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首页> 外文期刊>Journal of Biotechnology >Aqueous release and purification of poly(beta-hydroxybutyrate fromEscherichia coli
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Aqueous release and purification of poly(beta-hydroxybutyrate fromEscherichia coli

机译：大肠杆菌中聚（β-羟基丁酸酯）的水释放和纯化

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摘要

The poly(P-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon (phaCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% PHB/celldry mass when the cells are grown in Luria-Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.
机译：已在大肠杆菌中克隆了在单个操纵子（phaCAB）中组织的富营养小球藻（Ralstonia eutropha）的聚（P-羟基丁酸）（PHB）生物合成基因，其中来自质粒pTZ18U-PHB的野生型pha操纵子中基因的表达当细胞在补充了1％葡萄糖（w / v）的Luria-Bertani培养基中生长时，会导致50-80％PHB /细胞干质量的形成。与phaCAB基因结合，从质粒pSH2克隆噬菌体PhiX174的裂解基因E已被用于释放在大肠杆菌中产生的PHB颗粒。结果表明，半液体阶段的小PHB颗粒通过E-裂解通道结构从细胞中挤出，该结构的特征是在包膜中有一个小孔，内外膜融合在一起。 E-裂解后，所有包膜成分均保持完整，可通过密度梯度离心从释放的PHB颗粒混合物中除去。另外，描述了改进的E-裂解程序，其使得能够在纯水或低离子强度缓冲液中从细胞沉淀中释放PHB。水溶液中的PHB颗粒可被二价阳离子聚集。添加玻璃奶可加快PHB颗粒的团聚速度，并且与玻璃珠的结合可用于收集或进一步纯化水溶液中的PHB。

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