Nour Eddine El Gueddari

Complexation of copper(II) with chitosan nanogels: Toward control of microbial growth

Pure chitosan nanogels were produced, used to adsorb copper(II), and their antimicrobial activities were assessed. The complexation of copper(II) with chitosan solutions and dispersions was studied using UV-vis spectrometry. The adsorption capacity of chitosan nanogels was comparable to that of chitosan solutions, but copper(II)-loaded nanogels were more stable (i.e. no flocculation was observed while chitosan solutions showed macroscopic gelation at high copper concentration) and were easier to handle (i.e. no increase in viscosity). Adsorption isotherms of copper(II) onto chitosan were established and the impact of the pH on copper(II) release was investigated. The formation of a copper(II)-chitosan complex strongly depended on pH. Hence, release of copper(II) can be triggered by a decrease in pH (i.e. the protonation of chitosan amino groups). Furthermore, chitosan nanohydrogels were shown to be a suitable substrate for chitosan hydrolytic enzymes. Finally, a strong synergistic effect between chitosan and copper in inhibiting Fusarium graminearum growth was observed. The suitability of these copper(II)-chitosan colloids as a new generation of copper-based bio-pesticides, i.e. as a bio-compatible, bio-active and pH-sensitive delivery system, is discussed.

Chitinolytic enzymes from endophytic fungi

Fungal endophytes isolated from leaves of tree species of the forests of Western Ghats, southern India were screened for chitin modifying enzyme production. Thirty-one of the one hundred and sixty two isolates were positive for chitinase, while different isolates produced isoforms of the enzyme. Many isolates produced chitosanase that acted on chitosan with different degrees of acetylation. Modified chitin and different types of chitosans are used in biomedical applications including wound healing, drug delivery, gene delivery, tissue engineering, in the food industry as preservatives and emulsifying agents, and in biocatalysis. Horizontally transmitted endophytes appear to be a good source for a variety of chitin modifying enzymes with the potential to be used in biotechnology. The possibility of chitin modifying enzymes of endophytes in regulating plant defense against pathogens and pests in vivo should also be addressed.

Biotechnological approaches for field applications of chitooligosaccharides (COS) to induce innate immunity in plants

Abstract Plants have evolved mechanisms to recognize a wide range of pathogen-derived molecules and to express induced resistance against pathogen attack. Exploitation of induced resistance, by application of novel bioactive elicitors, is an attractive alternative for crop protection. Chitooligosaccharide (COS) elicitors, released during plant fungal interactions, induce plant defenses upon recognition. Detailed analyses of structure/function relationships of bioactive chitosans as well as recent progress towards understanding the mechanism of COS sensing in plants through the identification and characterization of their cognate receptors have generated fresh impetus for approaches that would induce innate immunity in plants. These progresses combined with the application of chitin/chitosan/COS in disease management are reviewed here. In considering the field application of COS, however, efficient and large-scale production of desired COS is a challenging task. The available methods, including chemical or enzymatic hydrolysis and chemical or biotechnological synthesis to produce COS, are also reviewed.

Partially Acetylated Chitosan Oligo-and Polymers Induce an Oxidative Burst in Suspension Cultured Cells of the Gymnosperm Araucaria angustifolia

Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

Growth rate inhibition of phytopathogenic fungi by characterized chitosans

The inhibitory effects of fifteen chitosans with different degrees of polymerization (DP) and different degrees of acetylation (FA) on the growth rates (GR) of four phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer) were examined using a 96-well microtiter plate and a microplate reader. The minimum inhibitory concentrations (MICs) of the chitosans ranged from 100 μg ×mL-1 to 1,000 μg ×mL-1 depending on the fungus tested and the DP and FA of the chitosan. The antifungal activity of the chitosans increased with decreasing FA. Chitosans with low FA and high DP showed the highest inhibitory activity against all four fungi. P. expansum and B. cinerea were relatively less susceptible while A. alternata and R. stolonifer were relatively more sensitive to the chitosan polymers. Scanning electron microscopy of fungi grown on culture media amended with chitosan revealed morphological changes.

A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation

ABSTRACT Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungus Puccinia graminis f. sp. tritici in Escherichia coli. The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein from P. graminis (PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed that PgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating that PgtCDA cannot deacetylate the two GlcNAc units closest to the oligomers nonreducing end. This unique property of PgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications. IMPORTANCE We successfully achieved heterologous expression of a fungal chitin deacetylase gene from the basidiomycete Puccinia graminis f. sp. tritici in the periplasm of E. coli as a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences.

Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.

Physical Properties and Stability of Soft Gelled Chitosan‐Based Nanoparticles

We addressed the role of the degree of acetylation (DA) and of Mw of chitosan (CS) on the physical characteristics and stability of soft nanoparticles obtained through either ionic cross-linking with sodium tripolyphosphate (TPP), or reverse emulsion/gelation. Each of these methods affords nanoparticles (NPs) or nanogels (NGs), respectively. The size of CS-TPP NPs comprising CS of high Mw (≈123-266 kDa) increases with DA (≈1.6%-56%), while it do not change for CS of low Mw (≈11-13 kDa); the zeta potential (ζ) decreases with DA regardless of Mw (ζ ≈+34.6 ± 2.6 to ≈+25.2 + 0.6 mV) and the NPs appear as spheres in transmission electron microscopy. Stability in various cell culture media (pH 7.4 at 37 °C) is greater for NPs made with CS of DA ≥ 27%. In turn, NGs exhibit larger sizes (520 ± 32 to 682 ± 27 nm) than do CS-TPP NPs, and can only be formed with CS of DA < 30%. The average diameter size for these NGs shows a monotonic increase with CSs Mw . The physical properties and stability of these systems in biological media depend mostly on the DA of CS and its influence on the balance between hydrophilic/hydrophobic interactions.

Production of bioactive chitosan oligosaccharides using the hypertransglycosylating chitinase-D from Serratia proteamaculans

The biological activities of chitosan and its oligosaccharides are greatly influenced by properties such as the degree of polymerization (DP), degree of acetylation (DA) and pattern of acetylation (PA). Here, structurally diverse chitosan oligosaccharides from chitosan polymers (DA=35% or 61%) were generated using Serratia proteamaculans wild-type chitinase D (SpChiD) and the W114A mutant which lacks transglycosylase activity. The crude oligosaccharide mixtures and purified fractions with specific DP and DA ranges were tested for their ability to induce an oxidative burst in rice cell suspension cultures. The crude mixtures were more active when produced by the W114A mutant whereas the purified fractions were more active when produced by wild-type SpChiD. Neither hydrolysis nor transglycosylation by SpChiD was inhibited in the presence of fully-deacetylated oligosaccharides, suggesting that SpChiD could be exploited to generate oligosaccharides with defined DA and PA values.

Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases

Summary We undertook production and inhibition studies of recombinant millet PGIP. Using computational mutagenesis, the most significant binding contact involved in pearl millet PGIP–AnPGII interaction was identified.