depending on the weight of the criteria, either a high initial phenylglycine amide concentration (yield on 7-ADCA) or a high initial 7-ADCA concentration (yield on phenylglycine amide) is beneficial.

Cephalexin↗

PubMed2000

Modeling solid-to-solid biocatalysis: integration of six consecutive steps.

A quantitative model for the conversion of a solid-substrate salt to a solid-product salt in a batch bioreactor seeded with product crystals is presented. The overall process consists of six serial steps (with dissolution and crystallization each in themselves complex multistep processes): solid-salt dissolution, salt dissociation into an ionic substrate and a counter-ion, bioconversion accompanied by biocatalyst inactivation, complexation of the ionic product with the counter-ion, and salt crystal growth. In the model, the consecutive steps are integrated, including biocatalyst inactivation and assuming that salt dissociation and complexation of ions are at equilibrium. Model parameters were determined previously in separate independent experiments. To validate the model, either dissolved or solid Ca-maleate was converted to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes in a batch bioreactor seeded with Ca-D-malate crystals. The model very well predicted the concentrations of all components in the liquid phase (Ca-maleate, Ca(2+), maleate(2-), D-malate(2-), and Ca-D-malate) and the amounts of the solid phases (Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O), especially when high initial amounts of Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O were present.

Bioreactors↗

PubMed2000

Growth of Ca-D-malate crystals in a bioreactor.

To develop a bioreactor for solid-to-solid conversions, the conversion of solid Ca-maleate to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes was studied. In a bioreactor seeded with product (Ca-D-malate) crystals, growth of Ca-D-malate crystals is the last step in the solid-to-solid conversion and is described here. Crystal growth is described as a transport process followed by surface processes. In contrast to the linear rate law obeyed by the transport process, the surface processes of a crystal-growth process can also obey a parabolic or exponential rate law. Growth of Ca-D-malate crystals from a supersaturated aqueous solution was found to be surface-controlled and obeyed an exponential rate law. Based on this rate law, a kinetic model was developed which describes the decrease in supersaturation due to Ca-D-malate crystal growth as a function of the constituent ions, Ca(2+) and D-malate(2-). The kinetic parameters depended on temperature, but, as expected (surface-controlled), they were hardly affected by the stirring speed.

Bioreactors↗

PubMed2000

D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity.

For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.

Calcium↗

PubMed1998

Ethene removal from gas by recycling a water-immiscible solvent through a packed absorber and a bioreactor.

Hydrophobic pollutants in waste gases are difficult to remove with the conventional biological treatment techniques because of the slow gas/water mass transfer rate. A two-stage system with a water-immiscible solvent as intermediate liquid was developed. This system consisted of a packed absorber for transfer of the model pollutant, ethene, from gas to solvent and a stirred-tank reactor (mixer) for solvent/water transfer and subsequent degradation by Mycobacterium parafortuitum. The solvent FC40, a perfluorocarbon, was recycled between these two compartments. The stability of the system was shown during a run of 10 days. The elimination efficiency was found to be a function of the solvent flow: 9% and 15% elimination were obtained at solvent flows of 6 x 10(-8) m3.s-1 and 11.3 x 10(-8) m3.s-1, respectively. Ethene removal remained constant at increasing solvent hold-ups up to 50% (v/v). In spite of the low elimination efficiencies caused by an inefficient use of the column, the feasibility of the system to remove ethene has been demonstrated. The system's performance was described by a steady-state mathematical model. Simulated ethene removal efficiencies agreed well with the experimental data. Based on this, the model was used to optimise the dimensions and operating conditions. Furthermore, the model was used to compare the performance of the combined system (PA/MS) with the performance of a similar system without solvent. It was found that the use of solvent as intermediate liquid can improve substantially the removal efficiency of hydrophobic gaseous pollutants compared with the system without solvent. This is dependent however, on the solubility of the pollutant in the solvent, on the dimensions of the system and on the operating conditions.

Absorption↗

PubMed1993

Two-liquid-phase bioreactors.

The application of two liquid phases that are poorly miscible is a fascinating research topic for biocatalytical conversions because of the promising results. Motives for application include an increase of productivity and achievement of continuous processing, but new limitations arise, e.g., interfacial effects such as biocatalyst accumulation and loss of activity, medium component accumulation, and slow coalescence. Centrifuges, membranes, and immobilization are tools that can overcome part of the problems, but more fundamental knowledge about interfaces and coalescence is still necessary for successful application. For scaleup and further development of processes based on the obtained results, a choice must be made for the configuration of the experimental setup of a bioreactor. Aspects like aeration, shear stress, batch or continuous processing, and immobilization can play an important role. This review article describes these aspects and the proposals that have been made in recent years concerning two-liquid-phase bioreactors. It shows some adaptations to existing bioreactors, such as loop reactors and stirred-tank reactors.

Biodegradation, Environmental↗