Chang R C, Chen J C, Shaw J F
Department of Sea-Food Technology, China Junior College of Marine Technology, Taipei, Taiwan.
Biochem Biophys Res Commun. 1996 Nov 21;228(3):774-9. doi: 10.1006/bbrc.1996.1730.
A fragment of Staphylococcus hyicus lipase gene (Ala248 to Ala640) was inserted into plasmid pET20(b+). The resulting His-tagged recombinant protein (49 kDa) was overexpressed in Escherichia coli BL21(DE3) as an highly active lipase and was purified by nickel-coupled resin. Site-directed mutagenesis showed that in comparison with wild type enzyme, the L326F and L326A enzymes showed a 3.4 and 5 fold increase in the K(m), respectively, but only a 44% and a 64% decrease in the kcat/K(m), respectively, suggesting that Leu326 of the putative lid participated in substrate-binding.
