title: 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.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, Republic of Korea.
authors.forename: Jung-Ung
authors.initials: JU
authors.lastname: An
authors.forename: Baek-Joong
authors.initials: BJ
authors.lastname: Kim
authors.forename: Seung-Hye
authors.initials: SH
authors.lastname: Hong
authors.forename: Deok-Kun
authors.initials: DK
authors.lastname: Oh
mesh_terms: D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism; D019117:Burkholderia / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019787:Linoleic Acid / Q000378:metabolism*; D008041:Linoleic Acids / Q000378:metabolism*; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008041:Linoleic Acids; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C024348:13-hydroxy-9,11-octadecadienoic acid; D019787:Linoleic Acid; C117433:13-lipoxygenase; D008084:Lipoxygenase