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Biotechnology and Applied Biochemistry (2002) 36, (63–70) (Printed in Great Britain)

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Purification and biochemical characterization of the carbamate hydrolase from Pseudomonas sp. 50432

G. Rasul Chaudhry*¹, A. Mateen*, B. Kaskar†, M. Bloda* and Sheikh Riazuddin‡

*Department of Biological Sciences, 327 SEB, Oakland University, Rochester, MI 48309-4401, U.S.A., †Ash Stevens Inc., Detroit, MI, U.S.A., and ‡National Center for Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan

Key words: carbofuran, benzofuran, pesticides.

Abbreviation used: DTT, dithiothreitol.

¹To whom correspondence should be addressed (e-mail chaudhry@oakland.edu).

A soluble carbamate hydrolase that had a wide specificity was purified 2032-fold from Pseudomonas sp. 50432. This was achieved using a combination of anion-exchange, gel-filtration and hydrophobic-interaction- chromatography techniques. Carbamate hydrolase cleaved the ester linkage of the N-methylcarbamates. The native enzyme was a monomer with a molecular mass of 88 kDa. The optimum pH and temperature of the enzyme activity were 8.5 and 37 °C respectively. The tested cations or EDTA did not affect the enzyme activity. However, 2-mercaptoethanol reversibly inhibited the enzyme activity. The enzyme showed the K_m values of 16 and 12 µM for carbofuran and carbaryl respectively. The purified enzyme did not hydrolyse o-nitrophenyl dimethylcarbamate but hydrolysed several N-methylcarbamates and 1-naphthyl acetate.

Received 4 April 2002/20 May 2002; accepted 28 May 2002

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