Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli
Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli
复制标题
红色红螺菌 (Rhodospirillum rubrum) 的 (R) 特异性反式 2,3-烯酰基辅酶 A 水合酶的表征和克隆，以及使用该酶在大肠杆菌中生产 PHA
DOI：
10.1007/s002530050010
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发表时间：
2000
影响因子：
5
通讯作者：
K. Gruys
中科院分区：
文献类型：
--
作者：
S. Reiser;T. Mitsky;K. Gruys
从红杜鹃（Rhodocellum rubrum）中纯化了一种（R）-反式-2，3-烯酰辅酶A水合酶。富集的蛋白质级分的蛋白质测序允许简并寡核苷酸的构建。用该寡核苷酸进行三轮菌落杂交后，克隆了编码（R）-特异性水合酶活性的基因，并在E.将酶纯化至均一。纯化的酶在水合反应中使用巴豆酰辅酶A、反式-2，3-戊烯酰辅酶A和反式-2，3-己烯酰辅酶A作为底物，具有大致相同的特异性。然而，使用反式-2，3-辛烯酰-CoA作为底物没有观察到活性，但该化合物确实部分抑制巴豆酰-CoA水合。基于核苷酸序列，该蛋白质具有15.4 kDa的单体分子量，并且如通过凝胶过滤色谱和非变性PAGE所确定的，在其天然形式下是同源四聚体。水合酶与PHA合成酶一起从Thiocapsa pfennigilinE中表达。大肠杆菌DH 5 α。这些菌株在油酸上的生长导致三元聚酯聚（3-羟基丁酸酯-共-3-羟基戊酸酯-共-3-羟基己酸酯）的产生。
An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity fromRhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encoding the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene inE. coliled to the purification of the enzyme to homogeneity. The purified enzyme used crotonyl-CoA,trans-2,3-pentenoyl-CoA, andtrans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction. However, no activity was observed usingtrans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration. Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel filtration chromatography and native PAGE. The hydratase was expressed together with the PHA synthase fromThiocapsa pfennigiiinE. colistrain DH5α. Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate).