一种来自金针菇（Agrocybe aegerita）的新型脂氧合酶：生化特性和动力学特性。
A new lipoxygenase from the agaric fungus Agrocybe aegerita: Biochemical characterization and kinetic properties.
机构信息
Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Giessen, Hesse, Germany.
Fraunhofer Institute for Molecular Biology and Applied Ecology IME Business Area Bioresources, Giessen, Hesse, Germany.
出版信息
PLoS One. 2019 Jun 19;14(6):e0218625. doi: 10.1371/journal.pone.0218625. eCollection 2019.
Oxylipins are metabolites with a variety of biological functions. However, the biosynthetic pathway is widely unknown. It is considered that the first step is the oxygenation of polyunsaturated fatty acids like linoleic acid. Therefore, a lipoxygenase (LOX) from the edible basidiomycete Agrocybe aegerita was investigated. The AaeLOX4 was heterologously expressed in E. coli and purified via affinity chromatography and gel filtration. Biochemical properties and kinetic parameters of the purified AaeLOX4 were determined with linoleic acid and linolenic acid as substrates. The obtained Km, vmax and kcat values for linoleic acid were 295.5 μM, 16.5 μM · min-1 · mg-1 and 103.9 s-1, respectively. For linolenic acid Km, vmax and kcat values of 634.2 μM, 19.5 μM · min-1 · mg-1 and 18.3 s-1 were calculated. Maximum activities were observed at pH 7.5 and 25 °C. The main product of linoleic acid conversion was identified with normal-phase HPLC. This analysis revealed an explicit production of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). The experimental regio specificity is underpinned by the amino acid residues W384, F450, R594 and V635 considered relevant for regio specificity in LOX. In conclusion, HPLC-analysis and alignments revealed that AaeLOX4 is a 13-LOX.
氧化脂类是具有多种生物学功能的代谢物。然而，其生物合成途径还不为人知。人们认为，第一步是对亚油酸等多不饱和脂肪酸进行氧化。因此，研究了可食用担子菌糙皮侧耳中的脂氧合酶（LOX）。通过亲和层析和凝胶过滤从大肠杆菌中异源表达 AaeLOX4 并进行纯化。用亚油酸和亚麻酸作为底物测定了纯化的 AaeLOX4 的生化性质和动力学参数。获得的亚油酸 Km、vmax 和 kcat 值分别为 295.5 μM、16.5 μM·min-1·mg-1 和 103.9 s-1。对于亚麻酸，Km、vmax 和 kcat 值分别为 634.2 μM、19.5 μM·min-1·mg-1 和 18.3 s-1。在 pH 7.5 和 25°C 时观察到最大活性。用正相 HPLC 鉴定了亚油酸转化的主要产物。该分析明确产生了 13-羟基-9,11-十八碳二烯酸（13-HPOD）。W384、F450、R594 和 V635 氨基酸残基被认为与 LOX 的区域特异性相关，实验的区域特异性得到了支持。综上所述，HPLC 分析和比对表明 AaeLOX4 是一种 13-LOX。
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