Discovery of the dual polysaccharide composition of emulsan and the isolation of the emulsion stabilizing component

Emulsan has been reported as an emulsion stabilizing amphipathic lipoheteropolysaccharide secreted by the oil-degrading bacterium Acinetobacter venetianus RAG-1. Previously, emulsan was regarded as a single polymer. As a result of developing a new purification process, we have discovered that emulsan is a complex of approximately 80% (w/w) lipopolysaccharide (LPS) and 20% (w/w) high molecular weight exopolysaccharide (EPS). The EPS was purified to 98% (w/w) using tangential flow filtration, Triton X-114 phase extraction, ammonium sulfate precipitation, and hydrophobic interaction chromatography. Several previously reported physical properties of emulsan can be attributed to the LPS fraction, such as charge, fatty acid profile, and solution behavior, while the EPS is responsible for the emulsion stabilization activity. The EPS is believed to be cationic in nature, thus providing an electrostatic binding mechanism for the formation of the emulsan complex.     

Uranium binding by emulsan and emulsanosols

Emulsan, the extracellular bioemulsifier of Acinetobacter RAG-1, bound up to 0.9 mum of uranium per 1 mg emulsan. The dissociation constant for the emulsan-uranium complex, K(app) (I) was 1.2 x 10(-4)M. Much larger amounts of uranium were bound to emulsan when the biopolymer occurred on hexadecane-water interfaces. Under these conditions, more than 3.5 microm uranium were bound per 1 mg emulsan and the dissociation constant K(app) (II) was 5.1 x 10(-5)M. At pH 2, more than 90% of the uranium bound to emulsan on the hexadecane-water interface was desorbed, while less than 10% bioemulsifier was released from the interface. The different binding parameters of emulsan when free in solution and while adsorbed onto the hexadecane water interface are discussed in view of potential applications and as a model system for studying the properties of an extracellular amphipathic polymer bound to a hydrophobic surface.     

Unmasking of surface components by removal of cell-associated emulsan from Acinetobacter Sp. RAG-1

Summary Acinetobacter calcoaceticusRAG-1 cells lacking the emulsan capsule on the cell surface were obtained by two methods; a) by selecting for mutants that lack emulsan with a specific phage and b) by removal of the emulsan capsule from wild type cells with a specific emulsan depolymerase. Emulsan deficient cells obtained by either method become deficient in the adsorption of phage ap3 and sensitive to a newly isolated bacteriophage, nø. When RAG-1 cells were first treated with emulsan depolymerase and subsequently incubated without the enzyme, regeneration of the cell-associated emulsan was correlated with an increase in phage ap3 adsorption and an inhibition in phage nø adsorption. By partial regeneration of cell surface emulsan, a physiological state was obtained in which RAG-1 cells were sensitive to and efficiently adsorbed found phages. Enzyme-treated RAG-1 cells were found to be more adherent to hexadecane than the untreated RAG-1 cells. The data indicate that in addition to its function as the ap3 receptor, cell-associated emulsan masks the expression of other cell-surface determinant(s) which function(s) as: (i) receptor for bacteriophage nø, and (ii) cell-surface sites which enhance adherence to hydrophobic surfaces.Present address: Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA     

Relationship between phage resistance and emulsan production,interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 10⁶) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.     

Engineering bacterial biopolymers for the biosorption of heavy metals; new products and novel formulations

Bioremediation of heavy metal pollution remains a major challenge in environmental biotechnology. One of the approaches considered for application involves biosorption either to biomass or to isolated biopolymers. Many bacterial polysaccharides have been shown to bind heavy metals with varying degrees of specificity and affinity. While various approaches have been adopted to generate polysaccharide variants altered in both structure and activity, metal biosorption has not been examined. Polymer engineering has included structural modification through the introduction of heterologous genes of the biosynthetic pathway into specific mutants, leading either to alterations in polysaccharide backbone or side chains, or to sugar modification. In addition, novel formulations can be designed which enlarge the family of available bacterial biopolymers for metal-binding and subsequent recovery. An example discussed here is the use of amphipathic bioemulsifiers such as emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1, that forms stable, concentrated (70%), oil-in-water emulsions (emulsanosols). In this system metal ions bind primarily at the oil/water interface, enabling their recovery and concentration from relatively dilute solutions. In addition to the genetic modifications described above, a new approach to the generation of amphipathic bioemulsifying formulations is based on the interaction of native or recombinant esterase and its derivatives with emulsan and other water-soluble biopolymers. Cation-binding emulsions are generated from a variety of hydrophobic substrates. The features of these and other systems will be discussed, together with a brief consideration of possible applications. Received: 2 February 2000 / Received revision: 2 June 2000 / Accepted: 3 June 2000     

Purification and properties of the myo-inositol-binding protein from a Pseudomonas sp.

Emulsan, the extracellular polyanionic emulsifying agent produced by Acinetobacter calcoaceticus RAG-1, has been implicated as a receptor for a specific virulent RAG-1 bacteriophage, ap3. Aqueous solutions of emulsan did not interfere with phage ap3 adsorption to RAG-1 cells. However, binding of phage ap3 occurred at the interfaces of hexadecane-in-water emulsions specifically stabilized by emulsan polymers. Binding of ap3 to emulsions was inhibited either in the presence of anti-emulsan antibodies or in the presence of a specific emulsan depolymerase. Moreover, when the phage was first bound to emulsan-stabilized emulsions and the emulsions subsequently treated with emulsan depolymerase, viable phage was released, indicating that phage ap3 DNA ejection was not triggered by binding. The results indicate that emulsan functions as the ap3 receptor and suggest that to function as a receptor, emulsan assumes a specific conformation conferred on it by its specific interaction with hydrophobic surfaces.     

The long‐lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein‐Barr virus

The Epstein‐Barr gammaherpesvirus (EBV) is the first oncogenic virus discovered in human. Indeed, EBV has been known for more than 50 years to be tightly associated with certain human cancers. As such, EBV has been the subject of extensive studies aiming at deciphering various aspects of its biological cycle, ranging from the regulation of its genome replication and maintenance to the induction of its lytic cycle, including the mechanisms that allow its immune evasion or that are related to its tumorogenicity. For more than 30 years the budding yeast Saccharomyces cerevisiae has fruitfully contributed to a number of these studies. The aim of this article is to review the various aspects of EBV biology for which yeast has been instrumental, and to propose new possible applications for these yeast‐based assays, as well as the creation of further yeast models dedicated to EBV. This review article illustrates the tremendous potential of S. cerevisiae in integrated chemobiological approaches for the biomedical research.     

Optimizing emulsan production of <Emphasis Type="Italic">A. venetianus</Emphasis> RAG-1 using response surface methodology

Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L ethanol, 8.2 g/L KH₂PO₄, 23.32 g/L K₂HPO₄, 5.77 g/L (NH₄)₂SO₄ and 0.354 g/L MgSO₄•7H₂O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production.     

Construction of a chimeric gene cluster for the biosynthesis of apoemulsan with altered molecular weight

Acinetobacter venetianus RAG-1 produces an extracellular protein/high-molecular-weight (HMW) polysaccharide complex termed emulsan. As an emulsion stabilizer, emulsan has potential industrial applications. To control the molecular weight of the polymer, a stable chromosomal mutant was generated where RAG-1 wza, wzb, wzc genes were replaced by Escherichia coli homologs. The heterologous Wza, Wzb, Wzc proteins restored production of HMW polysaccharide. The polymer produced was of higher molecular weight than from the parent strain and with the cells exhibiting modified hydrophobicity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression,purification and characterization of recombinant human interleukin-22 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Interleukin-22 (IL-22) is a member of the IL-10 family. Its potential in clinical use has been highlighted for its important roles in promoting antimicrobial defense and preventing epithelial damages. Previous studies have reported that IL-22 can be expressed using prokaryotic systems and purified from inclusion bodies, however the recovery rate was poor. To produce functional IL-22 with a high yield, human IL-22 was inserted into the eukaryotic expression vector pPICZαA and transformed into Pichia pastoris. The expression of recombinant human IL-22 (rhIL-22) was induced by methanol and accounted for about 85% of the total secreted proteins. A simple purification strategy was established to purify the rhIL-22 from the culture supernatant, yielding 100 mg/l at 90% purity by chromatography with a SP Sepharose FF column. Bioactivity analysis showed the purified rhIL-22 demonstrated a specific activity that was comparable with the commercial one. This study provides a new strategy for large-scale production of bioactive IL-22 for use in basic studies and therapeutic applications.     

Emulsan-based nanoparticles for in vivo drug delivery to tumors

Nanoparticles have been widely used as drug carriers, and finding new materials for them is important for efficient drug delivery. Herein, we developed a new nanoparticle using emulsan and flax seed oil. Emulsan is one of the representative biosurfactants obtained from Acinetobacter calcoaceticus RAG-1. The resulting nanoparticles have an emulsan shell and a hydrophobic oil core, into which pheophorbide a (Pba) was loaded as a model drug. The nanoparticles were about 165.7?nm and were stably dispersed in an aqueous condition for more than one week. They demonstrated fast uptake in SCC7 mouse squamous cell carcinoma cells and killed the tumor cells after laser irradiation due to the photodynamic effect of Pba. After injection into SCC7 tumor-bearing mice via the tail vein, the particles showed longer blood circulation and 3.04-fold higher tumor accumulation in tissue than free Pba. These results demonstrate that emulsan-based nanoparticles have promising potential in drug delivery.     

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.     

A Concise Review of Extraction and Characterization of Chondroitin Sulphate from Fish and Fish Wastes for Pharmacological Application

Chondroitin sulphate (CS) is one of the most predominant glycosaminoglycans (GAGs) available in the extracellular matrix of tissues. It has many health benefits, including relief from osteoarthritis, antiviral properties, tissue engineering applications, and use in skin care, which have increased its commercial demand in recent years. The quest for CS sources exponentially increased due to several shortcomings of porcine, bovine, and other animal sources. Fish and fish wastes (i.e., fins, scales, skeleton, bone, and cartilage) are suitable sources of CS as they are low cost, easy to handle, and readily available. However, the lack of a standard isolation and characterization technique makes CS production challenging, particularly concerning the yield of pure GAGs. Many studies imply that enzyme-based extraction is more effective than chemical extraction. Critical evaluation of the existing extraction, isolation, and characterization techniques is crucial for establishing an optimized protocol of CS production from fish sources. The current techniques depend on tissue hydrolysis, protein removal, and purification. Therefore, this study critically evaluated and discussed the extraction, isolation, and characterization methods of CS from fish or fish wastes. Biosynthesis and pharmacological applications of CS were also critically reviewed and discussed. Our assessment suggests that CS could be a potential drug candidate; however, clinical studies should be conducted to warrant its effectiveness.     

Enzymatic depolymerization of emulsan

Emulsan, the polyanionic emulsifying agent synthesized by Acinetobacter calcoaceticus RAG-1, was depolymerized by an enzyme obtained from a soil bacterium YUV-1. The extracellular emulsan depolymerase was produced when strains RAG-1 and YUV-1 were grown together on agar medium. The enzyme was extracted from the agar and concentrated by ultrafiltration and ammonium sulfate precipitation. The molecular weight of the enzyme was estimated to be 89,000. Emulsan depolymerase activity was due to an eliminase reaction which split glycosidic linkages within the heteropolysaccharide backbone of emulsan to generate reducing groups and alpha, beta-unsaturated uronides with an absorbance maximum of 233 nm. Deesterified emulsan was degraded by emulsan depolymerase at only 27% of the rate of the native polymer. The treatment of emulsan solutions with emulsan depolymerase for brief periods caused a rapid and parallel drop in viscosity and emulsifying activity. More than 75% of the viscosity and emulsifying activity was lost at a time when less than 0.5% of the glycosidic linkages were broken. These data indicate that (i) emulsan depolymerase is an endoglycosidase and (ii) the higher the molecular weight of emulsan, the greater its emulsifying activity. Exhaustive digestion of emulsan with emulsan depolymerase produced oligosaccharides with a number average molecular weight of about 3,000. The fractionation of the digest on Bio-Gel P-6 yielded four broad peaks. The pooled fractions from each of the peaks contained the same relative amounts of reducing sugar and had an absorbance at 233 nm. The molar ratio of esterified sugar to reducing groups was close to 2 in each fraction.     

Hydrocarbon degradation by Acinetobacter calcoaceticus RAG-1 using the self-cycling fermentation technique

The use of self-cycling fermentations (SCFs) as a method for dealing with insoluble carbon substrates was examined. The emulsan-producing Acinetobacter calcoaceticus RAG-1 was used as the test organism. Limiting concentrations of hexadecane, 1-hexadecene, or 1-chlorohexadecane were used as the carbon substrate. The parameters monitored were residual hydrocarbon concentration, cycle time (doubling time), biomass concentration and emulsan concentration. Cycle-to-cycle variations of the measured parameters were found to be samll. In all cases, no residual hydrocarbon was detected. The minimum dissolved oxygen concentration was found to correspond with the complete dissappearance of the carbon source. A correlation between minimum dissolved exygen concentration, biomass concentration, and emulsan concentration was noted, thus making it easy to determine when steady-state conditions had been reached with respect to biomass and emulsan concentrations. The specific emulsan and biomass yields were found to increase during early stages of the fermentation, attaining their respective maxima at steady-state. Foaming problems often associated with the complete utilization of the insoluble substrate were eliminated using SCF technology, because harvesting occurs immediately following carbon depletion. From the results, SCFs provide a convenient method by which to produce and harvest emulsan. (c) 1992 John Wiley & Sons, Inc.     

Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production

As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capacity has become the bottleneck in many existing manufacturing facilities. Single Pass Tangential Flow Filtration (SPTFF) is an emerging technology, which is potentially useful in debottlenecking downstream capacity, especially when the pool tank size is a limiting factor. It can be integrated as part of an existing purification process, after a column chromatography step or a filtration step, without introducing a new unit operation. In this study, SPTFF technology was systematically evaluated for reducing process intermediate volumes from 2× to 10× with multiple mAbs and the impact of SPTFF on product quality, and process yield was analyzed. Finally, the potential fit into the typical 3-column industry platform antibody purification process and its implementation in a commercial scale manufacturing facility were also evaluated. Our data indicate that using SPTFF to concentrate protein pools is a simple, flexible, and robust operation, which can be implemented at various scales to improve antibody purification process capacity.     

Properties of hydrocarbon-in-water emulsions stabilized by Acinetobacter RAG-1 emulsan

Emulsan is a polymeric extracellular emulsifying agent produced by Acinetobacter RAG-1. Hydrocarbon-in-water emulsions (V(f) of hydrocarbon of 0.01-0.10) were stabilized by small quantities of emulsan (0.02-0.2 mg/mL). Although both aliphatic and aromatic hydrocarbon emulsions were stabilized by emulsan, mixtures containing both aliphatics and aromatics were better substrates for emulsan than the individual hydrocarbon by themselves. The emulsan remained tightly bound to the hydrocarbon even after centrifugation as determined by (a) residual emulsan in the aqueous phase and (b) the fact that the resulting "cream" readily dispersed in water to reform stable emulsions. With hexadecane-to-emulsan weight ratio of 39 and 155, the noncoalescing oil droplets had average droplet diameters of 2.0 and 4.0 mum, respectively. Dialysis studies showed that the water-soluble dye Rhodamine B adsorbed tightly to the interface of hexadecane-emulsan droplets although the dye did not bind to either hexadecane or emulsan alone. At saturating concentrations of dye, 2.2 mumol of dye were bound per mg emulsan.     

Bacterial degradation of emulsan.

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Bacterial degradation of emulsan

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.