Amylose synthesized in vitro by amylosucrase: morphology, structure, and properties

The recombinant amylosucrase from Neisseria polysaccharea was used to synthesize in vitro amylose from sucrose as unique substrate. The morphology and structure of the insoluble residue were shown to depend only on the initial sucrose concentration (100, 300, or 600 mM), which controlled both the chain length and concentration at the precipitation stage. The average degree of polymerization (DP) in the precipitated product varied from 58 for the lowest initial sucrose concentration (100 mM) to 45 and 35 for higher sucrose concentrations (300 and 600 mM, respectively). The shorter chains (DP 35 and 45), produced in high yields (54 and 24 g/L respectively), precipitated as polycrystalline aggregates with exceptional crystallinity, without optimization of the reaction medium for crystallization. The longer chains (DP 58), produced in lower amount (2.9 g/L), formed networks similar to those observed for amylose gels. All synthesized products displayed a B-type crystal structure. Their melting behavior was also studied, the thermostability being higher for the precipitate containing the longer chains. Further thermal treatments were shown to still improve the crystallinity and yield substrates usable as new standards for the determination of the relative crystallinity of starchy products. The kinetics of chain elongation and aggregation were thoroughly investigated in order to explain how the action of amylosucrase resulted in such different amylose structures. These results emphasize the potentiality of amylosucrase in the design of amylodextrins with controlled morphology, structure, and physicochemical properties.     

Amylosucrase, a glucan-synthesizing enzyme from the alpha-amylase family

Amylosucrase (E.C. 2.4.1.4) is a member of Family 13 of the glycoside hydrolases (the alpha-amylases), although its biological function is the synthesis of amylose-like polymers from sucrose. The structure of amylosucrase from Neisseria polysaccharea is divided into five domains: an all helical N-terminal domain that is not similar to any known fold, a (beta/alpha)(8)-barrel A-domain, B- and B'-domains displaying alpha/beta-structure, and a C-terminal eight-stranded beta-sheet domain. In contrast to other Family 13 hydrolases that have the active site in the bottom of a large cleft, the active site of amylosucrase is at the bottom of a pocket at the molecular surface. A substrate binding site resembling the amylase 2 subsite is not found in amylosucrase. The site is blocked by a salt bridge between residues in the second and eight loops of the (beta/alpha)(8)-barrel. The result is an exo-acting enzyme. Loop 7 in the amylosucrase barrel is prolonged compared with the loop structure found in other hydrolases, and this insertion (forming domain B') is suggested to be important for the polymer synthase activity of the enzyme. The topology of the B'-domain creates an active site entrance with several ravines in the molecular surface that could be used specifically by the substrates/products (sucrose, glucan polymer, and fructose) that have to get in and out of the active site pocket.     

Structural investigation of the thermostability and product specificity of amylosucrase from the bacterium Deinococcus geothermalis

Amylosucrases are sucrose-utilizing α-transglucosidases that naturally catalyze the synthesis of α-glucans, linked exclusively through α1,4-linkages. Side products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes. Here, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis (DgAS). The three-dimensional structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying the DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea in complex with turanose. In the amylosucrase from N. polysaccharea (NpAS), key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanoid tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS. Our findings help to understand the inter-relationships between amylosucrase structure, flexibility, function, and stability and provide new insight for amylosucrase design.     

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme™, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.     

The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K _i = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.     

Combinatorial engineering to enhance thermostability of amylosucrase

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.     

Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)₈-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.     

Amylosucrase from Neisseria polysaccharea: novel catalytic properties

Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis - these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non-Michaelian profile never previously described.     

Crystal structure of the Glu328Gln mutant of Neisseria polysaccharea amylosucrase in complex with sucrose and maltoheptaose

Several enzymes acting on sucrose are found in glycoside hydrolase family 13 (the α–amylase family). They all transfer a glucosyl moiety from sucrose to an acceptor, but the products can be very different. The structure of a variant of one of these, the Glu328Gln mutant of Neisseria polysaccharea amylosucrase, has been determined in a ternary complex with sucrose and an oligosaccharide to 2.16 Å resolution using x-ray crystallography. Sucrose selectively binds in the active site and the oligosaccharide only binds at surface sites. When this structure is compared to structures of other enzymes acting on sucrose from glycoside hydrolase family 13, it is found that the active site residues are very similar around the glucose part of sucrose while much variation is seen around the fructose moiety.     

Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.     

Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase

A computer‐aided engineering approach recently enabled to deeply reshape the active site of N. polysaccharea amylosucrase for recognition of non‐natural acceptor substrates. Libraries of variants were constructed and screened on sucrose allowing the identification of 17 mutants able to synthesize molecules from sole sucrose, which are not synthesized by the parental wild‐type enzyme. Three of the isolated mutants as well as the new products synthesized were characterized in details. Mutants contain between 7 and 11 mutations in the active site and the new molecules were identified as being a sucrose derivative, named erlose (α‐d ‐glucopyranosyl‐(1→4)‐α‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐Fructose), and a new malto‐oligosaccharide named panose (α‐d ‐glucopyranosyl‐(1→6)‐α‐d ‐glucopyranosyl‐(1→4)‐α‐d ‐Glucose). These product specificities were never reported for none of the amylosucrases characterized to date, nor their engineered variants. Optimization of the production of these trisaccharides of potential interest as sweeteners or prebiotic molecules was carried out. Molecular modelling studies were also performed to shed some light on the molecular factors involved in the novel product specificities of these amylosucrase variants.     

Crystal structures of amylosucrase from Neisseria polysaccharea in complex with D-glucose and the active site mutant Glu328Gln in complex with the natural substrate sucrose

The structure of amylosucrase from Neisseria polysaccharea in complex with beta-D-glucose has been determined by X-ray crystallography at a resolution of 1.66 A. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 A. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly in the bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained (4)C(1) conformation, and one that binds in the packing interface to a symmetry-related molecule. A third weaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientation of the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrose complex shows one molecule bound in the active site, where the glucosyl moiety is located at the alpha-amylase -1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible access channel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtained through enzyme interactions with the glucosyl ring and that an important part of the enzyme function is a precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328. The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting beta-strand 7 and alpha-helix 7 that is much longer in amylosucrase compared to other enzymes from the alpha-amylase family (family 13 of the glycoside hydrolases).     

Glycogen phosphorylase, the product of the glgP Gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli

To understand the biological function of bacterial glycogen phosphorylase (GlgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression. glgP deletion mutants (DeltaglgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in DeltaglgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by DeltaglgP cells. The overall results thus show that GlgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.     

Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with alpha-amylases. Exactly how this enzyme catalyzes the formation of alpha-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg415, subsite +4) and the correct positioning (Asp394 and Arg446, subsite +1) of acceptor molecules. On the other hand, Arg226 (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.     

Alternansucrase acceptor reactions with methyl hexopyranosides

Alternansucrase (EC 2.4.1.140, sucrose: (1-->6), (1-->3)-alpha-D-glucan 6(3)-alpha-D-glucosyltransferase) is a D-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked D-glucan from sucrose. It also synthesizes oligosaccharides via D-glucopyranosyl transfer to various acceptor sugars. We have studied the acceptor products arising from methyl glycosides as model compounds in order to better understand the specificity of alternansucrase acceptor reactions. The initial product arising from methyl beta-D-glucopyranoside was methyl beta-isomaltoside, which was subsequently glucosylated to yield methyl beta-isomaltotrioside and methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside. These products are analogous to those previously described from methyl alpha-D-glucopyranoside. The major initial acceptor product from methyl alpha-D-mannopyranoside was methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-mannopyranoside, but several minor products were also isolated and characterized, including a 3,6-di-O-substituted mannopyranoside. Methyl alpha-D-galactopyranoside yielded two initial products, methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-galactopyranoside and methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside, in a 2.5:1 molar ratio. Methyl D-allopyranosides were glucosylated primarily at position 6, yielding methyl alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. The latter subsequently gave rise to methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. In general, the methyl alpha-D-hexopyranosides were better acceptors than the corresponding beta-glycosides.     

Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.     

Monomeric state and Ca2+ transport by sarcoplasmic reticulum Ca2(+)-ATPase, reconstituted with an excess of phospholipid

The structural basis for Ca2+ transport was examined in vesicles reconstituted with an excess of phospholipid by a cholate dialysis procedure. Unincorporated protein and vesicles with a relatively high protein content were removed by sucrose density centrifugation (3-12%), leaving a fraction of lipid-rich vesicles (lipid to protein weight ratio 800-900:1) with a high coupling ratio (1.0) and transport capacity (25 mumol/mg protein, after Ca-phosphate loading). Freeze-fracture analysis showed that the reconstituted vesicles had a remarkably narrow size distribution (diameter 794 +/- 77 A (S.D.], suitable for stereological analysis. Intramembranous particles were dispersed and occurred with a low frequency in the fractured shells, also before sucrose fractionation. It was calculated that the number of intramembranous particles corresponded to the number of Ca2(+)-ATPase polypeptide/vesicle. A ratio of unity between particles and polypeptide chains was also obtained from the density of particle distribution on flat surfaces of fused vesicles, prepared by sucrose fractionation. The size of the particles formed a broad distribution, having a peak value around 60-67 A, both in the reconstituted preparation and sarcoplasmic reticulum vesicles. No evidence for protein-protein interactions was found in chemical cross-linking experiments. It is concluded that the intramembranous particles in the reconstituted preparations are referable to monomeric Ca2(+)-ATPase which is capable of transporting Ca2+ inside the vesicles. The implications of the observations for the associational state of Ca2(+)-ATPase at high protein concentration are considered in relation to previous ultrastructural investigations of membranous Ca2(+)-ATPase in native and two-dimensional-crystalline forms.     

High level expression of a recombinant amylosucrase gene and selected properties of the enzyme

Two high-level heterologous expression systems for amylosucrase genes have been constructed. One depends on sigma-70 bacterial RNA polymerase, the other on phage T7 RNA polymerase. Translational fusions were formed between slightly truncated versions of the gene from Neisseria polysaccharea and sequences of expression vectors pQE-81L or pET33b(+), respectively. These constructs were introduced into different Escherichia coli strains. The resulting recombinants yielded up to 170 mg of dissolved enzyme per litre of culture at a moderate cell density of five OD₆₀₀. To our knowledge, this is the highest yield per cell described so far for amylosucrases. The recombinant enzymes could rapidly be purified through the use of histidine tags in the N-terminally attached sequences. These segments did not alter catalytic properties and therefore need not be removed for most applications. Investigations with glucose and malto-oligosaccharides of different lengths identified rate-limiting steps in the elongation (acceptor reaction) and truncation (donor reaction) of these substrates. The elongation of maltotriose and its reversal, the truncation of maltotetraose, were found to be particularly slow reactions. Potential reasons are discussed, based on the crystal structure of the enzyme. It is furthermore shown that amylosucrase is able to synthesise mixed disaccharides. All of the glucose epimers mannose, allose, and galactose served as acceptors, yielding between one and three main products. We also demonstrate that, as an alternative to the use of purified amylosucrase, cells of the constructed recombinant strains can be used to carry out glucosylations of acceptors.     

Oligosaccharide and sucrose complexes of amylosucrase. Structural implications for the polymerase activity

The glucosyltransferase amylosucrase is structurally quite similar to the hydrolase alpha-amylase. How this switch in functionality is achieved is an important and fundamental question. The inactive E328Q amylosucrase variant has been co-crystallized with maltoheptaose, and the structure was determined by x-ray crystallography to 2.2 A resolution, revealing a maltoheptaose binding site in the B'-domain somewhat distant from the active site. Additional soaking of these crystals with maltoheptaose resulted in replacement of Tris in the active site with maltoheptaose, allowing the mapping of the -1 to +5 binding subsites. Crystals of amylosucrase were soaked with sucrose at different concentrations. The structures at approximately 2.1 A resolution revealed three new binding sites of different affinity. The highest affinity binding site is close to the active site but is not in the previously identified substrate access channel. Allosteric regulation seems necessary to facilitate access from this binding site. The structures show the pivotal role of the B'-domain in the transferase reaction. Based on these observations, an extension of the hydrolase reaction mechanism valid for this enzyme can be proposed. In this mechanism, the glycogen-like polymer is bound in the widest access channel to the active site. The polymer binding introduces structural changes that allow sucrose to migrate from its binding site into the active site and displace the polymer.     

Synthesis and study of cross-linked chitosan-N-poly(ethylene glycol) nanoparticles

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan. Poly(ethylene glycol) dicarboxylic acid was used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. The prepared nanosystems were stable in aqueous media. The structure of the products was determined by nuclear magnetic resonance (NMR) spectroscopy, and the particle size was identified by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that biodegradable cross-linked chitosan nanoparticles experienced considerable swelling because of the length and flexibility of the cross-linking agent. The aqueous solutions or dispersions of nanoparticles were stable and clear or mildly opalescent systems depending on the ratio of cross-linking and molecular weight of chitosan, findings consistent with values of transmittance above 75%. Particle size measured by TEM varied in the range of 4-24 nm. In the swollen state, the average size of the individual particles measured by DLS was in the range of 50-120 nm depending on the molecular weight of chitosan and the ratio of cross-linking.