Donadio Giuliana, Sarcinelli Carmen, Pizzo Elio, Notomista Eugenio, Pezzella Alessandro, Di Cristo Carlo, De Lise Federica, Di Donato Alberto, Izzo Viviana
Department of Biology, University of Naples Federico II, Via Cinthia I, 80126, Napoli, Italy.
Department of Chemical Sciences, University of Naples Federico II, via Cinthia I, 80126, Napoli, Italy.
PLoS One. 2015 Apr 27;10(4):e0124427. doi: 10.1371/journal.pone.0124427. eCollection 2015.
Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.
单环酚类和儿茶酚是食品和制药行业重要的抗氧化化合物；通过低附加值起始化合物的生物转化来生产它们具有重大的生物技术意义。来自假单胞菌属OX1的甲苯邻二甲苯单加氧酶（ToMO）是一种细菌多组分单加氧酶（BMM），能够使多种芳香族化合物羟基化，并且已被证明是一种用于生产芳香族化合物单羟基和二羟基衍生物的通用生化工具。已经对其区域选择性和底物特异性的分子决定因素进行了深入研究，并开发了一种计算策略，该策略能够设计出能够使该酶的非天然底物羟基化以获得具有商业价值的高附加值化合物的突变体。在这项工作中，我们研究了在大肠杆菌JM109菌株细胞中表达的重组ToMO用于该酶的非天然底物（如2-苯氧基乙醇、酞内酯和2-茚醇）的生物转化，以生产六种羟基化衍生物。对获得的羟基化产物进行了鉴定、分离，并使用DPPH测定法在体外以及在大鼠心肌母细胞系H9c2上评估了它们的抗氧化潜力。用ToMO催化生物转化获得的羟基化化合物孵育H9c2细胞，对亚砷酸钠存在诱导的轻度氧化应激产生了不同的保护作用。所获得的结果再次证实了ToMO系统在具有生物技术重要性的氧官能化反应中的通用性。此外，获得的羟基化衍生物具有有趣的抗氧化潜力，这鼓励使用该酶进行进一步的官能化反应，并可能将其用作设计新型生物活性分子的支架。
