Z Naturforsch C J Biosci. 2014 Mar-Apr;69(3-4):149-54. doi: 10.5560/znc.2013-0133.
The lipoxygenase LOX(Psa) 1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOX(Psa) 1 were identified by de novo sequencing, and the postulated molecular mass of 72.5 kDa was confirmed. With linoleic acid as the substrate, the LOX(Psa)1 showed a specific activity of 113 U mg(-1) and maximal activity at pH 7.0 and 30 degrees C, respectively.
鲜美味雷蘑脂氧合酶 LOX(Psa)1 最初因能够将 (+)-柠檬烯氧化为具有重要经济价值的葡萄柚香气 (+)-诺卡酮而受到关注，本研究采用包括制备等电聚焦和层析技术的三步纯化方案，从富含肽酶的冻干物中分离出 LOX(Psa)1。经纯化酶的纳升液相色谱电喷雾串联质谱（nLC-ESI-MS/MS）和肽质量指纹图谱分析，得到来自鲜美味雷蘑的脂氧合酶的 38 个肽段。该 cDNA 编码的 643 个氨基酸长序列中近 50%被覆盖。通过从头测序鉴定了天然 LOX(Psa)1 的两个末端肽段，并证实了其推测的 72.5 kDa 分子质量。以亚油酸为底物时，LOX(Psa)1 的比活为 113 U mg(-1)，最适 pH 值和最适温度分别为 7.0 和 30°C。
