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 Cu(2+), 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.