关键词:
PHB polymerase
Azospirillum baldaniorum
PhbC activity
Biodegradable polymer
摘要:
Background: Azospirillum baldaniorum Sp245 produces poly-beta-hydroxybutyrate, a biodegradable polymer with characteristics similar to synthetic thermoplastics, including polypropylene. In the synthesis pathway, the poly-beta-hydroxybutyrate synthase enzyme uses thioesters of 3-hydroxy butyryl-CoA as a substrate and catalyzes their polymerization with HS-CoA release. Methods: A study was conducted using in silico analysis of the two phbC genes of A. baldaniorum Sp245. One was selected for amplification and cloning into the pEXP5- CT/TOPO (R) vector, which was analysed by restriction pattern, polymerase chain reaction, and sequencing. SDS-PAGE analysis determined the molecular weight of the PhbC1 protein from Azospirillum baldaniorum (AbPhbC1). The presence of the protein was confirmed by Western blotting using anti-polyhistidine monoclonal antibodies. The enzymatic activity in the crude extract of AbPhbC1 was determined by measuring the concentration of sulfhydryl groups using the Ellman method. A UV-Vis assay was performed. To confirm the presence of the poly-beta-hydroxybutyrate product, an NMR assay was performed. Results: In silico analyses, it is revealed that AbPhbC1 and the PhbC2 protein from Azospirillum baldaniorum (AbPhbC2) retain the poly-beta-hydroxybutyrate polymerase and alpha/beta hydrolase domain. The Cys-His-Asp catalytic triad is highly conserved in all four poly beta-hydroxyalkanoate synthases in the central subdomain, structurally similar to the reported crystallized proteins. The dimerization subdomain is different;in AbPhbC1, it is in the closed form;in AbPhbC2, it is in the open form;and in AbPhbC2, it lacks the EC region as class III and IV poly-beta-hydroxyalcanoate synthases. In vitro, the molecular weight of AbPhbC1 was 68 kDa. The polymerization of PHB by AbPhbC1 was detected by the release of HS-CoA from the quantification of SH. The UV-Vis scan showed a characteristic peak at 264 nm. A comparison of the NMR spectra of the bacterial and