The isolation and kinetics of penicillin amidase from Escherichia coli

Balasingham, K.; Warburton, Dorothy; Dunnill, P.; Lilly, M. D.

doi:10.1016/0005-2744(72)90027-7

Cited by 288 publications

(123 citation statements)

References 9 publications

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“…, [3] where k cat PenG is the catalytic constant for penicillin G. Phenylacetic acid is produced in both reactions, so the concentration of phenylacetic acid is described by:…”

Section: Determination Of Kinetic Parametersmentioning

confidence: 99%

“…phenylacetic acid and 6-aminopenicillanic acid (6-APA), 3 which is a building block in the synthesis of semi-synthetic ␤-lactam antibiotics (1). Since the enzyme can also be applied in the condensation reaction with a variety of acyl donors, penicillin acylase can potentially be used for biocatalytic synthesis of various semisynthetic antibiotics, which are of pharmaceutical importance.…”

mentioning

confidence: 99%

“…These parameters can in principle be determined with several methods. The 6-APA produced during hydrolysis of penicillin G can be detected directly using HPLC methods, by fluorescence measurements after reaction with fluorescamine (2), or by colorimetric measurements after derivatization with 4-(dimethylamino)benzaldehyde (3). The released phenylacetic acid can also be monitored by acid/base titration (4).…”

mentioning

confidence: 99%

“…Moreover, the classical steady-state method of measuring initial rates is prone to severe error because of the strong product inhibition by phenylacetic acid. Due to these complications, K m values for penicillin G and K i values for phenylacetic acid of penicillin acylase of E. coli have been reported, which differ by several orders of magnitude (3,(7)(8)(9)(10).…”

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confidence: 99%

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The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Alkema

Floris

Janssen

1999

Analytical Biochemistry

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Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K m values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower k cat /K m values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.

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“…, [3] where k cat PenG is the catalytic constant for penicillin G. Phenylacetic acid is produced in both reactions, so the concentration of phenylacetic acid is described by:…”

Section: Determination Of Kinetic Parametersmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Alkema

Floris

Janssen

1999

Analytical Biochemistry

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“…6 -APA reacted with p -dimethyl-amino-benzaldehyde (PDAB) in 10mM phosphate buffer, pH 8 (Balasingham et al, 1972). The difference between enzymatic activities of the supernatant (free enzyme) before and after immobilization was used to assess the enzymatic load of the gel.…”

Section: B) Enzyme Activitymentioning

confidence: 99%

Improving selectivity and productivity of the enzymatic synthesis of ampicillin with immobilized penicillin G acylase

Ferreira

Giordano

2004

Braz. J. Chem. Eng.

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-An experimental design was applied to improve the reaction conditions for enzymatic synthesis of ampicillin from phenylglycine methyl ester (PGME) and 6-aminopenicillanic acid (6-APA), catalyzed by penicillin G acylase from E. coli immobilized on an agarose-glyoxyl derivative. The presence and magnitude of interactions between reaction variables were estimated using a 2 5 factorial design. A batch reactor was employed to assess the influence of the following variables: pH, temperature, initial 6-APA concentration, buffer concentration, and the presence of methanol. Response variables were productivity, selectivity, and yield (based on initial 6-APA concentration). The best synthesis yield (56.9%) was at T = 4ºC and pH 6.5. The highest productivity (49.3 × 10 -3 mM of antibiotic/min) was achieved at T = 25ºC and pH 6.5. Our results indicate that it is possible to achieve high productivity for this system while maintaining a high selectivity and yield.

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Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products

Illanes

Altamirano

Zúñiga

1996

Biotechnol. Bioeng.

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The isolation and kinetics of penicillin amidase from Escherichia coli

Cited by 288 publications

References 9 publications

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Improving selectivity and productivity of the enzymatic synthesis of ampicillin with immobilized penicillin G acylase

Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products

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