13 research outputs found
Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans
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Discrimination of patterns of N-acetylation in chitooligosaccharides by gas phase IR spectroscopy integrated to mass spectrometry
International audienceWe propose a novel, bi-dimensional analysis of partially N-acetylated chitosan oligosaccharides based on gas phase Infra-Red spectroscopy integrated to mass spectrometry (MS). By providing simultaneously MS and IR fingerprints, this approach combines the advantages of MS with the refined structural detail offered by gas phase spectroscopy and provides robust signatures for the rapid discrimination of the patterns of N-acetylation. Four mono-N-deacetylated and two doubly-N-deacetylated chitosan tetramer standards with well-defined patterns of acetylation were produced and analyzed by IR integrated to MS. We show that each sequence displays a unique combination of MS and IR fingerprints, thus offering a rapid diagnostic for the pattern of acetylation without the need for reducing end labeling
Biotechnology-Derived Chitosans with Non-Random Patterns of Acetylation Differ from Conventional Chitosans in Their Properties and Activities
Chitosans are versatile biopolymers with multiple biological activities and potential applications. They are linear copolymers of glucosamine and N-acetylglucosamine defined by their degree of polymerization (DP), fraction of acetylation (FA), and pattern of acetylation (PA). Technical chitosans produced chemically from chitin possess defined DP and FA but random PA, while enzymatically produced natural chitosans are likely to have non-random PA. This natural process has not been replicated using biotechnology because chitin de-N-acetylases do not efficiently deacetylate crystalline chitin. Here, we show that such enzymes can partially N-acetylate polyglucosamine in the presence of excess acetate, yielding chitosans with FA up to 0.7 and an enzyme-dependent non-random PA. The biotech chitosans differ from technical chitosans both in terms of physicochemical and nanoscale solution properties and biological activities. As with synthetic block co-polymers, controlling the distribution of building blocks within the biopolymer chain will open a new dimension of chitosan research and exploitation
Work and Autonomy: Possibilities, Limits and Contradictions
Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 +/- 3 nm, and the zeta potential was -42 +/- 0.3 mV. The NCXs were stable in water, gastric buffer, intestinal buffer and HEPES buffered glycose, and at all pH values from 2 to 9 except pH 3, where they aggregated. When proteolytic enzymes were present in the buffer, the NCXs were degraded. Only at high concentrations the NCXs caused a decreased viability in HeLa and U2OS cell lines. The simple processing procedure and the high stability of the NCXs, makes them excellent candidates for use in the food and pharmaceutical industry
Chitosan Analysis by Enzymatic/Mass Spectrometric Fingerprinting and in Silico Predictive Modeling
Chitosans,
ÎČ-1,4-linked partially N-acetylated linear polyglucosamines,
are very versatile and promising functional biopolymers. Understanding
their structureâfunction relationships requires sensitive and
accurate structural analyses to determine parameters like degree of
polymerization (DP), fraction of acetylation (<i>F</i><sub>A</sub>), or pattern of acetylation (P<sub>A</sub>). NMR, the gold
standard for <i>F</i><sub>A</sub> analysis, requires large
amounts of sample. Here, we describe an enzymatic/mass spectrometric
fingerprinting method to analyze the <i>F</i><sub>A</sub> of chitosan polymers. The method combines the use of chitinosanase,
a sequence-specific hydrolase that cleaves chitosan polymers into
oligomeric fingerprints, ultrahigh-performance liquid chromatographyâelectrospray
ionization-mass spectrometry (UHPLCâESI-MS), and partial least-squares
regression (PLSR). We also developed a technique to simulate enzymatic
fingerprints in silico that were used to build the PLS models for <i>F</i><sub>A</sub> determination. Overall, we found our method
to be as accurate as NMR while at the same time requiring only microgram
amounts of sample. Thus, the method represents a powerful technique
for chitosan analysis
Chitosan Analysis by Enzymatic/Mass Spectrometric Fingerprinting and in Silico Predictive Modeling
Chitosans,
ÎČ-1,4-linked partially N-acetylated linear polyglucosamines,
are very versatile and promising functional biopolymers. Understanding
their structureâfunction relationships requires sensitive and
accurate structural analyses to determine parameters like degree of
polymerization (DP), fraction of acetylation (<i>F</i><sub>A</sub>), or pattern of acetylation (P<sub>A</sub>). NMR, the gold
standard for <i>F</i><sub>A</sub> analysis, requires large
amounts of sample. Here, we describe an enzymatic/mass spectrometric
fingerprinting method to analyze the <i>F</i><sub>A</sub> of chitosan polymers. The method combines the use of chitinosanase,
a sequence-specific hydrolase that cleaves chitosan polymers into
oligomeric fingerprints, ultrahigh-performance liquid chromatographyâelectrospray
ionization-mass spectrometry (UHPLCâESI-MS), and partial least-squares
regression (PLSR). We also developed a technique to simulate enzymatic
fingerprints in silico that were used to build the PLS models for <i>F</i><sub>A</sub> determination. Overall, we found our method
to be as accurate as NMR while at the same time requiring only microgram
amounts of sample. Thus, the method represents a powerful technique
for chitosan analysis
Enzymatic Production and Enzymatic-Mass Spectrometric Fingerprinting Analysis of Chitosan Polymers with Different Nonrandom Patterns of Acetylation
International audienceChitosans, a family of Ă-(1,4)-linked, partially N-acetylated polyglucosamines, are considered to be among the most versatile and most promising functional biopolymers. Chemical analysis and bioactivity studies revealed that the functionalities of chitosans strongly depend on the polymersâ degree of polymerization and fraction of acetylation. More recently, the pattern of acetylation (PA) has been proposed as another important parameter to influence functionalities of chitosans. We therefore carried out studies on the acetylation pattern of chitosan polymers produced by three recombinant fungal chitin deacetylases (CDAs) originating from different species, namely, Podospora anserina, Puccinia graminis f. sp. tritici, and Pestalotiopsis sp. We analyzed the chitosans by 1H NMR, 13C NMR, and SEC-MALS and established new methods for PA analysis based on enzymatic mass spectrometric fingerprinting and in silico simulations. Our studies strongly indicate that the different CDAs indeed produce chitosans with different PA. Finally, Zimm plot analysis revealed that enzymatically treated polymers differ with respect to their second virial coefficient and radius of gyration indicating an influence of PA on polymerâsolvent interactions