A new biocatalyst: Penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties

The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 μm, as determined by scanning electron microscopy. An immobilization yield of 95–100% and a recovered activity of 50–65% at 37°C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g_{dry weight}) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14°C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.     

Penicillin G acylase catalyzed acylation of 7-ACA in aqueous two-phase systems using kinetically and thermodynamically controlled strategies: improved enzymatic synthesis of 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ3-cephem-4-carboxylic acid

Penicillin G acylase (PGA) catalyzed acylation of 7-aminocephalosporanic acid (7-ACA) with R-mandelic acid and its derivatives gives 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ³-cephem-4-carboxylic acid. This compound is a useful intermediate for the synthesis of some 3′-functionalized cephalosporins. However, acylations catalyzed by PGA isolated from Escherichia coli give poor results both considering a kinetical or a thermodynamical approach. In order to improve this enzymatic acylation, polyethylene glycol (PEG 600)-ammonium sulphate aqueous two-phase systems have been studied with the aim to have, during the reaction, a continuous extraction of the acylation product outside of the enzyme environment (the ammonium sulphate phase). This strategy shifts the equilibrium in the thermodynamically controlled synthesis and prevents the hydrolysis of the synthesized antibiotic in the kinetically controlled synthesis. The best results were achieved using PEG 600 (80% in water) equilibrated with 4 M ammonium sulphate. In these conditions, the acylation product was completely partitioned in the PEG phase (K > 200), whereas the substrates maintained a suitable concentration in the enzyme environment. Both in the kinetic (88% yield) and the thermodynamic (75% yield) processes, the results obtained were sensitively improved in comparison with those achieved working in homogeneous solution (phosphate buffer). Using R-mandelic acid methyl ester, the yield increased from 65% (monophasic system) to 88%. The PEG solution, without isolation of the acylation product, was successfully used for the synthesis of Cefamandole and Cefonicid.     

Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzyme-binding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3 degrees C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions

The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated. Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports. Butyl methacrylate (BMA) was used as graft monomer. Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme. Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields. By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent. Cephalexin synthesis by the catalytic membrane was also performed in a non-isothermal bioreactor. Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained. The addition of methanol reduced the non-isothermal activity increase. The physical cause responsible for the non-isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis.     

Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH? group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.     

Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.     

Improved specific productivity in cephalexin synthesis by immobilized PGA in silica magnetic micro‐particles

There is a marked trend in pharmaceutical industry towards the replacement of classical organic methods by “green” alternatives that minimize or eliminate the generation of waste and avoid, where possible, the use of toxic and/or hazardous reagents and solvents. In this work the kinetically controlled synthesis of cephalexin by soluble and penicillin G acylase immobilized in sol–gel micro‐particles with magnetic properties was performed in aqueous media with PGME and 7‐ADCA as substrates, at different concentrations of substrate, temperature, pH, enzyme to substrate ratio and acyl donor to nucleophile ratio. Excess acyl donor had a strong effect on cephalexin productivity. A PGME/7‐ADCA ratio of 3 was considered optimum. A maximum specific productivity of at 160 mM 7‐ADCA, 480 mM PGME and low enzyme to substrate ratio at 32.5 U mmol^?1 7‐ADCA was obtained with immobilized PGA in full aqueous medium, suggesting that diffusional limitations were minimized when compared with other commercial biocatalysts. A half‐life of 133 h for the immobilized biocatalyst was estimated during cephalexin synthesis in the presence of 100 mM 7‐ADCA and 300 mM PGME, in 50 mM Tris/HCl at pH 7.2 and 14°C. These results compare quite favorably with those previously reported for the kinetically controlled synthesis of cephalexin. Biotechnol. Bioeng. 2010;107: 753–762. © 2010 Wiley Periodicals, Inc.     

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.     

A multicomponent reaction–diffusion model of a heterogeneously distributed immobilized enzyme

A physical model was derived for the synthesis of the antibiotic cephalexin with an industrial immobilized penicillin G acylase, called Assemblase. In reactions catalyzed by Assemblase, less product and more by-product are formed in comparison with a free-enzyme catalyzed reaction. The model incorporates reaction with a heterogeneous enzyme distribution, electrostatically coupled transport, and pH-dependent dissociation behavior of reactants and is used to obtain insight in the complex interplay between these individual processes leading to the suboptimal conversion. The model was successfully validated with synthesis experiments for conditions ranging from heavily diffusion limited to hardly diffusion limited, including substrate concentrations from 50 to 600 mM, temperatures between 273 and 303 K, and pH values between 6 and 9. During the conversion of the substrates into cephalexin, severe pH gradients inside the biocatalytic particle, which were previously measured by others, were predicted. Physical insight in such intraparticle process dynamics may give important clues for future biocatalyst design. The modular construction of the model may also facilitate its use for other bioconversions with other biocatalysts.     

Cephalexin synthesis by partially purified and immobilized enzymes

An enzyme which catalyzes the synthesis of cephalexin fromD -α phenylglycinemethylester (PGM) and 7-amino-3-desacetoxy-cephalosporanic acid (7-ADCA) was prepared from Xanthomonas citri (IFO 3835) and partially purified 30-fold by ammonium sulfate fractionation, DEAE-cellulose, and Sepharose-4B column chromatography. The K_m values for 7-ADCA, PGM, and cephalexin were determined as 11.1, 2.1, and 1.61 mM, respectively. The enzymatic cephalexin synthesis follows the reversible bi-uni reaction kinetics. The equilibrium constant is influenced by the initial mole ratios of 7-ADCA and PGM. The cephalexin hydrolysis is catalyzed by the same cephalexin synthesizing enzyme, but methanol does not participate in the hydrolytic reaction. The amount of enzyme in the reaction mixture affects the initial rate but does not influence the equilibrium product concentration. This cephalexin-synthesizing enzyme was immobilized onto several adsorbents. Among these, Kaolin and bentonite showed a higher retention of enzyme activity and stability for reuse. The immobilized-enzyme reaction kinetics were investigated and compared with those of the soluble enzyme. A rate expression for the enzymatic synthesis of cephalexin was derived. The results of computer simulation showed good agreement with the experimental results.     

Mandelic acid chiral separation utilizing a two-phase partitioning bioreactor built by polysulfone microspheres and immobilized enzymes

A novel two-phase partitioning bioreactor (TPPB) modified by polysulfone (PSF) microspheres and immobilized enzyme (novozym-435) was formed, and the resulting TPPB was applied into mandelic acid chiral separation. The PSF microspheres containing n-hexanol (named PSF/hexanol microspheres) was prepared by using the phase inversion method, which was used as the organic phase. Meanwhile, the immobilized enzyme novozym-435 was used as a biocatalyst. The water phase was composed of the phosphate buffer solution (PBS). (R, S)-Methyl mandelate was selected as the substrate to study enzymatic properties. Different reaction factors have been researched, such as pH, reaction time, temperature and the quantity of biocatalyst and PSF/hexanol microspheres added in. Finally, (S)-mandelic acid was obtained with an 80 % optical purity after 24 h in the two-phase partitioning bioreactor. The enantiomeric excess (ee_p) values were very low in the water phase, in which the highest ee_p value was only 46 %. The ee_p of the two-phase partitioning bioreactor had been enhanced more obviously than that catalyzed in the water phase.     

Production of lipase from Pseudomonas gessardii using blood tissue lipid and thereof for the hydrolysis of blood cholesterol and triglycerides and lysis of red blood cells

The study demonstrates the production of lipase (LIP) from Pseudomonas gessardii using blood tissue lipid as the substrate for the hydrolysis of blood cholesterol and triglycerides. The lipase was purified with the specific activity of 828 U/mg protein and the molecular weight of 56 kDa. The maximum lipase activity was observed at the pH 7.0 and the temperature 37 °C. The amino acid composition of purified lipase was determined by HPLC. The mesoporous activated carbon (MAC) was used for the immobilization of lipase for the repeated use of the enzyme catalyst. The K (m) value of immobilized lipase (MAC-LIP) and the free lipase (LIP) was 0.182 and 1.96 mM, respectively. The V (max) value of MAC-LIP and LIP was 1.33 and 1.26 mM/min, respectively. The MAC and MAC-LIP were characterized by scanning electron microscopy (SEM). The hydrolysis study showed 78 and 100% hydrolysis of triglycerides and cholesterol, respectively, for LIP and 84 and 100% hydrolysis of triglycerides and cholesterol, respectively, for MAC-LIP at the reaction time of 1 h. The effect of lipase on cell wall lysis was carried out on the RBCs of blood plasma. Interestingly, 99.9% lysis of RBCs was observed within 2 h. SEM images and phase contrast microscopy confirmed the lysis of RBCs. This work provides a potential biocatalyst for the hydrolysis of blood cholesterol and triglycerides.     

Preparation of optically active D-arylglycines for use as side chains for semisynthetic penicillins and cephalosporins using immobilized subtilisins in two-phase systems

Optically active D-arylglycines, which are of interest for preparation of semisynthetic penicillins and cephalosporins, were isolated from the racemic mixtures of their derivatives using immobilized proteolytic enzyme subtilisin (EC No. 3.4.4. 16). The performance of these reactions in two-phase systems, consisting of water and an immiscible organic solvent, improved the yield, purity, and economics of the process by increasing the substrate solubility and reducing the rate of nonenzymatic hydrolysis. The proportion of the organic phase can be as much as 75% of the overall volume without seriously impairing the enzymatic activity. The optically pure D-and L-arylglycines were liberated from their D- and L-derivatives by acid hydrolysis. The substituent influence of the various arylglycine derivatives on the rate of the enzymatic cleavage reaction was investigated.     

Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase

Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. In addition, the difficulty in the recovery of cellulase also leads to high cost for the enzymatic hydrolysis of cellulose. In this study, enzymatic degradation of cellulose was carried out in pH–pH recyclable aqueous two-phase systems (ATPS) composed by copolymers poly (AA-co-DMAEMA-co-BMA) (abbreviated P_ADB3.8) and poly (MAA-co-DMAEMA-co-BMA) (abbreviated P_MDB). In the systems, cellulase was immobilized on pH-response copolymer P_MDB by using 1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in the systems was 2.45, in the presence of 40 mM (NH₄)₂SO₄. Insoluble substrate and immobilized enzyme were biased to bottom phase, while the product was partitioned to top phase. Microcrystalline cellulose was hydrolyzed into reducing sugar, and the product entered into top phase. The yield of saccharification in ATPS could reach 70.57% at the initial substrate concentration of 0.5% (w/v), and the value was 9.3% higher than that in the single aqueous phase. Saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

Extraction of cephalosporin C from whole broth and separation of desacetyl cephalosporin C by aqueous two-phase partition

Cephalosporin C was extracted from diluted or whole broth by PEG/salt aqueous two-phase systems. Parameters such as PEG molecular weight, salt type, pH, and salt concentration were investigated for finding a suitable extraction system. In PEG 600/ammonium sulfate or phosphate systems, K(c) (partition coefficienct of cephalosporin C) was observed to be larger than 1, with K(d) (partition coefficient of desacetyl cephalosporin C) being smaller than 1. The particular values of these coefficients would imply that the difficult separation of cephalosporin C and desacetyl cephalosporin C could possibly be achieved via the aqueous two-phase extraction. The addition of surfactants, water-miscible solvents, and neutral salts for enhancement of the separation efficiency was also investigated. The addition of surfactants to the system did not affect the separation efficiency substantially. K(c) would increase whereas K(d) decreased as a result of the addition of acetone, MeOH, EtOH, IPA, and n-BuOH. Meanwhile both K(c) and K(d) would decrease whenever neutral salts, NaCl, KCl, Kl, or KSCN, were added. The partitioning behavior of cephalosporin C and desacetyl cephalosporin C in filtered, whole, and different batches of broth was notably quite similar to that of diluted broth. The recovery yield of cephalosporin C in whole broth extraction was observed to be a function of centrifugal force used in phase separation. (c) 1994 John Wiley & Sons, Inc.     

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.     

Hydrogel coated monoliths for enzymatic hydrolysis of penicillin G

The objective of this work was to develop a hydrogel-coated monolith for the entrapment of penicillin G acylase (E. coli, PGA). After screening of different hydrogels, chitosan was chosen as the carrier material for the preparation of monolithic biocatalysts. This protocol leads to active immobilized biocatalysts for the enzymatic hydrolysis of penicillin G (PenG). The monolithic biocatalyst was tested in a monolith loop reactor (MLR) and compared with conventional reactor systems using free PGA, and a commercially available immobilized PGA. The optimal immobilization protocol was found to be 5 g l(-1) PGA, 1% chitosan, 1.1% glutaraldehyde and pH 7. Final PGA loading on glass plates was 29 mg ml(-1) gel. For 400 cpsi monoliths, the final PGA loading on functionalized monoliths was 36 mg ml(-1) gel. The observed volumetric reaction rate in the MLR was 0.79 mol s(-1) m(-3) (monolith). Apart from an initial drop in activity due to wash out of PGA at higher ionic strength, no decrease in activity was observed after five subsequent activity test runs. The storage stability of the biocatalysts is at least a month without loss of activity. Although the monolithic biocatalyst as used in the MLR is still outperformed by the current industrial catalyst (immobilized preparation of PGA, 4.5 mol s(-1) m(-3) (catalyst)), the rate per gel volume is slightly higher for monolithic catalysts. Good activity and improved mechanical strength make the monolithic bioreactor an interesting alternative that deserves further investigation for this application. Although moderate internal diffusion limitations have been observed inside the gel beads and in the gel layer on the monolith channel, this is not the main reason for the large differences in reactor performance that were observed. The pH drop over the reactor as a result of the chosen method for pH control results in a decreased performance of both the MLR and the packed bed reactor compared to the batch system. A different reactor configuration including an optimal pH profile is required to increase the reactor performance. The monolithic stirrer reactor would be an interesting alternative to improve the performance of the monolith-PGA combination.     

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.