Chitin Oligosaccharides Research Articles

Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin

A chitin-degrading bacterial strain, KN1699, isolated from Yatsu dry beach (Narashino, Chiba Prefecture, Japan), was identified as Vibrio parahaemolyticus. Treatment of powdered chitin with crude enzyme solution prepared from the supernatant of KN1699 cultures yielded a disaccharide, beta-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN), as the primary chitin degradation product. The extracellular enzymes involved in the production of this heterodisaccharide, chitinase (Pa-Chi; molecular mass, 92 kDa) and chitin oligosaccharide deacetylase (Pa-COD; molecular mass, 46 kDa), were isolated from the crude enzyme solution, and their hydrolysis specificities were elucidated. These studies confirmed that (1) Pa-Chi hydrolyzes chitin to produce (GlcNAc)(2) and (2) Pa-COD hydrolyzes the acetamide group of reducing end GlcNAc residue of (GlcNAc)(2). These findings indicate that GlcNAc-GlcN is produced from chitin by the cooperative hydrolytic reactions of both Pa-Chi and Pa-COD.

Inhibitory analysis of the effect of polycyclic aromatic hydrocarbons on the activity of chitinase by means of liquid chromatography-mass spectrometry of chitin oligosaccharides

The analytical method of determining enzyme activity by liquid chromatography-mass spectrometry (LC/MS) was developed and applied for investigation of the effect of polycyclic aromatic hydrocarbons (PAHs) on the enzyme activity of chitinase. The measurement of chitinase activity by LC/MS is useful in order to use the nonderivatized substrate, which can show in vivo chitinase activity. Substrate consumption and product formation were monitored in order to determine chitinase activity. It was shown that, for the first time, in vitro addition of PAHs inhibited the activity of chitinase in a noncompetitive manner. The IC(50) value of benzo[a]pyrene was 1.4 microM, and PAHs containing four or more aromatic rings showed the same or higher inhibitory effect, whereas PAHs with a lower number of aromatic rings showed lower inhibition of the chitinase activity than benzo[a]pyrene.

Bacterial Lipopolysaccharides Induce Defense Responses Associated with Programmed Cell Death in Rice Cells

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.

Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

We used a comparative genomics approach implemented in the SEED annotation environment to reconstruct the chitin and GlcNAc utilization subsystem and regulatory network in most proteobacteria, including 11 species of Shewanella with completely sequenced genomes. Comparative analysis of candidate regulatory sites allowed us to characterize three different GlcNAc-specific regulons, NagC, NagR, and NagQ, in various proteobacteria and to tentatively assign a number of novel genes with specific functional roles, in particular new GlcNAc-related transport systems, to this subsystem. Genes SO3506 and SO3507, originally annotated as hypothetical in Shewanella oneidensis MR-1, were suggested to encode novel variants of GlcN-6-P deaminase and GlcNAc kinase, respectively. Reconstitution of the GlcNAc catabolic pathway in vitro using these purified recombinant proteins and GlcNAc-6-P deacetylase (SO3505) validated the entire pathway. Kinetic characterization of GlcN-6-P deaminase demonstrated that it is the subject of allosteric activation by GlcNAc-6-P. Consistent with genomic data, all tested Shewanella strains except S. frigidimarina, which lacked representative genes for the GlcNAc metabolism, were capable of utilizing GlcNAc as the sole source of carbon and energy. This study expands the range of carbon substrates utilized by Shewanella spp., unambiguously identifies several genes involved in chitin metabolism, and describes a novel variant of the classical three-step biochemical conversion of GlcNAc to fructose 6-phosphate first described in Escherichia coli.

Salicylic Acid and a Chitin Elicitor Both Control Expression of theCAD1Gene Involved in the Plant Immunity ofArabidopsis

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.

Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase

Bacillus subtilis W-118, a strain that produces antifungal materials, excreted a chitinase when cultured in a medium containing shrimp- and crab-shell powder as the major carbon source. This chitinase, purified by sequential chromatography, had a molecular mass of 20,600 Da and a p I of 6. The optimum pH, optimum temperature, and pH stability of the chitinase were pH 6, 37 °C, and pH 5–7, respectively. The unique characteristics of the purified chitinase include low molecular mass and acidic p I. In the investigation of the inhibitory activity, it was found that the growth of Fusarium oxysporum was 100% inhibited after incubation for 1 day with sterilized W-118 chitinase solution (5.6 units/mL). The chitinase hydrolyzates of chitin with low degrees of polymerization (DP 1–6) were analyzed by HPLC. Longer reaction times led to the generation of chitin oligosaccharides with lower DP. The chitin oligosaccharides were examined for their inhibitory effects on F. oxysporum and human leukemia cell lines.

Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor

Chitin is a major component of fungal cell walls and serves as a molecular pattern for the recognition of potential pathogens in the innate immune systems of both plants and animals. In plants, chitin oligosaccharides have been known to induce various defense responses in a wide range of plant cells including both monocots and dicots. To clarify the molecular machinery involved in the perception and transduction of chitin oligosaccharide elicitor, a high-affinity binding protein for this elicitor was isolated from the plasma membrane of suspension-cultured rice cells. Characterization of the purified protein, CEBiP, as well as the cloning of the corresponding gene revealed that CEBiP is actually a glycoprotein consisting of 328 amino acid residues and glycan chains. CEBiP was predicted to have a short membrane spanning domain at the C terminus. Knockdown of CEBiP gene by RNA interference resulted in the suppression of the elicitor-induced oxidative burst as well as the gene responses, showing that CEBiP plays a key role in the perception and transduction of chitin oligosaccharide elicitor in the rice cells. Structural analysis of CEBiP also indicated the presence of two LysM motifs in the extracellular portion of CEBiP. As the LysM motif has been known to exist in the putative Nod-factor receptor kinases involved in the symbiotic signaling between leguminous plants and rhizobial bacteria, the result indicates the involvement of partially homologous plasma membrane proteins both in defense and symbiotic signaling in plant cells.

Enzymatic hydrolysis of chitin in the production of oligosaccharides using Lecanicillium fungicola chitinases

Lecanicillium fungicola was selected among 15 strains as chitinase producer and it was used to obtain the crude enzyme (UFL) in submerged fermentation (SF) with added chitin. The UFL displayed at pH 6 and 40 °C the highest endochitinase (Endo) and N-acetylhexosaminidase (NHase) activities, 747 and 410 U/mg, respectively. Four bands of proteins with molecular weights of 123.1, 85.5, 33.1 and 23 kDa were detected in UFL by SDS-PAGE. In order to increase solubilities of the substrates, α and β-chitins were treated with alkali; degrees of deacetylation (DD) were determined 55 and 50%, respectively. Thereafter, chitin hydrolysis with UFL was carried out at 40 °C and pH 5, Endo and NHase at these conditions were 619 and 355 U/mg of protein, respectively. The partial deacetylation as well as the use of acidified reaction media improved significantly the enzyme efficiency in terms of yields of chitin oligosaccharides produced and process time. The maximum chitin oligosaccharides concentration ( P max) obtained from α- and β-deacetylated chitins were 2.77 and 4.44 mmol/l, respectively; whereas for α-chitin it was determined a very small amount of product (0.17 mmol/l). Despite of these results, the maximum production rate ( V max 0.0836 mmol/l h) for α-deacetylated chitin was significantly higher than β-deacetylated chitin (0.0363 mmol/l h).

A high-throughput evaluation system for Arabidopsis mutants for defense signaling

This paper describes an efficient evaluation system of Arabidopsis mutants for defense responses. Young A. thaliana seedlings were analyzed for the induction of reactive oxygen species (ROS) as well as defense gene expression by chitin oligosaccharide elicitor in microtiter plates. Combined with the analysis of ROS in microtiter plates, the system enabled to evaluate thousands of mutant plants within a couple of weeks. Pharmacological studies could be applied and provided information about the molecular machinery involved in the defense responses. Experiments with the dissected organs showed that the roots were responsible for most ROS generation while the gene activation was observed in all the organs. An example of the successful application of this system for the screening of Arabidopsis muntats is also presented. Thus, the system provides a promising approach to screen, or evaluate, novel mutants for a confined defense signaling cascade downstream of a specific elicitor.

Production of N,N′-diacetylchitobiose from chitin using temperature-sensitive chitinolytic enzyme preparations of Aeromonas sp. GJ-18

N,N′-diacetylchitobiose was produced from chitin as a major hydrolytic product by controlling the ratio of β-N-acetylglucosaminidase to N,N′-diacetylchitobiohydrolase activities in the crude enzyme preparation of Aeromonas sp. GJ-18. When the enzyme preparation was preincubated at 50 °C, β-N-acetylglucosaminidase was nearly inactivated, while the N,N′-diacetylchitobiohydrolase was still active. Thus, the composition of chitin oligosaccharides depended on the preincubation temperature of the crude enzyme preparations. Typically, after 7 days of incubation with the substrate chitin, 78.9 and 56.6% of N,N′-diacetylchitobiose yields were obtained from swollen α-chitin and powdered β-chitin, respectively, with enzyme preparations that had been pretreated at 50 °C for 60 min.

A β- N-acetylhexosaminidase from Symbiobacterium thermophilum; gene cloning, overexpression, purification and characterization

An ORF encoding the gene ( nahA)-related carbohydrate hydrolase was found in chromosomal DNA of the symbiotic thermophile, Symbiobacterium thermophilum. BLASTX results indicated that the product of this structural gene is β-glucosidase or β- N-acetylhexosaminidase. To investigate details of the nahA gene product, cloning of the gene, overproduction of the gene product in Escherichia coli, and purification and characterization of the resulting protein were conducted. The nahA gene was amplified by PCR using fragmented chromosomal DNA of S. thermophilum as a template, sequenced, and then ligated into the BamHI and NdeI sites of plasmid pET-25b(+) to construct the expression vector pST-BNAH-A for overproduction of the gene product. Results of studies on the hydrolytic activity of cell-free extracts against pNP-β-Glc, pNP-β-GlcNAc and pNP-β-GalNAc, obtained by disruption of cultured E. coli cells harboring pST-BNAH-A, suggested that the nahA gene product was β- N-acetylhexosaminidase. Comparison of the amino acid sequence of the recombinant protein with those of other β- N-acetylhexosaminidases indicated that the β- N-acetylhexosaminidase of S. thermophilum is a member of the 3-glycoside hydrolase family. The recombinant enzyme was purified to homogeneity from cell-free extract in an overall yield of 24%. This purified β- N-acetylhexosaminidase possessed thermostability, was stable in alkaline solution, and exhibited greater hydrolytic activity against chitin oligosaccharides than against pNP-β-GlcNAc.

Capillary electrophoresis separation of a mixture of chitin and chitosan oligosaccharides derivatized using a modified fluorophore conjugation procedure

A capillary electrophoresis (CE) method was developed for the simultaneous analysis of small chitin and chitosan oligosaccharides. For detection purposes, the oligomers were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS), a well known fluorophore for oligosaccharides analysis. The detection was performed by laser-induced fluorescence (LIF) with an argon ion laser having an excitation wavelength of 488 nm and with emission monitored at 520 nm. Derivatization parameters such as reaction time and conditions were examined. Separation conditions were also varied by testing a range of buffer pHs and concentrations. The best conditions were found using an 80 mM borate buffer at pH 8.4. This CE-LIF optimized method was used for the analysis of an enzymatically produced oligo-chitosan sample composed of a complex mixture and having an average degree of polymerization of 3.7 monomer units and 80% deacetylation. The oligo-chitosan sample was treated with a chitin deacetylase-like enzyme, the products were derivatized with APTS, and then analyzed without purification. The goal was to determine whether the deacetylase-like enzyme could increase the extent of deacetylation of the oligo-chitosan sample.

The Vibrio cholerae chitin utilization program.

Chitin, an insoluble polymer of GlcNAc, is an abundant source of carbon, nitrogen, and energy for marine microorganisms. Microarray expression profiling and mutational studies of Vibrio cholerae growing on a natural chitin surface, or with the soluble chitin oligosaccharides (GlcNAc)(2-6), GlcNAc, or the glucosamine dimer (GlcN)2 identified three sets of differentially regulated genes. We show that (i) ChiS, a sensor histidine kinase, regulates expression of the (GlcNAc)(2-6) gene set, including a (GlcNAc)2 catabolic operon, two extracellular chitinases, a chitoporin, and a PilA-containing type IV pilus, designated ChiRP (chitin-regulated pilus) that confers a significant growth advantage to V. cholerae on a chitin surface; (ii) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc; (iii) (GlcN)2 induces genes required for the transport and catabolism of nonacetylated chitin residues; and (iv) the constitutively expressed MSHA pilus facilitates adhesion to the chitin surface independent of surface chemistry. Collectively, these results provide a global portrait of a complex, multistage V. cholerae program for the efficient utilization of chitin.

Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls.

Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the beta -d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.

Enzymatic synthesis of complex glycosaminotrioses and study of their molecular recognition by hevein domains.

Hevein, a protein found in Hevea brasiliensis, has a CRD domain, which is known to bind chitin and GlcNAc-containing oligosaccharides. By using NMR and molecular modeling as major tools we have demonstrated that trisaccharides containing GalNAc and ManNAc residues are also recognized by hevein domains. Thus far unknown trisaccharides GlcNAcbeta(1-->4)GlcNAcbeta(1-->4)ManNAc (1) and GalNAcbeta(1-->4)GlcNAcbeta(1-->4)ManNAc (2) were synthesized with the use of beta-N-acetylhexosaminidase from Aspergillus oryzae. This method is based on the rather unique phenomenon that some fungal beta-N-acetylhexosaminidases cannot hydrolyze disaccharide GlcNAcbeta(1-->4)ManNAc (5) contrary to chitobiose GlcNAcbeta(1-->4)GlcNAc (4) that is cleaved and, therefore, cannot be used as an acceptor for further transglycosylation. Both trisaccharides 1 and 2 were prepared by transglycosylation from disaccharidic acceptor in good yields ranging from 35% to 40%. Our observations strongly indicate that the present nature of the modifications of chitotriose (GlcNAcbeta(1-->lcNAcbeta(1-->4)GlcNAc, 3) at either the non-reducing end (GalNAc instead of GlcNAc) or at the reducing end (ManNAc instead of GlcNAc) do not modify the mode of binding of the trisaccharide to hevein. The association constant values indicate that chitotriose (3) binding is better than that of 1 and 2, and that the binding of (with ManNAc at the reducing end) is favored with respect to that of 2 (with ManNAc at the reducing end with a non-reducing GalNAc moiety).

Molecular Characterization of an Intracellular β-N-Acetylglucosaminidase Involved in the Chitin Degradation System ofStreptomyces thermoviolaceusOPC-520

We purified and characterized an intracellular beta-N-acetylglucosaminidase (NagC) from a cytoplasmic fraction of Streptomyces thermoviolaceus OPC-520. The molecular mass of NagC was estimated to be 60 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the enzyme were 6.0 and 50 degrees C respectively. Purified NagC hydrolyzed chitin oligosaccharides from N,N'-diacetylchitobiose (GlcNAc)(2) to chitopentaose (GlcNAc)(5), hydrolyzed N,N'-diacetylchitobiose especially rapidly, and showed a tendency to decrease with increases in the degree of polymerization. But, NagC didn't hydrolyze chitohexaose (GlcNAc)(6). The gene encoding NagC was cloned and sequenced. The open reading frame of nagC encoded a protein of 564 amino acids with a calculated molecular mass of 62,076 Da. The deduced amino acid sequence of NagC showed homology with several beta-N-acetylglucosaminidases belonging to glycosyl hydrolase family 20. The expression plasmid coding for NagC was constructed in Escherichia coli. The recombinant enzyme showed pH and temperature optima and substrate specificity similar to those of the native enzyme. The gene arrangement near the nagC gene of S. thermoviolaceus OPC-520 was compared with that of S. coelicolor A3(2). Three genes, which appear to constitute an ABC transport system for sugar, were missing in the vicinity of the nagC gene.

The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase.

Chitin, a highly insoluble polymer of GlcNAc, is produced in massive quantities in the marine environment. Fortunately for survival of aquatic ecosystems, chitin is rapidly catabolized by marine bacteria. Here we describe a bacterial two-component hybrid sensor/kinase (of the ArcB type) that rigorously controls expression of approximately 50 genes, many involved in chitin degradation. The sensor gene, chiS, was identified in Vibrio furnissii and Vibrio cholerae (predicted amino acid sequences, full-length: 84% identical, 93% similar). Mutants of chiS grew normally on GlcNAc but did not express extracellular chitinase, a specific chitoporin, or beta-hexosaminidases, nor did they exhibit chemotaxis, transport, or growth on chitin oligosaccharides such as (GlcNAc)(2). Expression of these systems requires three components: wild-type chiS; a periplasmic high-affinity chitin oligosaccharide, (GlcNAc)(n) (n > 1), binding protein (CBP); and the environmental signal, (GlcNAc)(n). Our data are consistent with the following model. In the uninduced state, CBP binds to the periplasmic domain of ChiS and "locks" it into the minus conformation. The environmental signal, (GlcNAc)(n), dissociates the complex by binding to CBP, releasing ChiS, yielding the plus phenotype (expression of chitinolytic genes). In V. cholerae, a cluster of 10 contiguous genes (VC0620-VC0611) apparently comprise a (GlcNAc)(2) catabolic operon. CBP is encoded by the first, VC0620, whereas VC0619-VC0616 encode a (GlcNAc)(2) ABC-type permease. Regulation of chiS requires expression of CBP but not (GlcNAc)(2) transport. (GlcNAc)(n) is suggested to be essential for signaling these cells that chitin is in the microenvironment.

Specific activation of ERK pathways by chitin oligosaccharides in embryonic zebrafish cell lines.

Chitin oligosaccharides (COs) play a role in plant development and are presumed to affect body plan formation during vertebrate embryogenesis. The mechanisms of COs recognition and cellular processes underlying embryonic development are still not understood. We analyze the possible link with the mitogen-activated protein kinase pathway that is conserved in evolution through the plant and animal kingdom and has been implicated in diverse cellular processes, including cell growth, proliferation, differentiation, survival, and vertebrate development. We show that in vivo stimulation of embryonic zebrafish cells ZF13 and ZF29 with chitin tetrasaccharides at 10-9 M concentration transiently induced activation/phosphorylation of extracellular regulated kinases (ERKs), with a maximum after 15 min. Furthermore the biological specificity of chitin tetrasaccharides and various derivatives was examined. The replacement of one or two GlcNAc residues of the chitin backbone by glucose and fucosylation of chitin tetrasaccharides at the reducing terminus caused a complete loss of their activity. We also tested a chitin tetrasaccharide analogue in which the oxygen atoms in glycosidic linkages were replaced by sulfur atoms. This analog, which could not be enzymatically hydrolyzed, was as potent an inducer as chitin tetrasaccharide. These results suggest that the observed activation of ERKs is chitin tetrasaccharide-specific and does not require further enzymatic processing. We examined possible signaling pathways leading to ERK activation by COs by use of phosphospecific antibodies and inhibitors. We conclude that a high-affinity CO receptor system exists that links to the Raf, MEK, and ERK pathway in zebrafish cells.

Properties of catalytic, linker and chitin-binding domains of insect chitinase

Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the K m for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.

Effects of Dietary Supplementation of Fermented Chitin-chitosan (FERMKIT) on Toxicity of Mycotoxin in Ducks

Two experiments were conducted to evaluate the efficacy of dietary FERMKIT, a commercial toxin binder consisting of probiotic-fermented natural product containing chitin, chitosan and chitosan oligosaccharides (FERMKITO  , EASY-BIO SYSTEM, Inc., Korea), in binding aflatoxin (AF) and zearalenone (ZEN) and ameliorating their mycotoxicity in meat type ducks. FERMKIT was supplemented to AF contaminated diets (at 120 ppb) at either 0.3 or 0.6% in experiment 1 and to ZEN contaminated diets (at 150 ppb) at 0.6% in experiment 2. In experiment 1 body weight gains were reduced by 37% and mortality was increased by 18% in ducks fed diet contaminated with AF at 120 ppb compared to ducks fed control diet (<10 ppb AF) for the 4-wk experimental period. However, dietary FERMKIT supplementation effectively alleviated overall toxicity induced by AF. The significant treatment-related changes in feather growth, web-toe hemorrhage, leg deformity, liver paleness, organ weights, hematological values and serum biochemical values, as compared to the control, were observed. The FERMKIT supplementation significantly diminished the adverse effects of AF and restored all the parameters measured back (<0.05) toward the control values. These findings indicated that FERMKIT, when added at the levels of 0.3 or 0.6% in the 120 ppb AF diets, could modulate the toxicity of AF with percentage sorption capacity of 52.70% at the level 0.3% and 79.85% at the level 0.6% of the diets (experiment 1). In experiment 2, FERMKIT, when added at 0.6% to the 150 ppb ZEN diets for the 4-wk experimental period, diminished the toxicity as shown by body weight gain, weights of testicles, oviducts, Bursa of Fabricius and cloaca eversion score as compared with the controls (<10 ppb ZEN) and 150 ppb ZEN diet with no added FERMKIT. The findings indicated that FERMKIT could be protective against the effects of ZEN in young growing ducks with percentage sorption capacity of 67.11% as evaluated from toxicity index parameter measured when added at 0.6% of the diets containing 150 ppb ZEN. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 5 : 706-713)