Chitin nanocrystals prepared by TEMPO-mediated oxidation of alpha-chitin

Chitin nanocrystals dispersed in water were successfully prepared by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation of alpha-chitin in water at pH 10 under specific conditions, followed by ultrasonic treatment. When the amount of NaClO added as co-oxidant in the oxidation was 5.0 mmol/g of chitin, the weight percentage of the water-insoluble fraction in the TEMPO-oxidized chitin was 90%, and its carboxylate content reached 0.48 mmol/g. Since the TEMPO-oxidized chitin thus prepared had a crystallinity as high as that of the original alpha-chitin, the C6 carboxylate groups formed by TEMPO-mediated oxidation can be regarded as being present only on the chitin crystallite surfaces. No N-deacetylation occurred on the TEMPO-oxidized chitins. When the TEMPO-oxidized chitin was subjected to ultrasonic treatment in water, mostly individualized chitin nanocrystals were obtained, and the average nanocrystal length and width were 340 and 8 nm, respectively.     

Chitin binding by Thermobifida fusca cellulase catalytic domains

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose.     

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.     

Alpha-chitin nanocrystals prepared from shrimp shells and their specific surface area measurement

Alpha-chitin was isolated from shrimp shells. The chitin was subjected to extensive treatments of acid hydrolysis and mechanical disruption to yield nanocrystals. The goal of this article is to characterize alpha-chitin nanocrystals produced from shrimp shells in regard to crystallite properties and the specific surface area of the chitin nanoparticles. X-ray diffraction data indicate an increase in chitin crystallinity after hydrolysis, as less-ordered chitin domains are digested. Line broadening data were used to measure crystallite size and particle size in the hydrolyzed chitin nanocrystals. Dye adsorption with Congo red was used to measure the specific surface area of the particles, indicating values near 350 m2/g. This value was supported with calculations derived from X-ray crystallite size measurements. Particle surface area measurements were compared with similarly prepared cellulose nanocrystals.     

Chitin biosynthesis by a fungal membrane preparation. Evidence for a transient non-crystalline state of chitin

Chitin synthase activity of membrane preparations from hyphae of Schizophyllum commune was strongly inhibited by added chitinase because chitin immediately after its synthesis was highly susceptible to chitinase. In the absence of synthesis, chitin became more resistant to chitinase with time. Chitin synthesized in the presence of the optical brightener Calcofluor White M2R was extremely susceptible to degradation by chitinase and this susceptibility was maintained for a long time. X-ray diffraction analysis of chitin synthesized in the presence of Calcofluor revealed the absence of crystallinity as long as the material was kept in wet conditions. After drying, discrete deflections characteristic for alpha-chitin appeared concomitant with a decrease in the susceptibility for chitinase. These results strongly suggest the existence of a gap between polymerization and crystallization of chitin chains.     

First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (demospongia: Porifera)

The Porifera (sponges) are often regarded as the oldest, extant metazoan phylum, also bearing the ancestral stage for most features occurring in higher animals. The absence of chitin in sponges, except for the wall of peculiar resistance bodies produced by a highly derived fresh-water group, is puzzling, since it points out chitin to be an autapomorphy for a particular sponge family rather than the ancestral condition within the metazoan lineage. By investigating the internal proteinaceous (spongin) skeleton of two demosponges (Aplysina sp. and Verongula gigantea) using a wide array of techniques (Fourier transform infrared (FTIR), Raman, X-ray, Calcofluor White Staining, Immunolabeling, and chitinase test), we show that chitin is a component of the outermost layer (cuticle) of the skeletal fibers of these demosponges. FTIR and Raman spectra, as well as X-ray difractograms consistently revealed that sponge chitin is much closer to the alpha-chitin known from other animals than to beta-chitin. These findings support the view that the occurrence of a chitin-producing system is the ancestral condition in Metazoa, and that the alpha-chitin is the primitive form in animals.     

Dissociation of chitosomes by digitonin into 16 S subunits with chitin synthetase activity

Digitonin exerts profound effects on chitosomes (microvesicular structures with chitin synthetase activity isolated from the fungus Mucor rouxii). At low concentrations, it stimulates chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-beta acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) activity; at higher concentrations, it inhibits it. Digitonin also causes disintegration of the chitosome and the release of a homogeneous population of chitosome subunits with chitin synthetase activity. These chitosome subunits have a sedimentation coefficient of 16 S, compared to 105 S for whole chitosomes, as determined by centrifugation in sucrose density gradients, and measure 7--12 nm in diameter. After dissociation, chitin synthetase remains in a zymogenic state, and requires treatment with a protease for activation. No change in sedimentation coefficient of chitosome subunits was observed after proteolytic activation. The product synthesized by the chitosome subunits was characterized by X-ray diffractometry ad alpha-chitin and was by the criterion indistinfuishable from chitin made by preparations of undissociated chitosomes. However, in the electron microscope, the chitin microfibrils made from chitosome subunits were, in general, much shorter than those produced by undissociated chitosomes and often exhibited a needle-like appearance.     

Single crystals of alpha-chitin.

Single crystals of alpha-chitin were grown by the addition of precipitants to dilute solutions of low molecular weight chitin fractions dissolved in aqueous LiSCN. At temperatures around 200 degrees C, bundles of thin needle-shaped crystals were obtained. Each of these needles was an alpha-chitin single crystal, characterized by a spot electron diffraction pattern which could be indexed along the hk0 reciprocal net corresponding to the Minke and Blackwell unit cell [a = 0.474 nm, b = 1.88 nm, c (fibre axis) = 1.032 nm, space group P2(1)2(1)2(1)]. In a crystal, the a* parameter was along the crystal axis and the b* perpendicular to it.     

Two-dimensional magic angle spinning NMR investigation of naturally occurring chitins: precise 1H and 13C resonance assignment of alpha- and beta-chitin

13C homonuclear through-bond correlations of alpha- and beta-chitin were determined by using two-dimensional (2D) INADEQUATE spectra of these allomorphs purified from crab shell and squid pen, respectively. The 2D (13)C-(13)C correlation spectra where two directly bonded carbons share a common double-quantum frequency (DQ) enabled us to precisely assign all (13)C resonances of the chitin allomorphs for the first time. Following the complete (13)C assignment, (1)H chemical shifts of protons attached to each carbon nuclei were assigned by 2D frequency-switched Lee-Goldberg (FSLG) (1)H-(13)C heteronuclear correlation (HETCOR) spectra of the chitin allomorphs, recorded with a short mixing time (60 micros) to provide isotropic (1)H-(13)C chemical shift correlations between bonded pairs proton and carbon nuclei. From the (13)C and (1)H chemical shifts of chitin allomorphs, all 2-deoxy-2-acetamide-D-glucose (N-acetyl-D-glucosamine) monomer units in each allomorph were revealed to be an identical (13)C-(13)C backbone conformation and magnetically equivalent. In addition, it was strongly suggested that there are two different hydrogen-bonding patterns at the hydroxyl groups of alpha-chitin by comparing (1)H chemical shifts at the C6 site of alpha-chitin with those at the same site of beta-chitin.     

Identification and characterization of the gene cluster involved in chitin degradation in a marine bacterium, Alteromonas sp. strain O-7.

Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when alpha-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc)(2) from chitin. The optimum temperature and pH of ChiD were 50 degrees C and 7.0, respectively.     

Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction

A new method to determine the degree of deacetylation (DD) of alpha-chitin and chitosan in the range of 17-94% DD using X-ray powder diffraction (XRD) is proposed. The results were calibrated using (1)H NMR spectroscopy for chitosan and FTIR for chitin, in comparison with the potentiometric titration method. The results showed a good linear correlation between the CrI020 from XRD and the calibrated DD value. This method is found to be simple, rapid and nondestructive to the sample.     

Chitinolytic bacteria in the intestinal tract of Japanese coastal fishes

Intestinal bacteria from several coastal fish species were screened on 1/20 PYBG medium containing 0.2% colloidal chitin, and 361 bacteria capable of decomposing colloidal chitin were isolated. These isolates were subsequently screened on media containing either 0.5% alpha-chitin or 0.5% beta-chitin resulting in the identification of 31 alpha-chitinolytic and 275 beta-chitinolytic bacterial isolates. Partial 16S rRNA gene sequencing was carried out and homology searches of the resultant sequences against the DDBJ, EMBL, and GenBank databases revealed that the majority (99%) of the chitinolytic bacteria isolated belonged to the Vibrionaceae. Phylogenetic analysis using a Bayesian approach showed that the alpha-chitinolytic bacteria belonging to the Vibrionaceae formed a separate cluster from the non-alpha-chitinolytic bacteria in the Vibrionaceae.     

Solution properties of chitin in alkali

The solution properties of alpha-chitin dissolved in 2.77 M NaOH are discussed. Chitin samples in the weight-average molecular weight range 0.1 x 10(6) g/mol to 1.2 x 10(6) g/mol were prepared by heterogeneous acid hydrolysis of chitin. Dilute solution properties were measured by viscometry and light scattering. From dynamic light scattering data, relative similar size distributions of the chitin samples were obtained, except for the most degraded sample, which contained aggregates. Second virial coefficients in the range 1 to 2 x 10(-3) mL.mol.g(-2) indicated that 2.77 M NaOH is a good solvent to chitin. The Mark-Houwink-Sakurada equation and the relationship between the z-average radius of gyration (Rg) and the weight-average molecular weight (Mw) were determined to be [eta] = 0.10Mw0.68 (mL.g(-1)) and Rg = 0.17Mw0.46 (nm), respectively, suggesting a random-coil structure for the chitin molecules in alkali conditions. These random-coil structures have Kuhn lengths in the range 23-26 nm.     

Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system

Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to alpha-chitin and beta-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.     

An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.     

Characteristics of a Streptomyces coelicolor A3(2) extracellular protein targeting chitin and chitosan

Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets alpha-chitin, beta-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.     

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.     

Chitin hydrolysis by Listeria spp., including L. monocytogenes

Listeria spp., including the food-borne pathogen Listeria monocytogenes, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear beta-1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that L. monocytogenes and other Listeria spp. are able to hydrolyze alpha-chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30 degrees C than at 37 degrees C. In L. monocytogenes EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (chiB) and lmo1883 (chiA), but not on a gene encoding a putative chitin binding protein (lmo2467). The chiB and chiA genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of Listeria are discussed.     

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 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.     

Lattice resolution in alpha-chitin

The lattice images of the alpha-chitin microfibrils from lobster tendon were recorded with a transmission electron microscope operated at 120 keV. It was concluded that a close resemblance exists between alpha-chitin microfibrils and cellulose microfibrils. In both cases, the microfibrils are elongated single crystals (crystallites) of high perfection, with the chains aligned and probably fully extended along the microfibril axis.