Competitive Biosynthesis of Bacterial Alginate Using Azotobacter vinelandii 12 for Tissue Engineering Applications

This study investigated the effect of various cultivation conditions (sucrose/phosphate concentrations, aeration level) on alginate biosynthesis using the bacterial producing strain Azotobacter vinelandii 12 by the full factorial design (FFD) method and physicochemical properties (e.g., rheological properties) of the produced bacterial alginate. We demonstrated experimentally the applicability of bacterial alginate for tissue engineering (the cytotoxicity testing using mesenchymal stem cells (MSCs)). The isolated synthesis of high molecular weight (Mw) capsular alginate with a high level of acetylation (25%) was achieved by FFD method under a low sucrose concentration, an increased phosphate concentration, and a high aeration level. Testing the viscoelastic properties and cytotoxicity showed that bacterial alginate with a maximal Mw (574 kDa) formed the densest hydrogels (which demonstrated relatively low cytotoxicity for MSCs in contrast to bacterial alginate with low Mw). The obtained data have shown promising prospects in controlled biosynthesis of bacterial alginate with different physicochemical characteristics for various biomedical applications including tissue engineering.

Metabolic Model of the Nitrogen-Fixing Obligate Aerobe Azotobacter vinelandii Predicts Its Adaptation to Oxygen Concentration and Metal Availability

The world’s dependence on industrially produced nitrogenous fertilizers has created a dichotomy of issues. First, parts of the globe lack access to fertilizers, leading to poor crop yields that significantly limit nutrition while contributing to disease and starvation.

Growth, respiratory activity and chlorpyrifos biodegradation in cultures of Azotobacter vinelandii ATCC 12837

This study aimed to evaluate the growth, respiratory activity, and biodegradation of chlorpyrifos in cultures of Azotobacter vinelandii ATCC 12837. A strategy based on the modification of culture media and aeration conditions was carried out to increase the cell concentration of A. vinelandii , in order to favor and determine its tolerance to chlorpyrifos and its degradation ability. The culture in shaken flasks, using sucrose as a carbon source, significantly improved the growth compared to media with mannitol. When the strain was cultivated under oxygen-limited (5.5, 11.25 mmol L- 1 h- 1 ) and no-oxygen-limited conditions (22 mmol L -1 h -1 ), the growth parameters were not affected. In cultures in a liquid medium with chlorpyrifos, the bacteria tolerated a high pesticide concentration (500 ppm) and the growth parameters were improved even under conditions with a reduced carbon source (sucrose 2 g L -1 ). The strain degraded 99.6 % of chlorpyrifos at 60 h of cultivation, in co-metabolism with sucrose; notably, A. vinelandii ATCC 12837 reduced by 50% the initial pesticide concentration in only 6 h (DT 50 ).

Comparison of Biochemical Characteristics, Action Models, and Enzymatic Mechanisms of a Novel Exolytic and Two Endolytic Lyases with Mannuronate Preference

Recent explorations of tool-like alginate lyases have been focused on their oligosaccharide-yielding properties and corresponding mechanisms, whereas most were reported as endo-type with α-L-guluronate (G) preference. Less is known about the β-D-mannuronate (M) preference, whose commercial production and enzyme application is limited. In this study, we elucidated Aly6 of Flammeovirga sp. strain MY04 as a novel M-preferred exolytic bifunctional lyase and compared it with AlgLs of Pseudomonas aeruginosa (Pae-AlgL) and Azotobacter vinelandii (Avi-AlgL), two typical M-specific endolytic lyases. This study demonstrated that the AlgL and heparinase_II_III modules play indispensable roles in determining the characteristics of the recombinant exo-type enzyme rAly6, which is preferred to degrade M-enriched substrates by continuously cleaving various monosaccharide units from the nonreducing end, thus yielding various size-defined ΔG-terminated oligosaccharides as intermediate products. By contrast, the endolytic enzymes Pae-rAlgL and Avi-rAlgL varied their action modes specifically against M-enriched substrates and finally degraded associated substrate chains into various size-defined oligosaccharides with a succession rule, changing from ΔM to ΔG-terminus when the product size increased. Furthermore, site-directed mutations and further protein structure tests indicated that H195NHSTW is an active, half-conserved, and essential enzyme motif. This study provided new insights into M-preferring lyases for novel resource discoveries, oligosaccharide preparations, and sequence determinations.

Mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the monomer composition by catalysing the epimerization of β- d -mannuronic acid residues (M) into α- l -guluronic acid residues (G). The molecular weight is affected by alginate lyases, which catalyse a β-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii . Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that calcium promotes lyase activity whereas NaCl reduces the lyase activity of AlgE7. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activity where H154 functions as the catalytic base and Y149 as the catalytic acid. Importance Post-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

ABSTRACTAzotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe4-S4] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.

Growth, respiratory activity and chlorpyrifos biodegradation in cultures of Azotobacter vinelandii ATCC 12837

This study aimed to evaluate the growth, respiratory activity, and biodegradation of chlorpyrifos in cultures of Azotobacter vinelandii ATCC 12837. A strategy based on the modification of culture media and aeration conditions was carried out to increase the cell concentration of A. vinelandii, in order to favor and determine its tolerance to chlorpyrifos and its degradation ability. The culture in shaken flasks, using sucrose as a carbon source, significantly improved the growth compared to media with mannitol. When the strain was cultivated under oxygen-limited (5.5, 11.25 mmol L−1 h−1) and no-oxygen-limited conditions (22 mmol L−1 h−1), the growth parameters were not affected. In cultures in a liquid medium with chlorpyrifos, the bacteria tolerated a high pesticide concentration (500 ppm) and the growth parameters were improved even under conditions with a reduced carbon source (sucrose 2 g L−1). The strain degraded 99.6% of chlorpyrifos at 60 h of cultivation, in co-metabolism with sucrose; notably, A. vinelandii ATCC 12837 reduced by 50% the initial pesticide concentration in only 6 h (DT50). Graphical Abstract