Characterization and functional of four mutants of hydroxy fatty acid dehydrogenase from Lactobacillus plantarump-8

Abstract

Abstract In this study, the hydroxy fatty acid dehydrogenase CLA-DH from Lactobacillus plantarump-8 and its four mutant variants were expressed in Escherichia coli Rosetta (DE3). UV spectrophotometry was employed to verify the catalytic power of the purified CLA-DH to convert ricinoleic acid into 12-oxo-cis-9-octadecenoic acid in the presence of oxidized nicotinamide adenine dinucleotide (NAD+). The optimum reaction temperature for CLA-DH was 45°C, with a maintained stability between 20°C and 40°C. The optimal pH for CLA-DH catalytic activity was 6.0–7.0, with a maintained stability at a pH range of 6.0–8.0. In addition, Fe3+ promoted enzyme activity, whereas Cu2+, Zn2+, and Fe2+ inhibited enzyme activity (P < 0.05). The Km, Vmax, Kcat, and Kcat/Km of CLA-DH were determined as 2.19 ± 0.34 μM, 2.06 ± 0.28 μM min−1, 2.00 ± 0.27 min−1, and 0.92 ± 0.02 min−1μM−1, respectively. Site-directed mutagenesis and molecular dynamics simulations demonstrated that both Tyr156 and Ser143 residues play significant roles in the catalysis of CLA-DH, and its solubility is affected by Lys160 and Asp63. Moreover, Gas chromatography determined that recombinant CLA-DH could be successfully applied to Conjugated linoleic acids production.