Characterization of an omega-6 linoleate lipoxygenase from Burkholderia thailandensis and its application in the production of 13-hydroxyoctadecadienoic acid

Abstract

A recombinant putative lipoxygenase from Burkholderia thailandensis with a specific activity of 26.4 U mg⁻¹ was purified using HisTrap affinity chromatography. The native enzyme was a 75-kDa dimer with a molecular mass of 150 kDa. The enzyme activity and catalytic efficiency (k _cat/K _m) were the highest for linoleic acid (k _cat of 93.7 s⁻¹ and K _m of 41.5 μM), followed by arachidonic acid, α-linolenic acid, and γ-linolenic acid. The enzyme was identified as an omega-6 linoleate lipoxygenase (or a linoleate 13S-lipoxygenase) based on genetic and HPLC analyses as well as substrate specificity. The reaction conditions for the enzymatic production of 13-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) were optimal at pH 7.5, 25 °C, 20 g l⁻¹ linoleic acid, 2.5 g l⁻¹ enzyme, 0.1 mM Cu²⁺, and 6 % (v/v) methanol. Under these conditions, linoleate 13-lipoxygenase from B. thailandensis produced 20.8 g l⁻¹ 13-HODE (70.2 mM) from 20 g l⁻¹ linoleic acid (71.3 mM) for 120 min, with a molar conversion yield of 98.5 % and productivity of 10.4 g l⁻¹ h⁻¹. The molar conversion yield and productivity of 13-HODE obtained using B. thailandensis lipoxygenase were 151 and 158 % higher, respectively, than those obtained using commercial soybean lipoxygenase under the optimum conditions for each enzyme at the same concentrations of substrate and enzyme.