Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy.
A method based on Fourier transform infrared spectroscopy (FT-IR) has been developed for assaying the Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain. The kinetic of acetamide hydrolysis by the enzyme, in aqueous media, was monitored by measuring the intensity of the acetamide amide I band maximum at 1635 cm(-1) as a function of time. A value of 0.5mM(-1) cm(-1) was obtained for the extinction coefficient (epsilon) of acetamide at this frequency. The rate of the hydrolysis was found to be linear with the concentration of the enzyme up to 90 microM. The Michaelis-Menten kinetics parameters V and K(m) were determined as 30.7 U/mg and 4mM, respectively. These results were similar to those obtained using high-performance liquid chromatography analysis of the same hydrolytic reaction catalyzed by amidase either in water or in buffer. This suggests that the precision of the FT-IR method is suitable for the kinetic studies of amidase with the additional advantage of being able to perform a real-time measurement of the enzymatic activity.
Departamento de Engenharia Química, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1, 1949-014 Lisboa, Portugal.
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R
Pacheco
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Serralheiro
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A
Karmali
P I
PI
Haris
D000081:Acetamides / Q000032:analysis / Q000378:metabolism; D019342:Acetic Acid / Q000032:analysis / Q000378:metabolism; D000581:Amidohydrolases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002021:Buffers; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D000081:Acetamides; D002021:Buffers; D011994:Recombinant Proteins; C030686:acetamide; D000581:Amidohydrolases; C019665:amidase; D019342:Acetic Acid