Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase
Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase
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DOI：
10.1128/jb.185.12.3485-3490.2003
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发表时间：
2003-06
影响因子：
3.2
通讯作者：
Teruyuki Kobayashi;M. Shiraki;T. Abe;A. Sugiyama;Terumi Saito
中科院分区：
文献类型：
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作者：
Teruyuki Kobayashi;M. Shiraki;T. Abe;A. Sugiyama;Terumi Saito
从携带phaZ 2 Reu质粒的大肠杆菌中纯化了真养罗尔斯通氏菌胞内3-羟基丁酸酯（3 HB）寡聚体水解酶（PhaZ 2 Reu）。纯化的酶水解线性和环状3 HB-低聚物。虽然它没有降解结晶聚（3-羟基丁酸酯）（PHB），纯化的酶降解人工无定形的PHB在以前确定的细胞内的PHB（iPHB）解聚酶（PhaZ 1 Reu）的速率相似。该酶似乎是一种内切型水解酶，因为它积极水解环状3 HB-低聚物。然而，它降解各种线性3 HB-低聚物和无定形的PHB的方式外切型水解酶，释放一个单体单元的时间。发现PhaZ 2与PHB包涵体结合，并作为R.与此相反，PhaZ 1只与包涵体结合。当R.在营养丰富的培养基中培养真养菌H16，观察到了PHB的瞬时沉积：14%的细胞干重），并下降到一个非常低的水平，在稳定期（约。干细胞重量的1%）。在每个phaZ 1-null突变体和phaZ 2-null突变体中，细胞中的PHB含量增加到约100%。稳定期为5%。缺乏phaZ 1和phaZ 2的双突变体在对数期显示出增加的PHB含量（约100%）。20%）和升高的PHB水平（约。8%）。这些结果表明，PhaZ 2是一种新的iPHB解聚酶，参与了R.真养沿着与PhaZ 1。
ABSTRACT An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2Reu) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2Reu. The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1Reu). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.