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.

13‐Hydroxy‐9Z,11E‐Octadecadienoic Acid Production by Recombinant Cells Expressing Burkholderia thailandensis 13‐Lipoxygenase

“…S2) and was consistent with the previous reports for purified linoleate 13-lipoxygenase from B. thailandensis [7]. S2) and was consistent with the previous reports for purified linoleate 13-lipoxygenase from B. thailandensis [7].…”

“…3). The optimal concentration of methanol of permeabilized cells for 13-HODE production was 6 % (v/v) (data not shown), consistent with the previous report for the enzyme [7]. However, above 200 rpm, 13-HODE production did not increase.…”

“…Agitation is required to generate oxygen for the oxidation of linoleic acid by lipoxygenase [23]. The activity of purified linoleate 13-lipoxygenase from B. thailandensis increased approximately threefold with the addition of 0.1 mM Cu 2+ [7], while the enzymatic activity of recombinant cells increased approximately 1.2-fold with the addition of 0.15 mM Cu 2+ , which was the optimum concentration for 13-HODE production (Fig. 3).…”

“…They are widely used as starting materials for the synthesis of a variety of polymers and additives in the manufacturing of lubricants, emulsifiers, and stabilizers in the chemical, food, cosmetic, and pharmaceutical industries, owing to their high reactivities, solvent miscibilities, and stabilities compared to nonhydroxylated unsaturated fatty acids [2][3][4]. 13-Hydroperoxyoctadecadienoic acid (13-HPOD), which is easily reduced to 13-HODE, has been enzymatically produced from linoleic acid by linoleate 13-lipoxygenase from soybean, Gaeumannomyces graminis tritici [5], Pseudomonas aeruginosa [6], and Burkholderia thailandensis [7]. The specific activity of B. thailandensis 13-lipoxygenase for linoleic acid is the highest among the reported lipoxygenases, and B. thailandensis linoleate 13-lipoxygenase is a more efficient enzyme than commercial soybean linoleate 13-lipoxygenase [7].…”

“…13-Hydroperoxyoctadecadienoic acid (13-HPOD), which is easily reduced to 13-HODE, has been enzymatically produced from linoleic acid by linoleate 13-lipoxygenase from soybean, Gaeumannomyces graminis tritici [5], Pseudomonas aeruginosa [6], and Burkholderia thailandensis [7]. The specific activity of B. thailandensis 13-lipoxygenase for linoleic acid is the highest among the reported lipoxygenases, and B. thailandensis linoleate 13-lipoxygenase is a more efficient enzyme than commercial soybean linoleate 13-lipoxygenase [7]. Compared to enzymes, cells are more stable and show greater resistance to environmental perturbations.…”