N-acetylglucosamine 6-Phosphate Deacetylase (nagA) Is Required for N-acetyl Glucosamine Assimilation in Gluconacetobacter xylinus

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tetr; named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species

Emulsan, a tailorable biopolymer for controlled release

Microsphere hydrogels made with emulsan–alginate were used as carrier for the microencapsulation of blue dextran in order to study the effect of emulsan on the alginate bead stability. Blue dextran release studies indicated an increase of microsphere stability in presence of emulsan, as a coating agent. BSA adsorption by emulsan–alginate microspheres is also enhanced 40% compared to alginate alone. XPS studies confirm the presence of BSA adsorbed on emulsan microsphere surfaces. These results are in agreement with the equilibrium adsorption model of Freundlich. Studies of BSA adsorption using non-equilibrium Lagergren second-order and intraparticle models, are suggesting a complex mechanisms of protein adsorption by chemisorption and intraparticle diffusion. Also, enzymatic release of BSA from emulsan microspheres containing azo-BSA under physiological conditions is suggests the possibility of using microspheres as a depot for pre-proteins of medical interest.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Biosynthesis of emulsan biopolymers from agro-based feedstocks

Aims: The need for biocompatible, biodegradable, and versatile biopolymers permeates many fields including environmental and food technology. The goal of the study presented here is to establish the utility of agricultural oils as an inexpensive carbon source to produce materials useful for biomedical materials and offer positive attributes in terms of green chemistry. Methods and Results: Structural variants of the complex acylated polysaccha- ride, emulsan, secreted from Acinetobacter venetianus RAG-1, were biosynthe- sized in cultures supplemented with agricultural feedstocks to examine the feasibility of conversion of these substrates into value-added biopolymers. Acinetobacter venetianus produced chemically and biologically distinct emulsan variants in culture on soy molasses and tallow oil. These variants possess signi- ficant biological function, including macrophage activation and adjuvant activ- ity, in similar range to that observed for the standard emulsan formed on ethanol-fed A. venetianus. Conclusions: The results indicate that this novel family of biopolymers can be produced in significant quantities from the readily available renewable agricul- tural feedstocks and the resulting structures and functions can be correlated to the chemistry of these feedstocks. Significance and Impact of the Study: The significant quantities of agricultural oils produced annually represent an untapped source for bioconversion to valuable products. The results of this study confirm that the important polymer emulsan can be synthesized from this inexpensive carbon source.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Emulsan–alginate beads for protein adsorption

Emulsan–alginate beads were prepared and challenged using bovine serum albumin (BSA) to assess adsorption in comparison to alginate beads. BSA binding to the emulsan–alginate beads was improved over the alginate bead controls and protein adsorption was less sensitive to changes in ionic strength. BSA adsorption between pH 8.5 and 5.3 in alginate beads was 2–3-times lower compared to the emulsan–alginate beads in the same pH range. BSA adsorption and kinetic constants were at least 2-times higher for the emulsan–alginate beads compared to the alginate controls based on the Langmuir adsorption model. To further explore the utility of these novel emulsan–alginate bead systems, complex cell-free supernatants from some pathogenic microorganisms were exposed to the emulsan–alginate beads and increased protein adsorption was found when compared to controls. These trends were also confirmed with α-hemolysin toxicity studies. The data suggest that the protein-binding capacity of emulsan–alginate beads exceeds alginate controls, attributable to the unique binding features of emulsan.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Controlled release biopolymers for enhancing the immune response

Controlled release of biologically active compounds in the context of drug and vaccine delivery is an important area of research with broad implications in many areas of medicine. In particular, the challenges of oral delivery are of specific interest to reduce the cost and potential health risks related to parenteral administration of pharmaceuticals and vaccine formulations. We discuss the biological activities of two biopolymers, β-glucans and emulsans, both of which offer significant potential for individual formulations related to drug impact, while in combination offer synergistic opportunities in terms of formulation and delivery. β-Glucans have been established as potent immunomodulatory and biologically active compounds with application in a wide range of disease systems. The emulsan family of biopolymers also has significant potential in vaccine and drug delivery based on recent studies. Each of these biopolymers offers exciting opportunities to modulate biological responses via control of chemistry and physical properties achieved during biosynthesis or postsynthesis modifications. When combined into a delivery system for controlled release, synergistic outcomes may be achieved that offer new and exciting opportunities as described in the present paper. These outcomes represent the combined improvements of solubility in physiological environments and immunomodulation due to the specific chemistry and structures involved. Overall, this approach provides a new direction in controlled release wherein the biomaterial carrier, in this case emulsan, and the drug, in this case β-glucan, play an active role both in biological activation as well as in delivery profiles.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Emulsan quantitation by Nile red quenching fluorescence assay

A Nile red fluorescent technique to quantify 20– 200 μg ml⁻¹ of emulsan was developed. Nile red dissolved in DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm⁻¹ M⁻¹. Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG⁻¹.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Triggered release of proteins from emulsan–alginate beads

Emulsan/alginate beads were studied for protein adsorption and stability in the context of controlled release. The beads, 400F80 Am diameter with approximately 10% emulsan content, offer unusual opportunities for delivery of proteins due to the natural ability of emulsan to bind proteins, coupled with the selective biological activation features of this complex lipoheteropolysaccharide. The binding capacity of azo-bovine serum albumin by the emulsan/alginate beads was 0.637 ± 0.004 vs. 0.170 ± 0.007 μg/mg for beads formed from alginate alone. In additional protein adsorption experiments, the lipase and subtilisin maintained activity when adsorbed to the emulsan/alginate beads albeit with lower specific activity when compared to the enzyme free in solution. However, the half life of the adsorbed enzyme was significantly higher than the free forms. To explore functional utility of this system, two types of triggered release were studied in the context of these bead systems. First, azo-BSA as a model protein was physically bound to emulsan/alginate beads and then selectively released by triggering with subtilisin, a serine protease, which cleaves the azo dye, sulfanilic acid, from the bound protein. In absence of subtilisin no triggered release was observed. Second, azo-BSA as a prodrug model, was adsorbed to the emulsan/alginate beads and then release of the dye was demonstrated by lipase treatment which cleaves the fatty acid esters from the emulsan structure to release the bound protein. The results establish the versatility and utility of emulsan-based beads for protein binding and triggered release.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Emulsan-Alginate Microspheres as a New Vehicle for Protein Delivery

A solution containing emulsan, a lipoheteropolysaccharide, and calcium was used to produce emulsan-alginate microspheres (EAMs). Optical, scanning electron microscopy and EDX (Energy Dispersive X-ray) analysis of the microspheres suggested different morphologies and compositions, respectively, when compared with microspheres prepared only from alginate. The EAMs were twice as stable in phosphate solution compared to alginate alone when assessed with blue dextran encapsulation. The EAMs were able to adsorb about twice the amount of BSA (Bovine Serum Albumin) compared to alginate alone. When azo-BSA was adsorbed on the emulsan-alginate microspheres, protein release could be triggered with enzymes. BSA released from the EAMs retained about of 78% of the -helix structure.Centro de Investigación y Desarrollo en Fermentaciones IndustrialesLaboratorio de Nanobiomateriale

Triggered release of proteins from emulsan–alginate beads

Emulsan/alginate beads were studied for protein adsorption and stability in the context of controlled release. The beads, 400F80 Am diameter with approximately 10% emulsan content, offer unusual opportunities for delivery of proteins due to the natural ability of emulsan to bind proteins, coupled with the selective biological activation features of this complex lipoheteropolysaccharide. The binding capacity of azo-bovine serum albumin by the emulsan/alginate beads was 0.637F0.004 vs. 0.170F0.007 Ag/mg for beads formed from alginate alone. In additional protein adsorption experiments, the lipase andsubtilisin maintained activity when adsorbed to the emulsan/alginate beads albeit with lower specific activity when compared to the enzyme free in solution. However, the half life of the adsorbed enzyme was significantly higher than the free forms. To explore functional utility of this system, two types of triggered release were studied in the context of these bead systems. First, azo-BSA as a model protein was physically bound to emulsan/alginate beads and then selectively released by triggering with subtilisin, a serine protease, which cleaves the azo dye, sulfanilic acid, from the bound protein. In absence of subtilisin no triggered release was observed. Second, azo-BSA as a prodrug model, was adsorbed to the emulsan/alginate beads and then release of the dye was demonstrated by lipase treatment which cleaves the fatty acid esters from the emulsan structure to release the bound protein. The results establish the versatility and utility of emulsan-based beads for protein binding and triggered release.Fil: Castro, Guillermo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Kamdar, Romit R.. Tufts University. School of Engineering; Estados UnidosFil: Panilaitis, Bruce. Tufts University. School of Engineering; Estados UnidosFil: Kaplan, David L.. Tufts University. School of Engineering; Estados Unido

Emulsan quantitation by Nile red quenching fluorescence assay

A Nile red fluorescent technique to quantify 20–200 μg ml−1 of emulsan was developed. Nile red dissolvedin DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm−1 M−1. Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG-1.Fil: Castro, Guillermo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Larson, Bridget K.. Tufts University. School of Engineering; Estados UnidosFil: Panilaitis, Bruce. Tufts University. School of Engineering; Estados UnidosFil: Kaplan, David L.. Tufts University. School of Engineering; Estados Unido