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首页> 外文期刊>Applied Microbiology and Biotechnology >Pyranose 2-oxidase from Phanerochaete chrysosporium--further biochemical characterisation
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Pyranose 2-oxidase from Phanerochaete chrysosporium--further biochemical characterisation

机译：Phanerochaete chrysosporium的吡喃糖2-氧化酶-进一步的生化表征

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Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 degrees C. It is active over a broad pH range (5.0-9.0) with maximum activity at pH 8.0-8.5 and at 55 degrees C, and a broad substrate specificity. D-Glucose is the preferred substrate, but 1-beta-aurothioglucose, 6-deoxy-D-glucose, L-sorbose, D-xylose, 5-thioglucose, D-glucono-1,5-lactone, maltose and 2-deoxy-D-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (kcat) of 111.0 s-1 and an affinity constant (Km) of 1.43 mM for D-glucose and 83.2 microM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH > or = 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the kcat/K(m) value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the kcat decreased to 60.9 s-1 and the K(m) increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.
机译：使用苯基琼脂糖，Mono Q（两次）和苯基Superose液相色谱，从担子菌Phanerochaete chrysosporium中纯化吡喃糖2-氧化酶（P2O）43倍，使其具有明显的同质性。天然酶的分子量约为250 kDa（基于天然PAGE），由65 kDa的四个相同亚基组成。它包含等电点（pI）5.0、5.05和5.15的三种同工型，并且似乎不是糖蛋白。 P2O在pH 8.0和高达60摄氏度的温度下具有最佳稳定性。它在广泛的pH范围（5.0-9.0）内具有活性，在pH 8.0-8.5和55摄氏度下具有最大活性，并且具有广泛的底物特异性。 D-葡萄糖是优选的底物，但1-β-金硫代葡萄糖，6-脱氧-D-葡萄糖，L-山梨糖，D-木糖，5-硫代葡萄糖，D-葡萄糖基-1,5-内酯，麦芽糖和2-脱氧-D-葡萄糖也以相对高的速率被氧化。已证明P2O反应在pH 8.0时具有Ping Pong Bi Bi机制，其催化常数（kcat）为111.0 s-1，亲和常数（Km）对D-葡萄糖为1.43 mM，对氧为83.2 microM。在pH>或= 7.0的条件下，葡萄糖氧化的稳态动力学不受介质的影响，而在低pH下，pH和缓冲液组成均会影响P2O动力学，且kcat / K（m）值随pH的降低而降低。在乙酸盐缓冲液（0.1 M，pH 4.5）中观察到最大的影响，其中kcat降低至60.9 s-1，K（m）增加至240 mM。 P2O的活性被10 mM HgCl2，AgNO3和ZnCl2完全抑制，而50％被乙酸铅，CuCl2和MnCl2完全抑制。

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