最近,来自乳酸菌(LAB)的原核漆酶,可以降解生物胺,被发现了。从Oenococusoeni克隆了一种漆酶,一个非常重要的酿酒实验室,它已经在大肠杆菌中表达。该酶具有与先前从LAB中分离出的酶相似的特征,因为它能够氧化经典底物,例如2,2-叠氮基-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS),2,6-二甲氧基苯酚(2,6-DMP),和亚铁氰化钾K4[Fe(CN6)],和非常规底物作为生物胺。然而,它呈现出一些独特性,最大的特点是它的嗜冷行为,在这些酶中从未见过。由于其在各种生物技术过程中的潜在应用,能够在低温下有效催化的嗜冷酶引起了极大的兴趣。在这项研究中,我们报道了一种新的嗜冷漆酶的发现和表征,多铜氧化酶(MCO),来自Oenococusoeni细菌。嗜冷漆酶基因,命名为LcOe229,通过对O.Oeni的基因组分析鉴定,葡萄酒发酵中常见的革兰氏阳性菌。该基因成功克隆并在大肠杆菌中异源表达,重组酶纯化至均一。嗜冷漆酶的生化表征显示其在低温下的最佳活性,在10°C处有一个峰。据我们所知,这是迄今为止漆酶的最低最佳温度。此外,嗜冷漆酶在低pH(ABTS的最适pH2.5)下表现出显著的稳定性和活性,表明了它在各种生物技术应用中的潜力。测定了LcOe229的动力学性质,揭示了低温下几种底物的高催化效率(kcat/Km)。LcOe229的这种出色的冷适应性表明其在寒冷环境或需要低温过程的应用中作为生物催化剂的潜力。使用X射线晶体学确定了嗜冷漆酶的晶体结构,证明了与其他LAB漆酶相似的结构特征。例如扩展的N端和扩展的C端,后者含有二硫键。此外,该结构在T1Cu位点的入口处显示了两个Met残基,常见于乳酸菌漆酶,我们认为这可能与底物结合有关,从而扩大漆酶的底物结合袋。LcOe229与南极漆酶的结构比较尚未显示出赋予冷活性漆酶与嗜温的特定特征。因此,对这种嗜冷漆酶及其工程的进一步研究可能会导致增强的冷活性酶,具有改善的特性,可用于未来的生物技术应用。总的来说,这种来自O.oeni的新型嗜冷漆酶的发现扩展了我们对冷适应酶的理解,并为它们在寒冷环境中的工业应用提供了新的机会。
Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.