Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass.

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.

Molecular mass of Poly-3-hydroxybutyrate (P3HB) produced by Azotobacter vinelandii is influenced by the polymer content in the inoculum.

Poly-3-hydroxybutyrate (P3HB) is a biopolymer produced by Azotobacter vinelandii. The physicochemical properties and applications of P3HB are strongly influenced by its weight-average molecular mass (Mw), and in A. vinelandii, it could be influenced by the culture conditions. The aim of this study was to evaluate the effect of the P3HB content of the inoculum on the Mw of the polymer produced by A. vinelandii OP in bioreactor cultures. A. vinelandii cells containing 20, 50 and 70% of P3HB were used as inoculum. The P3HB content in the inoculum affected the volumetric P3HB productivity (qP3HB) and the Mw of P3HB. Those cultures inoculated with cells containing 20% of P3HB, achieved the highest qP3HB (0.17±0.018gP3HBL-1h-1); whereas a P3HB content of 70% was reflected as a low qP3HB (0.021±0.002gP3HBL-1h-1). On the other hand, using an inoculum with 70% of polymer content, the Mw of the biopolymer remained stable at values close to 3200kDa; whereas, when an inoculum with 20% of P3HB was used, the Mw decreased drastically during early stages of cultivation. These results show that manipulating the P3HB content of the inoculum is possible to produce biopolymers with a suitable Mw.