Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum
Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum
复制标题
丙酮丁醇梭菌诱导型辅酶 A 连接丁醛脱氢酶的纯化及性质
DOI：
10.1128/jb.170.7.2971-2976.1988
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发表时间：
1988
影响因子：
3.2
通讯作者：
P. Rogers
中科院分区：
文献类型：
--
作者：
N. Palosaari;P. Rogers
对丙酮丁醇梭菌辅酶A（CoA）连接的丁醛脱氢酶（BAD）进行了表征和纯化。该酶被诱导超过200倍，符合从产酸到产溶剂发酵的转变，在分批培养生长。发现酶活性的增加需要新的蛋白质合成，因为通过添加利福平阻断了诱导，并且针对纯化酶的抗体显示在发酵的转变时开始出现酶抗原，并且随着酶比活性的增加而协同增加。CoA连接的乙醛脱氢酶与BAD共纯化89倍纯化过程中，表明一种酶负责合成的两个醛中间体的丁醇和乙醇的生产。丁醇脱氢酶活性与TEAE纤维素上的BAD酶活性明显分开。测定天然酶的分子量为115，000，酶亚基的分子量为56，000，表明活性形式是同二聚体。在正向和反向方向的动力学常数进行了测定。在反向方向上，对丁醛和己醛的Vmax和表观亲和力均显著大于对乙醛的Vmax和表观亲和力，而在正向方向上，对丁酰-CoA的Vmax是对乙酰-CoA的Vmax的五倍。BAD的这些和其他性质表明，这种酶明显不同于其他报道的CoA依赖性醛脱氢酶。
The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases.