Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (kcat = 823 s−1, Km = 28.0 mM, and kcat/Km = 29.4 mM−1 s−1) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.
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机译:甘露醇脱氢酶(MDH)(EC 1.1.1.138)催化大果假丝酵母HH-01(KCCM-10252)中的甘露醇生物合成,该菌株目前用于工业生产甘露醇。在这项研究中,NAD(P)H依赖的MDH通过离子交换色谱,疏水相互作用色谱和亲和色谱从木兰假丝酵母HH-01纯化至均一。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和尺寸排阻色谱法测定的玉兰假丝酵母MDH的相对分子量分别为35和142kDa,表明该酶是四聚体。该酶催化果糖还原和甘露醇氧化。果糖还原和甘露醇氧化的最适pH和温度分别为7.5和37°C和10.0和40°C。木兰MDH显示出高底物特异性和高催化效率(kcat = 823 s -1 sup>,Km = 28.0 mM,kcat / Km = 29.4 mM -1 sup> s < sup> -1 sup>)代表果糖,这可能解释了该菌株中甘露醇的高产量。最初的速度和产物抑制研究表明,反应是通过顺序有序的Bi Bi机制进行的,木兰假单胞菌MDH特异于转移NADPH的4-pro-S氢,这是典型的短链脱氢酶还原酶(SDR) )。木兰假单胞菌MDH的内部氨基酸序列显示出与来自各种来源的SDR的显着同源性,表明木兰假单胞菌MDH是NAD(P)H依赖性四聚体SDR。尽管MDH已从其他几种来源中纯化和鉴定,但它们仍是C。木兰MDH与其他MDH的区别在于其高底物特异性和仅对果糖的催化效率,这使得 C。木兰MDH是工业应用的理想选择,包括酶法合成甘露醇和耐盐植物。
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