Odile Francesca Restaino

Structural analysis of bikunin glycosaminoglycan.

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production

BackgroundThe bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L-1 K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.ResultsThe inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L-1), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw-1), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.ConclusionThe increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.

Semi-Synthesis of Unusual Chondroitin Sulfate Polysaccharides Containing GlcA(3-O-sulfate) or GlcA(2,3-di-O-sulfate) Units

The extraction from natural sources of Chondroitin sulfate (CS), a polysaccharide used for management of osteoarthritis, leads to very complex mixtures. The synthesis of CS by chemical modification of other polysaccharides has seldom been reported due to the intrinsic complexity that arises from fine chemical modifications of the polysaccharide structure. In view of the growing interest in expanding the application of CS to pharmacological fields other than osteoarthritis treatment, we launched a program to find new sources of known or even unprecedented CS polysaccharides. As part of this program, we report herein on an investigation of the use of a cyclic orthoester group to selectively protect the 4,6-diol of N-acetyl-galactosamine residues in chondroitin (obtained from a microbial source), thereby facilitating its transformation into CSs. In particular, three CS polysaccharides were obtained and demonstrated to possess rare or hitherto unprecedented sulfation patterns by 2D NMR spectroscopy characterization. Two of them contained disaccharide subunits characterized by glucuronic acid residues selectively sulfated at position 3 (GlcA(3S)), the biological functions of which are known but have yet to be fully investigated. This first semi-synthetic access to GlcA(3S)-containing CS could greatly expedite such studies, since it can easily furnish considerable amounts of these polysaccharides, which are usually isolated with difficulty and in very low quantity from natural sources.

High-performance CE of Escherichia coli K4 cell surface polysaccharides.

A high‐performance CE application for a quick, reproducible, highly precise and sensitive determination of the lipopolysaccharide produced by Escherichia coli K4 (O5:K4:H4) and of its de‐lipid A form is described. The two species were separated within 30 min on an uncoated fused‐silica capillary, in normal polarity mode at 20 kV, using an SDS buffer. Detected at 190 nm, the de‐lipid A and the LPS species showed two peaks at distinctive migration times (10.45 and 16.10 min, respectively) and were quantified with high reproducibility and linearity (the correlation factors were 0.99 and 0.98, respectively) over the ranges from 60 to 600 ng (1–10 ng/nL) for de‐lipid A lipopolysaccharide and from 150 to 600 ng (2.5–10 ng/nL) for the LPS. The described method was also employed in the contemporary analysis and the determination of the two E. coli K4 cell surface polysaccharides, the LPS and the K4, and of their defructosylated and de‐lipid A species, respectively. The four molecules were detected and precisely quantified in complex matrices as fermentation broth supernatant or in samples withdrawn throughout the purification process, thus demonstrating the possibility to apply high‐performance CE as a reliable analytical tool in biotechnological processes.

Application of a 22L scale membrane bioreactor and cross-flow ultrafiltration to obtain purified chondroitin

Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed‐batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale‐up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨︁KTA cross‐flow instrument, was translated to a UNIFLUX‐10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C‐3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled‐up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild‐type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity.

Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing.

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed‐batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut‐offs for different phases of purification. The operational conditions (cross‐flow rate and transmembrane pressure) used for the process were determined on an ÄKTA cross‐flow instrument (GE Healthcare, USA), a lab‐scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.

Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4

Escherichia coli K4 is one of the bacteria expressing a surface polysaccharide, indicated as capsular polysaccharide (K-antigen), showing a chemical structure that resembles that of metabolites commonly used in pharmaceutical applications. In this study we provide evidence that homologous overexpression of the chondroitin polymerase, encoded by the kfoC gene, acts on a potential bottleneck for production of capsular polysaccharide, and increases productivity by 100%. However, we also demonstrate that genetic engineering and scale-up of the production process with E. coli K4 is not straight forward due to genetic instability of recombinant strains, partly overcome by multiple additions of antibiotic throughout fermentation that prove to increase plasmid maintenance inside the cells. A lower resistance to the antibiotic was nevertheless highlighted in the stationary phase suggesting other concomitant causes for plasmid instability. The latter might partly be related to a newly discovered endogenous mobile element that we indicate as pK4EC05. Sequencing and analysis of a 1900 bp fragment of pK4EC05 shows a high percentage of sequence similarity to large conjugative plasmids isolated from Shigella, Salmonella and E. coli strains.

New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors.

Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 μM·gcdw−1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced.

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.