Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria.
Microb Cell Fact. 2010 Jul 14;9:57. doi: 10.1186/1475-2859-9-57.
The flavin-dependent enzyme pyranose 2-oxidase (P2Ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. P2Ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. Recently, it was shown that P2Ox is useful as bioelement in biofuel cells, replacing glucose oxidase (GOx), which traditionally is used in these applications. P2Ox offers several advantages over GOx for this application, e.g., its much broader substrate specificity. Because of this renewed interest in P2Ox, knowledge on novel pyranose oxidases isolated from organisms other than white-rot fungi, which represent the traditional source of this enzyme, is of importance, as these novel enzymes might differ in their biochemical and physical properties.
We isolated and over-expressed the p2ox gene encoding P2Ox from the ectomycorrhizal fungus Lyophyllum shimeji. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in E. coli Rosetta 2. We obtained active, flavinylated recombinant P2Ox in yields of approximately 130 mg per L of medium. The enzyme was purified by a two-step procedure based on anion exchange chromatography and preparative native PAGE, yielding an apparently homogenous enzyme preparation with a specific activity of 1.92 U/mg (using glucose and air oxygen as the substrates). Recombinant P2Ox from L. shimeji was characterized in some detail with respect to its physical and catalytic properties, and compared to the well-characterised enzymes from Phanerochaete chrysosporium and Trametes multicolor.
L. shimeji P2Ox shows properties that are comparable to those of P2Ox from white-rot fungal origin, and is in general characterised by lower K(m) and k(cat) values both for electron donor (sugar) as well as electron acceptor (ferrocenium ion, 1,4-benzoquinone, 2,6-dichloroindophenol). While L. shimeji P2Ox is the least thermostable of these three enzymes (melting temperature T(m) of 54.9 degrees C; half-life time of activity tau1/2 of 0.12 at 50 degrees C and pH 6.5), P. chrysosporium P2Ox showed remarkable thermostability with T(m) of 75.4 degrees C and tau1/2 of 96 h under identical conditions.
黄素依赖酶吡喃糖 2-氧化酶（P2Ox）在过去几年中受到了越来越多的关注，因为这种酶有许多有吸引力的应用。P2Ox 是一种独特的生物催化剂，在碳水化合物的生物转化和合成碳水化合物化学中有很高的应用潜力。最近，人们发现 P2Ox 可用作生物燃料电池中的生物元素，替代传统应用中使用的葡萄糖氧化酶（GOx）。与 GOx 相比，P2Ox 在这些应用中具有许多优势，例如，它的底物特异性更广泛。由于对 P2Ox 的兴趣重新燃起，因此了解来自白腐真菌以外的生物体中分离出的新型吡喃糖氧化酶非常重要，因为这些新型酶在生化和物理性质上可能有所不同。
我们从外生菌根真菌 Lyophyllum shimeji 中分离并过表达了编码 P2Ox 的 p2ox 基因。将 p2ox cDNA 插入细菌表达载体 pET21a(+) 中，并在 E. coli Rosetta 2 中成功表达。我们以约 130mg/L 培养基的产量获得了活性、黄素化的重组 P2Ox。该酶通过基于阴离子交换层析和制备性天然 PAGE 的两步法进行纯化，得到了一种明显均一的酶制剂，比活为 1.92U/mg（以葡萄糖和空气氧为底物）。对来自 L. shimeji 的重组 P2Ox 进行了详细的物理和催化特性表征，并与来自 Phanerochaete chrysosporium 和 Trametes multicolor 的高度表征的酶进行了比较。
L. shimeji P2Ox 的性质与来自白腐真菌的 P2Ox 相似，并且通常表现出较低的 K(m)和 k(cat)值，无论是电子供体（糖）还是电子受体（铁氰化钾离子、1,4-苯醌、2,6-二氯靛酚）。虽然 L. shimeji P2Ox 是这三种酶中最不稳定的（熔点 T(m)为 54.9°C；半衰期 tau1/2 在 50°C 和 pH6.5 下为 0.12），但 P. chrysosporium P2Ox 在相同条件下表现出显著的热稳定性，T(m)为 75.4°C，tau1/2 为 96h。
