Cloning, expression, and purification of choline dehydrogenase from the moderate halophile Halomonas elongata
Cloning, expression, and purification of choline dehydrogenase from the moderate halophile Halomonas elongata
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DOI：
10.1128/aem.69.4.2126-2132.2003
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发表时间：
2003-04-01
影响因子：
4.4
通讯作者：
McAllister-Wilkins, EE
中科院分区：
文献类型：
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作者：
Gadda, G;McAllister-Wilkins, EE
胆碱脱氢酶(EC 1.1.99.1)通过甜菜碱醛中间体催化胆碱四电子氧化生成甘氨酸甜菜碱。这种反应在生物技术应用中具有相当大的兴趣，因为用细菌甘氨酸甜菜碱合成酶工程的转基因植物已经被证明增强了对各种环境胁迫的耐受性，如高盐度、冰冻和高温。到目前为止，胆碱脱氢酶的生化和动力学性质一直很差，主要是因为该酶在体外的不稳定性阻碍了其纯化。在本报告中，我们克隆并在大肠杆菌中表达了中度嗜盐长形嗜盐单胞菌的β基因，该基因编码一种假想的胆碱脱氢酶。重组酶经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和30~50%饱和硫酸铵处理后，再经DEAE-Sepharose柱层析，均一度达到70%以上。纯化的酶以胆碱或甜菜碱醛为底物具有相似的底物特异性，在pH为7和25℃时，O-2min(-1)mg(-1)mm(-1)的表观V/K值(V为最大速度，K为米氏常数)分别为0.9和0.6摩尔。以1 mM吩嗪甲硫酸盐为主要电子受体，胆碱和甜菜碱醛的表观V-max分别为10.9和5.7mgO-2min(-1)mg(-1)。当使用分子氧作为电子受体时，这些V-max值降低了四到七倍。总而言之，动力学数据与长柄链球菌编码胆碱脱氢酶的结论是一致的，当分子氧以外的电子受体不可用时，该酶也可以作为氧化酶。
Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 mumol of O-2 min(-1) mg(-1) mM(-1) at pH 7 and 25degreesC, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent V-max values for choline and betaine-aldehyde were 10.9 and 5.7 mumol of O-2 min(-1) mg(-1), respectively. These V-max values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available.