6-aminopenicillanic Acid Research Articles

Separation of phenyl acetic acid and 6-aminopenicillanic acid applying aqueous two-phase systems based on copolymers and salts

6-Aminopenicillanic acid (6-APA) is used for synthesis of semisynthetic antibiotics. Polymer-salt aqueous two-phase systems (ATPSs) were applied for separation of 6-APA and phenyl acetic acid (PAA), as the products of hydrolyzation reaction of Penicillin G/Penicillin V. The binodal curves of ATPS composed of a copolymer (reverse Pluronic 10R5, Pluronic L35 and PEG-ran-PPG) and a salt (Tri-sodium citrate, tri-potassium citrate, di-potassium phosphate, sodium sulphate and magnesium sulphate) were obtained. The results show that, at a fixed PPG/PEG ratio, block copolymers have larger two-phase region compared with random copolymer. After screening on the partition coefficient of PAA and 6-APA separately, Na2SO4 was selected for studying the effect of the copolymer structure and the composition of salt and copolymer on partitioning, considering higher selectivity of PAA and 6-APA. 10R5-Na2SO4 ATPS was selected as the most appropriate system for separation of 6-APA and PAA. This system was used for separation of mixture of 6-APA and PAA. The results show that selectivity was approx 53 and smaller in a system, containing a mixture of 6-APA and PAA. This observation can be justified by the interaction between 6-APA and PAA. Molecular interaction between these two molecules were investigated by the Flory–Huggins interaction parameter.

Extended release of 6-aminopenicillanic acid by silanol group functionalized vermiculite

The present study aimed to improve capability of vermiculite for extended release ofdrug such as: 6-Aminopenicillanic acid (6-APA). For this purpose, functionalization of vermiculite (VMT) was done by utilizing silanol through hydrolyzing of tetraethoxysilane. The synthesized samples characterized through the XRD, FTIR and BET analyses. The Maximum obtained 6-APA absorption capacity of VMT-Si was 243.9 mg/g, which was higher than other clays. The equilibrium studies demonstrate that drug absorption on VMT and VMT-Si is homogeneous and heterogeneous, respectively. Kinetic studies have been performed by applying reaction based models (such as: pseudo-first-order and pseudo-second-order) and diffusion based models (such as: Boyd, intra-particle diffusion and mass transfer models), which indicates the chemisorption interaction of both aforementioned compounds. The in-vitro release profile of VMT showed a release of about 52% after 5 hours. Whereas, the in-vitro release profile of 6-APA from VMT-Si showed substantially less release of 70% after 7 hours compare to VMT. The maximum obtained release amount of antibiotic (6-APA) per each gram of VMT-Si-6APA and VMT-6APA was 167 mg and 45 mg, respectively. Release kinetic studies show that the release mechanism of 6-APA is controlled by the diffusion process based on Fick's law. In conclusion, vermiculite functionalization with silanol, reduced the rate of drug release and increased the amount of released 6-APA.

A gossypol derivative as an efficient corrosion inhibitor for St2 steel in 1 M HCl + 1 M KCl: An experimental and theoretical investigation

Currently, there are several types of corrosion inhibitors were studied. The new type of corrosion inhibitor based on a gossypol derivative was first introduced. The anti-corrosion performance of 6-aminopenicillanic acid sodium gossypol (APASG) for St2 steel in a 1 M HCl + 1 M KCl solution was reported for the first time, and the inhibition properties of this material were fully characterized by gravimetric analysis, thermodynamic analysis, potentiodynamic polarization (PDP), electrochemical impedance spectroscopy (EIS), electrochemical noise (EN) analysis, electrochemical frequency modulation (EFM), density functional theory (DFT), molecular dynamics (MD) simulations, scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDX). The obtained results show that APASG is an excellent anti-corrosion inhibitor (97% at 100 mg/L/0.104 mM) at intermediate temperatures (303−333K). APASG adsorbed onto a metal surface by a physical and chemical adsorption mechanism, as shown by the Langmuir adsorption isotherms. Electrochemical analyses show that APASG acts as a mixed-type inhibitor. The thermodynamic adsorption data were measured and discussed. SEM-EDX tests of the inhibited steel sample show that the inhibitor significantly improved the surface morphology. DFT calculations indicated that the broadly stretched linked functional groups (hydroxyl and carboxyl) and heteroatoms (nitrogen, oxygen and sulfur) in the inhibitor structure are responsible for the inhibition efficiency of APASG. The obtained DFT and MD simulation data supported the experimental results.

Synthesis, Characterization and Antibacterial Activity of Some Penicillin Derivatives

This study illustrated the synthesis of two new di amidine compounds ([c] and [d]) by reaction of 6-amino penicillanic acid (6-APA) with diα-amino nitrile compounds ([a] and [b]). [a] and [b] di α-amino nitrile compounds synthesized from the condensation reaction on aldehyd and di amine in the presence of potassium cyanid as one pot-three components reaction. The new di amidine compounds ([c] and [d]) have been proven their efficiency by inhibiting some types of bacteria (Staphylococcus aurous, Streptococcus, Escherichia coliand klebsiella). Amidine compound [d] showed better effect than [c] against the selected bacteria.The synthesized compounds were characterized by conventional techniques using infrared spectrophotometer (IR) andproton-nuclear magnetic resonance (1H-NMR)

Process Development for 6-Aminopenicillanic Acid Production Using Lentikats-Encapsulated Escherichia coli Cells Expressing Penicillin V Acylase.

Penicillin V acylase (PVA, EC 3.5.1.11) hydrolyzes the side chain of phenoxymethylpenicillin (Pen V) and finds application in the manufacture of the pharmaceutical intermediate 6-aminopenicillanic acid (6-APA). Here, we report the scale-up of cultivation of Escherichia coli whole cells expressing a highly active PVA from Pectobacterium atrosepticum and their encapsulation in polyvinyl alcohol–poly(ethylene glycol) Lentikats hydrogels. A biocatalytic process for the hydrolysis of 2% (w/v) Pen V was set up in a 2 L reactor using the Lentikats-immobilized whole cells, with a customized setup to enable continuous downstream processing of the reaction products. The biocatalytic reaction afforded complete conversion of Pen V for 10 reaction cycles, with an overall 90% conversion up to 50 cycles. The bioprocess was further scaled up to the pilot-scale at 10 L, enabling complete conversion of Pen V to 6-APA for 10 cycles. The 6-APA and phenoxy acetic acid products were recovered from downstream processing with isolated yields of 85–90 and 87–92%, respectively. Immobilization in Lentikats beads improved the stability of the whole cells on storage, maintaining 90–100% activity and similar conversion efficiency after 3 months at 4 °C. The robust PVA biocatalyst can be employed in a continuous process to provide a sustainable route for bulk 6-APA production from Pen V.

Electric-field-enhanced selective separation of products of an enzymatic reaction in a membrane micro-contactor.

Processes employed in separations of products of enzyme reactions are often driven by diffusion, and their efficiency can be limited. Here, we exploit the effect of a direct current (DC) electric field that intensifies mass transfer through a semipermeable membrane for fast, continuous, and selective separation of electrically charged molecules. Specifically, we separate low-molecular-weight reaction products (phenylacetic acid, 6-aminopenicillanic acid) from the original reaction mixture containing a free enzyme (penicillin acylase). The developed microfluidic dialysis-membrane contactor allows a stable counter-current arrangement of the retentate and permeates liquid streams on which DC electric field is perpendicularly applied. The applied electric field significantly accelerates the transport of electrically charged products through the semipermeable membrane yielding high separation efficiencies at short residence times. The residence time of 5 min is sufficient to reach 100% separation yield in the electric field. The same residence time provides only a 50% yield in the diffusion-controlled experiments. We experimentally demonstrated that a combined microreactor-microextractor with a recycle of the soluble penicillin acylase can continuously produce both the reaction products at high concentrations. The developed membrane-contactor is a versatile platform allowing to tune its characteristics, such as selectivity given by the membrane, or the type of the retentate phase, for a specific application.

Laccase-catalyzed derivatization of 6-aminopenicillanic, 7-aminocephalosporanic and 7-aminodesacetoxycephalosporanic acid

Trametes spec. laccase (EC 1.10.3.2.) mediates the oxidative coupling of 6-aminopenicillanic, 7-aminocephalosporanic, and 7-aminodesacetoxycephalosporanic acid with 2,5-dihydroxybenzoic acid derivatives to form new penicillin and cephalosporin structures, respectively. The heteromolecular hybrid dimers are formed by nuclear amination of the p-hydroquinones with the primary amines and inhibited in vitro the growth of Staphylococcus species, including some multidrug-resistant strains.

Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis.

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b, or PBP4. Almost all penicillin scaffolds were coselective for all or combinations of PBP2a, 2b, and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin) and 2b (cefotaxime) or 2a, 2b, and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime, and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid), and various coselective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in β-lactam resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

Visualized characterization of bacterial penicillin G acylase for the hydrolysis of β-lactams using an activatable NIR fluorescent probe

Penicillin G acylase (PGA) identified from bacteria, is used as industrial biocatalyst for the hydrolysis of penicillins along with the production of 6-aminopenicillanic acid, a key intermediate of the semi-synthetic β-lactam antibiotics. Due to wide usage of PGA, some novel homology bioactive enzymes are investigated in various bacteria, and visualized analysis technique would be necessary and helpful for the development and utilization of PGA function in living systems. In the present work, an activated off-on near-infrared (NIR) fluorescent probe (DDAP) deduced from a novel fluorophore has been designed and developed for sensitively and selectively detecting endogenous PGA in real-time. The hydrolysis mechanism has been elucidated using in silico docking analysis, which confirmed DDAP as the specific substrate of PGA. Using DDAP as the fluorescent probe, the endogenous bacterial PGA was imaged in living systems successfully, which could be applied to identify bacteria with PGA expression from complex microorganism samples. Furthermore, using DDAP as the staining agent, the endogenous PGA in various bacteria has been profiled visually on native-PAGE gel, and the PGA activity in bacterial lysates could be effectively determined. The PAGE-gel for PGA analysis stained by DDAP was more sensitive than generally used silver and Coomassie Blue dyes. Finally, a series of β-lactams were hydrolyzed by recombinant PGA to further confirm its hydrolase property, corresponding to the hydrolysis of DDAP.

Insight into 6-aminopenicillanic acid structure and study of the quantum mechanical calculations of the acid–base site on γ-Fe2O3@SiO2core–shell nanocomposites and as efficient catalysts in multicomponent reactions

Magnetic 6-APA/γ-Fe2O3@Sio2nanocomposites have been developed by exploiting the potential of the acid–base bifunctional system to study the quantum mechanistic calculations.

Activating a Silver Lipoate Nanocluster with a Penicillin Backbone Induces a Synergistic Effect against S. aureus Biofilm.

Many antibiotic resistances to penicillin have been reported, making them obsolete against multiresistant bacteria. Because penicillins act by inhibiting cell wall production while silver particles disrupt the cell wall directly, a synergetic effect is anticipated when both modes of action are incorporated into a chimera cluster. To test this hypothesis, the lipoate ligands (LA) of a silver cluster (Ag29) of known composition (Ag29LA12)[3−] were covalently conjugated to 6-aminopenicillanic acid, a molecule with a β-lactam backbone. Indeed, the partially conjugated cluster inhibited an Staphylococcus aureus biofilm, in a dose–response manner, with a half-maximal inhibitory concentration IC50 of 2.3 μM, an improvement over 60 times relative to the unconjugated cluster (IC50 = 140 μM). An enhancement of several orders of magnitude over 6-APA alone (unconjugated) was calculated (IC50 = 10 000 μM). Cell wall damage is documented via scanning electron microscopy. A synergistic effect of the conjugate was calculated by the combination index method described by Chou–Talalay. This hybrid nanoantibiotic opens a new front against multidrug-resistant pathogens.

Impact of Bioorganic Additives of Different Nature on Aggregation Behavior and on Cloud Point Temperatures of Nonionic Surfactants Tergitol NP-7 and Triton X-114 in Buffer Solutions

Aqueous biphasic systems containing non-ionic ethoxylated surfactants are perspective as media for some biochemical reactions. A comprehensive study of phase and aggregation behavior of these micellar systems, as well as consideration of the effects of biocomponents, their nature and concentration, pH and temperature are evident. In this work, the influence of salt medium (phosphate buffer), pH and temperature, as well as the presence of different bioorganic additives on cloud point and structural-thermodynamic properties (CMC and aggregation number) were studied for micellar solutions of non-ionic surfactants Triton X-114 and Tergitol NP-7 in water and buffers with pH 6.8, 4.3, 2.7. As additives were used the components of a biocatalytic hydrolysis reaction of penicillin G, namely penicillin G potassium salt, phenylacetic acid, and 6-aminopenicillanic acid. The different effects of the biocomponents on the cloud point and CMC value of Triton X-114 in the buffer with pH 4.3 are discussed. In addition, it was shown that the CMC value and the aggregation number of Triton X-114 decrease in buffer solutions (salting-out effect) and the CMC value increase at growing the temperature to 37°C (what is typical for ethoxylated non-ionic surfactants). The addition of phenylacetic acid (10 mM) leads to a reduction in the aggregation number of Triton X-114 at pH 4.3; other bio-additives have practically no effect. Thus, the studied additives have different effects on the structure and phase behavior of investigated micellar solutions, and this is an important aspect for choosing an extraction system to carry out a bioreaction.

Optimization of penicillin G acylase immobilized on glutaraldehyde‐modified titanium dioxide

In this work, TiO2 , which was modified by glutaraldehyde, was adopted as the carrier; the penicillin G acylase (PGA) was immobilized and the influence of immobilized conditions, such as pH of solution, the concentration of PGA, the immobilization temperature, and the reaction time, on the catalytic performance of the immobilized PGA was investigated and optimized. During this process, potassium penicillin G (PG) was chosen as substrate, and the quantity of 6-aminopenicillanic acid (6-APA) produced by PG at the temperature of 25°C for 3Min in neutral solution was conscripted as the evaluation foundation, indexes, containing the loading capacity (ELC), the activity (EA), and activity retention rate (EAR), were calculated based on quantities of produced 6-APA and compared with finding out the suitable conditions. Results showed that when the solution pH, PGA concentration, immobilization temperature, and reaction time were 8.0, 2.5% (v/v), 35°C, and 24H, respectively, ELC, EA, and EAR presented optimal values of 9,190U, 14,969U/g, and 88.5% relatedly. After that, the stability and reusability of immobilized PGA were studied, and the results documented that the pH resistance, thermal stability, and storage stability of immobilized PGA were significantly improved. This work provided technique support for the practical application of immobilized PGA carrier.

Synthesis, Pharmacological and Toxicological Screening of Penicillin-Triazole Conjugates (PNTCs).

A series of hybrid antimicrobial compounds were prepared by carboxylic acid protection of 6-aminopenicillanic acid using benzyl alcohol and thionyl chloride succeeded by azide displacement using trifluoromethanesulfonyl azide in dichloromethane. The azide thus formed was reacted with substituted alkynes to furnish benzyl-protected penicillin–triazole conjugates. Benzyl deprotection of the conjugates resulted in furnishing PNTCs under water methanol mixture using Pd/C as a catalyst. The PNTCs (7a–j) formed were screened for in vitro antibacterial potency against pathogenic strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes and antifungal potency against Candida albicans, Aspergillus niger, and Aspergillus clavatus. Further antimicrobial evaluation revealed compounds 7c, 7d, 7e, 7g, and 7i to be the most compounds of the series with minimum inhibitory concentration value for antibacterial in the range 0.5–50 μg/mL and for antifungal in the range 9–300 μg/mL. Toxicological analysis documented for compounds 7c, 7d, 7e, 7g, and 7i revealed compound 7i to be the most promising member of the series with 1000 and 500 mg/kg LD50, and no-observed-adverse-effect level to facilitate future clinical studies of the same.

Fabricating CsPbX3/CN heterostructures with enhanced photocatalytic activity for penicillins 6-APA degradation

Fabrication of CsPbX3 (X = Cl, Br)/graphitic carbon nitride (CN) composite has proved to be an efficient way to improve the stability of CsPbX3 nanocrystals (NCs) and improve their photocatalytic activities. Here, the CsPbBr3/CN and CsPbCl3/CN heterostructures were successfully prepared, based on the band structure, the CsPbBr3/CN is indexed as a type I heterostructure, and CsPbCl3/CN is indexed as a type II heterostructure. Both CsPbX3/CN heterostructure could facilitate the electron-hole pairs (e−-h+) separation and exhibit the outstanding photocatalytic activity for penicillins 6-aminopenicillanic acid (6-APA) degradation under visible light, the degradation rates of 6-APA with CsPbCl3/CN and CsPbBr3/CN composite were about 78.11 and 83.31% in 120 min, respectively. The improved photocatalytic activity of the CsPbX3/CN heterostructures could be attributed to the favourable adsorbability, the enhanced light-harvesting ability, the efficient charge separation ability, as well as the synergistic effects of CsPbX3 NCs and CN. This work may not only open up a new insight for design of CsPbX3-based heterostructure as novel photocatalyst but also provide a new possibility for photocatalytic applications of perovskites-based NCs.

Synthesis of Antibiotic Penicillin-G Enzymatically by Penicillium chrysogenum

Penicillin-G antibiotic was used as the basic ingredient of making antibiotic type β-lactam such as tetracycline, amoxicillin, ampicillin and other antibiotics. Penicillin-G was splited into 6-amino penicillanic acid as the source of β-lactam. The biosynthetic pathway for the formation of penicillin-G in Penicillium chrysogenum cell through the formation of intermediates was carried out in the form of amino acids such as α-aminoadipate, L-cysteine, L-valine which are formed from glucose (food ingredients).The formation of 6-amino penicillanic acid is an amino acid combination of L-cysteine and L-valine, a step part of the formation of antibiotic penicillin-G in P. chrysogenum cells, thus, it is obvious that there are enzymes involved in its formation. The objective of this study was to examine the use of enzymes present in P. chrysogenum cells to produce penicillin-G and 6-amino penicillanic acid using the intermediate compounds α-aminoadipate, L-cysteine, L-valine and phenylacetic acid assisted by NAFA® coenzymes in P. chrysogenum cells which is more permeable. The research method started from producing biomass of P. chrysogenum cells that demonstrated penicillin-producing antibiotic capability, as the source of the enzyme, followed by addition of permeability treatment of P. chrysogenum cell membrane to get immobile of enzyme by its own cell therefore it can be used more than once. After that the enzyme activity was proven by adding α-aminoadipate, L-cysteine, L-valine, phenylacetic acid and NAFA® coenzyme for the formation of penicillin-G, whereas the addition of L-cystein, L-valine and NAFA® coenzyme were aimed to form 6-amino penicillanic acid. The results showed that P. chrysogenum is able to produce antibiotics with stationary early phase on day 6. The best increased permeability of P. chrysogenum cell membranes was obtained using a 1:4 of toluene:ethanol ratio mixture with the highest antibiotic concentration (130.06 mg/L) after testing for the enzymatic formation of antibacterial penicillin-G.

Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Ymax) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100 mM Tris/HCl buffer pH 7.0, 30 °C, 2.7% (v/v) DMSO, 20 mM MPOA, 0.3 UI/ml of SlPVA), Ymax was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Ymax and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.

Development of novel support for penicillin acylase and its application in 6-aminopenicillanic acid production

There is an ever-increasing demand for the β-lactam bulk intermediate 6-aminopenicillanic acid (6-APA) that has wide applications in the synthesis of newer generations of semisynthetic penicillins. It is commercially synthesized by biocatalytic transformation using penicillin acylase. Since the enzyme is soluble, immobilization on a solid porous support is necessary to make the catalyst recycleable and the process profitable. In this study, we developed a novel support of siliceous foam entrapped in a polymer matrix. Penicillin acylase was covalently immobilized on aminopropyl functionalized mesocellular foam silica (MCF) and was further cross-linked using glutaraldehyde without deactivation and upto 95% efficiency. The resulting biocatalyst had an activity of 1185 IU. mg−1 and demonstrated improved resistance to the substrate and product inhibition. These parameters along with improvement in pH and thermal stability enhanced 6-APA yield by 20% in beads. Intrinsic kinetic parameters were calculated from the developed rate equation to deduce enzyme catalytic mechanism.

Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability.

Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G to 6-aminopenicillanic acid and phenylacetic acid, which provides the precursor for most semisynthetic penicillins. Most applications rely on PGAs from Gram-negative bacteria. Here we describe the first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability. PGAs from Bacillus megaterium (BmPGA, Tm = 56.0°C), Bacillus thermotolerans (BtPGA, Tm = 64.5°C), and Bacillus sp. FJAT-27231 (FJAT-PGA, Tm = 74.3°C) were recombinantly produced with B. megaterium, secreted, purified to apparent heterogeneity, and crystallized. Structures with resolutions of 2.20Å (BmPGA), 2.27Å (BtPGA), and 1.36Å (FJAT-PGA) were obtained. They revealed high overall similarity, reflecting the high identity of up to approx. 75%. Notably, the active center displays a deletion of more than ten residues with respect to PGAs from Gram-negatives. This enlarges the substrate binding site and may indicate a different substrate spectrum. Based on the structures, ten single-chain FJAT-PGAs carrying artificial linkers were produced. However, in all cases, complete linker cleavage was observed. While thermostability remained in the wild-type range, the enzymatic activity dropped between 30 and 60%. Furthermore, four hybrid PGAs carrying subunits from two different enzymes were successfully produced. Their thermostabilities mostly lay between the values of the two mother enzymes. For one PGA increased, enzyme activity was observed. Overall, the three novel PGA structures combined with initial protein engineering experiments provide the basis for establishment of new PGA-based biotechnological processes.

Dynamic Modelling and Optimisation of the Batch Enzymatic Synthesis of Amoxicillin

Amoxicillin belongs to the β-lactam family of antibiotics, a class of highly consumed pharmaceutical products used for the treatment of respiratory and urinary tract infections, and is listed as a World Health Organisation (WHO) “Essential Medicine”. The demonstrated batch enzymatic synthesis of amoxicillin is composed of a desired synthesis and two undesired hydrolysis reactions of the main substrate (6-aminopenicillanic acid (6-APA)) and amoxicillin. Dynamic simulation and optimisation can be used to establish optimal control policies to attain target product specification objectives for bioprocesses. This work performed dynamic modelling, simulation and optimisation of the batch enzymatic synthesis of amoxicillin. First, kinetic parameter regression at different operating temperatures was performed, followed by Arrhenius parameter estimation to allow for non-isothermal modelling of the reaction network. Dynamic simulations were implemented to understand the behaviour of the design space, followed by the formulation and solution of a dynamic non-isothermal optimisation problem subject to various product specification constraints. Optimal reactor temperature (control) and species concentration (state) trajectories are presented for batch enzymatic amoxicillin synthesis.