Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass
Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass
复制标题
DOI：
10.1007/s00253-018-8806-y
复制
发表时间：
2018-03-01
影响因子：
5
通讯作者：
Segura, Daniel
中科院分区：
文献类型：
--
作者：
Adaya, Libertad;Millan, Modesto;Segura, Daniel
在棕色固氮菌中发现了一种新的聚-3-羟基丁酸酯解聚酶。这种酶，现在指定为PhbZ 1，是相关的聚-3-羟基丁酸酯（PHB）颗粒，当在大肠杆菌中表达，它显示在体外的天然或人工的聚羟基丁酸酯颗粒，但不对结晶聚羟基丁酸酯解聚活性。从PhbZ 1突变体中分离的天然PHB（nPHB）颗粒与野生型菌株的颗粒相比，具有减少的聚酯的内源性体外水解。还在游离辅酶A存在下测试了这种体外降解。在野生型的nPHB颗粒中观察到聚合物的硫解降解，导致3-羟基丁酰-CoA的形成，但在突变体的颗粒中不存在。以前有报道，A.在生物反应器中生长的vinelandii OP在培养20小时后显示出PHB的重均分子量（Mw）的降低，而较低分子量的聚合物部分的增加。这种减少与在培养过程中的PHB解聚酶活性的增加。在这里，我们表明，在phbZ 1突变体中，既没有降低分子量也没有出现低分子量聚合物。此外，在phbZ 1突变体的培养物中观察到较高的PHB积累。这些结果表明，PhbZ 1在生物反应器中的培养物中的PHB降解中具有作用，并且其失活允许产生均匀的高分子量的聚合物。
A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.