Characterization of the linoleic acid Δ9 hydratase catalyzing the first step of polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a

Michiki Takeuchi; Shigenobu Kishino; Akiko Hirata; Si-Bum Park; Nahoko Kitamura; Jun Ogawa

doi:10.1016/j.jbiosc.2014.10.022

Characterization of the linoleic acid Δ9 hydratase catalyzing the first step of polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a

J Biosci Bioeng. 2015 Jun;119(6):636-41. doi: 10.1016/j.jbiosc.2014.10.022. Epub 2014 Dec 1.

Authors

Michiki Takeuchi¹, Shigenobu Kishino², Akiko Hirata¹, Si-Bum Park³, Nahoko Kitamura¹, Jun Ogawa⁴

Affiliations

¹ Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
² Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan; Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
³ Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
⁴ Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan; Research Unit for Physiological Chemistry, Kyoto University, Kyoto, Japan. Electronic address: ogawa@kais.kyoto-u.ac.jp.

PMID: 25476761
DOI: 10.1016/j.jbiosc.2014.10.022

Abstract

Linoleic acid Δ9 hydratase, which is involved in linoleic acid saturation metabolism of Lactobacillus plantarum AKU 1009a, was cloned, expressed as a his-tagged recombinant enzyme, purified with an affinity column, and characterized. The enzyme required FAD as a cofactor and its activity was enhanced by NADH. The maximal activities for the hydration of linoleic acid and for the dehydration of 10-hydroxy-cis-12-octadecenoic acid (HYA) were observed at 37 °C in buffer at pH 5.5 containing 0.5 M NaCl. Free C16 and C18 fatty acids with cis-9 double bonds and 10-hydroxy fatty acids served as substrates for the hydration and dehydration reactions, respectively. The apparent Km value for linoleic acid was estimated to be 92 μM, with a kcat of 2.6∙10(-2) s(-1) and a Hill factor of 3.3. The apparent Km value for HYA was estimated to be 98 μM, with a kcat of 1.2∙10(-3) s(-1).

Keywords: Dehydration; Hydratase; Hydration; Hydroxy fatty acid; Lactic acid bacteria.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biocatalysis*
Flavin-Adenine Dinucleotide / metabolism
Hydrogen-Ion Concentration
Hydrolases / metabolism
Kinetics
Lactobacillus plantarum / enzymology*
Lactobacillus plantarum / metabolism*
Linoleic Acid / metabolism*
Lipid Metabolism
NAD / metabolism
Oleic Acids / chemistry
Oleic Acids / metabolism
Substrate Specificity
Temperature

Substances

Oleic Acids
NAD
Flavin-Adenine Dinucleotide
10-hydroxy-12-octadecenoic acid
Linoleic Acid
Hydrolases