7-aminocephalosporanic Acid Research Articles

Synthesis and Biological Activity Evaluation of Two Derivatives of 7-Aminocephalosporanic Acid

Synthesis and Biological Activity Evaluation of Two Derivatives of 7-Aminocephalosporanic Acid

Cephalosporin C acylase: Important role, obstacles, and strategies to optimize expression in E. coli

Cephalosporins have gained popularity due to the increasing resistance to penicillin antibiotics. Since the first discovery of cephalosporin C in 1945, the development of cephalosporin-class antibiotics has continued. Cephalosporin C was converted into a 7-aminocephalosporanic acid (7-ACA) compound which became a precursor for producing cephalosporin class antibiotics through side chain modification. The conversion of cephalosporin C to 7-ACA is more beneficial when performed enzymatically using the enzyme cephalosporin C acylase (CA) compared to chemical methods. This article will present the importance of cephalosporin CA enzymes in the development of cephalosporin antibiotics, the obstacles, and strategies to optimize the expression of these enzymes in Escherichia coli hosts.

Synthesis and Anti-Vibrio Activity of Some New 1,3,4-Thiadiazole Derivatives Containing 7-Aminocephalosporanic Acid

A series of new 7-aminocephalosporanic acid containing 1,3,4-thiadiazole derivatives (8a-f and 9) was synthesized in search of potential antibacterial agents. These structures were characterized by means of infrared, proton nuclear magnetic resonance, and electrospray ionization-high-resolution mass spectrometry. All the tested compounds demonstrated good inhibitory effects on anti-Vibrio activity. Among them, the minimum inhibitory concentration values of compounds 8a, 8c, 8d, and 9 against Vibrio harveyi and Vibrio parahaemolyticus reached 0.0039 mg/mL and 0.0078 mg/mL, respectively, showing better inhibitory activities even more than the standard seven streptomycin sulfate (0.016 mg/mL and 0.0625 mg/mL). Thus, these new compounds could serve as potential candidates to become leads for the development of new drugs eliciting anti-Vibrio agents effect.. KEYWORDS :1,3,4-Thiadiazole, 7-Aminocephalosporanic acid, Anti-vibrio activity.

A Comparative Study of Biocatalytic Acylation of 7-Aminocephalosporanic Acid and its C3 Derivatives

A Comparative Study of Biocatalytic Acylation of 7-Aminocephalosporanic Acid and its C3 Derivatives

Characterization and engineering of cephalosporin C acylases to produce 7-Aminocephalosporanic acid

7-Aminocephalosporanic acid (7-ACA) is the core nuclei for the synthesis of β-lactam antibiotics. Cephalosporin C acylases (CCAs) are a class of enzymes that enable the hydrolysis of cephalosporin C (CPC) to 7-ACA. In this study, we identified, characterized and engineered new CCAs to prepare 7-ACA. The best mutant A14 (L159S/A419V) was obtained after several rounds of semi-rational enzyme design using the combinatorial active-site saturation testing (CAST) and iterative evolution. A 2.8-fold increase in specific enzymatic activity (6.1 U/mg) was obtained compared to the wild-type enzyme. The engineered variant also enabled the hydrolysis of a variety of β-lactam antibiotics including Penicillin G and phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA). Overall, this study discovered new CCAs from distinct bacterial origins and enabled the synthesis of various core β-lactam antibiotics nuclei.

Bacterial conversion of Cephalosporin C: Optimization in Achromobacter xylosooxidans

Achromobacter xylosooxidans was isolated from the soil and studied for conversion of the cephalosporin C antibiotic. In order to synthesize broad-spectrum semi-synthetic cephalosporins 7-aminocephalosporanic acid (7-ACA) is a crucial intermediary product. This study identifies crucial variables and the best values for each for the synthesis of the cephalosporin C acylase (CCA) enzyme by Achromobacter xylosooxidans. An enzyme activity measured to be 31.1 and 32.2 U/ml using the substrates cephalosporin C (CPC) and glutaryl p-nitroanilide (GpNA), respectively, under optimal parameters. The CCA activity in Achromobacter xylosooxidans species was detected maximum at 72 h after incubation. HPLC revealed the species of Achromobacter xylosooxidans with the highest CCA activity (36.6 U/ml).

Properties and biotechnological applications of microbial deacetylase.

Deacetylases, a class of enzymes that can catalyze the hydrolysis of acetylated substrates to remove the acetyl group, used in producing various products with high qualities, are one of the most influential industrial enzymes. These enzymes are highly specific, non-toxic, sustainable, and eco-friendly biocatalysts. Deacetylases and deacetylated compounds have been widely applicated in pharmaceuticals, medicine, food, and the environment. This review synthetically summarizes deacetylases' sources, characterizations, classifications, and applications. Moreover, the typical structural characteristics of deacetylases from different microbial sources are summarized. We also reviewed the deacetylase-catalyzed reactions for producing various deacetylated compounds, such as chitosan-oligosaccharide (COS), mycothiol, 7-aminocephalosporanic acid (7-ACA), glucosamines, amino acids, and polyamines. It is aimed to expound on the advantages and challenges of deacetylases in industrial applications. Moreover, it also serves perspectives on obtaining promising and innovative biocatalysts for enzymatic deacetylation. KEYPOINTS: • The fundamental properties of microbial deacetylases of various microorganisms are presented. • The biochemical characterizations, structures, and catalyzation mechanisms of microbial deacetylases are summarized. • The applications of microbial deacetylases in food, pharmaceutical, medicine, and the environment were discussed.

Biochemical characterization of an acetylesterase from Bacillus subtilis and its application for 7-aminocephalosporanic acid deacetylation.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA), which could be converted from 7-aminocephalosporanic acid (7-ACA), is a crucial starting material that is used for synthesizing industrial semisynthetic β-lactam antibiotics. Enzymes involved in the conversion from 7-ACA to D-7-ACA present critical resources in the pharmaceutical industry. In the present study, a putative acetylesterase, EstSJ, identified from Bacillus subtilis KATMIRA1933, was first heterologously expressed in Escherichia coli BL21(DE3) cells and biochemically characterized. EstSJ belongs to carbohydrate esterase family 12 and is active on short-chain acyl esters from p-NPC2 to p-NPC6. Multiple sequence alignments showed that EstSJ was also an SGNH family esterase with a typical GDS(X) motif at its N-terminal end and a catalytic triad composed of Ser186-Asp354-His357. The purified EstSJ displayed the highest specific activity of 1,783.52 U mg-1 at 30°C and pH 8.0, and was stable within the pH range of 5.0-11.0. EstSJ can deacetylate the C3' acetyl group of 7-ACA to generate D-7-ACA, and the deacetylation activity was 4.50 U mg-1. Based on the structural and molecular docking with 7-ACA, the catalytic active sites (Ser186-Asp354-His357) together with four substrate-binding residues (Asn259, Arg295, Thr355, and Leu356) of EstSJ are revealed. This study provided a promising 7-ACA deacetylase candidate that could be applied to produce D-7-ACA from 7-ACA in the pharmaceutical industry.

Characterization of a novel carbohydrate esterase 7 family acetyl xylan esterase from Thermobifida halotolerans YIM 90462T

The newly semi-synthetic β-lactam antibiotics play critical roles in prevention and treatment of β-lactam antibiotic-resistant bacteria. Acetyl xylan esterases belong to carbohydrate esterase 7 family can specifically hydrolyze 7-aminocephalosporanic acid (7-ACA) to produce deacetyl-7-aminocephalosporanic acid (D-7ACA) which is one of the most important starting materials to synthesize newly semi-synthetic β-lactam antibiotics. Here, an effective acetyl xylan esterase, ThAXE, from Thermobifida halotolerans YIM 90462 T, was firstly identified and characterized. ThAXE exhibited its highest catalytic activity against p-nitrophenyl acetate (p-NPC2) at pH 8.5 and 50 °C, with Km and Kcat/Km were 1.0 ± 0.1 mM and 2262.9 mM−1 s−1, respectively. Additionally, ThAXE can hydrolyze the acetyl group of 7-ACA at its C3' position with the specific activity of 7.4 U mg−1. Furthermore, molecular docking combined with site-directed mutagenesis validation demonstrated that the pocket of catalytic residues Ser186, Asp272, and His301 could accommodate p-NPC2 and 7-ACA ligands. This is the first report of a novel acetyl xylan esterase from T. halotolerans that has considerable potential biotechnological values.

Controlled synthesis of 3D marigold-like ZnIn2S4/Ti3C2 for rapid and efficient removal of antibiotics

Developing of a rapid and efficient photocatalyst for the removal of antibiotics with high concentration in wastewater remediation is of great importance. 3D marigold-like ZnIn2S4/Ti3C2 was successfully fabricated by coupling 2D Ti3C2 nanosheets with hierarchical 3D ZnIn2S4 using a hydrothermal method. The degradation efficiency of ZnIn2S4/Ti3C2 composite for tetracycline (50 mg/L) and 7-Aminocephalosporanic acid (25 mg/L) within 120 min and 90 min can reach up to 91% and 95%, respectively. The introduction of Ti3C2 modulates the 3D marigold-like architecture of ZnIn2S4, which not only boosted its photon capture performance and adsorption capacity caused by the increased specific surface area, but also effectively separated photo-generated electrons and holes via the well-defined 2D/2D interface between ZnIn2S4 and Ti3C2. The synergistic effect of physical adsorption and photocatalytic degradation contributes to the high degradation efficiency and fast degradation performance. ZnIn2S4/Ti3C2 still exhibits high photocatalytic activity and excellent physicochemical stability after many cycles.

7-aminocephalosporanic acid, a novel HSP90β inhibitor, attenuates HFD-induced hepatic steatosis

Hepatic steatosis is one of the most important causes of liver disease worldwide. Heat shock protein 90 (HSP90) is essential for numerous client proteins. Recently, more attention was focused on increased HSP90 levels in hepatic steatosis, especially HSP90β. Thus, great efforts have been made to develop HSP90β inhibitors, and most natural inhibitors are derived from microorganisms. In this study, using microarray chips and surface pasmon resonance (SPR) technology, we screened 189 antibiotics in order to obtain an inhibitor directly binding to the non-N-terminal domain of HSP90β. Finally, we discovered an antibiotic, 7-aminocephalosporanic acid (7ACA), with a KD value of 6.201 μM between 7ACA and non-N-terminal domain of HSP90β. Besides, 7ACA was predicted to interact with the middle domain (MD) of HSP90β. In HepG2 cells, we found that 7ACA reduced cellular total cholesterol (TC) and triglyceride (TG) by decreasing sterol regulatory element-binding proteins (SREBPs). In HFD fed mice, administration of 7ACA (5, 10, and 25 mg kg−1 d−1, ig, for 12 weeks) dose-dependently decreased serum TC and TG and played an important role in protecting liver and adipose tissue from lipid accumulation. In conclusion, our study demonstrated that antibiotic 7ACA, as an HSP90β middle domain inhibitor, was promising for the development of lipid-lowering drugs.

Alternative Synthesis of Cefamandole with Biocatalytic Acylation Catalyzed by Immobilized Cephalosporin-Acid Synthetase

The processes of the biocatalytic acylation of 1-methyl-5-mercapto-1,2,3,4-tetrazolil-7-amino-cephalosporanic acid (7-TMCA) and 7-aminocephalosporanic acid (7-ACA) by methyl ester of mandelic acid (MEMA) were optimized with an immobilized cephalosporin-acid synthetase as the biocatalyst. Under optimized conditions in water-organic medium containing 43% (vol/vol) of ethylene glycol at 30°С with a spontaneous pH gradient in the range of 8.0–6.0, the following yields of biocatalytic transformations were reached: (80.8 ± 1.9)% for 7‑TMCA acylation (at a concentration of 100–120 mМ) resulted in cefamandole (CFM) production, and (88.6 ± 2.0)% for 7-ACA acylation of (at concentration of 140–170 mМ) resulted in a semiproduct of CFM (S-p CFM) formation. In the second process, the concentration of the target β-lactam product in the final reaction mixture is one and a half times higher than that with the first one. In light of the undoubted environmental benefits of the chemical transformation of S-p CFM to CFM over the process of the chemical production of 7-TMCA from 7-ACA, we conclude that the second pathway of combined chemical and biocatalytic CFM synthesis is preferable.

A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum.

The pharmaceutical industry has developed various highly effective semi-synthetic cephalosporins, which are generated by modifying the side chains of the core molecule 7-aminocephalosporanic acid (7-ACA). In industrial productions, the 7-ACA nucleus is obtained in vitro from cephalosporin C (CPC) by chemical or enzymatic processes, which are waste intensive and associated with high production costs. Here, we used a transgenic in vivo approach to express bacterial genes for cephalosporin C acylase (CCA) in the CPC producer Acremonium chrysogenum. Western blot and mass spectrometry analyses verified that the heterologous enzymes are processed into α- and β-subunits in the fungal cell. Extensive HPLC analysis detected substrates and products of CCAs in both fungal mycelia and culture supernatants, with the highest amount of 7-ACA found in the latter. Using different incubation times, temperatures, and pH values, we explored the optimal conditions for the active bacterial acylase to convert CPC into 7-ACA in the culture supernatant. We calculated that the best transgenic fungal strains exhibit a one-step conversion rate of the bacterial acylase of 30%. Our findings can be considered a remarkable contribution to supporting future pharmaceutical manufacturing processes with reduced production costs.

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.

Optimization of cephalosporin C acylase immobilization using crosslinked enzyme aggregates technique

Cephalosporin C acylase (CCA) is an essential enzyme for the one-step conversion of cephalosporin C into 7-aminocephalosporanic acid (7-ACA), an intermediate compound used to synthesize various semi-synthetic cephalosporin antibiotics. The industrial process prefers to use enzymes in immobilized form rather than soluble. A crosslinked enzyme aggregate (CLEAs) is a potential matrix-less enzyme immobilization technique to produce stable immobilized enzymes with high activity and low production costs. This study aimed to optimize the CCA immobilization using the CLEAs technique with Chitosan as a co-aggregate. The CCA lysate was obtained from harvesting CCA fermentation broth using a mutant strain of Escherichia coli through cell separation and lysis steps. Partially purified CCA by ammonium sulfate addition was conducted to obtain an active fraction of 20-60% saturation, followed by co-aggregation with Chitosan to form physical CCA aggregates. The aggregates were then immobilized by a crosslinking technique using glutaraldehyde to form CLEAs-CCA. Optimization of the immobilization process was carried out by Response Surface Methodology in three steps, (i) screening of the influencing factors, (ii) determining the level of the significant factors, and (iii) optimizing the immobilization condition. The CLEAs-CCA activity was used as a response parameter. Under optimum conditions, CLEAs-CCA activity obtained was 85.91 Ug-1.

Evaluation of Ceftriaxone Residue in Cow Milk and its Toxicity on Danio rerio

Ceftriaxone (CEFT), a widely used broad-spectrum beta -lactam cephalosporin antibiotic, is used to treat bovine mastitis, caused by a variety of bacteria. If used injudiciously, this antibiotic leaves a residue that persists after pasteurization. Antibiotic residue contamination occurs when antibiotic residue exceeds its Maximum Residue Limits (MRLs). This has negative impacts on both public health and the environment. The aim of a recent study was to determine the concentration of ceftriaxone residue (CEFTR) in raw and pasteurized mastitis cow milk, and its role in developmental toxicity and genotoxicity in the zebra fish model. The CEFTR concentrations in raw and pasteurized milk were several times higher than CEFT's MRL. CEFTR showed a decrease in body length and yolk sac region of zebra fish larvae 7-amino cephalosporanic acid (7-ACA), C3 and C7 are the cephalosporin components produced by the degradation of CEFT that may present in CEFTR, and have an impact on the zebra fish embryo in this stage of development. Comet Assay or Single Cell Gel Electrophoresis (SCGE) also exhibited highest percentage of tail DNA, and tail moment (DNA migration) that is the ultimate indicator of DNA damage by breaking DNA strands and incorporating guanine residue into the genome that ultimately damages the DNA. As a result, the CEFTR is extremely concerning for public health and the environment. The toxic effects of the CEFTR in zebra fish model have not yet been studied. This may be the first comprehensive study.

Optimization of Cephalosporin C Acylase Expression in Escherichia coli by High-Throughput Screening a Constitutive Promoter Mutant library.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.

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.

Functional Expression and Characterization of Acetyl Xylan Esterases CE Family 7 from Lactobacillus antri and Bacillus halodurans.

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50°C, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana β-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.

Multiple draft tubes airlift loop membrane bioreactor as an efficient system for acidic 7-amino cephalosporanic acid (7-ACA) wastewater treatment

In this study, a lab-scale multiple draft tubes airlift loop membrane bioreactor (Mt-ALMBR) was used for treating acidic 7-amino cephalosporanic acid (7-ACA) wastewater under different pHs (3.54–6.20) and hydraulic retention time (HRT) (48 h, 36 h, 24 h and 16 h). During about 200 days operation, under HRT of 48 h and pH condition about 6.0, the optimum average COD and BOD5 removal rates were reach to 84.4 ± 2.1% and 94.9 ± 0.8%, and the highest 7-ACA removal rate also observed as 77.6%. Biodegradation, membrane rejection, hydrolysis and sludge adsorption were the four main pathways of 7-ACA removal. With the increase of pH, biodegradation, membrane rejection and hydrolysis had significant positive impacts on 7-ACA removal, while adsorption had a negative impact. Moreover, mathematical models for 7-ACA removal rate and pH were calculated to guide the operation of Mt-ALMBR. Biodegradation was the main pathway to remove 7-ACA when pH was >4.17.