Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257:: A versatile enzyme for the oxidative fermentation of various ketoses
Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257:: A versatile enzyme for the oxidative fermentation of various ketoses
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DOI：
10.1271/bbb.65.2755
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发表时间：
2001-12-01
影响因子：
1.6
通讯作者：
Matsushita, K
中科院分区：
文献类型：
--
作者：
Adachi, O;Fujii, Y;Matsushita, K
用葡萄糖酸杆菌IFO 3257的膜组分成功地对膜结合的藜麦蛋白D-阿拉伯醇脱氢酶进行了增溶。在酶的溶解和随后的酶纯化步骤中，经过多次令人失望的试验，人们特别注意提纯具有天然膜中活性的ARDH。选择最佳的洗涤剂，通过添加PQQ和Ca~(2+)使ARDH保持全酶活性，并选择含有醋酸盐缓冲液和Ca~(2+)的缓冲体系，对于处理这种高度疏水的不稳定的酶是至关重要的。在洗涤剂和Ca~(2+)存在下，DEAE-Toyota opearl和CM-Toyota opearl两步柱层析对酶进行纯化。ArdH均一，在分析超速离心法中呈单一沉淀峰。亚基I和亚基II的相对分子质量分别为82 kDa和14 kDa，形成异二聚体结构。用高效液相色谱检测到经十二烷基硫酸钠处理后的ArdH释放出的PQQ，证明ArdH为藜麦蛋白。EDTA处理的膜组分在加入PQQ和Ca~(2+)后失去了酶活性，而ARDH活性恢复到原来的水平。ARDH最突出的独特特性是底物专一性，它具有高度的通用性，可以被多种底物不可逆地氧化，不仅是戊醇，还包括D-山梨醇、D-甘露醇、甘油、新赤藓糖醇和2，3-丁二醇。ArdH可能在醋酸菌的氧化发酵产酮糖过程中起主要作用。与III型或III型藜麦蛋白乙醇脱氢酶(ADH)不同，ARDH不含血红素成分，也不与伯醇反应。
Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purify ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.