Moonmangmee Duangtip, Adachi Osao, Shinagawa Emiko, Toyama Hirohide, Theeragool Gunjana, Lotong Napha, Matsushita Kazunobu
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Biosci Biotechnol Biochem. 2002 Feb;66(2):307-18. doi: 10.1271/bbb.66.307.
Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from mesoerythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized mesoerythritol to L-erythrulose irreversibly with almost 100% of recovery at 37 degrees C. L-Erythrulose was also produced efficiently by the resting cells at 37 degrees C with 85% recovery. The enzyme responsible for mesoerythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L-erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound mesoerythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between mesoerythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0-10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production.
筛选出耐热的弗氏葡萄糖杆菌CHM 43用于在较高温度下由内消旋赤藓糖醇生产L-赤藓酮糖。该菌株的生长细胞和膜组分能将内消旋赤藓糖醇快速不可逆地氧化为L-赤藓酮糖，在37℃时回收率几乎达到100%。静止细胞在37℃时也能高效产生L-赤藓酮糖，回收率为85%。发现负责内消旋赤藓糖醇氧化的酶位于该生物体的细胞质膜中。经EDTA处理的酶在形成L-赤藓酮糖时需要PQQ和Ca2+，这表明催化内消旋赤藓糖醇氧化的酶是一种醌蛋白。将膜结合的醌蛋白内消旋赤藓糖醇脱氢酶（QMEDH）溶解并纯化至同质。纯化后的酶在SDS-PAGE中显示出一条单一的条带，其分子量对应于80 kDa。发现QMEDH的最适pH为5.0。以内消旋赤藓糖醇为底物时，该酶的米氏常数为25 mM。QMEDH对C3-C6糖醇表现出广泛的底物特异性，其中两个羟基的赤藓糖形式存在于伯醇基团相邻位置。另一方面，将同一生物体的胞质NAD依赖性内消旋赤藓糖醇脱氢酶（CMEDH）纯化至结晶状态。CMEDH的分子量为60 kDa，由两个相同的亚基组成，表观沉降常数为3.6 s。CMEDH催化内消旋赤藓糖醇和L-赤藓酮糖之间的氧化还原反应。在碱性pH（如9.0 - 10.5）下，在NAD存在时观察到氧化反应是可逆的。在pH 6.0时，以NADH为辅酶可发现L-赤藓酮糖的还原反应。从催化特性判断，胞质组分中的NAD依赖性酶被视为典型的NAD依赖性戊糖醇脱氢酶，且该酶与L-赤藓酮糖生产的氧化发酵无关。
