Penicillin G Acylase Research Articles

Expanding the genetic code with a lysine derivative bearing an enzymatically removable phenylacetyl group.

We report the genetically encoded incorporation of phenylacetyl protected lysine (PacK) into proteins in Escherichia coli. This unnatural side-chain modification can be enzymatically removed using either penicillin G acylase (PGA) or, surprisingly, the sirtuin SrtN from Bacillus subtilis. Our approach expands the toolbox to reversibly control protein structure and function under very mild and non-denaturing conditions, as demonstrated by triggering the activity of the nonribosomal peptide synthetase GrsA.

Quest for novel fluorogenic xanthene dyes: Synthesis, spectral properties and stability of 3-imino-3H-xanthen-6-amine (pyronin) and its silicon analog

To expand the range of primary aniline fluorophores available and suitable for the design of fluorogenic protease probes, the synthesis of 3-imino-3H-xanthen-6-amine (known as pyronin) and its silicon analog (Si-pyronin) was explored and presented here. A comprehensive photophysical study of these two fluorescent anilines, confirms the effectiveness of the heteroatom-substitution approach (O → SiMe2) to yield dramatic red-shifts in absorption and fluorescence maxima of the xanthene scaffold (+85 nm). However, it also revealed its adverse effect on the hydrolytic stability of the Si-pyronin, especially at physiological pH. The pro-fluorescent character and utility of these two fluorogenic (hetero)xanthene dyes are also proved by the preparation and in vitro validation of activatable fluorescence “turn-on” probes for penicillin G acylase (PGA).

The study of titanium dioxide modification by glutaraldehyde and its application of immobilized penicillin acylase

In this work, titanium dioxide particle was firstly modified with glutaraldehyde, and modification conditions, i.e. particle size (d), active temperature (Ta), modification temperature (Tm), modification time (tm), pH value, aldehyde ethanol ratio (Ra) and ethanol ratio (Re), were studied and optimized adopting grafting rate (Gr) as the optimizing index, the ultraviolet-visible spectrophotometry as characterization method, and the obtained optimal modification conditions were d = 3.3 μm, Ta = 120 °C, Tm = 45 °C, tm = 30 h, pH = 6.0, Ra = 1:10 and Re = 100%. Under the optimal conditions, Gr of glutaraldehyde arrived at 50%. Thereafter, a series of modified titanium dioxide with Gr in the range of [8%, 46%] and with Gr = 30% obtained from different modification conditions were adopted to study the immobilization of penicillin G acylase (PGA), during this process, the ultraviolet-visible spectrophotometry of 6-aminopenicillanic acid was conscripted as characterization method, the enzyme loading capacity (ELC), the enzyme activity (EA) and enzyme activity retaining ratio (EAR) were explored, and results documented with the increase of Gr, ELC, EA and EAR increased. As Gr kept as a similar level, ELC, EA and EAR also showed a similar value. Finally, the structure of titanium dioxide, glutaraldehyde modified titanium dioxide and PGA immobilized titanium dioxide was characterized by the flourier transform infrared spectroscopy (FTIR), and results exhibited aimed samples were obtained successfully.

Mathematical model based optimization of enzymatic aldol addition of propanal to formaldehyde

A multi-enzyme route for the production of an atorvastatin side-chain was proposed. The approach includes the consecutive double aldol addition of acetaldehyde to the phenylacetamide amino-protected propanal by 2-deoxyribose-5-phosphate aldolase (DERA), the oxidation by ketoreductase, the lipase-catalysed acylation and amino group deprotection by penicillin G-acylase. To explore the feasibility of the process, the DERA reaction was studied into detail. Based on the kinetic and stability studies, a mathematical model was developed and validated in a batch and fed-batch reactor. The highest productivity was obtained in repetitive batch reactor (229.1 g/(L day)). The highest final product concentration of 124 g/L was obtained in fed-batch reactor. The mathematical model-based optimisation provided insight into the possibilities for process metrics improvement. Further, the second step was explored. The results showed that the DERA reaction and the oxidation reaction can be carried out as the multi-enzyme one pot process in the sequential manner.

The engineering and immobilization of penicillin G acylase onto thermo‐sensitive tri‐block copolymer system

In this work, the relationships between catalytic performances of penicillin G acylase (PGA) and the molar ratio of carrier, thermo‐sensitive tri‐block polymer, poly (N,N‐diethylacrylamide‐b‐ β‐hydroxyethyl methacrylate‐b‐glycidyl methacrylate) (PDEA‐b‐PHEMA‐b‐PGMA) were studied firstly, and result documented the optimal molar ratio was nDEA:nHEMA:nGMA = 100:47:24, which presented a suitable lower critical solution temperature (LCST) of 35°C and the activity retention ratio of 80.62% (±0.50%). Based on the suitable carrier, immobilization conditions were investigated and optimized. When pH of solution, concentration of PGA, immobilized time, and immobilization temperature were 8.0, 1/10 (m/v), 16 hours, and 36°C, respectively, enzyme loading capacity (L), enzyme activity (Ea), and activity retention ratio (Ar) of PGA arrived at the highest value of 21 223 U, 16 199 U/g, and 93.50% (±0.50%), respectively. Besides, the response rate (Rr) of immobilized PGA was the same as free PGA, the reusable stability (Rs) was 77.00% (±1.00%) after using for 11 times, which indicated that the carrier has better compatibility with L, Ar, Rs, and Rr.

Directed evolution of a penicillin V acylase from Bacillus sphaericus to improve its catalytic efficiency for 6-APA production

Penicillin acylase is commonly used to produce the medical intermediates of 6-Aminopenicillanic acid (6-APA) and 7-Aminodesacetoxycephalosporanic acid (7-ADCA) in industrial process. Nowadays, Penicillin G acylase (PGA) has been widely applied for making pharmaceutical intermediates, while penicillin V acylase (PVA) has been less used for that due to its low activity and poor conversion. In this study, a PVA from Bacillus sphaericus (BspPVA) was employed for directed evolution study with hoping to increase its catalytic efficiency. Finally, a triple mutant BspPVA-3 (T63S/N198Y/S110C) was obtained with 12.4-fold specific activity and 11.3-fold catalytic efficiency higher than BspPVA-wt (wild type of BspPVA). Moreover, the conversion yields of 6-APA catalyzed by BspPVA-3 reached 98% with 20% (w/v) penicillin V as substrate, which was significantly higher than that of the BspPVA-wt (85%). Based on the analysis of modeling, the enhancement of specific activity of mutant BspPVA-3 was probably attributed to the changes in the number of hydrogen bonds within the molecules. The triple mutant PVA developed in this study has a potential for large-scale industrial application for 6-APA production.

Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. coli.

High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20°C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA)U/L/OD600, respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable β-lactam antibiotics.

Synthesis, stability and spectral behavior of fluorogenic sulfone-pyronin and sulfone-rosamine dyes

The first synthesis of sulfone-pyronin and sulfone-rosamine dyes bearing optically tunable primary amino groups (acting as fluorogenic centers) is presented. Sulfone analogs of xanthene-based fluorophores have recently been highlighted as a new class of near-infrared (NIR) fluorescent dyes (Liu et al. ACS Appl Mater Interfaces 2016; 8(35):22953-62), and the availability of fluorogenic derivatives is essential for the rapid construction of “turn-on” reactive probes for chemoselective bioimaging. However, these fluorescent anilines have been found to be unstable in aqueous physiological conditions due to the marked electrophilicity of their meso-position and hence its propensity to undergo nucleophilic attack by a water molecule or a hydroxyde anion. Further investigations have helped us to determine the acidic pH range at which the sulfone-rosamine 2 is fully-stable and a complete assessment of the photophysical properties could be performed. Its pro-fluorescent character and utility were then proved by the preparation and in vitro enzymatic activation of a penicillin G acylase (PGA) sensitive fluorogenic probe.

Kinetic model discrimination of penicillin G acylase thermal deactivation by non-isothermal continuous activity assay

A novel approach for the determination of a kinetic model of enzyme deactivation is presented incorporating time and temperature dependence into a single, continuous assay. A generic method for a non-isothermal, continuous activity assay is developed. Unique temperature profiles that enhance model discrimination with fewer experiments compared to linear temperature scans were used to differentiate kinetic models of penicillin G acylase (PGA) deactivation. Three models are examined in depth but the technique is generalizable to any kinetic deactivation model. Using the Akaike information criterion (AIC), the Lumry-Eyring model was found to best capture PGA deactivation behavior and the corresponding kinetic parameters are presented for the first time. Additionally, simulated experiments on lysozyme and TEM-1 beta-lactamase were used to develop temperature profiles that best differentiate the studied kinetic models. The results from the proposed approach were consistent with conventional, but tedious, isothermal batch experiments.

Kinetics improvement of protease-mediated formation of pyronin dyes

A fluorescent probe for protease sensing and based on the “covalent-assembly” principle is reported. The basic rational for this unusual class of chemodosimeters proposed by the Anslyn and Yang groups entails the synthesis of non-fluorophore caged precursors full-stable and reactive towards the targeted analyte. Unlike the first generation of protease-sensitive “covalent-assembly” type probes recently published by ourselves (Org. Biomol. Chem.2017, 15, 2575–2584), the availability of dicyanomethylidenyl and enzyme-labile phenylacetamide moieties within the core structure of mixed bis-aryl ether 2 enables its rapid conversion into a fluorescent pyronin dye at physiological pH and upon activation with penicillin G acylase (PGA). This is real progress towards the practical implementation of this ingenious activation mechanism to the detection of enzymes in their native environment (in cellulo or in vivo).

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

In this study, a novel kind of flower-like monodisperse silica nanoparticles (silica nanoflowers) were successfully synthesized based on the microemulsion phase composed of water-surfactant-oil ternary systems. The obtained silica nanoflowers were used to immobilize penicillin G acylase (PGA). The maximum adsorption capacity of immobilized PGA (PGA@nanoflowers) was 207.3 mg/support and the specific activity was 539.1 U/support. In addition, based on the mussel-inspired chemistry, we demonstrated a simple and highly effective strategy to stabilize the enzymes that immobilized on silica nanoflowers by the poly(dopamine) (PDA) coating through in situ polymerization. The effects of pH and temperature on the activity of free PGA, PGA@nanoflowers and immobilized PGA with PDA coating (PDA-coated PGA@nanoflowers) were investigated. The operational stability, storage stability and kinetic properties of the different preparation of PGA were also tested. Compared to free PGA and PGA@nanoflowers, the stabilities of PDA-coated PGA@nanoflowers were all improved significantly. This work demonstrated that the PDA coating presented a new way for surface modification of immobilized enzyme to improve the properties of enzyme. The approach of enzyme@silica hybrid nanoflowers shielding in PDA layer would have great potential in various enzyme immobilization.

Enzyme@silica nanoflower@metal-organic framework hybrids: A novel type of integrated nanobiocatalysts with improved stability

A novel integrated nanobiocatalyst system based on an enzyme@silica nanoflower@metal-organic framework (enzyme@SNF@ZIF-8) structure with improved stability is fabricated for the first time. The versatility of this system is validated using penicillin G acylase (PGA) and catalase (CAT) as model enzymes. The microporous ZIF-8 layer can be controlled by varying the number of ZIF-8 coating cycles, which produces PGA@SNF@ZIF-8 nanobiocatalysts with different ZIF-8 layer thicknesses. After the second ZIF-8 coating cycle, a PGA@SNF@ZIF-8(2) structure with a homogeneous and well-intergrown ZIF-8 layer is formed, which possesses excellent mechanical and chemical stability. Moreover, PGA@SNF@ZIF-8(2) shows improved thermal/storage stability and reusability compared with free PGA and PGA immobilized on silica nanoflowers (PGA@SNF). The obtained CAT-based nanobiocatalysts (CAT@SNF@ZIF-8(2)) also show excellent catalytic performance.

Efficient synthesis of β-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase.

Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of β-lactam antibiotics. Site-directed mutagenesis was performed to increase the process efficiency. Molecular docking was carried out to speculate the key mutant positions corresponding with synthetic activity, which resulted in the achievement of an efficient mutant, βF24G. It yielded higher conversions than the wild-type enzyme in the synthesis of amoxicillin (95 versus 17.2%) and cefadroxil (95.4 versus 43.2%). The reaction time for achieving the maximum conversion decreased from 14 to 16h to 2-2.5h. Furthermore, the secondary hydrolysis of produced antibiotics was hardly observed. Kinetic analysis showed that the (kcat/Km)AD value for the activated acyl donor D-hydroxyphenylglycine methyl ester (D-HPGME) increased up to 41 times. In contrast, the (kcat/Km)Ps values for the products amoxicillin and cefadroxil decreased 6.5 and 21 times, respectively. Consequently, the α value (kcat/Km)Ps/(kcat/Km)AD, which reflected the relative hydrolytic specificity of PGA for produced antibiotics with respect to the activated acyl donor, were only 0.028 and 0.043, respectively. The extremely low hydrolytic activity for the products of the βF24G mutant enabled greater product accumulation to occur during synthesis, which made it a promising enzyme for industrial applications.

Immobilization of penicillin G acylase on paramagnetic polymer microspheres with epoxy groups

Paramagnetic polymer microspheres were synthesized by the inverse suspension polymerization method through polymerization of glycidyl methacrylate, ally glycidyl ether and methacrylamide on the surface of silica-coated Fe3O4 nanoparticles using N,N’-methylene-bis(acrylamide) as a cross-linking agent. Penicillin G acylase (PGA) was covalently immobilized on the surface of the paramagnetic microspheres by reacting the amino groups of the PGA molecules with the epoxy groups of the paramagnetic polymer microspheres. The effect of the SiO2 coating and the amount of paramagnetic Fe3O4 nanoparticles on the initial activity and the operational stability of the immobilized PGA was investigated. The results indicated that SiO2 played an important role in the polymerization process and paramagnetic polymer microspheres with a SiO2-coated Fe3O4 nanoparticles mass content of 7.5% are an optimal support material for PGA immobilization. Immobilized PGA on the paramagnetic polymer microspheres shows a high initial activity of 430 U/g (wet) and retains 99% of its initial activity after recycling 10 times. Furthermore, immobilized PGA exhibits high thermal stability, pH stability and excellent reusability, which can be rapidly recycled by the aid of magnet.

Magnetic mesoporous microspheres modified with hyperbranched amine for the immobilization of penicillin G acylase

Fe3O4@SiO2@m-SiO2 magnetic mesoporous (SmSMM) microspheres were synthesized by two steps Stöber hydrolysis. The surface of the SmSMM microspheres were modified with γ-(2,3-epoxypropoxy)propytrimethoxysilane (KH560) and dendritic hyperbranched amine (DHA) to obtain SmSMM-DHA microspheres, which were used as carriers for the immobilization of penicillin G acylase (PGA). The diameter of SmSMM-DHA microspheres was around 500nm and the specific saturation magnetization was 61.25emug−1. The advantages of the as-prepared carries were large specific surface area and high amino content, the values were 323.1m2g−1 and 2.1mmolg−1, respectively. The influences of the factors on amino content, immobilization rate and enzyme activity were investigated. The research results of PGA immobilized on SmSMM-DHA microspheres indicated that compared with free enzyme, the immobilized PGA showed excellent pH and thermo stability. Meanwhile, after being used for ten times, the enzyme activity retention rate was 94.5%. The designed immobilized enzyme shows potential application value in the synthesis of antibiotics.

<italic>In vivo</italic> imaging of penicillin G acylase in mice via a near-infrared fluorescent probe

Penicillin G acylase (PGA) is a hydrolytic enzyme, which is also industrially employed in the preparation of semisynthetic penicillin. Abuse of PGA might occur in milk products to decompose the possible excessive antibiotics. Detection of PGA, including in vivo imaging of PGA, is necessary to understand the potential function of this enzyme in living biosystems; however, a near-infrared (NIR) fluorescent probe for PGA is still lacking. On the basis of amino hemicyanine as well as the ability of PGA to hydrolyze phenylacetamide, in this work we have developed a new selective and sensitive NIR fluorescent probe for such a purpose. Combined with in vivo fluorescence imaging system, the probe has been preliminarily used to image the change of the PGA activity in mice. In addition, the NIR feature of our probe makes it of potential use in studying the function of PGA in vivo .

Potential of Pichia pastoris for the production of industrial penicillin G acylase.

This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880U/L of an active enzyme after 142h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.

Enzymatic reactive crystallization for improving ampicillin synthesis

The enzyme penicillin G acylase (PGA) catalyzes the condensation of phenylglycine methyl ester (PGME) with 6-aminopenicillanic acid (6-APA) to form ampicillin. We improved the selectivity of ampicillin synthesis with PGA by running simultaneous reaction and crystallization. However, the enzyme also catalyzes two undesirable side reactions: the hydrolysis of PGME to phenylglycine and the hydrolysis of ampicillin to phenylglycine and 6-APA. We demonstrate that a fifty percent improvement in selectivity for ampicillin over phenylglycine is achieved by combining reaction and crystallization in batch at pH value of 6 with saturated 6-APA and equimolar PGME. The enhancement in selectivity is mainly attributed to the decreased rates of enzymatic ampicillin hydrolysis; however, the course of the pH value during the reaction also has an effect on enzyme activity that improved selectivity. In addition to showing experimental results, we developed a new kinetic process model that predicts the observed improvement. The new model accounts for the solubility limits of different species as functions of pH value as well as the large change in pH value at high conversion. Previous work does not account for changes in activity with conversion. The pH-dependent activity for the specific enzyme used in this system, Assemblase® from DSM-Sinochem, is well-realized by the model and generalization to other PGAs is possible within the model framework; the selectivity parameters α, β0, and γ for Assemblase® are compared to PGA from E. coli as evidence.

A Facile Preparation Process of Magnetic Aldehyde-Functionalized Ni(0.5)Zn(0.5)Fe₂O4@SiO₂ Nanocomposites for Immobilization of Penicillin G Acylase (PGA).

A facile sol combustion and gel calcination process has been reported for the preparation of core– shell magnetic Zn0.5Fe₂O₄@SiOnanocomposites. The morphology, chemical composition, structure and magnetic property of as-prepared nanocomposites were investigated by XRD, VSM, BET, SEM, and TEM, and the magnetic Ni0.5Zn0.5Fe₂O₄@SiO₂ nanocomposites were characterized with average size of about 25 nm, saturation magnetization of 90.8 Am²/kg and the specific surface area of 67.1 m2/g. The surface of Ni0.5Zn0.5Fe₂O₄@SiO₂ nanocomposites was functionalized with glutaraldehyde to form the aldehyde-functionalized magnetic Ni0.5Zn0.5Fe₂O4@SiO₂ nanocomposites, and penicillin G acylase (PGA) was successfully immobilized onto them. And the immobilized PGA exhibited high effective activity, good stability of enzyme catalyst and good reusability, and could retain 63.5% of initial activity after 12 consecutive operations. The kinetic parameters were determined, and the value of K m for the immobilized PGA (161.7 mmol/L) is higher than that of the free PGA (3.5 mmol/L), while v max (1.626 mmol/min) is also larger than that of the free PGA (0.838 mmol/min), which revealed that the immobilization of PGA onto Ni0.5Zn0.5Fe₂O₄@SiO₂ nanocomposites was an efficient and simple way for preparation of stable PGA.

Batch and in-flow kinetic resolution of racemic 1-(N-acylamino)alkylphosphonic and 1-(N-acylamino)alkylphosphinic acids and their esters using immobilized penicillin G acylase

The kinetic resolution of racemic 1-(N-acylamino)alkylphosphonic acids 3 (R3=OH) and their dimethyl esters 1, as well as 1-(N-acylamino)alkylphosphinic acids 4 (R3=H or Ph) using penicillin G acylase (PGA) immobilized on three types of mesoporous silicas in both a batch slurry system and in a continuous-flow reactor was studied. The initial hydrolytic deacylation rates in the presence of those catalysts were measured and the relationships between the substrate structure and the enzyme efficiency are discussed. The stereospecific hydrolysis of the N-acyl group of both racemic N-acylated phosphorus analogues of amino acids and their esters catalyzed by the immobilized PGA proved to be a highly effective method for the kinetic resolution of all the investigated compounds, with the stereochemical preference of PGA for (R)-substrates.