63 research outputs found
Chondroitin sulfate: are the purity and the structural features well assessed? A review on the analytical challenges
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Biotechnological transformation of hydrocortisone to 16?-hydroxy hydrocortisone by Streptomyces roseochromogenes
No full textStreptomyces roseochromogenes is able to hydroxylate
steroid compounds in different positions of their cycloalkane rings thanks to a cytochrome P-450 multienzyme complex. In this paper, the hydroxylation of the hydrocortisone in the 16α position, performed by bacterial whole cells, was investigated in both shake flask and fermentation conditions; the best settings for both cellular growth and transformation reaction were studied by investigating the optimal medium composition, the kinetic of conversion, the most suitable substrate concentration and the preferred addition timing. Using newly formulated malt extract- and yeast extract-based media, a 16α-hydrohydrocortisone concentration of 0.2±0.01 g L−1 was reached in shake flasks. Batch experiments in a 2-L fermentor established the reproducibility and robustness of the biotransformation, while a pulsed batch fermentation strategy allowed the production to increase up to 0.508±0.01 g L−1. By-product formation was investigated, and two new derivates of the hydrocortisone obtained during the bacterial transformation reaction and unknown so far, a C-20 hydroxy derivate and a C-21 N-acetamide one, were determined by NMR analyses
Streptomycetes as Microbial Cell Factories for the Biotechnological Production of Melanin
No full textMelanins are complex, polymeric pigments with interesting properties like UV-light absorbance ability, metal ion chelation capacity, antimicrobial action, redox behaviors, and scavenging properties. Based on these characteristics, melanins might be applied in different industrial fields like food packaging, environmental bioremediation, and bioelectronic fields. The actual melanin manufacturing process is not environmentally friendly as it is based on extraction and purification from cuttlefish. Synthetic melanin is available on the market, but it is more expensive than animal-sourced pigment and it requires long chemical procedures. The biotechnological production of microbial melanin, instead, might be a valid alternative. Streptomycetes synthesize melanins as pigments and as extracellular products. In this review, the melanin biotechnological production processes by different Streptomyces strains have been revised according to papers in the literature. The different fermentation strategies to increase melanin production such as the optimization of growth conditions and medium composition or the use of raw sources as growth substrates are here described. Diverse downstream purification processes are also reported as well as all the different analytical methods used to characterize the melanin produced by Streptomyces strains before its application in different fields
High-performance CE of Escherichia coli K4 cell surface polysaccharides
No full textA 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 W, 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
New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors
No full textEscherichia 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
Production and purification of capsular polysaccharide from Escherichia coli K4 for potential biotechnological applications.
No full textSaccharomyces pastoranius as cell factory to improve glutathione production using novel fermentation strategies
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