An improved microtiter plate assay to monitor the oxidative burst in monocot and dicot plant cell suspension cultures

BACKGROUND: A screening method for elicitor and priming agents does not only allow detecting new bioactive substances, it can also be used to understand structure–function relationships of known agents by testing different derivatives of them. This can not only provide new lead compounds for the development of novel, more environment-benign, bio-based agro-chemicals, it may eventually also lead to a better understanding of defense mechanisms in plants. Reactive oxygen species (ROS) are sensitive indicators of these mechanisms but current assay formats are not suitable for multiplex screening, in particularly not in the case of monocot systems. RESULTS: Here we describe continuous monitoring of ROS in 96-well microtiter plates using the chemiluminescent probe L012, a luminol derivative producing chemiluminescence when oxidised by ROS like hydrogen peroxide, superoxide, or hydroxyl radical that can thus be used as an indicator for these ROS. We were able to measure ROS in both monocot (Oryza sativa) and dicot (Medicago truncatula) cell suspension cultures and record dose dependencies for the carbohydrate elicitors and priming agents ulvan and chitosan at low substrate concentrations (0.3–2.5 µg/ml). The method was optimized in terms of cell density, L012 concentration, and pre-incubation time. In contrast to the single peak observed using a cuvette luminometer, the improved method revealed a double burst in both cell systems during the 90-min measuring period, probably due to the detection of multiple ROS rather than only H2O2. CONCLUSION: We provide a medium throughput screening method for monocot and dicot suspension-cultured cells that enables direct comparison of monocot and dicot plant systems regarding their reaction to different signaling molecules.<br

Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture

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. extensively deacetylated derivatives of chitin, is more limited. Unequivocal evidence for its presence is only available for fungi where chitosans are produced from chitin by the action of chitin deacetylases. However, neither the structural details such as fraction and pattern of acetylation nor the physiological roles of natural chitosans are known at present. We hypothesise that the chitin deacetylases are generating chitins and chitosans with specific acetylation patterns and that these provide information for the interaction with specific chitin‑ and chitosan‑binding proteins. These may be structural proteins involved in the assembly of the complex chitin‑ and chitosan‑containing matrices such as fungal cell walls and insect cuticles, chitin‑ and chitosan‑modifying and ‑degrading enzymes such as chitin deacetylases, chitinases, and chitosanases, but also chitin‑ and chitosan‑recognising receptors of the innate immune systems of plants, animals, and humans. The acetylation pattern, thus, may constitute a kind of 'ChitoCode', and we are convinced that new in silico, in vitro, and in situ analytical tools as well as new synthetic methods of enzyme biotechnology and organic synthesis are currently offering an unprecedented opportunity to decipher this code. We anticipate a deeper understanding of the biology of chitin‑ and chitosan‑containing matrices, including their synthesis, assembly, mineralisation, degradation, and perception. This in turn will improve chitin and chitosan biotechnology and the development of reliable chitin‑ and chitosan‑based products and applications, e.g. in medicine and agriculture, food and feed sciences, as well as cosmetics and material sciences

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. We here present data on the enzymatic production of all 14 possible partially acetylated chitosan tetramers, out of which four were purified (>95%) regarding DP, DA, and pattern of acetylation (PA). We used bacterial, fungal, and viral chitin deacetylases (CDAs), either to partially deacetylate the chitin tetramer; or to partially re-N-acetylate the glucosamine tetramer. Both reactions proceeded with surprisingly strong and enzyme-specific regio-specificity. These pure and fully defined chitosans will allow to investigate the particular influence of DP, DA, and PA on the biological activities of chitosans, improving our basic understanding of their modes of action, e.g. their molecular perception by patter recognition receptors, but also increasing their usefulness in industrial applications.</p

A High Diversity in Chitinolytic and Chitosanolytic Species and Enzymes and Their Oligomeric Products Exist in Soil with a History of Chitin and Chitosan Exposure

Chitin is one of the most abundant biomolecules on earth, and its partially de-N-acetylated counterpart, chitosan, is one of the most promising biotechnological resources due to its diversity in structure and function. Recently, chitin and chitosan modifying enzymes (CCMEs) have gained increasing interest as tools to engineer chitosans with specific functions and reliable performance in biotechnological and biomedical applications. In a search for novel CCME, we isolated chitinolytic and chitosanolytic microorganisms from soils with more than ten-years history of chitin and chitosan exposure and screened them for chitinase and chitosanase isoenzymes as well as for their patterns of oligomeric products by incubating their secretomes with chitosan polymers. Of the 60 bacterial strains isolated, only eight were chitinolytic and/or chitosanolytic, while 20 out of 25 fungal isolates were chitinolytic and/or chitosanolytic. The bacterial isolates produced rather similar patterns of chitinolytic and chitosanolytic enzymes, while the fungal isolates produced a much broader range of different isoenzymes. Furthermore, diverse mixtures of oligosaccharides were formed when chitosan polymers were incubated with the secretomes of select fungal species. Our study indicates that soils with a history of chitin and chitosan exposure are a good source of novel CCME for chitosan bioengineering

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 (FA). Recent evidence suggests that the pattern of acetylation (PA), i.e., the sequence of acetylated and non-acetylated residues along the linear polymer, is equally important, but chitosan polymers with defined, non-random PA are not yet available. One way in which the PA will influence the bioactivities of chitosan polymers is their enzymatic degradation by sequence-dependent chitosan hydrolases present in the target tissues. The PA of the polymer substrates in conjunction with the subsite preferences of the hydrolases determine the type of oligomeric products and the kinetics of their production and further degradation. Thus, the bioactivities of chitosan polymers will at least in part be carried by the chitosan oligomers produced from them, possibly through their interaction with pattern recognition receptors in target cells. In contrast to polymers, partially acetylated chitosan oligosaccharides (paCOS) can be fully characterised concerning their DP, FA, and PA, and chitin deacetylases (CDAs) with different and known regio-selectivities are currently emerging as efficient tools to produce fully defined paCOS in quantities sufficient to probe their bioactivities. In this review, we describe the current state of the art on how CDAs can be used in forward and reverse mode to produce all of the possible paCOS dimers, trimers, and tetramers, most of the pentamers and many of the hexamers. In addition, we describe the biotechnological production of the required fully acetylated and fully deacetylated oligomer substrates, as well as the purification and characterisation of the paCOS products

Revised domain structure of ulvan lyase and characterization of the first ulvan binding domain

Biomass waste products from green algae have recently been given new life, as these polysaccharides have potential applications in industry, agriculture, and medicine. One such polysaccharide group called ulvans displays many different, potentially useful properties that arise from their structural versatility. Hence, performing structural analyses on ulvan is crucial for future applications. However, chemical reaction–based analysis methods cannot fully characterize ulvan and tend to alter its structure. Thus, better methods require well-characterized ulvan-degrading enzymes. Therefore, we analysed a previously sequenced ulvan lyase (GenebankTM reference number JN104480) and characterized its domains. We suggest that the enzyme consists of a shorter than previously described catalytic domain, a newly identified substrate binding domain, and a C-terminal type 9 secretion system signal peptide. By separately expressing the two domains in E. coli, we confirmed that the binding domain is ulvan specific, having higher affinity for ulvan than most lectins for their ligands (affinity constant: 105 M−1 ). To our knowledge, this is the first description of an ulvan-binding domain. Overall, identifying this new binding domain is one step towards engineering ulvan enzymes that can be sed to characterize ulvan, e.g. through enzymatic/mass spectrometric fingerprinting analyses, and help unlock its full potential.</p

Customized Chitooligosaccharide Production – Controlling the Length via Engineering of Rhizobial Chitin Synthases and the Choice of Expression System

Chitooligosaccharides (COS) can be produced by partial depolymerisation of chitin or in microbial cell factories expressing a chitin synthase (CHS) from Rhizobia. As different CHS have been reported to produce COS of different chain lengths, we here show the results of molecular dynamics simulations of two CHS with their bound COS products of different chain lengths. The PyMOL file comprises representative frames visualizing the binding of membrane embedded NodC from Rhizobium sp. GRH2 (NodC_GRH2) and NodC from Sinorhizobium (Ensifer) meliloti (NodC_Sm) to their natural products. Shown are the fully acetylated chitin tetramer (A4) and pentamer (A5) in NodC_Sm as well as pentamer (A5) and hexamer (A6) in NodC_GRH2. The designations indicate which NodC (GRH2 or Sm), which substrate (A4-A6) and which simulation (I-II) as well as which frame is depicted

Design and Characterization of Chitosan Nanoformulations for the Delivery of Antifungal Agents

Among different Candida species triggering vaginal candidiasis, Candida albicans is the most predominant yeast. It is commonly treated using azole drugs such as Tioconazole (TIO) and Econazole (ECO). However, their low water solubility may affect their therapeutic efficiency. Therefore, the aim of this research was to produce a novel chitosan nanocapsule based delivery system comprising of TIO or ECO and to study their suitability in vaginal application. These systems were characterized by their physicochemical properties, encapsulation efficiency, in vitro release, storage stability, cytotoxicity, and in vitro biological activity. Both nanocapsules loaded with TIO (average hydrodynamic size of 146.8 ± 0.8 nm, zeta potential of +24.7 ± 1.1 mV) or ECO (average hydrodynamic size of 127.1 ± 1.5 nm, zeta potential of +33.0 ± 1.0 mV) showed excellent association efficiency (99% for TIO and 87% for ECO). The analysis of size, polydispersity index, and zeta potential of the systems at 4, 25, and 37 °C (over a period of two months) showed the stability of the systems. Finally, the developed nanosystems presented fungicidal activity against C. albicans at non-toxic concentrations (studied on model human skin cells). The results obtained from this study are the first step in the development of a pharmaceutical dosage form suitable for the treatment of vaginal candidiasis