Enhancing immobilized PGA catalytic performance and reusability via magnetic core-shell nanocomposite carriers with a microenvironment optimization strategy

Introduction: Penicillin G amidase is an industrially significant enzyme widely employed in the production of semi-synthetic β-lactam antibiotics through the hydrolysis of Penicillin G to 6- aminopenicillanic acid. Owing to its commercial importance, extensive research has focused on improving the operational stability, reusability, and catalytic efficiency of PGA through various immobilization strategies. Methods: Optimization of multiple parameters for free and immobilized Penicillin G Acylase (PGA) is critical for improving the enzyme';s catalytic effectiveness, stability, and reusability in industrial and medicinal applications. This procedure entails methodically altering and analyzing variables such as substrate concentration, mechanical stability, cycle number, and storage conditions, and their effects on operational stability, pH, and temperature. PGA was optimized by entrapment on collagen hydrogel beads, resulting in collagen hydrogel + gelatin hybrid gel beads. Result: Immobilized PGA in Collagen Hydrogel + gelatin hybrid beads showed superior thermal stability, reusability, and storage stability as compared to gelatin-immobilized PGA. The entrapment of PGA onto Collagen Hydrogel + gelatin hybrid beads revealed several advantages and could be used in the production of 6-aminopenicillanic acid (6APA). Discussion: The study investigated the biochemical behavior of Penicillin G amidase (PGA) immobilized on collagen hydrogel and a collagen–gelatin bio-composite. Relative analysis focused on enzyme activity, stability, and mechanical strength, revealing insights into their appropriateness as immobilization matrices for enhanced PGA performance in industrial biocatalysis applications. Conclusion: Hydrogel + gelatin hybrid beads are more beneficial in industrial applications due to their greater stability and usability. PGA entrapment onto Hydrogel + gelatin hybrid beads has shown numerous advantages and may be useful in the manufacture of 6APA (6-aminopenicillanic acid).

Synthesis of polymer-functionalized β-cyclodextrin, Mg2+ doped, coating magnetic Fe3O4 nanoparticle carriers for penicillin G acylase immobilization

Enzyme@silica hybrid nanoflowers shielding in polydopamine layer for the improvement of enzyme stability

Immobilization of penicillin G acylase on macrocellular heterogeneous silica-based monoliths

Sandwich-likely structured, magnetically-driven recovery, biomimetic composite penicillin G acylase-based biocatalyst with excellent operation stability

L-aspartate-alpha-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the decisive step in the biosynthesis of β-alanine. However, the low catalytic stability and efficiency of ADC limit its industrial applications. In this study, a variant of ADC from Bacillus subtilis were used as a starting point for engineering. After constructing a random mutagenesis library by error-prone PCR, followed by high-throughput screening,four substitutions (S7 N, K63 N, A99T, and K113R) were identified. By screening saturation mutagenesis libraries on these positions and computational analysis, two recombined variants N3(S7 N/K63 N/I88 M/A99E/K113R/I126*) and Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*) with improved performance were obtained. Compared to the wild type, the catalytic efficiency and catalytic stability of the best two variants were enhanced up to 95 %(variant N3) and up to 89 %(variant Y1), respectively. In addition, Y1 exhibited 3.37 times improved half-life and 2-fold improved total turnover number. Hydrophilicity analysis and molecular dynamics (MD) simulation revealed that the increased hydrophilicity and steric hindrance of key amino acid residues would affect the catalytic activity and stability. The improved catalytic performance of the variants could be attributed to their enhanced binding capacity to the substrate within the active pocket and the alleviation of mechanism-based inactivation.

Direct synthesis of Cerium(Ⅳ)-Incorporated mesostructured cellular foam for immobilization of penicillin G acylase

The immobilization of penicillin G acylase on modified TiO2 with various micro-environments

In this work, Fe3O4 nanoparticles (NPs) was synthesized by inverting microemulsion method. After that, based on the physical and chemical properties of tannic acid (TA), poly tannic acid (PTA) was coated on Fe3O4 NPs surface. Fe3O4 NPs coated with PTA, on the one hand, was used to immobilize Penicillin G acylase (PGA) by physical adsorption. On the other hand, it was modified by glutaraldehyde (GA). GA grafting rate (Gr-GA ) was optimized, and the Gr-GA was 30.0% under the optimum conditions. Then, through the Schiff base reaction between the glutaraldehyde group and PGA amino group, this covalent immobilization of PGA was further realized under mild conditions. Finally, the structures of every stage of magnetic composites were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibration magnetometer (VSM) and transmission electron microscopy (TEM), respectively. The results indicated that the enzyme activity (EA), enzyme activity recovery (EAR) and maximum load (ELC) of the immobilized PGA were 26843 U/g, 80.2% and 125 mg/g, respectively. Compared to the physical immobilization of PGA by only coating PTA nanoparticles, further modified nanoparticles by GA showed higher catalytic stability, reusability and storage stability.

One-pot synthesis of aldehyde-functionalized mesoporous silica-Fe3O4 nanocomposites for immobilization of penicillin G acylase

Epoxy-functionalized mesostructured cellular foams as effective support for covalent immobilization of penicillin G acylase

Improved stability and altered selectivity of tyrosinase based graphite electrodes for detection of phenolic compounds

Abstract: Penicillin G Acylase (PGA) has emerged as a critical biocatalyst in pharmaceutical sciences, exceeding its traditional role in penicillin synthesis. Despite its industrial significance, challenges, including substrate specificity, stability under industrial conditions, and efficiency in immobilization, persist. Engineering enhanced enzyme variants and developing advanced immobilization techniques along with process optimization shall be possible solutions to further improve reaction efficiency and scalability. Green chemistry integration can make PGA-based processes more sustainable. Moreover, the use of computational tools, including AI-driven optimization, can guide enzyme design and reaction condition refinement. A review synthesizing these advancements not only consolidates existing knowledge but also identifies opportunities for further innovation, ensuring the enzyme’s continued industrial and scientific relevance. The review discusses the structure and functionality of PGA, highlighting its diverse applications beyond penicillin production. Beyond antibiotic synthesis, PGA's usefulness extends to ester synthesis, resolving racemic mixtures and peptide bond formation, underlining its importance in various bioconversions and synthetic reactions. This adaptability is crucial for green chemistry, promoting sustainable practices in industrial processes. The kinetic parameters of PGA are discussed, providing insights into its operational efficiency. Despite its significant potential, PGA faces limitations in commercial applications, primarily due to stability issues under industrial conditions. Efforts to enhance PGA's stability, including engineering approaches, are explored to improve its industrial applicability. The review concludes by emphasizing PGA's role as a catalyst with vast implications in science and medicine, particularly in an era of rising antibiotic resistance. It underscores the enzyme's interconnected roles in production and therapeutics, its broad spectrum of applications, and the shift from traditional penicillin synthesis to broad-spectrum bioconversions. The scope of PGA engineering is also highlighted, indicating future directions for research and application in the pharmaceutical industry.

Penicillin G Acylase (PGA) plays a central role in the synthesis of β- lactam antibiotics. While certain variants have been extensively studied, their catalytic efficiency remains suboptimal for industrial application, necessitating further enzyme engineering to enhance substrate binding and reaction kinetics. This study aims to rationally design and engineer PGA variants with improved catalytic efficiency and stability toward β-lactam antibiotics, using an integrated approach of 4D QSAR modeling and neural network-guided mutation prediction. A dataset of 30 enzyme-substrate complexes involving three PGA variants and diverse β-lactam substrates was compiled. Ten complexes were randomly selected for external validation. The binding conformation of Cefotaxime to a Bacillus thermotolerans PGA variant was used as a reference for molecular docking and structural alignment. Binding site analyses identified optimal substrate orientations, followed by 4D grid-based energy profiling, which revealed 15 high-energy hotspot residues per variant. These positions were systematically mutated in silico, generating 1130 variants through a neural network-based residue substitution algorithm. Subsequent docking studies with Cefotaxime showed a strong positive correlation between predicted docking energies and Ki values derived from the 4D QSAR model, validating the model's predictive capability. Molecular dynamics simulations (2 × 100 ns) for selected variants, particularly Sequence Id_0, Id_2, Id_5, and Id_7, demonstrated stable binding interactions and favourable atomic distances, indicative of improved substrate affinity. In Sequence Id_11, the hotspot is Phe148. Chain A showed the best results with Val and Leu as single mutants, followed by Met56 in Chain B with Leu, and Ser144 in Chain A with Glu, Ala, Ile, and Arg. In the case of Sequence Id_03, the hotspot is Phe147. Chain A showed good results with Ala, Lys, Thr, and Ser, whereas Tyr71 in Chain B showed good results with Glu, Lys, and Thr, and Arg266 in Chain B showed good results with Ala, Thr, and Val. Those that showed the highest sum of docking scores and Ki were chosen for further studies. The study highlights the critical role of residue Phe148 in mediating stable interactions with Cefotaxime and other β-lactam substrates. The integrated computational strategy establishes a robust framework for engineering catalytically superior PGA variants, offering a valuable basis for further experimental validation and application in antibiotic biosynthesis.

The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (ΔGi values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower Km and higher kcat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.