Abstract:The enzymatic synthesis of β-lactam semi-synthetic antibiotics has been receiving increasing attention as a
green-chemistry alternative for the industrial production of these drugs, because mild reaction conditions
may be used. A nonconventional fed-batch reactor is presented here, using a bi-disperse gel matrix for
immobilization of the enzyme penicillin G acylase (PGA) [EC 3.5.1.11]. The catalyst particles are suspended
within Taylor−Couette vortices, performing the kinetically controlled synthesis of ampici… Show more
“…For example, the maximal conversion and yield were 61% and 53%, respectively, with the substrate molar ratio of 1.0, while being 90% and 83%, respectively, with the substrate molar ratio of 2.0. Nonetheless, using lower substrate molar ratios seemed to be favorable for synthesis, resulting in the higher S/H ratio, which is in good agreement with the previous results (Ferreira et al 2007; Ribeiro et al 2005). Fig.…”
Section: Resultssupporting
confidence: 92%
“…Therefore, the enzymatic method exhibited promising application potential for the chemoenzymatic production of cefathiamidine. However, many problems associated with this enzymatic process such as relatively low substrate concentrations and unsatisfactory operational stability for its large-scale application may be addressed by the corresponding measures (e.g., using the supersaturated substrate solutions and fed-batch feeding strategy, and improving enzyme immobilization methods) (Volpato et al 2010; Ferreira et al 2007; Youshko et al 2004; Illanes et al 2007; Montes et al 2007). …”
Section: Discussionmentioning
confidence: 99%
“…The enzyme activities of various immobilized PAs were determined according to the previous methods (Ferreira et al 2007; Shewale et al 1987), with some modifications. Briefly, the immobilized enzyme was added into 5 mL of phosphate buffer (100 mmol/L, pH 7.5) containing PGK (1.08 mmol, 216 mmol/L).…”
BackgroundEnzymatic approaches have become promising alternatives to chemical methods for the production of semi-synthetic β-lactam antibiotics. In this work, enzymatic synthesis of N-bromoacetyl-7-aminocephalosporanic acid (N-bromoacetyl-7-ACA), the key intermediate for the production of cefathiamidine, was reported for the first time.ResultsOf the immobilized penicillin acylases (PAs) tested, PGA-750 was the best biocatalyst. Optimization of the biocatalytic process was conducted. The optimal acyl donor, molar ratio of acyl donor to 7-ACA, pH, temperature, 7-ACA concentration, and enzyme dosage were methyl bromoacetate, 3, 7.5, 20 °C, 50 mmol/L and 4 U/mL, respectively. Under the optimal conditions, enzymatic N-acylation of 7-ACA with methyl bromoacetate afforded the desired product with the yield of 85% in 2 h, where the synthesis/hydrolysis (S/H) ratio was approximately 1.5. The immobilized enzyme PGA-750 exhibited good operational stability, and the relative yields of approximately 90% and 63% were achieved, respectively, when it was reused in 7th and 11th batch.ConclusionsAn enzymatic approach to N-bromoacetyl-7-ACA, the key intermediate for the industrial production of cefathiamidine, has been developed successfully in a fully aqueous medium. The present work may open up a novel opportunity for the production of cefathiamidine through a simple and green process.Graphical abstractEnzymatic synthesis of N-bromoacetyl-7-ACA, the key intermediate for the production of cefathiamidine, was reported for the first time.
Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-016-0127-3) contains supplementary material, which is available to authorized users.
“…For example, the maximal conversion and yield were 61% and 53%, respectively, with the substrate molar ratio of 1.0, while being 90% and 83%, respectively, with the substrate molar ratio of 2.0. Nonetheless, using lower substrate molar ratios seemed to be favorable for synthesis, resulting in the higher S/H ratio, which is in good agreement with the previous results (Ferreira et al 2007; Ribeiro et al 2005). Fig.…”
Section: Resultssupporting
confidence: 92%
“…Therefore, the enzymatic method exhibited promising application potential for the chemoenzymatic production of cefathiamidine. However, many problems associated with this enzymatic process such as relatively low substrate concentrations and unsatisfactory operational stability for its large-scale application may be addressed by the corresponding measures (e.g., using the supersaturated substrate solutions and fed-batch feeding strategy, and improving enzyme immobilization methods) (Volpato et al 2010; Ferreira et al 2007; Youshko et al 2004; Illanes et al 2007; Montes et al 2007). …”
Section: Discussionmentioning
confidence: 99%
“…The enzyme activities of various immobilized PAs were determined according to the previous methods (Ferreira et al 2007; Shewale et al 1987), with some modifications. Briefly, the immobilized enzyme was added into 5 mL of phosphate buffer (100 mmol/L, pH 7.5) containing PGK (1.08 mmol, 216 mmol/L).…”
BackgroundEnzymatic approaches have become promising alternatives to chemical methods for the production of semi-synthetic β-lactam antibiotics. In this work, enzymatic synthesis of N-bromoacetyl-7-aminocephalosporanic acid (N-bromoacetyl-7-ACA), the key intermediate for the production of cefathiamidine, was reported for the first time.ResultsOf the immobilized penicillin acylases (PAs) tested, PGA-750 was the best biocatalyst. Optimization of the biocatalytic process was conducted. The optimal acyl donor, molar ratio of acyl donor to 7-ACA, pH, temperature, 7-ACA concentration, and enzyme dosage were methyl bromoacetate, 3, 7.5, 20 °C, 50 mmol/L and 4 U/mL, respectively. Under the optimal conditions, enzymatic N-acylation of 7-ACA with methyl bromoacetate afforded the desired product with the yield of 85% in 2 h, where the synthesis/hydrolysis (S/H) ratio was approximately 1.5. The immobilized enzyme PGA-750 exhibited good operational stability, and the relative yields of approximately 90% and 63% were achieved, respectively, when it was reused in 7th and 11th batch.ConclusionsAn enzymatic approach to N-bromoacetyl-7-ACA, the key intermediate for the industrial production of cefathiamidine, has been developed successfully in a fully aqueous medium. The present work may open up a novel opportunity for the production of cefathiamidine through a simple and green process.Graphical abstractEnzymatic synthesis of N-bromoacetyl-7-ACA, the key intermediate for the production of cefathiamidine, was reported for the first time.
Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-016-0127-3) contains supplementary material, which is available to authorized users.
“…[6][7][8][9] Among these, reaction with product crystallization seems to be a promising option due to the advantage of obtaining high concentrations of product and its recovery in the solid phase. Significant work has been done in this area, including building kinetic models, optimization of pH and temperature of the reaction, heterogeneous reactions, and complex reactor design.…”
Seeded reactive crystallization in the manufacture of semi-synthetic β-lactam antibiotics is described and the beneficial effects on yield are discussed. Conventional enzymatic synthesis of β-lactam is limited by secondary hydrolysis reactions that consume the desired product as it is being produced. Recent work in this area has pointed to the potential advantage of performing reactions at conditions that allow product crystallization to reduce the rate of secondary hydrolysis by protecting ampicillin in the solid phase. However, these approaches led to crystallization of both D-phenylglycine and ampicillin, which will greatly increase downstream processing. In the work described here, seeded crystallization is used to promote secondary nucleation of the desired ampicillin while it is being produced by the synthesis reaction, thereby selectively crystallizing ampicillin. Quantification of the solid phase confirmed selective crystallization of ampicillin with purities greater than 95% wt in all runs.React. Chem. Eng. This journal is
“…3 Namely, the usage of the immobilized PGA enables better operational stability and control of reactions, more efficient recovery and reuse of the biocatalyst, and application of different reactor designs. 4 Various meth-ods for immobilization of PGA have already been described in literature including adsorption and covalent attachment to porous organic 5 and inorganic carriers, 6,7 inclusion in and attachment to biopolymer gels, 8 and carrier-free techniques 3 ; among them, the covalent immobilization of PGA onto epoxy-activated carriers has drawn considerable interest. 9 Epoxy-carriers are very convenient for the multipoint covalent attachment of enzymes considering that the present epoxy groups could bind to amino, thiol, and hydroxyl groups of the enzyme molecules.…”
The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low-cost, easy-to-prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA-co-EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA-co-EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA.
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