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In this work, we aimed to tune cellulose nanocrystals (CNCs) properties by introducing different functional groups (aldehyde, carboxyl, silane, and ammonium groups) on the surface through different chemical modifications. These functional groups were obtained by combining: the periodate oxidation with TEMPO-oxidation, aminosylation or cationization. CNCs produced and their films were characterized to elucidate their performances. The results showed that the properties of obtained CNCs varied depending on the grafted functionalities on the surface. The results reveal that after each modification a colloidal stability is preserved. Interestingly, Periodate oxidation of cellulose nanocrystals results in film components that interact through intra- and intermolecular hemiacetals and lead to films with a tensile strength of 116 MPa compared to the pristine CNCs, in contrast the subsequent modifications led to lower tensile strength. Of note, remarkable thermal stability has been achieved after modifications reaching a maximum of 280 °C. The oxygen barrier properties of the films after modifications varied between 0.48 and 0.54 cm

Photopolymerization is an effective method to covalently cross-link polymer chains that can be shaped into several biomedical products and devices. Additionally, polymerization reaction may induce a fluid-solid phase transformation under physiological conditions and is ideal for in vivo cross-linking of injectable polymers. The photoinitiator is a key ingredient able to absorb the energy at a specific light wavelength and create radicals that convert the liquid monomer solution into polymers. The combination of photopolymerizable polymers, containing appropriate photoinitiators, and effective curing based on dedicated light sources offers the possibility to implement photopolymerization technology in 3D bioprinting systems. Hence, cell-laden structures with high cell viability and proliferation, high accuracy in production, and good control of scaffold geometry can be biofabricated. In this review, we provide an overview of photopolymerization technology, focusing our efforts on natural polymers, the chemistry involved, and their combination with appropriate photoinitiators to be used within 3D bioprinting and manufacturing of biomedical devices. The reviewed articles showed the impact of different factors that influence the success of the photopolymerization process and the final properties of the cross-linked materials.

International audience; TEMPO-Oxidized cellulose nanofibrils (toCNF), in the form of highly entangled network such as membrane or cryogels, have proven to be of interest for various applications, including drug release or purification by pollutant adsorption. β-Cyclodextrins (β-CDs) have the ability to form inclusion complexes with large amount of hydrophobic molecules, and are considered as a promising way to bring new functionalities to these materials, by reducing drug burst release effect or improving the pollutant adsorption properties. The study of the adsorption β-CDs onto toCNF is then crucial to design toCNF/β-CDs materials, but is very complex due to the chemical proximity between these compounds. In this study, we develop toCNF cryogels containing various types of β-CDs derivatives by physical adsorption. Different protocols for analyzing the interactions between these compounds, such as Isothermal Titration Calorimetry (ITC), Quartz-Crystal Microbalance with dissipation monitoring (QCM-d) and a Phenolphthalein-based protocol (PhP protocol) have been performed. Adsorption between β-CD and toCNF was proven at two different temperatures with ITC. QCM-d measurements allowed measuring adsorption of different β-CDs derivatives onto toCNF, with higher adsorption measured for the modified β-CDs, and with estimated binding capacity ranging from 13.4 to 47.6 μmol/g toCNF. PhP protocol allowed us to monitor the amount of β-CDs released in aqueous environment, highlighting a lower release for modified β-CDs onto toCNF, and the results were consistent with the estimated binding capacity. This quantification of the binding adsorption capacity of various β-CDs is key results for optimizing the design of toCNF/β-CDs materials.

Many studies show how biomaterial properties like stiffness, mechanical stimulation and surface topography can influence cellular functions and direct stem cell differentiation. In this work, two different natural materials, gelatin (Gel) and cellulose nanofibrils (CNFs), were combined to design suitable 3D porous biocomposites for soft-tissue engineering. Gel was selected for its well-assessed high biomimicry that it shares with collagen, from which it derives, while the CNFs were chosen as structural reinforcement because of their exceptional mechanical properties and biocompatibility. Three different compositions of Gel and CNFs, i.e., with weight ratios of 75:25, 50:50 and 25:75, were studied. The biocomposites were morphologically characterized and their total- and macro- porosity assessed, proving their suitability for cell colonization. In general, the pores were larger and more isotropic in the biocomposites compared to the pure materials. The influence of freeze-casting and dehydrothermal treatment (DHT) on mechanical properties, the absorption ability and the shape retention were evaluated. Higher content of CNFs gave higher swelling, and this was attributed to the pore structure. Cross-linking between CNFs and Gel using DHT was confirmed. The Young&rsquo, s modulus increased significantly by adding the CNFs to Gel with a linear relationship with respect to the CNF amounts. Finally, the biocomposites were characterized in vitro by testing cell colonization and growth through a quantitative cell viability analysis performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, the cell viability analysis was performed by the means of a Live/Dead test with Human mesenchymal stem cells (hMSCs). All the biocomposites had higher cytocompatibility compared to the pure materials, Gel and CNFs.

3D printed polycaprolactone (PCL) has potential as a scaffold for bone tissue engineering, but the hydrophobic surface may hinder optimal cell responses. The surface properties can be improved by coating the scaffold with cellulose nanofibrils material (CNF), a multiscale hydrophilic biocompatible biomaterial derived from wood. In this study, human bone marrow-derived mesenchymal stem cells were cultured on tissue culture plates (TCP) and 3D printed PCL scaffolds coated with CNF. Cellular responses to the surfaces (viability, attachment, proliferation, and osteogenic differentiation) were documented. CNF significantly enhanced the hydrophilic properties of PCL scaffolds and promoted protein adsorption. Live/dead staining and lactate dehydrogenase release assays confirmed that CNF did not inhibit cellular viability. The CNF between the 3D printed PCL strands and pores acted as a hydrophilic barrier, enhancing cell seeding efficiency, and proliferation. CNF supported the formation of a well-organized actin cytoskeleton and cellular production of vinculin protein on the surfaces of TCP and PCL scaffolds. Moreover, CNF-coated surfaces enhanced not only alkaline phosphatase activity, but also collagen Type-I and mineral formation. It is concluded that CNF coating enhances cell attachment, proliferation, and osteogenic differentiation and has the potential to improve the performance of 3D printed PCL scaffolds for bone tissue engineering.

The current study aims to demonstrate the influence of the surface chemistry of wood-derived cellulose nanofibril (CNF) hydrogels on fibroblasts for tissue engineering applications. TEMPO-mediated oxidation or carboxymethylation pretreatments were employed to produce hydrogels with different surface chemistry. This study demonstrates the following: first, the gelation of CNF with cell culture medium and formation of stable hydrogels with improved rheological properties; second, the response of mouse fibroblasts cultured on the surface of the hydrogels or sandwiched within the materials with respect to cytotoxicity, cell attachment, proliferation, morphology, and migration. Indirect cytotoxicity tests showed no toxic effect of either hydrogel. The direct contact with the carboxymethylated hydrogel adversely influenced the morphology of the cells and limited their spreading, while typical morphology and spreading of cells were observed with the TEMPO-oxidized hydrogel. The porous fibrous structure may be a key to cell proliferation and migration in the hydrogels.

Recent studies have discovered a substantial viscosity increase of aqueous cellulose nanocrystal (CNC) dispersions upon heat aging at temperatures above 90 &deg, C. This distinct change in material properties at very low concentrations in water has been proposed as an active mechanism for enhanced oil recovery (EOR), as highly viscous fluid may improve macroscopic sweep efficiencies and mitigate viscous fingering. A high-temperature (120 &deg, C) core flood experiment was carried out with 1 wt. % CNC in low salinity brine on a 60 cm-long sandstone core outcrop initially saturated with crude oil. A flow rate corresponding to 24 h per pore volume was applied to ensure sufficient viscosification time within the porous media. The total oil recovery was 62.2%, including 1.2% oil being produced during CNC flooding. Creation of local log-jams inside the porous media appears to be the dominant mechanism for additional oil recovery during nano flooding. The permeability was reduced by 89.5% during the core flood, and a thin layer of nanocellulose film was observed at the inlet of the core plug. CNC fluid and core flood effluent was analyzed using atomic force microscopy (AFM), particle size analysis, and shear rheology. The effluent was largely unchanged after passing through the core over a time period of 24 h. After the core outcrop was rinsed, a micro computed tomography (micro-CT) was used to examine heterogeneity of the core. The core was found to be homogeneous.

Pretreatment, fractionation and hydrolysis remains costly and challenging process steps in biochemical conversion of softwoods. Here, ionic liquid pretreatment using 1-ethyl-3-methylimidazolium acetate (EMIM-OAc) at high temperature (100 °C, 6 h) and alkali based (NaOH/urea) pretreatment at sub-zero temperature (−18 °C, 24 h) were compared and combined in studies of Norway Spruce biomass deconstruction. Both treatments significantly improved the enzymatic digestibility of the biomass. EMIM-OAc gave higher glucan than mannan digestibility, indicating a more pronounced effect on the cellulose polymer than on the hemicellulose polymer. In contrast, low temperature alkali pretreatment using NaOH or NaOH + urea gave a more pronounced effect on mannan than on glucan digestibility. By combining the two methods the total monosugar yield after enzymatic hydrolysis was improved by 20–50% as compared to using ionic liquid or alkali based pretreatment alone. Lignin dissolution was low for both methods under the conditions studied.

The applicability of cellulose nanofibrils (CNFs) as viscosifying agent in a starch-reduced low-fat mayonnaise and in an oil-reduced full-fat mayonnaise has been considered. For low-fat mayonnaise a 50 wt% reduction in the ordinary starch content was performed, while for full-fat mayonnaise, the oil content was reduced from 79 to 70 wt%. To study if the stability was affected when CNFs were added, analyses as visual and accelerated stability tests, droplet size measurements and rheology studies, determining the shear viscosity, and the loss and storage moduli, were conducted after 1 day, 1 week and 1 month of storage in room temperature. Even though changes in droplet size distributions and rheological properties indicated some coalescence, the visual stability was not changed after 1 month of storage for any of the samples. The decrease in viscosity and moduli inflicted by reduction of starch or fat, could be regained by the addition of CNFs at 0.75 wt % and 0.42 wt %, respectively. Based on the results in this work, mayonnaise with reduced starch or fat content can be produced when CNFs are used as a viscosifying agent.

Cellulose nanocrystals (CNCs) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanofibrils (T-CNFs) were tested as enhanced oil recovery (EOR) agents through core floods and microfluidic experiments. Both particles were mixed with low salinity water (LSW). The core floods were grouped into three parts based on the research objectives. In Part 1, secondary core flood using CNCs was compared to regular water flooding at fixed conditions, by reusing the same core plug to maintain the same pore structure. CNCs produced 5.8% of original oil in place (OOIP) more oil than LSW. For Part 2, the effect of injection scheme, temperature, and rock wettability was investigated using CNCs. The same trend was observed for the secondary floods, with CNCs performing better than their parallel experiment using LSW. Furthermore, the particles seemed to perform better under mixed-wet conditions. Additional oil (2.9&ndash, 15.7% of OOIP) was produced when CNCs were injected as a tertiary EOR agent, with more incremental oil produced at high temperature. In the final part, the effect of particle type was studied. T-CNFs produced significantly more oil compared to CNCs. However, the injection of T-CNF particles resulted in a steep increase in pressure, which never stabilized. Furthermore, a filter cake was observed at the core face after the experiment was completed. Microfluidic experiments showed that both T-CNF and CNC nanofluids led to a better sweep efficiency compared to low salinity water flooding. T-CNF particles showed the ability to enhance the oil recovery by breaking up events and reducing the trapping efficiency of the porous medium. A higher flow rate resulted in lower oil recovery factors and higher remaining oil connectivity. Contact angle and interfacial tension measurements were conducted to understand the oil recovery mechanisms. CNCs altered the interfacial tension the most, while T-CNFs had the largest effect on the contact angle. However, the changes were not significant enough for them to be considered primary EOR mechanisms.

Wood-based TEMPO-oxidised cellulose nanofibrils (toCNF) are promising materials for biomedical applications. Cyclodextrins have ability to form inclusion complexes with hydrophobic molecules and are considered as a method to bring new functionalities to these materials. Water sorption and mechanical properties are also key properties for biomedical applications such as drug delivery and tissue engineering. In this work, we report the modification with &beta, cyclodextrin (&beta, CD) of toCNF samples with different carboxyl contents viz. 756 &plusmn, 4 &micro, mol/g and 1048 &plusmn, 32 &micro, mol/g. The modification was carried out at neutral and acidic pH (2.5) to study the effect of dissociation of the carboxylic acid group. Films processed by casting/evaporation at 40 &deg, C and cryogels processed by freeze-drying were prepared from &beta, CD modified toCNF suspensions and compared with reference samples of unmodified toCNF. The impact of modification on water sorption and mechanical properties was assessed. It was shown that the water sorption behaviour for films is driven by adsorption, with a clear impact of the chemical makeup of the fibres (charge content, pH, and adsorption of cyclodextrin). Modified toCNF cryogels (acidic pH and addition of cyclodextrins) displayed lower mechanical properties linked to the modification of the cell wall porosity structure. Esterification between &beta, CD and toCNF under acidic conditions was performed by freeze-drying, and such cryogels exhibited a lower decrease in mechanical properties in the swollen state. These results are promising for the development of scaffold and films with controlled mechanical properties and added value due to the ability of cyclodextrin to form an inclusion complex with active principle ingredient (API) or growth factor (GF) for biomedical applications.

The assessment of several ink formulations for 3D printing based on two natural macromolecular compounds is presented. In the current research we have exploited the fast crosslinking potential of pectin and the remarkable shear-thinning properties of carboxylated cellulose nanofibrils, which is known to induce a desired viscoelastic behavior. Prior to 3D printing, the viscoelastic properties of the polysaccharide inks were evaluated by rheological measurements and injectability tests. The reliance of the printing parameters on the ink composition was established through one-dimensional lines printing, the base units of 3D-structures. The performance of the 3D-printed structures after ionic cross-linking was evaluated in terms of mechanical properties and rehydration behavior. MicroCT was also used to evaluate the morphology of the 3D-printed objects regarding the effect of pectin/nanocellulose ratio on the geometrical features of scaffolds. The proportionality between the two polymers proved to be the determining factor for the firmness and strength of the printed objects.

Inks for 3D printing based on cellulose nanofibrils (CNFs) or mixtures of CNFs and either cellulose nanocrystals (CNCs) or alginate were assessed by determining their viscoelastic properties i.e. complex viscosity and storage and loss moduli (G′ and G″). Two types of alginates were used, i.e. from Laminaria hyperborea stipe and Macrocystis pyrifera. Shape fidelity of 3D printed grids were qualitatively evaluated and compared to the viscoelastic properties of the inks. The biocomposite gels containing alginate were post stabilized by crosslinking with Ca2+. Mechanical properties of the crosslinked biocomposite gels were assessed. The complex viscosity, G′ and G″ of CNF suspensions increased when the solid content was increased from 3.5 to 4.0 wt%, but levelled off by further increase in CNF solid content. The complex viscosity at low angular frequency at 4 wt% was as high as 104 Pa·s. This seemed to be the necessary viscosity level for obtaining good shape fidelity of the printed structures for the studied systems. By replacing part of the CNFs with CNCs, the complex viscosity, G′ and G″ were reduced and so was also the shape fidelity of the printed grids. The changes in complex viscosity and moduli when CNFs was replaced with alginate depended on the relative amounts of CNFs/alginate. The type of alginate (from either L. hyp. stipe or M. pyr.) did not play a role for the viscoelastic properties of the inks, nor for the printed grids before post stabilization. Replacing CNFs with up to 1.5 wt% alginate gave satisfactory shape fidelity. The effect of adding alginate and subsequent crosslinking with Ca2+, strongly affected the strength properties of the gels. By appropriate choice of relative amounts of CNFs and alginate and type of alginate, the Young’s modulus and rupture strength could be controlled within the range of 30–150 kPa and 1.5–6 kg, respectively. The deformation at rupture was around 55%. The alginate from L. hyp. stipe yields higher Young’s modulus and lower syneresis compared to M. pyr. This shows that the choice of alginate plays a significant role for the mechanical properties of the final product, although it does not influence on the viscoelastic properties of the ink. The choice of alginate should be L. hyp. stipe if high strength is desired.

Biopolymers such as gelatin (Gel) and cellulose nanofibrils (CNF) have many of the essential requirements for being used as scaffolding materials in tissue regeneration; biocompatibility, surface chemistry, ability to generate homogeneous hydrogels and 3D structures with suitable pore size and interconnection, which allows cell colonization and proliferation. The purpose of this study was to investigate whether the mechanical behaviour of the Gel matrix can be improved by means of functionalization with cellulose nanofibrils and proper cross-linking treatments. Blending processes were developed to achieve a polymer nanocomposite incorporating the best features of both biopolymers: biomimicry of the Gel and structural reinforcement by the CNF. The designed 3D structures underline interconnected porosity achieved by freeze-drying process, improved mechanical properties and chemical stability that are tailored by CNF addition and different cross-linking approaches. In vitro evaluations reveal the preservation of the biocompatibility of Gel and its good interaction with cells by promoting cell colonization and proliferation. The results support the addition of cellulose nanofibrils to improve the mechanical behaviour of 3D porous structures suitable as scaffolding for tissue regeneration.

Pickering o/w emulsions prepared with 40 wt % rapeseed oil were stabilized with the use of low charged enzymatically treated cellulose nanofibrils (CNFs) and highly charged 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized CNFs. The emulsion-forming abilities and storage stability of the two qualities were tested in the presence of NaCl and acetic acid, at concentrations relevant to food applications. Food emulsions may be an important future application area for CNFs due to their availability and excellent viscosifying abilities. The emulsion characterization was carried out by visual inspection, light microscopy, viscosity measurements, dynamic light scattering and mild centrifugation, which showed that stable emulsions could be obtained for both CNF qualities in the absence of salt and acid. In addition, the enzymatically stabilized CNFs were able to stabilize emulsions in the presence of acid and NaCl, with little change in the appearance or droplet size distribution over one month of storage at room temperature. The work showed that enzymatically treated CNFs could be suitable for use in food systems where NaCl and acid are present, while the more highly charged TEMPO-CNFs might be more suited for other applications, where they can contribute to a high emulsion viscosity even at low concentrations. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of polysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes. The catalytic domains of a considerable fraction of LPMOs and other carbohydrate-active enzymes (CAZymes) are tethered to carbohydrate-binding modules (CBMs) by flexible linkers. These linkers preclude X-ray crystallographic studies, and the functional implications of these modular assemblies remain partly unknown. Here, we used NMR spectroscopy to characterize structural and dynamic features of full-length modular ScLPMO10C from Streptomyces coelicolor. We observed that the linker is disordered and extended, creating distance between the CBM and the catalytic domain and allowing these domains to move independently of each other. Functional studies with cellulose nanofibrils revealed that most of the substrate-binding affinity of full-length ScLPMO10C resides in the CBM. Comparison of the catalytic performance of full-length ScLPMO10C and its isolated catalytic domain revealed that the CBM is beneficial for LPMO activity at lower substrate concentrations and promotes localized and repeated oxidation of the substrate. Taken together, these results provide a mechanistic basis for understanding the interplay between catalytic domains linked to CBMs in LPMOs and CAZymes in general. © 2018 Courtade et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. This is the authors' accepted and refereed manuscript to the article. The final authenticated version is available online at: http://doi.org/10.1074/jbc.RA118.004269

The application of nanotechnology to the petroleum industry has sparked recent interest in increasing oil recovery, while reducing environmental impact. Nanocellulose is an emerging nanoparticle that is derived from trees or waste stream from wood and fiber industries. Thus, it is taken from a renewable and sustainable source, and could therefore serve as a good alternative to current Enhanced Oil Recovery (EOR) technologies. However, before nanocellulose can be applied as an EOR technique, further understanding of its transport behavior and retention in porous media is required. The research documented in this paper examines retention mechanisms that occur during nanocellulose transport. In a series of experiments, nanocellulose particles dispersed in brine were injected into sandpacks and Berea sandstone cores. The resulting retention and permeability reduction were measured. The experimental parameters that were varied include sand grain size, nanocellulose type, salinity, and flow rate. Under low salinity conditions, the dominant retention mechanism was adsorption and when salinity was increased, the dominant retention mechanism shifted towards log-jamming. Retention and permeability reduction increased as grain size decreased, which results from increased straining of nanocellulose aggregates. In addition, each type of nanocellulose was found to have significantly different transport properties. Experiments with Berea sandstone cores indicate that some pore volume was inaccessible to the nanocellulose. As a general trend, the larger the size of aggregates in bulk solution, the greater the observed retention and permeability reduction. Salinity was found to be the most important parameter affecting transport. Increased salinity caused additional aggregation, which led to increased straining and filter cake formation. Higher flow rates were found to reduce retention and permeability reduction. Increased velocity was accompanied by an increase in shear, which is believed to promote breakdown of nanocellulose aggregates. © 2018 by the authors. Funding details: CERC35, CERC, Canada Excellence Research Chairs, Government of Canada; Funding details: 974 767 880; Funding details: 262644, I-CORE, Israeli Centers for Research Excellence; Funding details: RGPAS/477902-2015, NSERC, Natural Sciences and Engineering Research Council of Canada; Funding details: 244615/E30, Norges Forskningsråd; Funding details: 244615/E30; Funding details: 262644, CERC, Canada Excellence Research Chairs, Government of Canada; Funding details: NSERC, Natural Sciences and Engineering Research Council of Canada

Alginate and cellulose nanofibrils (CNF) are attractive materials for tissue engineering and regenerative medicine. CNF gels are generally weaker and more brittle than alginate gels, while alginate gels are elastic and have high rupture strength. Alginate properties depend on their guluronan and mannuronan content and their sequence pattern and molecular weight. Likewise, CNF exists in various qualities with properties depending on, e.g., morphology and charge density. In this study combinations of three types of alginate with different composition and two types of CNF with different charge and degree of fibrillation have been studied. Assessments of the composite gels revealed that attractive properties like high rupture strength, high compressibility, high gel rigidity at small deformations (Young’s modulus), and low syneresis was obtained compared to the pure gels. The effects varied with relative amounts of CNF and alginate, alginate type, and CNF quality. The largest effects were obtained by combining oxidized CNF with the alginates. Hence, by combining the two biopolymers in composite gels, it is possible to tune the rupture strength, Young’s modulus, syneresis, as well as stability in physiological saline solution, which are all important properties for the use as scaffolds in tissue engineering. (c) 2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

Cellulose nanofibrils (CNF) have potential as rheology modifiers of water based fluids, e.g. drilling fluids for use in oil wells or as additives in injection water for enhanced oil recovery (EOR). The temperature in oil wells can be high (>100°C), and the retention time long; days for drilling fluids and months for EOR fluids. Hence, it is important to assess the temperature stability over time of nanocellulose dispersions to clarify their suitability as rheology modifiers of water based fluids at such harsh conditions. Dispersions of CNF produced mechanically, by using TEMPO mediated oxidation and by using carboxymethylation as pretreatment, in addition to cellulose nanocrystals (CNC), have been subjected to heat aging. Temperature stability was best for CNC and for mechanically produced CNF that were stable after heating to 140°C for three days. The effect of additives was evaluated; cesium formate and sodium formate increased the temperature stability of the dispersions, while there was no effect of using phosphate buffer.

We have studied the degradation of well-characterized soluble heteropolymeric chitosans by a novel family 46 chitosanase, ScCsn46A from Streptomyces coelicolor A3(2), to obtain insight into the enzyme's mode of action and to determine its potential for production of different chitooligosaccharides. The degradation of both a fully deacetylated chitosan and a 32% acetylated chitosan showed a continuum of oligomeric products and a rapid disappearance of the polymeric fraction, which is diagnostic for a nonprocessive endomode of action. The kinetics of the degradation of the 32% acetylated chitosan demonstrated an initial rapid phase and a slower second phase, in addition to a third and even slower kinetic phase. The first phase reflects the cleavage of the glycosidic linkage between two deacetylated units (D-D), the primary products being fully deacetylated dimers, trimers, and tetramers, as well as longer oligomers with increasing degrees of acetylation. In the subsequent slower kinetic phases, oligomers with a higher degree of acetylated units (A) appear, including oligomers with A's at the reducing or nonreducing end, which indicate that there are no absolute preferences for D in subsites -1 and +1. After maximum degradation of the chitosan, the dimers DA and DD were the dominant products. The degradation of chitosans with varying degrees of acetylation to a maximum degree of scission showed that ScCsn46A could degrade all chitosan substrates extensively, although to decreasing degrees of scission with increasing F(A). The potential use of ScCsn46A to prepare fully deacetylated oligomers and more highly acetylated oligomers from chitosan substrates with varying degrees of acetylation is discussed.

Chitooligosaccharides (CHOS) are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine. CHOS can be produced using chitin or chitosan as a starting material, using enzymatic conversions, chemical methods or combinations thereof. Production of well-defined CHOS-mixtures, or even pure CHOS, is of great interest since these oligosaccharides are thought to have several interesting bioactivities. Understanding the mechanisms underlying these bioactivities is of major importance. However, so far in-depth knowledge on the mode-of-action of CHOS is scarce, one major reason being that most published studies are done with badly characterized heterogeneous mixtures of CHOS. Production of CHOS that are well-defined in terms of length, degree of N-acetylation, and sequence is not straightforward. Here we provide an overview of techniques that may be used to produce and characterize reasonably well-defined CHOS fractions. We also present possible medical applications of CHOS, including tumor growth inhibition and inhibition of T(H)2-induced inflammation in asthma, as well as use as a bone-strengthener in osteoporosis, a vector for gene delivery, an antibacterial agent, an antifungal agent, an anti-malaria agent, or a hemostatic agent in wound-dressings. By using well-defined CHOS-mixtures it will become possible to obtain a better understanding of the mechanisms underlying these bioactivities.

We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin-remazol-brilliant violet) and a small, presumably amorphous, subfraction of alpha-chitin and beta-chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472-484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (- 2 to + 2), as opposed to six (- 3 to + 3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site-directed mutagenesis study.

Chitooligosaccharides (CHOS) are oligomers composed of glucosamine and N-acetylglucosamine with several interesting bioactivities that can be produced from enzymatic cleavage of chitosans. By controlling the degree of acetylation of the substrate chitosan, the enzyme, and the extent of enzyme degradation, CHOS preparations with limited variation in length and sequence can be produced. We here report on the degradation of chitosans with a novel family 75 chitosanase, SaCsn75A from Streptomyces avermitilis . By characterizing the CHOS preparations, we have obtained insight into the mode of action and subsite specificities of the enzyme. The degradation of a fully deacetylated and a 31% acetylated chitosan revealed that the enzyme degrade these substrates according to a nonprocessive, endo mode of action. With the 31% acetylated chitosan as substrate, the kinetics of the degradation showed an initial rapid phase, followed by a second slower phase. In the initial faster phase, an acetylated unit (A) is productively bound in subsite -1, whereas deacetylated units (D) are bound in the -2 subsite and the +1 subsite. In the slower second phase, D-units bind productively in the -1 subsite, probably with both acetylated and deacetylated units in the -2 subsite, but still with an absolute preference for deacetylated units in the +1 subsite. CHOS produced in the initial phase are composed of deacetylated units with an acetylated reducing end. In the slower second phase, higher amounts of low DP fully deacetylated oligomers (dimer and trimer) are produced, while the higher DP oligomers are dominated by compounds with acetylated reducing ends containing increasing amounts of internal acetylated units. The degradation of chitosans with varying degrees of acetylation to maximum extents of degradation showed that increasingly longer oligomers are produced with increasing degree of acetylation, and that the longer oligomers contain sequences of consecutive acetylated units interspaced by single deacetylated units. The catalytic properties of SaCsn75A differ from the properties of a previously characterized family 46 chitosanase from S. coelicolor (ScCsn46A).

Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the details of its enzymatic properties have not yet been fully unraveled. We have studied the properties of HCHT by analyzing how the enzyme acts on high-molecular weight chitosans, soluble copolymers of β-1,4-linked N-acetylglucosamine (GlcNAc, A), and glucosamine (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial family 18 enzymes, we show that HCHT degrades chitosan primarily via an endoprocessive mechanism, as would be expected on the basis of the structural features of its substrate-binding cleft. The preferences of HCHT subsites for acetylated versus nonacetylated sugars were assessed by sequence analysis of obtained oligomeric products showing a very strong, absolute, and a relative weak preference for an acetylated unit in the −2, −1, and +1 subsites, respectively. The latter information is important for the design of inhibitors that are specific for the human chitinases and also provides insight into what kind of products may be formed in vivo upon administration of chitosan-containing medicines or food products. Copyright © 2012 American Chemical Society. This is the authors’ accepted and refereed manuscript to the article.

We have studied the degradation of soluble heteropolymeric chitosans with a bacterial family 19 chitinase, ChiG from Streptomyces coelicolor A3(2), to obtain insight into the mode of action of ChiG, to determine subsite preferences for acetylated and deacetylated sugar units, and to evaluate the potential of ChiG for production of chito-oligosaccharides. Degradation of chitosans with varying degrees of acetylation was followed using NMR for the identity (acetylated/deacetylated) of new reducing and nonreducing ends as well as their nearest neighbors and using gel filtration to analyze the size distribution of the oligomeric products. Degradation of a 64% acetylated chitosan yielded a continuum of oligomers, showing that ChiG operates according to a nonprocessive, endo mode of action. The kinetics of the degradation showed an initial rapid phase dominated by cleavage of three consecutive acetylated units (A; occupying subsites -2, -1, and +1), and a slower kinetic phase reflecting the cleavage of the glycosidic linkage between a deacetylated unit (D, occupying subsite -1) and an A (occupying subsite +1). Characterization of isolated oligomer fractions obtained at the end of the initial rapid phase and at the end of the slower kinetic phase confirmed the preference for A binding in subsites -2, -1, and +1 and showed that oligomers with a deacetylated reducing end appeared only during the second kinetic phase. After maximum conversion of the chitosan, the dimers AD/AA and the trimer AAD were the dominating products. Degradation of chitosans with varying degrees of acetylation to maximum degree of scission produced a wide variety of oligomer mixtures, differing in chain length and composition of acetylated/deacetylated units. These results provide insight into the properties of bacterial family 19 chitinases and show how these enzymes may be used to convert chitosans to several types of chito-oligosaccharide mixtures.