Dissecting and optimizing bioactivities of chitosans by enzymatic modification.

Chitosans are versatile biopolymers with antimicrobial and plant-strengthening properties relevant to agriculture. However, a limited understanding of molecular structure-function relationships and cellular modes of action of chitosans hampers the development of effective chitosan-based agro-biologics. We partially hydrolyzed a chitosan polymer (degree of polymerization DP 800, fraction of acetylation F 0.

Chitins and chitosans-A tale of discovery and disguise, of attachment and attainment.

Chitin polymers are an essential structural component of fungal cell walls, but host chitinases can weaken them, contributing to disease resistance in fungal pathogens. Chitin oligomers thus produced are immunogenic signal molecules eliciting additional disease resistance mechanisms. Fungi may counteract these, e.

Transcriptomics reveal a mechanism of niche defense: two beneficial root endophytes deploy an antimicrobial GH18-CBM5 chitinase to protect their hosts.

Effector secretion is crucial for root endophytes to establish and protect their ecological niche. We used time-resolved transcriptomics to monitor effector gene expression dynamics in two closely related Sebacinales, Serendipita indica and Serendipita vermifera, during symbiosis with three plant species, competition with the phytopathogenic fungus Bipolaris sorokiniana, and cooperation with root-associated bacteria. We observed increased effector gene expression in response to biotic interactions, particularly with plants, indicating their importance in host colonization.

Heterogeneously deacetylated chitosans possess an unexpected regular pattern favoring acetylation at every third position.

Chitosans are promising biopolymers for diverse applications, with material properties and bioactivities depending i.a. on their pattern of acetylation (PA).

Fast insights into chitosan-cleaving enzymes by simultaneous analysis of polymers and oligomers through size exclusion chromatography.

The thorough characterization of chitosan-cleaving enzymes is crucial to unveil structure-function relationships of this promising class of biomolecules for both, enzymatic fingerprinting analyses and to use the enzymes as biotechnological tools to produce tailor-made chitosans for diverse applications. Analyzing polymeric substrates as well as oligomeric products has been established as an effective way to understand the actions of enzymes, but it currently requires separate, rather laborious methods to obtain the full picture. Here, we present ultra high performance size exclusion chromatography coupled to refractive index and mass spectrometry detection (UHPSEC-RI-MS) as a straightforward method for the semi-quantitative analysis of chitosan oligomers of up to ten monomers in length.

Engineering of a chitin deacetylase to generate tailor-made chitosan polymers.

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions.

Composition and Charge Compensation in Chitosan/Gum Arabic Complex Coacervates in Dependence on pH and Salt Concentration.

In this study, complex coacervates of the biopolyelectrolytes chitosan and gum arabic were investigated with respect to their composition and charge compensation depending on the pH and salt concentration. Individual polyelectrolyte yields were deduced from thermogravimetric analysis and chitosan quantification via enzymatic hydrolysis/HPLC-ELSD. The polyelectrolyte mass ratio in the complex coacervate is found to remain approximately constant irrespective of the pH, despite the latter's effect on the polyelectrolyte charge ratio.

Quantitative enzymatic-mass spectrometric analysis of the chitinous polymers in fungal cell walls.

Chitin is an essential structural component of complex and dynamic fungal cell walls. It may be converted by partial or full deacetylation to yield chitosan. Here, we describe a method to quantify N-acetyl d-glucosamine (GlcNAc, A) and d-glucosamine (GlcN, D) units and, thus, total amount and average fraction of acetylation (x̅ F) of the chitinous polymers by complete enzyme hydrolysis of the polymers followed by mass spectrometric analyses of the monomers.

Quantification of chitosan in aqueous solutions by enzymatic hydrolysis and oligomer analysis via HPLC-ELSD.

A new method for quantitative analysis of chitosan in aqueous solution is introduced, comprising an enzyme-driven cleavage to water-soluble chitooligosaccharides (COS), N-acetylation, separation via UHPLC and detection by use of an evaporative light scattering detector (ELSD). Chitosans with different fractions of acetylation (F) and molecular weights (M) were hydrolyzed using a chitosanase/chitinase mixture. By subsequent N-acetylation with isotopically labelled acetic anhydride, COS mixtures with F = 1 were obtained allowing for chromatographic separation solely based on their degree of polymerization (DP).

Deciphering the ChitoCode: fungal chitins and chitosans as functional biopolymers.

Chitins and chitosans are among the most widespread and versatile functional biopolymers, with interesting biological activities and superior material properties. While chitins are evolutionary ancient and present in many eukaryotes except for higher plants and mammals, the natural distribution of chitosans, i.e.

In silico and in vitro analysis of an Aspergillus niger chitin deacetylase to decipher its subsite sugar preferences.

Chitin deacetylases (CDAs) are found in many different organisms ranging from marine bacteria to fungi and insects. These enzymes catalyze the removal of acetyl groups from chitinous substrates generating various chitosans, linear copolymers consisting of N-acetylglucosamine (GlcNAc) and glucosamine. CDAs influence the degree of acetylation of chitosans as well as their pattern of acetylation, a parameter that was recently shown to influence the physicochemical properties and biological activities of chitosans.

The non-sulfated ulvanobiuronic acid of ulvans is the smallest active unit able to induce an oxidative burst in dicot cells.

Ulvans from green algae are promising compounds for plant protection because they are environmentally friendly and induce plant defense responses. We analyzed the structure-function relationship of ulvan polymers and oligomers for their elicitor activity in suspension-cultured cells of three dicot species. The polysaccharide from Ulva fasciata was characterized regarding its monosaccharide composition, degree of sulfation, and molecular mass.

Chitosan and Chitin Deacetylase Activity Are Necessary for Development and Virulence of Ustilago maydis.

The biotrophic fungus harbors a chitin deacetylase (CDA) family of six active genes as well as one pseudogene which are differentially expressed during colonization. This includes one secreted soluble CDA (Cda4) and five putatively glycosylphosphatidylinositol (GPI)-anchored CDAs, of which Cda7 belongs to a new class of fungal CDAs. Here, we provide a comprehensive functional study of the entire family.

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases.

During the past decade, detailed studies using well-defined 'second generation' chitosans have amply proved that both their material properties and their biological activities are dependent on their molecular structure, in particular on their degree of polymerisation (DP) and their fraction of acetylation (). Recent evidence suggests that the pattern of acetylation (PA), i.e.

Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation.

Chitosan obtained by enzymatic deacetylation of chitin using chitin deacetylase (CDA) holds promise primarily due to the possibility to yield chitosan with non-random patterns of acetylation and more environmentally friendly process compared to chemical deacetylation. In the present study, a sustainable bioprocess is reported for over-expression of a bacterial CDA in E. coli pLysS cells.

Enzymatic Production and Enzymatic-Mass Spectrometric Fingerprinting Analysis of Chitosan Polymers with Different Nonrandom Patterns of Acetylation.

Chitosans, a family of ß-(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 polymers' degree of polymerization and fraction of acetylation. More recently, the pattern of acetylation ( P) has been proposed as another important parameter to influence functionalities of chitosans.

'Slipped Sandwich' Model for Chitin and Chitosan Perception in Arabidopsis.

Chitin, a linear polymer of N-acetyl-d-glucosamine, and chitosans, fully or partially deacetylated derivatives of chitin, are known to elicit defense reactions in higher plants. We compared the ability of chitin and chitosan oligomers and polymers (chitin oligomers with degree of polymerization [DP] 3 to 8; chitosan oligomers with degree of acetylation [DA] 0 to 35% and DP 3 to 15; chitosan polymers with DA 1 to 60% and DP approximately 1,300) to elicit an oxidative burst indicative of induced defense reactions in Arabidopsis thaliana seedlings. Fully deacetylated chitosans were not able to trigger a response; elicitor activity increased with increasing DA of chitosan polymers.

Endochitinase 1 (Tv-ECH1) from Trichoderma virens has high subsite specificities for acetylated units when acting on chitosans.

Chitosans with defined characteristics have been shown to possess reproducible bioactivities for numerous applications. A promising approach for producing chitosans with defined degrees of polymerization (DP), degrees of acetylation (DA), and patterns of acetylation (PA) involves using chitin-modifying enzymes. One such enzyme, the chitinase Tv-ECH1 belonging to the glycoside hydrolase (GH) family 18, seems to have an important role in the biocontrol properties of the fungus Trichoderma virens, suggesting its potential in generating novel chitosans for plant health applications.

A chitin deacetylase of Podospora anserina has two functional chitin binding domains and a unique mode of action.

Chitosan is a structurally diverse biopolymer that is commercially derived from chitin by chemical processing, but chitin deacetylases (CDAs) potentially offer a sustainable and more controllable approach allowing the production of chitosans with tailored structures and biological activities. We investigated the CDA from Podospora anserina (PaCDA) which is closely related to Colletotrichum lindemuthianum CDA in the catalytic domain, but unique in having two chitin-binding domains. We produced recombinant PaCDA in Hansenula polymorpha for biochemical characterization and found that the catalytic domain of PaCDA is also functionally similar to C.

Enzymatic production of all fourteen partially acetylated chitosan tetramers using different chitin deacetylases acting in forward or reverse mode.

Some of the most abundant biomolecules on earth are the polysaccharides chitin and chitosan of which especially the oligomeric fractions have been extensively studied regarding their biological activities. However, most of these studies have not been able to assess the activity of a single, defined, partially acetylated chitosan oligosaccharide (paCOS). Instead, they have typically analyzed chemically produced, rather poorly characterized mixtures, at best with a single, defined degree of polymerization (DP) and a known average degree of acetylation (DA), as no pure and well-defined paCOS are currently available.

Reassessment of chitosanase substrate specificities and classification.

Chitosanases can be used to produce partially acetylated chitosan oligosaccharides (paCOS) for different applications, provided they are thoroughly characterized. However, recent studies indicate that the established classification system for chitosanases is too simplistic. Here, we apply a highly sensitive method for quantitatively sequencing paCOS to reassess the substrate specificities of the best-characterized class I-III chitosanases.