Statistical optimization of synthetic azo dye (orange II) degradation by azoreductase from Pseudomonas oleovorans PAMD_1.

Pseudomonas oleovorans PAMD_1 produced an intracellular azoreductase as the more prominent enzyme that reduces the azo bridge during the azo dye decolorization process. In order to optimize the expression of azoreductase, statistically based experiments were applied. Eleven significant factors were screened on decolorization activity using Plackett-Burman design. Dye, NADH, glucose, and peptone were identified as having highest positive influence on the decolorization activity. Central composite design of response surface methodology was employed for the concerted effect of these four factors on decolorization activity. This method showed that the optimum medium containing dye (200 mg L(-1)), NADH (1.14 mM), glucose (2.07 g L(-1)), and peptone (6.44 g L(-1)) for the decolorization of Orange II up to 87% in 48 hr. The applied methodology was validated through the adequacy and accuracy of the overall experiments, and the results proved that the applied methods were most effective. Further, the enzyme was purified ninefold with 16% yield by anion-exchange chromatography and a specific activity of 26 U mg(-1). The purified enzyme with a molecular mass of 29,000 Da gave a single band on sodium dodecyl sulfate (SDS) gel, and the degradation products sulfanilic acid and 1-amino-2-napthol of Orange II by azoreductase were analyzed by using an ultraviolet-visible (UV-Vis) spectrophotometer and hish-performance liquid chromatography (HPLC).

Genetic characterization of the poly(hydroxyalkanoate) synthases of various Pseudomonas oleovorans strains.

We identified the poly(hydroxyalkanoate) synthase (PHAS) genes of three strains of Pseudomonas oleovorans by using polymerase chain reaction (PCR)-based detection methods. P. oleovorans NRRL B-14682 contains Class I PHA synthase gene (phaC), NRRL B-14683 harbors Class II phaC1 and phaC2 genes, and NRRL B-778 contain both the Class I and II PHA synthase genes. Inverse-PCR and chromosomal walking techniques were employed to obtain the complete sequences of the Class I phaCs of NRRL B-778 (phbC778; 1698 bps) and B-14682 (phbC14682; 1899 bps). BLAST search indicated that these genes are new and had not been previously cloned. The gene product of phbC778 (i.e., PhbC778; 566 amino acid residues) is homologous to the Class I PHA synthases of Pseudomonas sp. HJ-2 and Pseudomonas sp. strain 61-3, and that of phbC14682 (PhbC14682; 632 amino acids) is homologous to PHAS of Delftia acidovorans. The PhbC14682 contains an extra sequence of 33 amino acids in its conserved alpha/beta-hydrolase domain, making it only the second Class I PHA synthase found to contain this cellular proteolytic sequence. Consistent with their Pseudomonas origin, the codon-usage profiles of PhbC778 and PhbC14682 are similar to those of Pseudomonas Class II PHASs. These new Pseudomonas Class I phbC genes provide valuable addition to the gene pool for the construction of novel PHASs through gene shuffling.

Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase.

Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.