Enzymatic Synthesis Of Cephalexin Research Articles

Crystallization Kinetics of Cephalexin Monohydrate in the Presence of Cephalexin Precursors

The solubility and crystallization kinetics of cephalexin monohydrate, an important β-lactam antibiotic, are reported for the first time for an aqueous solution. The nucleation and growth kinetics are determined with online process analytical technologies (PAT) and offline image analysis to support implementation of a continuous process. Effects of two cosolutes, 7-aminodesacetoxycephalosporanic acid (7-ADCA) and phenylglycine methyl ester (PGME), on nucleation and growth are examined. These cosolutes are precursors in the enzymatic synthesis of cephalexin. Primary nucleation of cephalexin was unaffected by the cosolutes, but 7-ADCA, even in small quantities (<10 mM), significantly decreased the cephalexin monohydrate growth rate; PGME had a less significant impact on growth rate. Possible mechanisms of growth inhibition are discussed; a model wherein cosolutes slow growth by pinning advancing growth steps and halting step advancement below a critical supersaturation best explains the data. Impact on crys...

Enzymatic synthesis of Cephalexin in recyclable aqueous two-phase systems composed by two pH responsive polymers

The synthesis of Cephalexin in novel recyclable aqueous two-phase systems was studied, using immobilized penicillin acylase as biocatalyst, 7-amino-3-desacetoxy cephalosporanic acid (7-ADCA) as nucleophile and phenyl glycine methyl ester (PGME) as acyl donor. The aqueous two-phase system (ATPS) was composed by two pH-response copolymers PADBA and PMDB, which could be recycled efficiently. In the ATPS, high partition coefficient (2.57) for Cephalexin and low partition coefficient (0.21) for 7-ADCA were obtained. Then the synthesis of Cephalexin in ATPS and conventional one-phase system was compared. Higher Cephalexin yield was obtained in ATPS. In addition, synthesis in ATPS was improved at higher substrates concentrations, obtaining yield of 98.2% with 50mM 7-ADCA and 150mM PGME at pH 5.8, 20°C. Even reducing acyl donor to nucleophile ratio to 2, the yield can still above 90%. PADBA/PMDB recycling aqueous two-phase system is suitable for synthesis of Cephalexin.

Batch reactor performance for enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size

Batch reactor performance for enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size

Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems

Abstract The kinetically controlled synthesis of cephalexin in aqueous two-phase systems was studied, using immobilized penicillin acylase, 7-amino 3-desacetoxycephalosporanic acid as nucleophile and phenylglycine methyl ester as acyl donor. The organic phases used were 80% (v/v) polyethyleneglycol 400 and 600 and the aqueous phase was 2.5 M (NH 4 ) 2 SO 4 . 7-amino 3-desacetoxycephalosporanic acid and cephalexin partition coefficients were determined at pH 7.4 and 7.8, at 14 °C and 20 °C. Highest partition coefficient for cephalexin was obtained for polyethyleneglycol 400–(NH 4 ) 2 SO 4 at pH 7.4 and 20 °C, while the lowest partition coefficient for 7-amino desacetoxycephalosporanic acid was obtained in the same system at pH 7.8 and 14 °C. No significant effect of pH was observed on conversion yield and productivity of cephalexin synthesis; however, higher values were obtained with polyethyleneglycol 400 as organic phase. Higher conversion yields with both biphasic systems were obtained at the lowest temperature, where product hydrolysis was lower; volumetric productivity was higher for the fully aqueous medium (control), being higher at 20 °C. All parameters of synthesis were improved at higher substrates concentrations, obtaining conversion yields of 78.2% and 65.4%, with 60 mM 7-amino desacetoxycephalosporanic acid for the polyethyleneglycol 400–(NH 4 ) 2 SO 4 system and the control, respectively.

Enzymatic synthesis of cephalexin in aqueous two-phase systems

Cephalexin was synthesized in aqueous two-phase systems (ATPSs) by using penicillin G acylase (PGA) (penicillin amidohydrolase, EC3.5.1.11) as a catalyst and 7-aminodeacetoxicephalosporanic acid (7-ADCA) and phenylglycine methyl ester (PGME) as substrates. In the ATPS composed of 20% (w/w) PEG 400 and 15% (w/w) magnesium sulfate, the partition coefficient of PGA ( K E) was less than 0.01, PGA biased to the magnesium sulfate-rich bottom phase. The partition coefficient of cephalexin ( K C) was 6.7, and the partition coefficients of PGME ( K P) and 7-ADCA ( K A) were 1.5 and 1.2, respectively. The stability of PGA in 20% (w/w) PEG 400 and 15% (w/w) magnesium sulfate ATPS was investigated. The activity of PGA was remained 80.6% after PGA was retained in the ATPS for 35 h and reserved 59% in the 20% magnesium sulfate solution at pH 7.0. When free enzyme was used for bioconversion, a high yield of cephalexin (60%) was achieved at the concentration of 100 mM 7-ADCA and 100 mM PGME in this ATPS than that of in an aqueous system (21%).

Enhancement effect of reverse micelles on enzymatic synthesis of cephalexin.

Enhancement effect of reverse micelles on enzymatic synthesis of cephalexin.

Enhancement effect of water activity on enzymatic synthesis of cephalexin

The effect of water activity (a(w)) of the reaction medium on the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenylglycine methyl ester (PGM) was investigated using the alpha-amino acid ester hydrolase enzyme from Xanthomonas citri. It was found that the synthetic activity of the enzyme and the conversion yield were markedly improved when the a(w) of the reaction medium was lowered to about 0.97. The water activity depressing agents evaluated were glycerol, sucrose, and sorbitol, and the conversion yields were improved up to 170% with 15% glycerol, 230% with 30% sucrose, and 270% with 20% sorbitol, respectively. The extent of favorable effect of a(w) on the conversion yield was not the same among the a(w) depressors, probably due to other unknown interactions between the enzyme and depressors. However, optimal a(w) values corresponding to the maximum conversion yield coincided for all a(w) depressors used. The conversion yield of CEX showed an increasing trend with increasing a(w) up to the optimal a(w) value (0.96-0.97) which corresponds to the maximum conversion yield and a decreasing trend beyond the optimal a(w). There appears to be a delicate balance between the hydrolytic reaction of PGM and synthetic reaction of CEX. The increasing a(w)-[E . PGM] complex and the branched reaction pathway fluxes from [E . PGM] to PG (D-alpha-phenyl glycine) and CEX are balanced in such a way that the maximum CEX conversion yield is obtained at a(w) value of 0.96-0.97. The a(w) depressors stabilized the enzyme somewhat, but this positive effect was considered to be only a minor contribution to the substantial yield enhancement. The a(w) depressor effect on viscosity and in turn the mass transfer rate limitation was ruled out since the change in conversion due to the viscosity change was found to be insignificant.

Enhancement effect of polyethylene glycol on enzymatic synthesis of cephalexin

In an enzymatic synthesis of cephalexin (CEX) using an acylase from Xanthomonas citri, the effect of polyethylene glycol (PEG) on the synthetic reaction of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenyl-glycine methyl ester (PGM) to CEX was investigated. The addition of PEG (MW 300-20,000) increased the yield significantly. This yield enhancement effect tended to increase with the increasing molecular weight of PEG. Addition of PEG to the reaction system did not affect both the CEX and PGM hydrolytic reactions. The PEG added to the reaction medium used in these experiments did not depress the water activity significantly, and the product yield improvement could not be explained by the activity alone. The PEG stabilized the enzyme activity to some extent, but this stabilizing effect was only partially attributable to the yield enhancement of CEX. The enhancing effect of PEG on the synthetic yield increased with the increasing PEG molecular weight or the length of the poly(oxy-1,2-ethanediyl) chain, which increases the hydrophobicity of PEG. This finding consequently has led to the conclusion that the PEG structure renders the affinity between enzyme and 7-ADCA, which is a hydrophobic substrate. The microenvironmental hydrophobicity of PEG and its interaction with the hydrophobic substrate was found to be the main reason for the improvement of the CEX yield. In fact, the Michaelis-Menten kinetic constant for 7-ADCA, K(7-ADCA) in the presence of PEG was smaller than that in the control system (without PEG addition).

Effect of water activity on enzymatic synthesis of cephalexin

In enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-α-phenylglycine methyl ester (PGM) using an acylase fromXanthomonas citri, it was found that the synthetic activity and conversion yield were enhanced markedly by depressing the water activity (a w ) of reaction system. This enhancement was probably resulted from the change of thermodynamic equilibrium and maximized at a range ofa w from 0.96 to 0.97.

Reaction kinetics of cephalexin synthesizing enzyme from Xanthomonas citri

In enzymatic synthesis of cephalexin from D-alpha-phenylglycine methyl ester (PGM) and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) using alpha-acylamino-beta-lactam acylhydrolase from Xanthomonas citri, it was found that this enzyme catalyzes all three reactions including PGM hydrolysis, cephalexin synthesis, and cephalexin hydrolysis. Based on our experimental results, a mechanistic kinetic model for cephalexin synthesizing enzyme system having acyl-enzyme intermediate was proposed. From this kinetic model, the reaction rate equations for three reactions were derived, and the kinetic parameters were evaluated. A good agreement between the simulation results and the experimental results was found.

Enzymatic biosynthesis of cephalexin.

The reaction kinetics of the enzymatic synthesis of cephalexin from 7-aminodeacetoxy cephalosporanic acid and phenylglycine methylester was studied using the synthesizing enzyme obtained from Xanthomonas citri. The activation energy, Km values for 7-aminodeacetoxy cephalosporanic acid and phenylglycine methylester, and Ki value for phenylglycine methylester were determined as 8.63 kcal/mol, 3.7mM, 14.5mM, and 70mM, respectively. The enzyme was found to be constitutive and susceptible to deactivation.