Hydrolysis Of Penicillin Research Articles

Label-Free Photonic Crystal-Based β-Lactamase Biosensor for β-Lactam Antibiotic and β-Lactamase Inhibitor

A simple, label-free, and visual photonic crystal-based β-lactamase biosensor was developed for β-lactam antibiotic and β-lactamase inhibitor in which the penicillinase (a β-lactamase) was immobilized on the pH-sensitive colloidal crystal hydrogel (CCH) film to form penicillinase colloidal crystal hydrogel (PCCH) biosensing film. The hydrolysis of penicillin G (a β-lactam antibiotic) can be catalyzed by penicillinase to produce penicilloic acid, leading to a pH decrease in the microenvironment of PCCH film, which causes the shrink of pH-sensitive CCH film and triggers a blue-shift of the diffraction wavelength. Upon the addition of β-lactamase inhibitor, the hydrolysis reaction is suppressed and no clear blue-shift is observed. The concentrations of β-lactam antibiotic and β-lactamase inhibitor can be sensitively evaluated by measuring the diffraction shifts. The minimum detectable concentrations for penicillin G and clavulanate potassium (a β-lactamase inhibitor) can reach 1 and 0.1 μM, respectively. Furthermore, the proposed method is highly reversible and selective, and it allows determination of penicillin G in fish pond water samples.

Kinetic characterization of GES-22 β-lactamase harboring the M169L clinical mutation.

The class A β-lactamase GES-22 has been identified in Acinetobacter baumannii isolates in Turkey, and subsequently shown to differ from GES-11 by a single substitution (M169L). Because M169 is part of the omega loop, a structure that is known to have major effects on substrate selectivity in class A β-lactamases, we expressed, purified and kinetically characterized this novel variant. Our results show that compared with GES-116 × His, GES-226 × His displays more efficient hydrolysis of penicillins, and aztreonam, but a loss of efficiency against ceftazidime. In addition, the M169L substitution confers on GES-22 more efficient hydrolysis of the mechanistic inhibitors clavulanic acid and sulbactam. These effects are highly similar to other mutations at the homologous position in other class A β-lactamases, suggesting that this methionine has a key structural role in aligning active site residues and in substrate selectivity across the class.

A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase.

Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.

Identification of peptide inhibitors of penicillinase using a phage display library

There is a constant need to identify novel inhibitors to combat β-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM−1) and binding affinity (KD = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV–vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a Ki of 9.22 μM and a Ki′ of 33.12 μM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of β-lactamases and development of novel inhibitors of β-lactamases.

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

This work presented a highly sensitive bacterial antibiotic susceptibility test through β-lactamase assay using Parylene-matrix chip. β-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to β-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate β-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/z<500). The β-lactamase assay for the Escherichia coli antibiotic susceptibility test was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a β-lactamase assay with minimal interference of low molecular weight noise peaks. The β-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay.

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.

Application of capillary electrophoresis to the simultaneous determination and stability study of four extensively used penicillin derivatives

The applicability of capillary electrophoresis for the analysis of four extensively used penicillin derivatives (benzylpenicillin, ampicillin, amoxicillin, oxacilllin) has been studied. Because of structural similarities, the electrophoretic behavior of these derivatives is very similar; consequently an efficient separation using the conventional capillary zone electrophoresis is hard to be achieved. Their simultaneous separation was solved by using micellar electrokinetic capillary chromatography, the separation being based on the differential partition of the analytes between the micellar and aqueous phase. Using a buffer solution containing 25 mM sodium tetraborate and 100 mM sodium dodecyl sulfate as surfactant, at a pH of 9.3, applying a voltage of + 25 kV at a temperature of 25 °C, we achieved the simultaneous separation of the studied penicillin derivatives in less then 5 minutes. The separation conditions were optimized and the analytical performance of the method was evaluated in terms of precision, linearity, limit of detection, and quantification. Also, a simple capillary zone electrophoresis method was applied to study the stability of the studied penicillin derivatives in water at different temperatures, using ciprofloxacin hydrochloride as internal standard. It was observed that the extent of the hydrolysis of penicillins in water is highly dependent on the time and also temperature.

Enzymatic hydrolysis of penicillin and in situ product separation in thermally induced reversible phase-separation of ionic liquids/water mixture

Enzymatic hydrolysis of penicillin G to produce 6-aminopenicillanic acid, key intermediate for the production of semisynthetic β-lactam antibiotics, is one of the most relevant example of industrial implementation of biocatalysts. The hydrolysis reaction is traditionally carried out in aqueous buffer at pH 7.5–8. However, the aqueous rout exhibits several drawbacks in enzyme stability and product recovery. In this study, several ionic liquids (ILs) have been used as media for enzymatic hydrolysis of penicillin G. The results indicated that hydrophobic ILs/water two-phase system were good media for the reaction. In addition, a novel aqueous two-phase system based on the lower critical solution temperature type phase changes of amino acid based ILs/water mixture was developed for in situ penicillin G hydrolysis and product separation. For instance, hydrolysis yield of 87.13% was obtained in system containing 30wt% [TBP][Tf-ILe] with pH control (pH 7.6). Since the phase-separation of this medium system can be reversible switched from single to two phases by slightly changing the solution temperature, enzymatic hydrolytic reaction and product recovery were more efficient than those of aqueous system. In addition, the ILs could be reused for at least 5 cycles without significant loss in hydrolysis efficiency.

Substitutions in the Ω‐Loop of TEM β‐Lactamase Change the Substrate Profile of the Enzyme (LB140)

Serine β‐lactamases are bacterial enzymes that hydrolyze β‐lactam antibiotics. Mechanistically, serine β‐lactamases are similar to serine proteases such as chymotrypsin. They both utilize acylation and deacylation of an active site serine in their mechanism of catalysis. TEM‐1, a common plasmid‐encoded serine β‐lactamase, catalyzes the hydrolysis of early penicillins and cephalosporins. Here we examine a previously identified triple mutant of TEM‐1, 165‐TyrTyrGly‐167 (wild type165‐TrpGluPro‐167) with substrate specificity altered from preferential hydrolysis of ampicillin to that of ceftazidime. When compared to chymotrypsin, the Glu166Tyr substitution in the TEM‐1 triple mutant is analogous to a substitution of the general base His57 in chymotrypsin that results in an enzyme that maintains function. Our findings agree with previous observations of altered substrate specificity of the triple mutant, which displays increased hydrolysis of ceftazidime. Additionally, enzyme kinetic analyses show that the hydrolysis of ceftazidime follows a branched pathway characteristic of substrate‐induced reversible inactivation. Structural analysis of the triple mutant reveals enlargement of the active site making it more accessible for larger substrates. Furthermore, in the crystal structure of the triple mutant, Tyr166 is within hydrogen bond distance to Ser70 suggesting involvement in the catalytic mechanism of the enzyme. These findings indicate that Glu166 general base residue can be substituted in the mechanism of serine β‐lactamases. More generally, this provides insights into alternate mechanisms for reactions catalyzed by serine hydrolases.Grant Funding Source: Supported by NIH

Thin-Layer Polymer Wrapped Enzymes Encapsulated in Hierarchically Mesoporous Silica with High Activity and Enhanced Stability

A novel soft-hard cooperative approach was developed to synthesize bioactive mesoporous composite by pre-wrapping Penicillin G amidase with poly(acrylaimde) nanogel skin and subsequently incorporating such Penicillin G amidase nanocapsules into hierarchically mesoporous silica. The as-received bioactive mesoporous composite exhibited comparable activity and extraordinarily high stability in comparison with native Penicillin G amidase and could be used repetitively in the water-medium hydrolysis of penicillin G potassium salt. Furthermore, this strategy could be extended to the synthesis of multifunctional bioactive mesoporous composite by simultaneously introducing glucose oxidase nanocapsules and horseradish peroxidase nanocapsules into hierarchically mesoporous silica, which demonstrated a synergic effect in one-pot tandem oxidation reaction. Improvements in the catalytic performances were attributed to the combinational unique structure from soft polymer skin and hard inorganic mesoporous silica shell, which cooperatively helped enzyme molecules to retain their appropriate geometry and simultaneously decreased the enzyme-support negative interaction and mass transfer limitation under heterogeneous conditions.

A low detection limit penicillin biosensor based on single graphene nanosheets preadsorbed with hematein/ionic liquids/penicillinase

In this study, we reported on a low detection limit penicillin biosensor with layer-by-layer (LbL) film containing single-graphene nanosheets (SGNs) preadsorbed with hematein, ionic liquids (ILs) and penicillinase. The penicillinase catalyzes the hydrolysis of penicillin to penicilloic acid, where H+ is liberated and monitored amperometrically with hematein as a pH indicator. The SGN-hematein/ILs/penicillinase biosensor exhibited excellent performance for penicillin in PBS with a wide range from 1.25×10−13 to 7.5×10−3M, and a low detection limit of 10−13M (0.04ppt, S/N≥3). Furthermore, the detection of penicillin concentration in real sample (milk) had acceptable accuracy with the assay system.

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

BackgroundPenicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits.ResultsThis work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively.ConclusionsTo the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.

Penicillinase-based amperometric biosensor for penicillin G

A biosensor for penicillin G was created by immobilizing penicillinase to a gold electrode by means of a cysteine self-assembled monolayer. The biosensor amperometrically monitored the catalytic hydrolysis of penicillin in a very sensible manner. Furthermore, it was successfully used to measure the Michaelis–Menten enzymatic constant and a low limit of detection of 4.5nM was obtained.

Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila.

Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and β chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, β = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, β = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.

Covalent immobilization of penicillin G acylase onto amine‐functionalized PVC membranes for 6‐APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

AbstractThis article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde‐activated NH2‐PVC membranes. The immobilized enzyme was used for 6‐aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters KM and VM were determined. KM of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Covalent immobilization of penicillin G acylase onto chemically activated surface of poly(vinyl chloride) membranes for 6‐penicillic acid production from penicillin hydrolysis process I. Optimization of surface modification and its characterization

AbstractThe covalent immobilization of penicillin G acylase (PGA) onto the surface of NH2–poly(vinyl chloride) (PVC) membranes was studied. PGA was chosen because it plays a relevant role in the pharmaceutical industry, catalyzing the production of an important intermediate for the industrial production of semisynthetic penicillin and cephalosporine. Because PVC has no functional groups in its structure, in this work, we focused on the functionalization of PVC with primary amine functional groups for the covalent immobilization of PGA. This goal was achieved through an aminoalkylation process of the surface of the PVC membranes with ethylene diamine followed by activation with glutaraldehyde to finally immobilize the enzyme. Different factors affecting the modification and activation processes were studied, and their impacts on the catalytic activity of the immobilized PGA were followed. The functionalized membranes were characterized with Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy to verify the modification process. In addition, the changes resulting from the modification in physical characteristics, such as surface roughness, water uptake, and mechanical properties, were monitored. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Improved X-ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration.

Penicillin G acylase from Bacillus megaterium (BmPGA) is currently used in the pharmaceutical industry as an alternative to PGA from Escherichia coli (EcPGA) for the hydrolysis of penicillin G to produce 6-aminopenicillanic acid (6-APA), a penam nucleus for semisynthetic penicillins. Despite the significant differences in amino-acid sequence between PGAs from Gram-positive and Gram-negative bacteria, a representative PGA structure of Gram-positive origin has never been reported. In this study, crystallization and diffraction studies of BmPGA are described. Poor diffraction patterns with blurred spots at higher resolution were typical for BmPGA crystals cryocooled after a brief immersion in cryoprotectant solution. Overnight soaking in the same cryo-solution substantially improved both the mosaicity and resolution limit through the establishment of a new crystal-packing equilibrium. A crystal of BmPGA diffracted X-rays to 2.20 Å resolution and belonged to the monoclinic space group P2(1) with one molecule of BmPGA in the asymmetric unit.

Characterization of a new Bacillus cereus ATUAVP1846 strain producing penicillin V acylase, and optimization of fermentation parameters

Penicillin acylases are involved chiefly in the industrial production of semisynthetic penicillins, which remain the most widely used group of antibiotics. We have isolated a new bacterial strain ATUAVP1846 that produces penicillin V acylase (PVA). Phylogenetic analysis using 16S rRNA sequencing showed 99.37% homology with Bacillus cereus. Maximum PVA production was observed with B. cereus ATUAVP1846 at 30°C, pH 7 after 24 h fermentation time under submerged conditions. Highest enzyme productivity was achieved using sucrose as carbon source, and tryptone and ammonium hydrogen phosphate as nitrogen sources. Minimal medium containing 0.4% glucose and 0.3% ammonium hydrogen phosphate was found to be optimal for maximum PVA production from B. cereus ATUAVP1846. The crude enzyme from B. cereus ATUAVP1846 was partially purified using ammonium sulfate fractionation and showed highest enzymatic activity in the hydrolysis of penicillin V at 40°C and pH 6. The crude enzyme preparation also showed unique substrate specificity, preferring ampicillin and cephalexin over penicillin V.

Ceftazidime-hydrolysing -lactamase OXA-145 with impaired hydrolysis of penicillins in Pseudomonas aeruginosa

To describe a novel extended-spectrum oxacillinase, named OXA-145, differing from narrow-spectrum OXA-35 (from the OXA-10 group) by deletion of residue Leu-165. The genetic environment of bla(OXA-145) and the biochemical properties of OXA-145 are reported. We also assessed the impact of the Leu-165 deletion on the hydrolysis spectrum of the ancestor OXA-10. Extended-spectrum β-lactamase OXA-145 was identified in the multidrug-resistant clinical Pseudomonas aeruginosa 08-056, and characterized by isoelectric focusing, PCR and DNA sequencing. Antibiotic susceptibility tests were performed by agar dilution. The resistance profiles conferred by cloned bla(OXA-10), bla(OXA-35), bla(OXA-145) and a bla(OXA-10) derivative obtained by site-directed mutagenesis were determined in Escherichia coli. Kinetic parameters of OXA-35 and OXA-145 were established after purification of His-tagged proteins. The sequence of OXA-145, encoded by a class 1 integron-borne gene in strain 08-056, differed from that of narrow-spectrum penicillinase OXA-35 by a single amino acid deletion (Leu-165) located in the highly conserved omega loop. Deletion of Leu-165 from OXA-35 (yielding OXA-145) or OXA-10 (the progenitor of OXA-35) extended the hydrolysis spectrum to third-generation cephalosporins and to monobactams, while reducing that for penicillins. OXA-145 showed biphasic hydrolysis curves for all the substrates tested. Its activity against nitrocefin was 10-fold higher in the presence of sodium hydrogen carbonate. OXA-145 is a new extended-spectrum β-lactamase from the OXA-10 group. The deletion of Leu-165 is responsible for a shift in the hydrolysis spectrum from penicillins to third-generation cephalosporins, as well as monobactams. The loss of penicillin hydrolysis was due to a non-carboxylated Lys-73.

Effect of Internal Diffusional Restrictions on the Hydrolysis of Penicillin G: Reactor Performance and Specific Productivity of 6-APA with Immobilized Penicillin Acylase

A mathematical model that describes the heterogeneous reaction-diffusion process involved in penicillin G hydrolysis in a batch reactor with immobilized penicillin G acylase is presented. The reaction system includes the bulk liquid phase containing the dissolved substrate (and products) and the solid biocatalyst phase represented by glyoxyl-agarose spherical porous particles carrying the enzyme. The equations consider reaction and diffusion components that are presented in dimensionless form. This is a complex reaction system in which both products of reaction and the substrate itself are inhibitors. The simulation of a batch reactor performance with immobilized penicillin G acylase is presented and discussed for the internal diffusional restrictions impact on effectiveness and productivity. Increasing internal diffusional restrictions, through increasing catalyst particle size and enzyme loading, causes impaired catalyst efficiency expressed in a reduction of effectiveness factor and specific productivity. High penicillin G initial concentrations decrease the impact of internal diffusional restrictions by increasing the mass transfer towards porous catalyst until product inhibition becomes significant over approximately 50 mM of initial penicillin G, where a drop in conversion rate and a maximum in specific productivity are then obtained. Results highlight the relevance of considering internal diffusional restrictions, reactor performance, and productivity analysis for proper catalyst and reactor design.