Chitin Oligosaccharide Deacetylase Research Articles

Impact of chitin-derived β-N-acetyl-D-glucosaminyl-(1,4)-D-glucosamine on chitinase up-regulation in Shewanella baltica.

The first steps in chitin degradation in marine bacteria involve chitinase, which produces N,N'-diacetylchitobiose (GlcNAc)2 from chitin. Moreover, in Vibrio bacteria, chitinase activity is enhanced by heterodisaccharide β-N-acetyl-D-glucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN) produced from (GlcNAc)2 by chitin oligosaccharide deacetylase (COD). However, the role of COD in other marine bacteria, such as Shewanella, remains unexplored. This study investigates GlcNAc-GlcN's impact on chitinase gene expression and enzyme production in S. baltica ATCC BAA-1091, drawing parallels with Vibrio parahaemolyticus RIMD2210633. Using real-time quantitative PCR, the study assesses the up-regulation of chitinase gene expression in S. baltica in response to GlcNAc-GlcN, informed by COD's known ability to produce GlcNAc-GlcN from (GlcNAc)2. In Vibrio, GlcNAc-GlcN considerably up-regulates chitinase gene expression. This study posits a similar regulatory mechanism in S. baltica, with preliminary investigations indicating COD's capacity to produce GlcNAc-GlcN. This study highlights the importance of exploring GlcNAc-GlcN's regulatory role in chitin metabolism across diverse marine bacteria. The potential induction of chitinase production in S. baltica suggests broader ecological implications. Further research is crucial for a comprehensive understanding of chitin utilization and regulatory pathways in marine bacterial genera.

Chitin Heterodisaccharide, Released from Chitin by Chitinase and Chitin Oligosaccharide Deacetylase, Enhances the Chitin-Metabolizing Ability of Vibrio parahaemolyticus.

Vibrio parahaemolyticus RIMD2210633 secretes both chitinase and chitin oligosaccharide deacetylase and produces β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) from chitin. Previously, we reported that GlcNAc-GlcN induces chitinase production by several strains of Vibrio harboring chitin oligosaccharide deacetylase genes (T. Hirano, K. Kadokura, T. Ikegami, Y. Shigeta, et al., Glycobiology 19:1046-1053, 2009). The metabolism of chitin by Vibrio was speculated on the basis of the findings of previous studies, and the role of chitin oligosaccharide produced from chitin has been well studied. However, the role of GlcNAc-GlcN in the Vibrio chitin degradation system, with the exception of the above-mentioned function as an inducer of chitinase production, remains unclear. N,N'-Diacetylchitobiose, a homodisaccharide produced from chitin, is known to induce the expression of genes encoding several proteins involved in chitin metabolism in Vibrio strains (K. L. Meibom, X. B. Li, A. Nielsen, C. Wu, et al., Proc Natl Acad Sci U S A 101:2524-2529, 2004). We therefore hypothesized that GlcNAc-GlcN also affects the expression of enzymes involved in chitin metabolism in the same manner. In this study, we examined the induction of protein expression by several sugars released from chitin using peptide mass fingerprinting and confirmed the expression of genes encoding enzymes involved in chitin metabolism using real-time quantitative PCR analysis. We then confirmed that GlcNAc-GlcN induces the expression of genes encoding many soluble enzymes involved in chitin degradation in Vibrio parahaemolyticus Here, we demonstrate that GlcNAc-GlcN enhances the chitin-metabolizing ability of V. parahaemolyticusIMPORTANCE We demonstrate that β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) enhances the chitin-metabolizing ability of V. parahaemolyticus Members of the genus Vibrio are chitin-degrading bacteria, and some species of this genus are associated with diseases affecting fish and animals, including humans (F. L. Thompson, T. Iida, and J. Swings, Microbiol Mol Biol Rev 68:403-431, 2004; M. Y. Ina-Salwany, N. Al-Saari, A. Mohamad, F.-A. Mursidi, et al., J Aquat Anim Health 31:3-22, 2019). Studies on Vibrio are considered important, as they may facilitate the development of solutions related to health, food, and aquaculture problems attributed to this genus. This report enhances the current understanding of chitin degradation by Vibrio bacteria.

Chitin oligosaccharide deacetylase from Shewanella baltica ATCC BAA-1091.

Chitin oligosaccharide deacetylase (COD) from bacteria that have been examined so far typically comprise two carbohydrate-binding domains (CBDs) and one polysaccharide deacetylase domain. In contrast, Shewanella baltica ATCC BAA-1091 COD (Sb-COD) has only one CBD, yet exhibits chitin-binding properties and substrate specificities similar to those of other CODs.

Review: Symposium on Applied Glycoscience] Chitin Oligosaccharide Deacetylase from Marine Bacteria : Enzymological Characters, Three-dimensional Structure, and the Roles of Its Product

Review: Symposium on Applied Glycoscience] Chitin Oligosaccharide Deacetylase from Marine Bacteria : Enzymological Characters, Three-dimensional Structure, and the Roles of Its Product

【ミニレビュー-第36回糖質科学懇話会-】 <i>Vibrio</i>属細菌のキチンオリゴ糖デアセチラーゼの機能と酵素化学

【ミニレビュー-第36回糖質科学懇話会-】 <i>Vibrio</i>属細菌のキチンオリゴ糖デアセチラーゼの機能と酵素化学

Heterodisaccharide 4-O-(N-acetyl-β-d-glucosaminyl)-d-glucosamine is an effective chemotactic attractant for Vibrio bacteria that produce chitin oligosaccharide deacetylase

To investigate the attractant effect of 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine (GlcNAc-GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc-GlcN from N,N'-diacetylchitobiose (GlcNAc)(2). The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane-migration method. The results demonstrated that GlcNAc-GlcN functions as an effective chemoattractant in the CE family 4 COD-producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc-GlcN appears to stimulate polar flagellum rotation. GlcNAc-GlcN is a specific chemoattractant for the CE family 4 COD-producing vibrios, V. parahaemolyticus and V. alginolyticus. It was clarified for the first time that GlcNAc-GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc-GlcN from (GlcNAc)(2).

Heterodisaccharide 4-O-(N-acetyl-β-d-glucosaminyl)-d-glucosamine is a specific inducer of chitinolytic enzyme production in Vibrios harboring chitin oligosaccharide deacetylase genes

Vibrio parahaemolyticus KN1699 produces 4-O-(N-acetyl-beta-d-glucosaminyl)-d-glucosamine (GlcNAc-GlcN) as a major end product from chitin using two extracellular hydrolases: glycoside hydrolase family 18 chitinase, which produces (GlcNAc)(2) from chitin, and carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), which hydrolyzes the N-acetyl group at the reducing-end GlcNAc residue of (GlcNAc)(2). In this study, we clarified that this heterodisaccharide functions as an inducer of the production of the two above-mentioned chitinolytic enzymes, particularly chitinase. Similar results for chitinase production were obtained with other chitin-decomposing Vibrio strains harboring the CE family 4 COD gene; however, such an increase in chitinase production was not observed in chitinolytic Vibrio strains that did not harbor the COD gene. These results suggest that GlcNAc-GlcN is a unique inducer of chitinase production in Vibrio bacteria that have the COD-producing ability and that the COD involved in the synthesis of this signal compound is one of the key enzymes in the chitin catabolic cascade of these bacteria.

A Chitin Oligosaccharide Deacetylase from Vibrio sp. SN184: Gene Cloning, Overexpression, Purification and Characterization

The chitinolytic bacterial strain SN184, which produces chitin oligosaccharide deacetylase (COD), was identified a species of the genus Vibrio. The COD gene was cloned from the chromosomal DNA of SN184 and its nucleotide sequence was analyzed. The gene encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence of this protein showed high similarity to those of several bacterial CODs belonging to carbohydrate esterase family 4. An expression plasmid containing the COD gene of strain SN184 was constructed and inserted into Escherichia coli cells; the recombinant enzyme was secreted into the culture medium with the aid of the signal sequence. The recombinant enzyme was purified from the culture supernatant and its substrate specificity was investigated. These studies confirmed that the recombinant COD hydrolyzed N-acetyl groups of the second N-acetyl-D-glucosamine residue from the nonreducing-ends of di-N-acetylchitobiose and tri-N-acetylchitotriose.

Purification, Characterization and Cloning of Vibrio parahaemolyticus Chitinolytic Enzymes and Application to Oligosaccharide Production

Chitinase (Pa-Chi) and chitin oligosaccharide deacetylase (Pa-COD) are involved in the production of a heterodisaccharide, β-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN). These enzymes were recovered from the supernatant of Vibrio parahaemolyticus KN1699 cell culture and purified and characterized. For each enzyme, an ORF encoding gene and its signal sequence were cloned from genomic DNA of strain KN1699. In addition, the expression plasmid was constructed for each enzyme gene and inserted into Escherichia coli cells, and recombinant Pa-Chi and Pa-COD (Pa-rChi and Pa-rCOD) were secreted into the culture medium with the aid of signal peptides. Di-N-acetylchitobiose [(GlcNAc)2] was produced in 60% (w/w) yield by cultivating the Pa-rChi-secreting E. coli cells in 2% (w/v) β-chitin-containing medium. Moreover, GlcNAc-GlcN was produced in high yield by treating (GlcNAc)2 with the culture supernatant of Pa-rCOD-secreting E. coli cells.

The Chitin Catabolic Cascade in the Marine Bacterium Vibrio Cholerae: Characterization of a Unique Chitin Oligosaccharide Deacetylase

Chitin, one of the most abundant organic substances in nature, is consumed by marine bacteria, such as Vibrio cholerae, via a multitude of tightly regulated genes (Li and Roseman 2004, Proc Natl Acad Sci USA. 101:627-631). One such gene, cod, is reported here. It encodes a chitin oligosaccharide deacetylase (COD), when cells are induced by chitobiose, (GlcNH(2))(2), or crude crab shells. COD was molecularly cloned (COD-6His), overproduced, and purified to apparent homogeneity. COD is secreted at all stages of growth by induced V. cholerae. The gene sequence predicts a 26 N-terminal amino acid signal peptide not found in the isolated protein. COD is very active with chitin oligosaccharides, is virtually inactive with GlcNAc, and slightly active with colloidal ([(3)H]-N-acetyl)-chitin. The oligosaccharides are converted almost quantitatively to products lacking one acetyl group. The latter were characterized by mass spectrometry (ESI-MS), and treatment with nitrous acid. COD catalyzes the following reactions (n = 2-6): (GlcNAc)(n)--> GlcNAc-GlcNH(2)-(GlcNAc)(n-2) + Ac(-). That is, COD hydrolyzes the N-acetyl groups attached to the penultimate GlcNAc residue. The gene bank sequence data show that cod is highly conserved in Vibrios and Photobacteria. One such gene encodes a deacetylase isolated from V. alginolytics (Ohishi et al. 1997, Biosci Biotech Biochem. 61:1113-1117; Ohishi et al. 2000, J Biosci Bioeng. 90:561-563), that is specific for (GlcNAc)(2), but inactive with higher oligosaccharides. The COD enzymatic products, GlcNAc-GlcNH(2)-(GlcNAc)(n), closely resemble those obtained by hydrolysis of the chitooligosaccharides with Nod B: GlcNH(2)-(GlcNAc)(3-4). The latter are key intermediates in the biosynthesis of Nod factors, critically important in communications between the symbiotic nitrogen fixing bacteria and plants. Conceivably, the COD products play equally important roles in cellular communications that remain to be defined.

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.

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.

Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum.

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosaccharides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three. In vivo radioactive labelling experiments, in which D-[1-14C]-glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo-chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N-acetyl group on the non-reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O-acetyl substituent on the non-reducing terminal sugar residue (designated NodBCL metabolites). The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP-N-acetyl-D-[U-14C]-glucosamine was added using thin-layer chromatography. The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites. The same results were obtained when the NodB and NodC proteins were produced separately in two different strains. On the basis of these and other recent results, we propose that NodB is a chitin oligosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase and, by default, NodA is involved in lipid attachment.