Kinetics and function of two mutants of CLA hydrase from Lactobacillus plantarum p-8.

In this work, the gene of conjugated linoleic acid hydrase (CLA-HY) was cloned from L. plantarum p-8, and the protein of CLA-HY was expressed in Escherichia coli BL21. Gas chromatography-mass spectrometry was employed to verify that the purified CLA-HY can convert linoleic acid (LA) into 10-hydroxy-cis-12-octadecenoic add (10-HOE) in the presence of flavin adenine dinucleotide (FAD). The optimal pH and temperature for maximizing CLA-HY catalytic activity were found to be 6.0 and 35 degrees C, respectively. In addition, the catalytic ability of CLA-HY can be inhibited by a number of cations such as me, Mn2+, Zn2+, Cu2+, Fe2+, Fe3+, Ni2+ and Ca2+. Finally, the Km, Vmax, Kcat and Kcat/Km of CLA-HY were determined as 7.62 mM, 2.59 mM(-1) h(-1), 8.33 x 10(3) h(-1) and 1.09 x 10(3) mM(-1) h(-)(1), respectively. Moreover, it was demonstrated that both M76 and G74 residues played significant roles in catalysing the conversion of LA into 10-HOE using site-directed mutation technology and molecular simulations.