Ellinor B. Heggset

Production of Chitooligosaccharides and Their Potential Applications in Medicine

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 TH2-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.

Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes

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 α‐chitin and β‐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.

Degradation of chitosans with a family 46 chitosanase from Streptomyces coelicolor A3(2).

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 enzymes 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 As 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.

Degradation of Chitosans with Chitinase G from Streptomyces coelicolor A3(2) : Production of Chito-oligosaccharides and Insight into Subsite Specificities

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.

Human Chitotriosidase-Catalyzed Hydrolysis of Chitosan

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.

Mode of action of a family 75 chitosanase from Streptomyces avermitilis.

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).

Temperature stability of nanocellulose dispersions.

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.

Cytocompatibility of Wood-Derived Cellulose Nanofibril Hydrogels with Different Surface Chemistry

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.

Mechanical Properties of Composite Hydrogels of Alginate and Cellulose Nanofibrils

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.

Polymeric 3D scaffolds for tissue regeneration: Evaluation of biopolymer nanocomposite reinforced with cellulose nanofibrils

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.