PDF(Pubmed)
Abstract：
Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive.
OBJECTIVE: Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.
OBJECTIVE: Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.
摘要：
染料脱色过氧化物酶是具有广泛底物特异性的血红素过氧化物酶。它们的生理功能在很大程度上仍然未知，但是植物细胞壁聚合物在解聚中的作用已被广泛提出。这里,报道了一种用于细菌染料脱色过氧化物酶的新表达系统以及以前未开发的植物分子的活性。来自Amycolatopsis75iv2（DyP2）的染料脱色过氧化物酶是在革兰氏阳性细菌LligidansTK24中以细胞内和细胞外形式异源产生的，没有外部血红素补充。该酶在一系列O-糖苷上进行了测试，它们是具有苯基糖苷键的植物次生代谢产物。O-糖苷引起了人们的极大兴趣,用于研究化合物本身以及作为研究特定木质素-碳水化合物复合物的潜在模型。水杨苷的主要DyP反应产物，熊果苷,fraxin,柚皮苷,芦丁,和gossypin是氧化偶联的低聚物。当使用熊果苷时，观察到自由基聚合时聚糖部分的裂解，fraxin,芦丁,和gossypin作为底物。熊果苷和fraxin释放的葡萄糖量达到总底物的23%和3%，分别。所提出的机理表明，由于自由基在对位的定位，醚键的不稳定。此外,DyP2在复杂的木质纤维素材料如小麦秸秆上进行了测试，云杉,柳树,和纯化的水溶性木质素馏分，但是没有观察到碳水化合物的显著变化，尽管有明显的氧化活性。因此DyP2对这种木质素-碳水化合物复合物的确切作用仍然难以捉摸。
目的：过氧化物酶需要正确掺入血红素辅因子才能发挥活性。由于宿主生物体的血红素合成不足，过氧化物酶的异源过量生产通常会导致酶失活。因此,过氧化物酶在纯化过程中或纯化后与过量血红素一起孵育以重建活性。作为生产宿主的S.lividans可以在细胞内和细胞外产生完全活性的过氧化物酶，而不需要补充血红素。这减少了下游加工步骤的数量,并且有利于更可持续地生产工业相关的酶。此外，本研究通过研究天然相关的植物次生代谢产物和分析形成产物，扩展了染料脱色过氧化物酶的应用范围。揭示了以前被忽视的导致糖基部分释放的自由基聚合伪影，探讨DyP过氧化物酶的作用机制。关键方面是连续添加，而不是更常见的单一添加方法，共底物，过氧化氢.这种连续添加允许过氧化物酶在没有自氧化的情况下完成大量的周转。
目的：过氧化物酶需要正确掺入血红素辅因子才能发挥活性。由于宿主生物体的血红素合成不足，过氧化物酶的异源过量生产通常会导致酶失活。因此,过氧化物酶在纯化过程中或纯化后与过量血红素一起孵育以重建活性。作为生产宿主的S.lividans可以在细胞内和细胞外产生完全活性的过氧化物酶，而不需要补充血红素。这减少了下游加工步骤的数量,并且有利于更可持续地生产工业相关的酶。此外，本研究通过研究天然相关的植物次生代谢产物和分析形成产物，扩展了染料脱色过氧化物酶的应用范围。揭示了以前被忽视的导致糖基部分释放的自由基聚合伪影，探讨DyP过氧化物酶的作用机制。关键方面是连续添加，而不是更常见的单一添加方法，共底物，过氧化氢.这种连续添加允许过氧化物酶在没有自氧化的情况下完成大量的周转。
