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Journal of Biochemistry Advance Access published online on December 13, 2006
Journal of Biochemistry, doi:10.1093/jb/mvm013
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© 2006 The Japanese Biochemical Society

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

M Helmy1,* and N El-Adl1

1Biotechnology Dept., Institute of Graduate Studies and Research, Alexandria University. 163 El Horraya Avenue, El Shatby 21526, Alexandria, Egypt

*Corresponding author Email: esmailgadmoh{at}yahoo.fr URL: http//:www.alexbiotechnologycenter.com Tel: 00203-4295007 ext. 259 Fax: 00203-4285792

Received September 20, 2006; Accepted November 18, 2006


  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.

Key Words: antithrombin, Lipase, substrate inducer, isolation and purification, ion exchange chromatography, Pseudomonas pseudoalcaligenes


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