Thermally reversible hydration of beta-chitin

Thermally induced transition between anhydrous and hydrated forms of highly crystalline beta-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet beta-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 degrees C on heating and one broad exothermic peak at between 60 and 0 degrees C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet beta-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the beta-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process.     

Preparation of chitin nanofibers from squid pen beta-chitin by simple mechanical treatment under acid conditions

A procedure for preparing individualized chitin nanofibers 3-4 nm in cross-sectional width and at least a few microns in length was developed. The key factors to prepare the chitin nanofibers with such high aspect ratios are as follows: (1) squid pen beta-chitin is used as the starting material and (2) ultrasonication of the beta-chitin in water at pH 3-4 and 0.1-0.3% consistency for a few minutes. Transparent and highly viscous dispersions of squid pen beta-chitin nanofibers in water can be obtained by this method. No N-deacetylation occurs on the chitin molecules during the nanofiber conversion procedure. Moreover, the original crystal structure of beta-chitin is maintained, although crystallinity index decreases from 0.51 to 0.37 as a result of the nanofiber conversion. Cationization of the C2 amino groups present on the crystallite surfaces of the squid pen beta-chitin under acid conditions is necessary for preparing the nanofibers.     

Esterification of beta-chitin via intercalation by carboxylic anhydrides

beta-chitin is known to form intercalation complexes with aliphatic alcohols and amines. We found that it also forms complexes with carboxylic anhydrides. When the beta-chitin-acetic anhydride complex was heated to 105 degrees C, the hydroxyl groups of chitin were acetylated by a host-guest reaction, maintaining the host's crystal structure. Structures of complex and acetylated products were analyzed by X-ray diffraction, (13)C CP/MAS NMR, and infrared spectroscopy. The maximum degree of substitution (DS) was close to 1.0, suggesting regioselective esterification at the C6 position of chitin. Partially acetylated beta-chitin with a DS of 0.4 could incorporate various guest species that are difficult to be incorporated by original beta-chitin. In contrast, beta-chitin acetate with a DS of 1 lost the ability to form a complex. Intercalation complexes of beta-chitin with cyclic anhydrides (succinic and maleic) also underwent esterification by heating, and the products with a DS of approximately 1 dissolved in aqueous alkali, apparently as the result of the dissociation of introduced carboxyl groups. These phenomena are potentially useful in controlling the complexation ability of beta-chitin and the preparation of regioselectively esterified chitin derivatives.     

Structural data on the intra-crystalline swelling of beta-chitin

The intra-crystalline swelling of the highly crystalline beta-chitin from Tevnia jerichonana was investigated by X-ray crystallography and Fourier transform infrared (FTIR) spectroscopy, using hydrogenated and deuterated hydrochloric acids as swelling agents. Three levels of swelling were identified that could be defined as inter- and intra-sheet swelling. A moderate and reversible swelling in water and methanol gave crystalline beta-chitin cystallosolvates, namely dihydrate and methanolate, respectively. In these, an inter-sheet swelling was observed, corresponding to an expansion of only the b parameter of the unit cell of beta-chitin. Under these swelling conditions, the use of deuterated reagents had no effect on the amide N&z.sbnd;H⋯O&z.dbnd6;C hydrogen bonds that hold the structure of beta-chitin together, but only induced a partial and reversible deuteration of the chitin hydroxymethyl groups. A more severe swelling - but still reversible - occurred with 6 N HCl or DCl, which converted the crystals of beta-chitin into a paracrystalline gel-like product resulting from inter-sheet+intra-sheet swelling. With this acid strength, the deuteration pattern indicated that a fraction of the amide hydrogen bonds was broken and became susceptible to an irreversible deuteration. A very severe and irreversible swelling occurred with 8 N HCl or DCl. In that case, the inter- and intra-sheet swelling was extensive to the point where all memory of the parallel-chain beta-chitin was lost. In addition, this swelling was accompanied by a drastic and rapid depolymerization. The treatment with 8 N HCl led invariably to crystalline alpha-chitin when the samples were neutralized.     

Preparation and characterization of novel beta-chitin-hydroxyapatite composite membranes for tissue engineering applications

Beta-chitin is a biopolymer principally found in shells of squid pen. It has the properties of biodegradability, biocompatibility, chemical inertness, wound healing, antibacterial and anti-inflammatory activities. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has osteoconductive property. In this work, beta-chitin-HAp composite membranes were prepared by alternate soaking of beta-chitin membranes in CaCl2 (pH 7.4) and Na2HPO4 solutions for 2 h in each solution. After 1, 3 and 5 cycles of immersion, beta-chitin membranes were characterized using the SEM, FT-IR, EDS and XRD analyses. The results showed the presence of apatite layer on surface of beta-chitin membranes, and the amounts of size and deposition of apatite layers were increased with increasing number of immersion cycles. Human mesenchymal stem cells (hMSCs) were used for evaluation of the biocompatibility of pristine as well as composite membranes for tissue engineering applications. The presence of apatite layers on the surface of beta-chitin membranes increased the cell attachment and spreading suggesting that beta-chitin-HAp composite membranes can be used for tissue engineering applications.     

Formation of a lamellar compound by reaction of acrylic acid crystallosolvated in highly crystalline beta-chitin

A lamellar compound resulted from reaction of acrylic acid inside crystalline beta-chitin and the structure was investigated. Beta-chitin acts like a layered crystal, having stacked molecular sheets composed of parallel chains bound in one direction by intermolecular amide hydrogen bonding. Small guest molecules can be inserted between the molecular sheets, and a crystallosolvate can be formed. By immersion of beta-chitin in acrylic acid, a crystallosolvate was formed, which was then changed into the more stable lamellar compound by heat treatment at 105 degrees C. NMR measurement and IR spectroscopy showed that during the heat treatment there was a reaction between acrylic acid and the beta-chitin molecular sheet, but the sheet structure was maintained. By IR with deuteration, it was shown that the accessibility of solvents to this lamellar compound was greater than that for the initial beta-chitin. The lamellar compound is considered a kind of "pillared" structure related to the lamellar crystal.     

Beta-chitin from the pens of Loligo sp.: extraction and characterization

Recently the squid pens, a rich source of beta-chitin containing low contents of inorganic compounds, have become available in considerable amounts as a refuse of the fishery industries in Brazil. Thus, the aim of this work is to use squid pens from Loligo sanpaulensis and Loligo plei, species found in the Brazilian coast, as the raw material for the extraction of beta-chitin. The squid pens were submitted to the usual sequence of treatments used for chitin extraction - demineralization and deproteinization - but due to its low content of inorganic compounds a two-step alkaline treatment was enough to produce beta-chitin with low contents of ash (< or = 0.7%). Indeed, the low contents of ash and metals, such as Ca (< or = 10.4 ppm), Mg (< or = 2.5 ppm), Mn (< or = 3.1 ppm) and Fe (< or = 1.8 ppm), are lower than those reported in most of the papers found in the literature. Also, the beta-chitin extracted by employing only the alkaline treatment was more acetylated than the other samples prepared in this work. Regardless of the treatment employed for the extraction of the beta-chitin from the squid pens, its infrared spectra and X-ray diffraction pattern presented only minor differences, however they were clearly distinguished from commercial alpha-chitin.     

Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase

Finely powdered alpha- and beta-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and beta-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and Bacillus licheniformis SK-1 were used to digest alpha- and beta-chitin powder. Chitinase from B. cepacia TU09 produced GlcNAc in greater than 85% yield from beta- and alpha-chitin within 1 and 7 days, respectively. B. licheniformis SK-1 chitinase completely hydrolyzed beta-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose. However, only a 41% yield of GlcNAc was achieved from digesting alpha-chitin with B. licheniformis SK-1 chitinase.     

Alkali-induced conversion of beta-chitin to alpha-chitin

Crystal conversion of beta-chitin to alpha-chitin by aq. NaOH treatment was studied for a highly crystalline beta-chitin sample from diatom spine. The minimum NaOH concentration to cause swelling was between 25% and 30% w/w. The alkali-swollen material was poorly crystalline and was regenerated as alpha-chitin on washing with water. This conversion caused total collapse of the original microfibrillar morphology. These features are similar to those of 7 N-8 N HCl treatment reported earlier, but alkali treatment was free from depolymerization or deacetylation.     

Synthesis, characterization and bioactivity studies of novel beta-chitin scaffolds for tissue-engineering applications

Chitin is a biopolymer and it is non-toxic, biodegradable and biocompatible. Chitin has many potential industrial applications because of its abundance, biodegradability, non-toxicity, chemical inertness. beta-Chitin scaffolds were prepared by using saturated calcium chloride alcoholic solution (CaCl(2).6H(2)O/EtOH) and then followed by dialysis with lyophilization. The prepared beta-chitin scaffolds were characterized by FT-IR, scanning electron microscopy (SEM) and thermogravimetric (TGA). The preliminary bioactivity studies of beta-chitin scaffolds were studied by using simulated body fluid (SBF) solution for 7, 14 and 21 days. We also immersed the beta-chitin scaffolds in saturated aqueous CaCl(2) and Na(2)HPO(4) solution over 12h. After 7, 14 and 21 days, the scaffolds were characterized by SEM and FT-IR studies. The SEM studies showed that there is a calcium phosphate layer in the surface as well as in the cross-section of beta-chitin scaffolds. It seems that the beta-chitin scaffolds are useful in the tissue-engineering field.     

Innovative crystal transformation of dihydrate trehalose to anhydrous trehalose using ethanol

The crystal transformation of dihydrate trehalose to anhydrous trehalose was investigated using ethanol and a new type of crystal particle with porous structure could be obtained. The specific surface area of the anhydrous crystal transformed at 50 degrees C was 3.3 m(2)/g, with a median pore diameter of 0.21 microm, and void volume of 0.22 mL/g. The crystal transformation was monitored by measuring the crystal moisture content. The crystal transformation rates could be correlated with the Avrami equation, using the mechanism parameter n=11.5, suggesting that the change of surface area occurred during crystal transformation from dehydrate to anhydrous trehalose. The apparent activation energy of the crystal transformation was 132 kJ/mol.     

Structure of the nacreous organic matrix of a bivalve mollusk shell examined in the hydrated state using cryo-TEM

During mollusk shell formation, the mineral phase forms within an organic matrix composed of beta-chitin, silk-like proteins, and acidic glycoproteins rich in aspartic acid. The matrix is widely assumed to play an important role in controlling mineralization. Thus, understanding its structure is of prime importance. Cryo-transmission electron microscopy (Cryo-TEM) studies of the matrix of the bivalve Atrina embedded in vitrified ice show that the interlamellar sheets are composed mainly of highly ordered and aligned beta-chitin fibrils. The silk, which is quantitatively an important component of the matrix, could not be imaged within the sheets. Organic material was, however, observed between sheets. We infer that this is the location of the silk. As this material reveals no regular structure, we suggest that at least prior to mineralization the silk is in the form of a hydrated gel. This is supported by cryo-TEM structural observations of an artificial assembly of beta-chitin with and without silk. This view of the nacreous organic matrix significantly changes previous models of the matrix structure and hence hypotheses pertaining to the mechanisms by which mineral formation occurs.     

Crystallization of calcium carbonate salts into beta-chitin scaffold

Composites of beta-chitin with calcium carbonate polymorphs were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. The three main polymorphs of calcium carbonate: aragonite, calcite and vaterite, were observed. Their location within the matrix is a function of the polymorph. The supersaturation inside the compartmentalized space in the chitin governs the location and polymorphism of the crystals.     

Inclusion complex of beta-chitin and aliphatic amines

Inclusion complexation of beta-chitin with linear aliphatic amines was studied by X-ray diffraction. All tested amines, C3 to C8 monoamines and C2 to C7 diamines with terminal amino groups, reversibly formed crystalline complexes with beta-chitin by immersion of dry chitin in pure liquid. Complex formation caused linear increase in the 010 sheet spacing of beta-chitin depending on the carbon number of amine. The complexes could be classified as type I and type II according to the increment of sheet spacing against carbon number. All monoamines formed type II complexes. In dry conditions, diamine formed a type I complex though the type of diamine complex differed for guest species in wet conditions. Based on the unit cell dimension and thermogravimetry, type II and type I are likely to correspond to guest-host (amine-chitobiose) ratios of 2:1 and 1:1, respectively. These differences seem to arise from varied interactions between functional groups of chitin and amines.     

Oriented crystallization of octacalcium phosphate into beta-chitin scaffold

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.     

Importance of exposed aromatic residues in chitinase B from Serratia marcescens 2170 for crystalline chitin hydrolysis

Chitinase B (ChiB) of S. marcescens has five exposed aromatic residues linearly aligned toward the catalytic cleft, Tyr481 and Trp479 in the C-terminal domain, and Trp252, Tyr240 and Phe190 in the catalytic domain. To determine the contribution of these residues to the hydrolysis of crystalline beta-chitin, site-directed mutagenesis, to replace them by alanine, was carried out. The Y481A, W479A, W252A, and Y240A mutations all decreased the binding activity and hydrolyzing activity toward beta-chitin microfibrils. Substitution of Trp residues affected the binding activity more severely than that of Tyr residues. The F190A mutation decreased neither the binding activity nor the hydrolyzing activity. None of the mutations decreased the hydrolyzing activity toward soluble substrates. These results suggest that ChiB hydrolyzes crystalline beta-chitin via a mechanism in which four exposed aromatic residues play important roles, similar to the mechanism of hydrolysis by ChiA of this bacterium, although the directions of hydrolysis of the two chitinases are opposite.     

Thermodynamic aspects of the heterogeneous deacetylation of beta-chitin: reaction mechanisms

This paper aims at giving a better understanding of the reaction mechanisms involved in the heterogeneous deacetylation of beta-chitin in relation with the influence of soda concentration (30-55% (w/v)) and the type of sodium hydroxide hydrates formed in solution. The role of temperature (35-110 degrees C) and of the amount of sodium acetate generated in the reaction medium was also investigated. We demonstrated that the type of soda hydrate formed before deacetylation starts and its relative abundance drive the reaction efficiency. Thus, in the first part of this work, we evidenced that activation energies and the global reaction order associated to sodium hydroxide varied as a function of soda concentration. Therefore, we revealed that deacetylation efficiency was emphasized when the less hydrated soda was used, whereas anhydrous soda showed no or very low activity. We also pointed out that various parameters could be responsible for the progressive dehydration of the reaction medium, responsible for the transformation of the most reactive hydrates into less effective species. We underlined that this progressive dehydration could be caused by either one or all of the three following phenomena: alkaline hydrolysis of the polymer, the delivery of sodium acetate in the medium, and the evaporation of water when we process deacetylation at high temperatures and in open reactors. Beside kinetics reasons, we revealed that the transformation of soda hydrates as the deacetylation proceeded was also ascribable for the low reaction efficiency at long reaction times. Thanks to our investigations, we concluded that the amount of water present in the system chitin/soda/water/sodium acetate was the angle stone of complex equilibriums governing the reaction, and we propose soda mono- and dihydrates to be the most active reactants for the chitin deacetylation.     

Substrate specificity of chitinases from two species of fish, greenling, Hexagrammos otakii, and common mackerel, Scomber japonicus, and the insect, tobacco hornworm, Manduca sexta

Three chitinase isozymes, HoChiA, HoChiB, and HoChiC, were purified from the stomach of the greenling, Hexagrammos otakii, by ammonium sulfate fractionation, followed by column chromatography on Chitopearl Basic BL-03 and CM-Toyopearl 650S. The molecular masses and pIs of HoChiA, HoChiB, and HoChiC are 62 kDa and pH 5.7, 51 kDa and pH 7.6, and 47 kDa and pH 8.8, respectively. Substrate specificities of these chitinases were compared with those of another fish stomach chitinase from the common mackerel, Scomber japonicus (SjChi), as well as two from the tobacco hornworm, Manduca sexta (MsChi535 and MsChi386). The efficiency parameters, kcat/Km, toward glycolchitin for HoChiA and SjChi were larger than those for HoChiB and HoChiC. The relative activities of HoChiA and SjChi toward various forms of chitin were as follows: shrimp shell or crab shell alpha-chitin > beta-chitin > silkworm cuticle alpha-chitin. On the other hand, the relative activities of HoChiB and HoChiC were beta-chitin > silkworm alpha-chitin > shrimp and crab alpha-chitin. MsChi535 preferred silkworm alpha-chitin to shrimp and crab alpha-chitins, and no activity was observed toward beta-chitin. MsChi386, which lacked the C-terminal linker region and the chitin-binding domain, did not hydrolyze silkworm alpha-chitin. These results demonstrate that fish and insect chitinases possess unique substrate specificities that are correlated with their physiological roles in the digestion of food or cuticle.     

Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12

Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD(ChiA1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(ChiA1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(ChiA1) was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD(ChiA1) to regenerated chitin. These observations suggest that ChBD(ChiA1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(ChiA1) exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD(ChiA1) is mediated mainly by hydrophobic interactions. Hydrolysis of beta-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta-chitin microcrystals but greatly enhances the efficiency of degradation.     

Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1

We describe the overexpression and characterization of a new 30 kDa family 18 chitinase (Ech30) from Trichoderma atroviride strain P1. Sequence alignments indicate that the active site architecture of Ech30 resembles that of endochitinases such as hevamine from the rubber tree (Hevea brasiliensis). The ech30 gene was overexpressed in Escherichia coli without its signal peptide and with an N-terminal His-tag. The enzyme was produced as inclusion bodies, from which active chitinase could be recovered using a simple refolding procedure. The enzyme displayed an acidic pH-optimum (pH 4.5-5.0), probably due to the presence of a conserved Asn residue near the catalytic glutamate, which is characteristic for acidic family 18 chitinases. Studies with oligomers of N-acetylglucosamine [(GlcNAc)(n)], 4-methylumbelliferyl (4-MU) labelled GlcNAc oligomers and beta-chitin reveal enzymatic properties typical of an endochitinase: 1) low activity towards short substrates (kinetic parameters for the hydrolysis of 4-MU-(GlcNAc)2 were K(m), 149+/-29 microM and k(cat), 0.0048+/-0.0005 s(-1)), and 2) production of relatively large amounts of trimers and tetramers during degradation of beta-chitin. Detailed studies with GlcNAc oligomers indicated that Ech30 has as many as seven subsites for sugar binding. As expected for a family 18 chitinase, catalysis proceeded with retention of the beta-anomeric configuration.