Authors: Adel Sayari Habib Mosbah Youssef Gargouri
Publish Date: 2007/04/17
Volume: 36, Issue: 1, Pages: 14-22
Abstract
In addition to their physiological importance microbial lipases like staphylococcal ones are of considerable commercial interest for biotechnological applications such as detergents food production and pharmaceuticals and industrial synthesis of fine chemicals The gene encoding the extracellular lipase of Staphylococcus simulans SSL was subcloned in the pET14b expression vector and expressed in Esherichia coli BL21 DE3 The wildtype SSL was expressed as amino terminal His6tagged recombinant protein Onestep purification of the recombinant lipase was achieved with nickel metal affinity column The purified Histagged SSL His6SSL is able to hydrolyse triacylglycerols without chain length selectivity The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds and their respective capacity to hydrolyse substrates having different physicochemical properties It has been proposed using homology alignment that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate To evaluate the importance of this environment the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using sitedirected mutagenesis The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column The substitution of Asp290 by Ala was accompanied by a significant shift of the acylchain length specificity of the mutant towards short chain fatty acid esters Kinetic studies of wildtype SSL and its mutant D290A were carried out and show essentially that the catalytic efficiency k cat /K M of the mutant was affected Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase
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