Cloning and Sequence Analysis of Fatty Acid Hydroxylase Gene in Lactobacillus Sakei Y-20 Strain and Characteristics of Fatty Acid Hydroxylase

Abstract

Lactic acid bacteria (LAB) are used to produce food alcoholic beverages. Yeast and LAB produce aroma lactone, with a sweet fruit smell, and convert hydroxyl fatty acids during whiskey production. Hydroxyl fatty acids, including ricinoleic acid, also show analgesic and anti-inflammatory effects. This study investigated fatty acid hydroxylase (FAH) characteristics and the genomic sequence in Lactobacillus sakei Y-20 strain. The enzyme locates the cell membrane fraction as a cell membrane protein in the strain. Hydroxylase was used for cloning from the LAB Y-20 strain genome. The genetic region excised by restrictive enzyme Sau3A was ligated in plasmid DNA and was transformed to competent cells. A clone from LAB Y-20 strain has 1,218 base pairs. The enzyme, a 42.9 kDa protein comprising 405 amino acids, has 90% homology with nlpC/P60 family protein in Lactobacillus spp. SDS-PAGE analysis showed that FAH produced by recombinant cells was 46 kDa. Its optimum pH was 6.0. It was activated by manganese ion, iron ion, cobalt ion, and calcium ion. However, mercury ion and azide ion inhibited it. Its active site was a heme-iron ligand signature motif. Although FAH hydroxylated oleic acid and linoleic acid with dihydronicotinamide adenine dinucleotide (NADH), it did not hydroxylate saturated fatty acids.

• Cloning
• Sequence analysis
• Lactobacillus sakei
• Fatty acid hydroxylase
• Linoleic acid