One-step purification of a P. pseudoalcaligenes lipase induced by a specific substrate

One-step purification of a P. pseudoalcaligenes lipase induced by a specific substrate Abstract The present study reports a new, simple and fast method of purification based on specific substrate inducers. Monoglycerides (MG) were used as inducers and the only carbon source for Pseudomonas pseudoalcaligenes. This provoked the bacterial cells to produce a lipase enzyme that showed a single band of 30 KDa in gel electrophoresis under reducing and non-reducing conditions. This was not the case when Tween20, a synthetic substrate, was used. The MG-induced lipase was passed over a carboxymethyl cellulose column. Isoelectric focusing of the lipase fraction revealed a single band with a pI of 8.0. P- nitrophenyl acetate proved to be a good substrate for the routine measurement of lipolytic activity since it had a good signal to noise ratio of 140 mg −1 . The enzyme was found to have an apparent Vmax of 4.5 IU, an apparent Km of 20 mM, a kcat of 0.69 sec −1 and a specific activity of 2.3 IU/mg protein on this substrate. antithrombin, Lipase, substrate inducer, isolation and purification, ion exchange chromatography, Pseudomonas pseudoalcaligenes © 2006 The Japanese Biochemical Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

One-step purification of a P. pseudoalcaligenes lipase induced by a specific substrate

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Publisher
Oxford University Press
Copyright
© 2006 The Japanese Biochemical Society
ISSN
0021-924X
eISSN
1756-2651
D.O.I.
10.1093/jb/mvm013
Publisher site
See Article on Publisher Site

Abstract

Abstract The present study reports a new, simple and fast method of purification based on specific substrate inducers. Monoglycerides (MG) were used as inducers and the only carbon source for Pseudomonas pseudoalcaligenes. This provoked the bacterial cells to produce a lipase enzyme that showed a single band of 30 KDa in gel electrophoresis under reducing and non-reducing conditions. This was not the case when Tween20, a synthetic substrate, was used. The MG-induced lipase was passed over a carboxymethyl cellulose column. Isoelectric focusing of the lipase fraction revealed a single band with a pI of 8.0. P- nitrophenyl acetate proved to be a good substrate for the routine measurement of lipolytic activity since it had a good signal to noise ratio of 140 mg −1 . The enzyme was found to have an apparent Vmax of 4.5 IU, an apparent Km of 20 mM, a kcat of 0.69 sec −1 and a specific activity of 2.3 IU/mg protein on this substrate. antithrombin, Lipase, substrate inducer, isolation and purification, ion exchange chromatography, Pseudomonas pseudoalcaligenes © 2006 The Japanese Biochemical Society

Journal

The Journal of BiochemistryOxford University Press

Published: Jan 1, 2006

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