TH Köln - Campus Leverkusen, Campusplatz 1, 51379, Leverkusen, Germany.
Appl Biochem Biotechnol. 2022 Dec;194(12):6194-6212. doi: 10.1007/s12010-022-04095-0. Epub 2022 Jul 29.
Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPL) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPL, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (k/K) of 2.73 × 10 s·M towards 13S-HPODE. Interestingly, HPL could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPL. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPL reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis.
过氧化物裂解酶（HPLs）催化 13S-过氧氧代十八碳二烯酸（13S-HPODE）分解为具有绿色气味的己醛和 12-氧代-9(Z)-十二烯酸，后者尚未在工业上使用。在这里，我们从番木瓜（Carica papaya）中克隆并在大肠杆菌中功能表达了 HPL，以详细研究 12-氧代-9(Z)-十二烯酸的合成。为了提高全长 HPL 的低催化活性，我们删除了疏水性、非保守的 N 端序列。这将酶活性从初始的 10 U/l 提高到 40 U/l。通过优化溶解缓冲液，使表达培养基中的酶活性提高到 2700 U/l。四聚体酶在 1.5 L 发酵罐中产生，并通过亲和层析进行富集。该酶制剂的 pH 最适值略偏酸性，对 13S-HPODE 的催化效率（k/K）为 2.73×10 s·M。有趣的是，HPL 可用于合成 12-氧代-9(Z)-十二烯酸，1 mM 的 13S-HPODE 在 10 s 内转化，产率为 90%。在 10 mg/ml 的蛋白浓度下，观察到 10(E)-异构体 traumatin 的缓慢形成，表明存在非酶促异构化过程。考虑到这一点，从红花油开始开发了一种连续添加荧光假单胞菌脂肪酶、大豆脂肪氧合酶（LOX-1）和 HPL 的一锅酶级联反应。在 HPL 反应 1 分钟后，快速提取反应混合物，得到 43%的产率。这项工作首次深入了解了 12-氧代-9(Z)-十二烯酸的酶级联合成，它可以作为生物基聚合物合成的双功能前体。
