Redirecting the inactivation pathway of penicillin amidase and increasing amoxicillin production via a thermophilic molecular chaperone

We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol-water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol-water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the beta-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37 degrees C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol-water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.     

Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis.

An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.     

Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates

Penicillin G acylase (PGA) catalyzes the synthesis/hydrolysis of acyl derivatives of phenylacetic acid through the formation of a covalent intermediate (the acyl–enzyme complex). When used for the kinetically controlled synthesis of β-lactam antibiotics, this enzyme promotes two undesired side reactions: the hydrolysis of the acyl side-chain precursor and of the antibiotic. Therefore, a high selectivity (synthesis/hydrolysis, S/H ratio) is very important for the process economics. Here, the enzymatic synthesis of ampicillin from d-phenylglycine methyl ester (PGME) and 6-aminopenicillic acid (6-APA), using PGA from Escherichia coli (EC 3.5.1.11) is studied. Kinetic assays provided S/H for high concentrations of substrates (up to 200 mM of 6-APA and 500 mM of PGME), using soluble PGA, at 25 °C, pH 6.5. S/H increased with 6-APA concentration, in accordance with the literature. However, when the concentration of 6-APA approached saturation, the rate of enzymatic hydrolysis tended towards zero (i.e., S/H tended to infinity). On the other hand, when the concentration of ester was augmented, S/H consistently decreased. This behavior, to the best of our knowledge still not reported, indicates that the acylation step may occur with 6-APA already positioned for the nucleophilic attack.     

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.     

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin.     

Poly-gamma-glutamate in bacteria

Poly-gamma-glutamate (PGA), a natural polymer, is synthesized by several bacteria (all Gram-positive), one archaea and one eukaryote. PGA has diverse biochemical properties, enabling it to play different roles, depending on the organism and its environment. Indeed, PGA allows bacteria to survive at high salt concentrations and may also be involved in virulence. The minimal gene sets required for PGA synthesis were recently defined. There are currently two nomenclatures depending on the PGA final status: cap, for 'capsule', when PGA is surface associated or pgs, for 'polyglutamate synthase', when PGA is released. The minimal gene sets contain four genes termed cap or pgs B, C, A and E. The PGA synthesis complex is membrane-anchored and uses glutamate and ATP as substrates. Schematically, the reaction may be divided into two steps, PGA synthesis and PGA transport through the membrane. PGA synthesis depends primarily on CapB-CapC (or PgsB-PgsC), whereas PGA transport requires the presence, or the addition, of CapA-CapE (or PgsAA-PgsE). The synthesis complex is probably responsible for the stereochemical specificity of PGA composition. Finally, PGA may be anchored to the bacterial surface or released. An additional enzyme is involved in this reaction: either CapD, a gamma-glutamyl-transpeptidase that catalyses anchorage of the PGA, or PgsS, a hydrolase that facilitates release. The anchoring of PGA to the bacterial surface is important for virulence. All cap genes are therefore potential targets for inhibitors specifically blocking PGA synthesis or anchorage.     

Enzymatic synthesis of high-molecular-mass poly-gamma-glutamate and regulation of its stereochemistry

For the first time, we succeeded in synthesizing in vitro poly-gamma-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookjang. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be approximately 7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg(2+) was essential in catalysis and that Zn(2+) additionally boosted the activity. In contrast, Fe(2+) and Ca(2+) acted as inhibitors. Mn(2+) did not apparently influence the in vitro formation of PGA. DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; d-Glutamate was preferable to the L isomer as a substrate. When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B. subtilis cells, were also indispensable for enzymatic PGA synthesis.     

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.     

Immobilization of the acylase from Escherichia coli on glyoxyl-agarose gives efficient catalyst for the synthesis of cephalosporins

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli, when used in kinetically controlled N-acylation (kcNa) of cephalosporanic nuclei, can be strongly influenced by the moiety in 3-position of the cephem structure. In the synthesis of Cefonicid (1c), the adsorption of the cephalosporanic nucleus (7-SACA) in the PGA active site appeared sensitively increased by a positive ionic interaction between an arginine (ArgA145) in the enzyme active site and the sulphonic group of the β-lactam structure. Interestingly, when PGA was immobilized on solid supports, any effect depending on the substrate structure resulted minimized; the catalytic properties of this enzyme were affected with different outcomes depending on the type of matrix and binding chemistry. The PGA immobilized on glyoxyl-agarose (hydrophilic support activated with aldehyde groups) resulted in a good catalyst when used in kinetically controlled N-acylation of different cephalosporanic nuclei. This derivatives allow much better Vs/Vh(1) (defined as the ratio between the rate of synthesis and the rate of hydrolysis of the acylating agent) than the same enzyme immobilized on Eupergit C, an acrylic hydrophobic supports activated with epoxy groups. The synthetic performances of the Eupergit derivative versus different nuclei were always much poorer if compared with glyoxyl-agarose or the soluble protein. The use of PGA immobilized on glyoxyl-agarose allowed the development of efficient processes for the preparation of Cefazolin in high yield and purity. The results obtained in the optimization of this process are presented.     

Studies on the chemical stability of propylene glycol alginate esters

The chemical stability of propylene glycol alginates (PGAs) has been examined. Under acidic conditions the ester groups in PGA are stable to hydrolysis but hydrolytic degradation of the glycosidic linkages in the polysaccharide backbone occurs. Under alkaline conditions the ester groups are hydrolysed with the primary 2-hydroxyprop-1-yl ester groups being more susceptible than secondary 1-hydroxyprop-2-yl ester groups, with little degradation of the polysaccharide backbone. Sodium carbonate-bicarbonate buffer was a much more effective hydrolysing reagent than sodium hydroxide at the same concentration and pH, and the rate of hydrolysis was greatly accelerated by increasing the hydrolysis temperature. Acetate, citrate and phosphate ions accelerated the rate of hydrolysis of the ester groups in PGA when added to the sodium hydroxide hydrolysing reagent. Hydrolysis of the ester groups in PGA with sodium hydroxide was unaffected by the addition of imidazole. However hydrolysis of the ester groups in PGA with sodium hydroxide in the presence of 1-aminobutane led to the formation of an alginate amide in which only the primary 2-hydroxylprop-1-yl ester groups were present, suggesting that a nucleophilic substitution of primary ester groups by amine groups is involved in the reaction.     

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.     

Modeling of product removal during enzymatic conversions by using affinity molecules

The feasibility of using magnetic particles for in-line product isolation during enzymatic conversion was studied. A comparison was made between a process based on magnetic particles and a conventional adsorption column. The enzymatic reaction was described by two consecutive first-order reactions (synthesis and subsequent hydrolysis), while the adsorption of substrate and product was described by multicomponent Langmuir isotherms. The yield as well as synthesis/hydrolysis ratio were calculated for various system characteristics. The results show that magnetic particles are very effective when the affinity with the particles is specific and for enzymatic conversions involving low ratios of the rate of synthesis versus the rate of hydrolysis. For slow conversions and for low specific affinity molecules column separations are more appropriate.     

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.     

Bisphenol derivative of allysine for high-performance liquid chromatographic analysis of allysine residue of proteins

Allysine is the most important precursor of physiologically essential cross-links formation in collagen and elastin and is formed by enzymatic oxidative deamination of lysine residues. Because it is a highly reactive aldehyde, many cross-linking amino acid residues may arise from its reaction with other allysine residues or lysine or even histidine residues. We purified and isolated an allysine bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenylpentane (ACPP), from the reaction products of phenol and allysine residue of bovine ligamentum nuchae by acid hydrolysis in 6 M HCl. The structure of ACPP was verified by UV, fast atom bombardment-MS, ¹H- and ¹³C-nuclear magnetic resonance spectroscopies. The optimal reaction condition for ACPP synthesis accompanied by hydrolysis of such proteins was investigated and an ion-paired high-performance liquid chromatographic method for determination of allysine as ACPP was also developed.     

Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction

Amoxicillin can be produced in an enzymatic suspension-to-suspension reaction in which the substrate(s) and product(s) are mainly present as solid particles, while the reaction takes place in the liquid phase. During these suspension-to-suspension reactions different subprocesses take place, such as dissolution/crystallization of substrates and products, enzymatic synthesis of the product(s), and undesired enzymatic hydrolysis of substrates and/or products. All these subprocesses are influenced by pH and also influence the pH because the reactants are weak electrolytes. This paper describes a quantitative model for predicting pH and concentrations of reactants during suspension-to-suspension reactions. The model is based on mass and charge balances, pH-dependent solubilities of the reactants, and enzyme kinetics. For the validation of this model, the kinetically controlled synthesis of amoxicillin from 6-aminopenicillanic acid and D-(p)hydroxyphenylglycine methyl ester was studied. The pH and the dissolved concentrations took a very different course at different initial substrate amounts. This was described quite reasonably by the model. Therefore, the model can be used as a tool to optimize suspension-to-suspension reactions of weak electrolytes.     

A New Enzymatic Reaction: Enzyme Catalyzed Ammonolysis of Carboxylic Esters

A new enzymatic reaction of carboxylic esters and ammonia (ammonolysis) was studied. This reaction provides a synthetically useful and mild alternative for the synthesis of amides. Several lipases and one esterase acted as catalyst. Ammonolysis of esters of chiral carboxylic acids gave higher ee values than hydrolysis under comparable reaction conditions. Furthermore, consecutive enzymatic esterification and ammonolysis provided a convenient one-pot synthesis of carboxylic amides from carboxylic acids.     

Insight into molecular mechanism underlying the transesterification catalysed by penicillin G amidase (PGA) using a combination protocol of experimental assay and theoretical analysis

Penicillin G amidase (PGA) is one of the most recognised biocatalysts because of its critical application in the antibiotic industry. Herein, the additive effects involved in transesterification catalysed by PGA are explored in detail using a combination of experimental analysis and theoretical modelling. The transesterification ability of PGA is experimentally determined with 17 N-containing compounds as additives, and, on this basis, a series of quantitative structure–activity relationship (QSAR) models are developed from various physicochemical parameters characterising structural variation over the additives. The resulting models exhibit both good stability and predictive power, from which five most important properties that highlight structural basis and reaction mechanism underlying the transesterification are extracted, revealing that the topological property and electrostatic profile of additives exert a significant effect on reaction yield; the charge distribution around additive molecules is the most significant factor controlling reaction yield, and then the topological structure. Furthermore, it is inferred that the additive imidazole might constitute the catalytic triad of Ser, Glu or Asp involved in PGA active site, which appears similar to lipase, rendering PGA with the catalytic ability of transesterfication. The study highlights the potential application of QSAR methodology in the field of enzymatic regulator design.     

A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation

Alkyl glycosides are surfactants with good biodegradability and low toxicity, attractive to produce by an enzymatic method to get a well-defined product. In this paper, we report a novel thermostable variant of a family 3 beta-glucosidase to be an efficient catalyst in alkyl-glucoside forming reactions using transglycosylation with hexanol or octanol as the acceptor molecule. The enzyme has an apparent optimum for hydrolysis at 90 degrees C, which coincides with its unfolding temperature. The total activity is lower at lower temperature (60 degrees C), but the ratio of alcoholysis/hydrolysis is slightly more favourable. This ratio is however more heavily influenced by the water content and the pH. Optimal reaction conditions for hexyl glucoside synthesis from p-nitrophenyl-beta-glucopyranoside were a water/hexanol two-phase system containing 16% (v/v) water, pH 5.8, and a temperature of 60 degrees C. Under these conditions, the total initial reaction rate was 153 micromol min(-1)mg(-1) and the alcoholysis/hydrolysis ratio was 5.1. Comparing with alcoholysis/hydrolysis ratios of other beta-glycosidases, TnBgl3B can be considered to be a very promising catalyst for alkyl glucoside production.     

Triazene drug metabolites. Part 17: Synthesis and plasma hydrolysis of acyloxymethyl carbamate derivatives of antitumour triazenes

A series of 3-acyloxymethyloxycarbonyl-1-aryl-3-methyltriazenes 5 was synthesised by the sequential reaction of 1-aryl-3-methyltriazenes with (i) chloromethyl chloroformate, (ii) NaI in dry acetone, and (iii) either the silver carboxylate or the carboxylic acids in the presence of silver carbonate. The hydrolysis of these compounds was studied in pH 7.7 isotonic phosphate buffer and in human plasma. Triazene acyloxycarbamates demonstrated their ability to act as substrates for plasma enzymes. For compound 5f, a pH-rate profile was obtained which showed the hydrolysis to involve acid-base catalysis. The reaction is also buffer catalysed. Thus, at pH 7.7, pH-independent, base-catalysed and buffer-catalysed processes all contribute to the hydrolysis reaction. The sensitivity of the hydrolysis reaction to various structural parameters in the substrates indicates that hydrolysis occurs at the ester rather than the carbamate functionality. In plasma, the rates of hydrolysis correlate with partition coefficients, the most lipophilic compounds being the most stable. An aspirin derivative suffers two consecutive enzymatic reactions, the scission of the aspirin acetyl group being followed by the scission of the acyloxy ester group. These results indicate that triazene acyloxymethyl carbamates are prodrugs of the antitumour monomethyltriazenes. They combine chemical stability with a rapid enzymatic hydrolysis, and are consequently good candidates for further prodrug development. Moreover, this type of derivative allowed the synthesis of mutual prodrugs, associating the antitumour monomethyltriazenes with anti-inflammatory NSAIDs as well as with the anticancer agent butyric acid.     

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.