The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis.
A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide ((2)H(2)O). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm(-1) and of the amide I band at 1605 cm(-1) were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients epsilon of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM(-1)cm(-1) at 1625, 1605, and 1365 cm(-1), respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P. aeruginosa. The kinetic constants (V(max), K(m), and K(cat)) determined by Lineweaver-Burk plot were 532.2 U mg(-1) protein, 6.4mM, and 806.36 s(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.
Karmali
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Centro de Investigação de Engenharia Biotecnológica do Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1, 1949-014 Lisbon, Portugal. akarmali@deq.isel.ipl.pt
Karmali
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Teixeira
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Curto
M J Marcelo
MJ
D032449:Canavalia / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017550:Spectroscopy, Fourier Transform Infrared; D014508:Urea / Q000737:chemistry*; D014510:Urease / Q000737:chemistry*
D014508:Urea; D014510:Urease