6-aminopenicillanic Acid Research Articles

Strategies to enhance the hydrolytic activity of Escherichia coli BL21 penicillin G acylase based on heterologous expression and targeted mutagenesis

Penicillin G acylase (PGA) serves as a critical biocatalyst for the hydrolysis of penicillin G, yielding 6-aminopenicillanic acid, a vital precursor for β-lactam semi-synthetic antibiotics. The catalytic efficiency of PGA, however, remains suboptimal in native Escherichia coli strains. To improve this, E. coli BL21 was engineered as a microbial cell factory via heterologous expression and site-directed mutagenesis to enhance PGA activity. The heterologous pga gene from Providencia rettgeri was integrated into E. coli BL21 (DE3) for the biosynthesis of PGA, achieving a PGA activity of 253 ± 2 U/mL after 16 hours of fermentation. The N167 site underwent mutation, producing the sites N167A and N167I. Plasmids carrying these mutations were introduced into E. coli BL21(DE3), and the enzymatic activities were recorded as 293 ± 3 U/mL for the N167A mutant and 238 ± 2 U/mL for the N167I mutant. This study not only introduces a novel approach to enhancing PGA activity but also illustrates the potential for catalytic optimization through targeted modifications of the enzyme's active site.

Causal Effects of Gut Microbiota on Gout and Hyperuricemia: Insights from Genome-Wide Mendelian Randomization, RNA-Sequencing, 16S rRNA Sequencing, and Metabolomes.

This study investigated the causal relationship between gut microbiota (GM), serum metabolome, and host transcriptome in the development of gout and hyperuricemia (HUA) using genome-wide association studies (GWAS) data and HUA mouse model experiments. &#160;Methods: Mendelian randomization (MR) analysis of GWAS summary statistics was performed using an inverse variance weighted (IVW) approach to determine predict the causal role of the gut microbiota on gout. The HUA mouse model was used to characterize changes in the gut microbiome, host metabolome, and host kidney transcriptome by integrating cecal 16S rRNA sequencing, untargeted serum metabolomics, and host mRNA sequencing.</p> &#160;Results: Our analysis demonstrated causal effects of seven gut microbiota taxa on gout, including genera of Ruminococcus, Odoribacter, and Bacteroides. Thirty-eight, immune cell traits were associated with gout. Dysbiosis of Dubosiella, Lactobacillus,Bacteroides, Alloprevotella, and Lachnospiraceae_NK4A136_group genera were associated with changes in the serum metabolites and kidney transcriptome of the HUA model mice. The changes in the gut microbiome of the HUA model mice correlated significantly with alterations in the levels of serum metabolites such as taurodeoxycholic acid, phenylacetylglycine, vanylglycol, methyl hexadecanoic acid, carnosol, 6-aminopenicillanic acid, sphinganine, p-hydroxyphenylacetic acid, pyridoxamine, and de-o-methylsterigmatocystin, and expression of kidney genes such as CNDP2, SELENOP, TTR, CAR3, SLC12A3, SCD1, PIGR, CD74, MFSD4B5, and NAPSA.</p> &#160;Conclusion: Our study demonstrated a causal relationship between GM, immune cells, and gout. HUA development involved alterations in the vitamin B6 metabolism because of gut microbiota dysbiosis that resulted in altered pyridoxamine and pyridoxal levels, dysregulated sphingolipid metabolism, and excessive inflammation.</p>.

Analysis of dissolved organic matter characteristics in pharmaceutical wastewater via spectroscopy combined with Fourier-transform ion cyclotron resonance mass spectrometry

Studying the changes in organic matter and characteristic pollutants during the treatment of penicillin-containing pharmaceutical wastewater, which can be reflected by changes in dissolved organic matter (DOM), is crucial for improving the effectiveness of wastewater treatment units and systems. Herein, water quality indicators, spectroscopic methods, and Fourier-transform ion cyclotron resonance mass spectrometry were utilized to characterize the general molecular compositions and specific molecular changes in DOM during the treatment of typical penicillin-containing pharmaceutical wastewater, including in each of the influent, physicochemical treatment, biological treatment, oxidation treatment, and effluent stages. The influent exhibited a high organic matter content (concentration of dissolved organic carbon >10,000 mg·L−1), its DOM mainly contained protein- and lignin-like substances composed of CHON and CHONS molecules, and the relative intensity (RI) of penicillin was extremely high (RI = 0.220). Compared with the influent, the abundance of CHON and CHONS molecules detected after physicochemical treatment decreased by 70.3 % and 62.5 %, respectively, and the RI of penicillin decreased by 85.5 %. Biological treatment caused substantial changes in DOM components through oxidation, dealkylation, and denitrification reactions, accounting for 36.8 %, 28.9 %, and 14.8 % of the total identified reactions, respectively. Additionally, lignin-like substances were generated in large quantities, the overall humification level significantly increased, and the RI value increased for the penicillin intermediate, 6-aminopenicillanic acid (6-APA). Oxidation treatment effectively removed phosphorus-containing substances and some lignin-like substances produced by biological treatment; however, it was not effective in removing characteristic pollutants such as 6-APA. Such characteristic substances continued to be present in the effluent, and the DOM mainly contained protein- and humus-like substances, accounting for 30.8 % and 47.3 %, respectively. The study findings reveal the changes in organic matter and characteristic pollutants during the treatment of penicillin-containing wastewater from the perspective of the general molecular composition and specific molecular changes in DOM, providing support for further exploration of wastewater treatment mechanisms and improvements in treatment unit efficiency.

Preparation of alginate/vermiculite composite functionalized with silanol group for controlled drug delivery: Effect of CaCl2 concentration, release kinetics, cytotoxicity, and antimicrobial activities

In this study, alginate/vermiculite (Alg/VMT) hydrogel with 3-aminopropyl triethoxysilane (Alg/VSN) and tetraethoxysilane (Alg/VS) synthesized with various concentrations of CaCl2 (10 %–15 %–20 % M) to extend the release of 6-Aminopenicillanic acid (AP). Composites characterized by XRD, FTIR and BET. The result of Alg/VS composite shows an excellent loading of 243.90 mg/g through AP intercalated in the VMT layer. The equilibrium and Kinetic studies indicated that AP adsorption on Alg/VS and Alg/VSN was heterogeneous with chemical interaction. The in-vitro release Alg/VS showed a rapid burst release of 14 % in the first half an hour and only 75 % of the drug remained in the composite. Whereas, the in-vitro release Alg/VSN showed substantially less burst release with the cumulative release of 9 % (in the first 0.5 h). In-vitro release kinetics in the presence of CaCl2 concentrations showed that maximum 19 % of AP released within 12 h. The kinetic release was followed by a controlled release pattern (Korsmeyer-Peppas model) with Fick's law mechanism. The composites behaved as barriers against cell growth and had better biocompatibility against standard strains of Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus. MTT assay results from per cent cell viability composites modified by silanol groups were 96 % the means samples were nontoxic. The types of newly synthesized composites were able to finely decrease cell toxicity and improve AP release in vitro.

Microbial regulatory mechanisms of disease-resistant tobacco varieties in the prevention and control of bacterial wilt disease

Bacterial wilt disease poses a significant threat to the production and quality of tobacco. Disease-resistant varieties of tobacco are the primary means of preventing bacterial wilt, but the soil microbial mechanisms that alleviate this disease require further in-depth research. In our study, we analyzed the physicochemical and microbial properties of rhizosphere soil in four different disease-resistant varieties of tobacco. The treatments included: T1 (Chang Bo Huang; 100 % infection rate); T2 (Yun Yan 87; 80 % infection rate); T3 (K326; 50 % infection rate) and T4 (YAN YAN 97; 0 % infection rate). Our results showed that the T4 treatment improved the fresh biomass of tobacco by 35.42 % compared to the T1 treatment. In addition, a significant increase in soil pH and soil organic matter (SOM) from 4.98 and 1.24 % under T1 treatment to 5.86 and 1.38 % under T4 treatment, respectively. As the disease resistance of the tobacco variety improved was found, there was a noticeable upward trend in the number of operational taxonomic units (OTUs) in the rhizosphere soil, showing higher sensitivity compared to fungi. Additionally, rhizosphere soil from susceptible varieties exhibited higher levels of metabolites, resulting in the accumulation of acidic substances (6-aminopenicillanic acid, garcinia acid, and 4-decan-4-ylbenzenesulfonic acid). Moreover, these soil metabolites might have attracted certain pathogenic fungi (Monoblepharomycota), contributing to the spread of soil diseases. This study suggests that the rhizosphere soil of disease-resistant tobacco variety provides a better microecological environment which could inhibit accumulation of allelopathic substances and reduce the spread of bacterial wilt disease.

Revealing the Structure of 6-Aminopenillanic Acid: The Active Nucleus of Penicillins.

6-Aminopenicillanic acid is a penicillanic acid compound and is the active nucleus common to all penicillins. Using laser ablation techniques, we transformed the solid into the gas phase and characterized its conformational panorama by combining supersonic expansions and Fourier transform microwave techniques. Five conformers were determined, adopting different spatial configurations. Among them, the axial and equatorial forms, which are biologically relevant, have been observed. The structural similarity to d-Ala-d-Ala and the detection of both axial and equatorial forms could explain its potential as a penicillin core and its capability as an antibiotic.

Site-directed mutagenesis of recombinant penicillin-G acylase from Escherichia coli and effect on the hydrolytic activity

Penicillin-derived antibiotics such as amoxicillin are one of the antibiotics that are widely used. Amoxicillin is synthesized through the availability of raw materials, 6-aminopenicillanic acid (6-APA), with penicillin-G acylase (PGA) as the catalyst. Previous studies revealed a recombinant PGA through the utilization of expression hosts. However, the low activity of the expressed enzyme is still an obstacle to the optimum utilization of its enzyme. In this study, we applied site-directed mutagenesis computationally for initial prediction by creating 36 PGA mutants through homology modeling and predicted their binding affinity as well as bounding interaction mode to penicillin-G through molecular docking. Our research found that four mutants had better potential activity than wild-type PGA. In the first experiment, only the βThr68Tyr PGA mutant was selected to be explored further. The mutant PGA gene inserted into pET-22b plasmid was transformed to Escherichia coli BL21(DE3) by heat shock method. For enzyme expression, IPTG and arabinose were used as inducers. As a result, the specific enzyme activity of mutant PGA showed insignificantly lower values in comparison to wild-type PGA. This activity appeared to be influenced by the formation of inclusion bodies during post-translational protein maturation, as described in this paper.

Synthesis, Characterization and Spectral Studies of Some Metal Ions Complexes with New Schiff Base Ligand Derived From 6- Amino Penicillanic Acid And Biological Screening Study For The Pt(II) Complex.

The complexes of Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Hg (II) and Pt(II)were prepared from the complexation reaction between the new Schiff base ligand and Selected Metal Ions. Ligand was derived from the condensation reaction of Oxo(4-(pyridin-2-yl)phenyl) methyline, 4-amino antipyrine, and 6-amino pencillic acid ). The ligand and its complexes were characterized by C.H.N elemental analysis, Uv-Vis, FT-IR, and molar conductivity, Atomic absorption, magnetic moment measurements and mass spectra studies. The results of this studies show the coordination sites for the ligand with the metal ion were to be through oxygen, nitrogen atoms of 6-amino pencillic acid, and the nitrogen atom of 4-amino antipyrine. The complexes were found to have the general formula [(M)(L)2]Cl2 where M = Co(II), Ni(II), Cu(II), Zn(II), Hg(II), and Cd(II), except the [Pt(L)Cl]Cl. The electronic spectral and magnetic measurement data indicated that the complexes exhibited octahedral geometry, except the Pt(II) complex suggested a square planar geometry around the central metal ion. The ligand forms shape as natural tridentate manner. The biological activity results showed the inhibitory effect for platinum(II) the complex. In this study the cytotoxicity of Pt(II) complex on human colon cancer cell line LS 174 T CRC was investigated and African green monkey kidney cells normal cells were MTT assay. The metal complexes showed selective cytotoxicity against colorectal, this metal Complex excelled in halting proliferation of LS 174 T CRC cancer cells line with median inhibitory concentration (IC50) values of Pt(II) complex. The results indicate undoubtedly the possibility of using them as antitumor drugs in the field of pharmacy colon cancer.

Predicting bioactivity of antibiotic metabolites by molecular docking and dynamics

Antibiotics enter the environment through waste streams, where they can exert selective pressure for antimicrobial resistance in bacteria. However, many antibiotics are excreted as partly metabolized forms, or can be subject to partial breakdown in wastewater treatment, soil, or through natural processes in the environment. If a metabolite is bioactive, even at sub-lethal levels, and also stable in the environment, then it could provide selection pressure for resistance. (5S)-penicilloic acid of piperacillin has previously been found complexed to the binding pocket of penicillin binding protein 3 (PBP3) of Pseudomonas aeruginosa. Here, we predicted the affinities of all potentially relevant antibiotic metabolites of ten different penicillins to that target protein, using molecular docking and molecular dynamics simulations. Docking predicts that, in addition to penicilloic acid, pseudopenicillin derivatives of these penicillins, as well as 6-aminopenicillanic acid (6APA), could also bind to this target. MD simulations further confirmed that (5R)-pseudopenicillin and 6APA bind the target protein, in addition to (5S)-penicilloic acid. Thus, it is possible that these metabolites are bioactive, and, if stable in the environment, could be contaminants selective for antibiotic resistance. This could have considerable significance for environmental surveillance for antibiotics as a means to reduce antimicrobial resistance, because targeted mass spectrometry could be required for relevant metabolites as well as the native antibiotics.

Isolation and Molecular Characterization of Indigenous Penicillium chrysogenum/rubens Strain Portfolio for Penicillin V Production.

Beta (β)-lactam antibiotic is an industrially important molecule produced by Penicillium chrysogenum/rubens. Penicillin is a building block for 6-aminopenicillanic acid (6-APA), an important active pharmaceutical intermediate (API) used for semi-synthetic antibiotics biosynthesis. In this investigation, we isolated and identified Penicillium chrysogenum, P. rubens, P. brocae, P. citrinum, Aspergillus fumigatus, A. sydowii, Talaromyces tratensis, Scopulariopsis brevicaulis, P. oxalicum, and P. dipodomyicola using the internal transcribed spacer (ITS) region and the β-tubulin (BenA) gene for precise species identification from Indian origin. Furthermore, the BenA gene distinguished between complex species of P. chrysogenum and P. rubens to a certain extent which partially failed by the ITS region. In addition, these species were distinguished by metabolic markers profiled by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Secalonic acid, Meleagrin, and Roquefortine C were absent in P. rubens. The crude extract evaluated for PenV production by antibacterial activities by well diffusion method against Staphylococcus aureus NCIM-2079. A high-performance liquid chromatography (HPLC) method was developed for simultaneous detection of 6-APA, phenoxymethyl penicillin (PenV), and phenoxyacetic acid (POA). The pivotal objective was the development of an indigenous strain portfolio for PenV production. Here, a library of 80 strains of P. chrysogenum/rubens was screened for PenV production. Results showed 28 strains capable of producing PenV in a range from 10 to 120 mg/L when 80 strains were screened for its production. In addition, fermentation parameters, precursor concentration, incubation period, inoculum size, pH, and temperature were monitored for the improved PenV production using promising P. rubens strain BIONCL P45. In conclusion, P. chrysogenum/rubens strains can be explored for the industrial-scale PenV production.

New Semisynthetic Penicillins Obtained by Coupling of the 6-Aminopenicillanic Acid with 5-Mercapto-1,2,4-triazoles-3,4-disubstituted.

In a basic medium, 5-Mercapto-1,2,4-triazoles pass into the thiol form, allowing their transformation into sodium salts, which, in reaction with sodium monochloroacetate, lead to sodium 5-thioacetates of 1,2,4-triazoles-3,4-disubstituted. Sulfur derivatives converted to pivalic mixed anhydrides were used as active forms in the acylation of 6-amino penicillanic acid (6-AP) to obtain new semisynthetic penicillins. They contain in the molecule, together with the β-lactam ring, the nucleus 3-[(5-nitroindazol-1'-yl-methyl)]-4-aryl-5-mercapto-1,2,4-triazole, both contributing to an important antibacterial effect. The structure of the new antibiotics was confirmed by the results of elemental and spectral analysis (FT-IR, 1H- and 13C-NMR). The synthetic penicillins were tested for toxicological action and antibacterial activity and the obtained results were close to those for amoxicillin, the reference drug.

Synthesis of New Derivatives of Leaf-Eating Ant Toxic Gland Pheromone Compounds and Investigation of their Biological Properties

‌Background: The anti-mycobacterial characteristics of 2-pyrazinoic acid esters have been discovered through recent research. Research has demonstrated that the pyrazine ring and the alkyl part of these compounds interact with the enzyme phytynetase, which in turn inhibits the interaction between NADPH and mycobacterial fatty acids synthase, the enzyme responsible for synthesizing the fatty acids required for the cell wall of mycobacteria. Materials and Methods: Targeted pyrazinoic acid molecules have been synthesized, their structure verified by spectroscopic data, and their anti-mycobacterial activity against Mycobacterium TB H37Rv at a dose of 10 micrograms per milliliter assessed in order to test this theory. Significant anti-mycobacterial capabilities were shown by several compounds, including 3c, 3J, and 3M, which inhibited mycobacterial growth by 45.7%, 45.4%, and 51%, respectively. Findings: The results showed that the compounds exhibited little toxicity and did not inhibit macrophage cell proliferation. Pyrazinamide had significant antibacterial and antifungal activity, despite its lack of fatal action against non-tubercular mycobacteria and both Gram-positive and Gram-negative bacteria. Compound 3B, for example, exhibited excellent antibacterial properties against Gram-positive bacteria, such as S. aureus, with a minimum inhibitory concentration (MIC) of 125 micrograms per milliliter. Conversely, the compounds showed equally potent antibacterial properties against Gram-negative bacteria. Interestingly, compared to Gram-positive bacteria, the ester produced from maltol exhibited greater antibacterial efficacy against Gram-negative bacteria. Compounds synthesized with biodegradable groups also exhibited time-dependent toxicity against K562 leukemia cancer cells in MTT measurements, with compounds 3e and 3J displaying IC50 values of 25 micromolars. Conclusion: After synthesizing pyrazinoic acid monoterpene esters, spectral data was used to confirm their structures. When their biological characteristics were assessed, toxicity studies against K562 leukemia cells produced encouraging results. Through the use of Thio Ester-mediated activation of 2-pyrazinoic acid by 2, 2-dibenzothiazole disulfide and triphenylphosphine, 6-aminopenicillanic acid (6-APA) was coupled with pyrazinoic acid.

Molecular Modeling of Penicillin Acylase Binding with a Penicillin Nucleus by High Performance Computing: Can Enzyme or its Mutants Possess beta-lactamase Activity?

High-performance computing has been used for molecular modeling of penicillin acylase interaction with a penicillin nucleus 6-aminopenicillanic acid (6-APA) to assess whether the wild-type enzyme or its mutants could possess β-lactamase activity. Applying parallel hybrid GPU/CPU computing technologies for metadynamics calculations with the PLUMED library in conjunction with AMBER software suite it has been shown that trace amounts of wild-type penicillin acylase6-APA complexes leading to a β-lactamase reaction can be formed. Higher β-lactamase activity can be observed in enzyme mutants by introducing charged residue in the substrate binding pocket and its proper positioning with respect to a catalytic nucleophile, including stabilization of the tetrahedral intermediate in the oxyanion hole. Thus, it has been shown that the certain mutations facilitate the orientation of the substrate required for the manifestation of β-lactamase activity in the penicillin acylase active center.

Insights into the anticancer activity of chiral alkylidene-β-lactams and alkylidene-γ-lactams: Synthesis and biological investigation

Chiral alkylidene-β-lactams and alkylidene-γ-lactams were synthesized and screened for their in vitro activity against four human cancer cell lines (melanoma, esophageal, lung and fibrosarcoma carcinoma). Alkylidene-β-lactams were synthesized via Wittig reaction of diverse phosphorus ylides with benzhydryl 6‐oxopenicillanate, derived from 6-aminopenicillanic acid. Moreover, novel chiral alkylidene-γ-lactams were synthesized through a multistep strategy starting from a chiral substrate (d-penicillamine). The in vitro assays allowed the identification of four compounds with IC50 values < 10 μM for A375 cell line, and three compounds with IC50 values < 10 μM for OE19 cell line. The effect of the most promising compounds on cell death mechanism, reactive oxygen species generation as well as the evaluation of their ability to act as MMP-9 inhibitors were studied. The reported results unveil the potential of alkylidene-β-lactams as anticancer agents.

Design and synthesis of iron(II) complexes with Schiff bases derived from 6-aminopenicillanic acid and heterocyclic aldehydes

The reaction of iron (II) acetate with Schiff bases derived from the condensation reaction of benzaldehyde or indole-3-carboxaldehyde with (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1azabi cyclo[3.2.0] hepta -ne-2-carboxylic acid (6- Aminopenicillanic Acid) in 1:1 molar ratio gave a new series of iron(II) complexes. The coordination bond formation behaviour of Schiff bases with iron atom has been characterised by elemental analysis, conductance measurements, molecular weight determinations, IR, UV and 1H NMR spectral studies.

Positive Effects of Neutrophil Elastase Inhibitor (Sivelestat) on Gut Microbiome and Metabolite Profiles of Septic Rats.

BackgroundNeutrophil elastase (NE) is associated with sepsis occurrence and progression. We hypothesized that the NE inhibitor Sivelestat might modulate abnormal gut microbiota and metabolites during sepsis.MethodsSixty Sprague-Dawley (SD) rats were randomly divided into sham control (SC), sepsis (CLP), and sepsis+Sivelestat (Sive) groups. The rats’ survival status was monitored for 24 hours postoperatively, and feces were collected for microbiome and non-targeted metabolomics analyses.ResultsSivelestat administration significantly improved the survival of septic rats (80% vs 50%, P = 0.047). Microbiome analysis showed that the microbiota composition of rats in the CLP group was significantly disturbed, as potential pathogens such as Escherichia-Shigella and Gammaproteobacteria became dominant, and the beneficial microbiota represented by Lactobacillus decreased. These changes were reversed in Sive group, and the overall microbial status was restored to a similar composition to SC group. Differential analysis identified 36 differential operational taxonomic units and 11 metabolites between the Sive and CLP groups, such as 6-Aminopenicillanic acid, gamma-Glutamyl-leucine, and cortisone (variable importance in projection>1and P<0.05). These discriminatory metabolites were highly correlated with each other and mainly involved in the phenylalanine, tyrosine, and tryptophan biosynthesis pathways. Integrated microbiome and metabolome analyses found that almost all Sivelestat-modulated microbes were associated with differential metabolites (P < 0.05), such as Lactobacillus and some amino acids, suggesting that the Sivelestat-induced metabolic profile differences were in part due to its influence on the gut microbiome.ConclusionSivelestat administration in septic rats improved survival, gut microbiota composition and associated metabolites, which could provide new options for sepsis treatment.

Biosynthesis of β-lactam nuclei in yeast

We have engineered brewer's yeast as a general platform for de novo synthesis of diverse β-lactam nuclei starting from simple sugars, thereby enabling ready access to a number of structurally different antibiotics of significant pharmaceutical importance. The biosynthesis of β-lactam nuclei has received much attention in recent years, while rational engineering of non-native antibiotics-producing microbes to produce β-lactam nuclei remains challenging. Benefited by the integration of heterologous biosynthetic pathways and rationally designed enzymes that catalyze hydrolysis and ring expansion reactions, we succeeded in constructing synthetic yeast cell factories which produce antibiotic cephalosporin C (CPC, 170.1 ± 4.9 μg/g DCW) and the downstream β-lactam nuclei, including 6-amino penicillanic acid (6-APA, 5.3 ± 0.2 mg/g DCW), 7-amino cephalosporanic acid (7-ACA, 6.2 ± 1.1 μg/g DCW) as well as 7-amino desacetoxy cephalosporanic acid (7-ADCA, 1.7 ± 0.1 mg/g DCW). This work established a Saccharomyces cerevisiae platform capable of synthesizing multiple β-lactam nuclei by combining natural and artificial enzymes, which serves as a metabolic tool to produce valuable β-lactam intermediates and new antibiotics.

Changes in Oxygen Availability during Glucose-Limited Chemostat Cultivations of Penicillium chrysogenum Lead to Rapid Metabolite, Flux and Productivity Responses.

Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors.

Improving the 6-Aminopenicillanic acid release process using vermiculite-alginate biocomposite bead on drug delivery system

The present study deals with developing vermiculite (VMT)-alginate (Alg) composites with different cross-linker concentrations (CaCl2) to deliver the controlled 6-aminopenicillin acid (6-APA). The Characterization of synthesized composites were conducted by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analyses. Optimization attempts were explored via the response surface method (RSM) to best predict the actual amount of compound. The adsorption capacity of 6-APA onto this adsorbent was found to be 208.33 mg/g, which was higher than that for other clays. The equilibrium and Kinetic studies (chemical reaction and diffusion-based models) indicated that drug absorption on VMT-Alg is homogeneous with chemical interaction. An increase in cross-linker (CaCl2) concentration leads to improvement in the drug encapsulation efficiency while has no significant effect on loading efficiency. The in-vitro release of the pure drug shows a rapid burst release followed by 100% cumulative release within 6 hours. Whereas, the synthesized drug with Alg substantially showed less release of 43% after 8 hours. Release experiments revealed that the presence of the CaCl2 delayed the release of the 6-APA less than 35% after 12 hours. The kinetic release of 6-APA followed by the Korsmeyer-Peppas model based on Fick's law mechanism due to the kinetic exponent (n < 0.5). All studied composites antibacterial activity after 24 h exposure against E. Coli and S. aureus. The antibacterial activities of composites were evaluated by the halo of no growth. The results showed that VMT-Alg-6APA composite had strong activity against Gram-positive and Gram-negative bacteria.

Development and Validation of Ecofriendly HPLC-MS Method for Quantitative Assay of Amoxicillin, Dicloxacillin, and Their Official Impurity in Pure and Dosage Forms.

Novel, accurate, selective, and rapid high-performance liquid chromatography mass spectrometry method was developed for simultaneous analysis of amoxicillin trihydrate, dicloxacillin sodium, and their official impurity 6-aminopenicillanic acid. The chromatographic separation was carried out by applying the mixture on a C18 column (3.5 µm ps, 100 mm × 4.6 mm id) using acetonitrile:water (65 : 35 by volume) as a mobile phase within only 4 min. The quantitative analysis was executed using single quadrupole mass spectrometer in which electrospray ionization, selected ion monitoring, and negative mode were operated. The retention times were 1.61, 2.54, and 3.50 mins for amoxicillin, 6-aminopenicillanic acid, and dicloxacillin, respectively. The method was validated in linear ranges of 2–28 µg mL−1, 2–35 µg mL−1, and 1–10 µg mL−1 for amoxicillin, dicloxacillin, and 6-aminopenicillanic acid, respectively. The results obtained from the suggested HPLC/MS were statistically compared with those obtained from the reported HPLC method, where no significant difference appeared respecting accuracy and precision. According to the analytical eco-scale assessment method, the proposed method was proved to be greener than the reported one, where the analysis time and the amount of the wasted effluent decreased.