N,N'-Diacetylchitobiose

Insights into Chitin-Degradation Potential of Shewanella khirikhana JW44 with Emphasis on Characterization and Function of a Chitinase Gene SkChi65.[Pubmed:38674717]

Microorganisms. 2024 Apr 11;12(4):774.

Chitin, a polymer of beta-1,4-linked N-acetylglucosamine (GlcNAc), can be degraded into valuable oligosaccharides by various chitinases. In this study, the genome of Shewanella khirikhana JW44, displaying remarkable chitinolytic activity, was investigated to understand its chitin-degradation potential. A chitinase gene SkChi65 from this strain was then cloned, expressed, and purified to characterize its enzymatic properties and substrate hydrolysis. Genome analysis showed that, of the 14 genes related to chitin utilization in JW44, six belonged to glycoside hydrolase (GH) families because of their functional domains for chitin binding and catalysis. The recombinant chitinase SkChi65, consisting of 1129 amino acids, was identified as a member of the GH18 family and possessed two chitin-binding domains with a typical motif of [A/N]KWWT[N/S/Q] and one catalytic domain with motifs of DxxDxDxE, SxGG, YxR, and [E/D]xx[V/I]. SkChi65 was heterologously expressed as an active protein of 139.95 kDa best at 37 degrees C with 1.0 mM isopropyl-beta-d-thiogalactopyranoside induction for 6 h. Purified SkChi65 displayed high stability over the ranges of 30-50 degrees C and pH 5.5-8.0 with optima at 40 degrees C and pH 7.0. The kinetic parameters K(m), V(max), and k(cat) of SkChi65 towards colloidal chitin were 27.2 muM, 299.2 muMs(-1), and 10,203 s(-1), respectively. In addition to colloidal chitin, SkChi65 showed high activity towards glycol chitosan and crystalline chitin. After analysis by thin-layer chromatography, the main products were N,N'-diacetylchitobiose, and GlcNAc with (GlcNAc)(2-6) used as substrates. Collectively, SkChi65 could exhibit both exo- and endochitinase activities towards diverse substrates, and strain JW44 has a high potential for industrial application with an excellent capacity for chitin bioconversion.

Luminescence detection of peptide:N-glycanase activity using engineered split inteins.[Pubmed:36373598]

Chem Commun (Camb). 2022 Nov 29;58(95):13282-13285.

A split intein-based method has been developed to detect peptide:N-glycanase (PNGase) activity in live cells. PNGase cleaves the linkage between N,N'-diacetylchitobiose and the Asn side-chain of N-intein peptides and the products react rapidly with C-intein by protein trans-splicing to generate an active luciferase.

Crystal structure of ChbG from Klebsiella pneumoniae reveals the molecular basis of diacetylchitobiose deacetylation.[Pubmed:36002585]

Commun Biol. 2022 Aug 24;5(1):862.

The chitobiose (chb) operon is involved in the synthesis of chitooligosaccharide and is comprised of a BCARFG gene cluster. ChbG encodes a chitooligosaccharide deacetylase (CDA) which catalyzes the removal of one acetyl group from N,N'-diacetylchitobiose. It is considered a novel type of CDA due to its lack of sequence homology. Although there are various structural studies of CDAs linked to the kinetic properties of the enzyme, the structural information of ChbG is unavailable. In this study, the crystal structure of ChbG from Klebsiella pneumoniae is provided. The molecular basis of deacetylation of diacetylchitobiose by ChbG is determined based on structural analysis, mutagenesis, biophysical analysis, and in silico docking of the substrate, diacetylchitobiose. This study contributes towards a deeper understanding of chitin and chitosan biology, as well as provides a platform to engineer CDA biocatalysts.

Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N'-diacetylchitobiose.[Pubmed:35323464]

Mar Drugs. 2022 Feb 24;20(3):165.

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401(T), with an ability to degrade crystalline chitin into (GlcNAc)(2) (N,N'-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline alpha-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline alpha-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline alpha-chitin, with temperature optima of 45-50 degrees C and pH optima of 7.0-7.5. They are all stable at 40 degrees C and in the pH range of 5.0-11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 degrees C. When acting on crystalline alpha-chitin, the main products of the three chitinases are all (GlcNAc)(2), which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)(2).

Characterization of a beta-N-acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34.[Pubmed:35289418]

J Food Sci. 2022 Apr;87(4):1466-1474.

In applications of chitin, one of the most abundant resources on earth, human milk oligosaccharides with many health functions were synthesized by transglycosylation of beta-N-acetylhexosaminidase. Synthesis of new transfer products can be expected by other beta-N-acetylhexosaminidases in nature. A total of 38 microorganisms that secrete beta-N-acetylhexosaminidases with transglycosylation activity were isolated from a soil screen. Using N,N'-diacetylchitobiose as the substrate, the transfer ratio increased with a decrease in substrate degradation when it was less than 60%. Metarhizium sp. A34 beta-N-acetylhexosaminidase had high transglycosylation activity and showed a maximum production of the oligosaccharides against the substrate degradation where (GlcNAc)(5) and (GlcNAc)(4) were produced in addition to (GlcNAc)(3) . The maximum curve was attributed to a sequential reaction of transglycosylation followed by hydrolysis where oligosaccharides are an intermediate product and are hydrolyzed in a second step. The purified beta-N-acetylhexosaminidase from Metarhizium sp. A34 had an optimal pH of 5 and was stable from pH 7 to 8. At pH 5, it had an optimal temperature of 40 degrees C and was stable up to 30 degrees C for 30 min. This enzyme had high thermostability up to 55 degrees C when bound to the cell wall. The acceptor specificity for the transglycosylation reaction was enhanced for lower molecular weight sugar alcohols in the order of glycerin (C3), erythritol (C4), and xylitol (C5). The transfer product with glycerin was identified as 1-O-beta-d-N-acetylglucosaminyl glycerin, which may prove useful as a starting material for new glycolipids in food applications. PRACTICAL APPLICATION: Metarhizium sp. A34 beta-N-acetylhexosaminidase produced 1-O-beta-d-N-acetylglucosaminyl glycerin through the transglycosylation. Chitin oligosaccharides of the donor are obtained by hydrolysis of chitin. 1-O-beta-d-N-Acetylglucosaminyl glycerin may be useful to start material for the synthesis of new glycolipids. High thermostability of this enzyme is useful to prevention of contamination in the transglycosylation reaction.

Occurrence of free N-glycans with a single GlcNAc at the reducing termini in animal sera.[Pubmed:34939097]

Glycobiology. 2022 Mar 31;32(4):314-332.

Recent studies demonstrated the occurrence of sialyl free N-glycans (FNGs) in sera from a variety of animals. Unlike the intracellular FNGs that mainly carry a single N-acetylglucosamine at their reducing termini (Gn1-type), these extracellular FNGs have an N,N'-diacetylchitobiose at their reducing termini (Gn2-type). The detailed mechanism for how they are formed, however, remains unclarified. In this study, we report on an improved method for isolating FNGs from sera and found that, not only sialyl FNGs, but also neutral FNGs are present in animal sera. Most of the neutral oligomannose-type FNGs were found to be Gn1-type. We also found that a small portion of sialyl FNGs were Gn1-type. The ratio of Gn1-type sialyl FNGs varies between species, and appears to be partially correlated with the distribution of lysosomal chitobiase activity. We also identified small sialylated glycans similar to milk oligosaccharides, such as sialyl lactose or sialyl N-acetyllactosamine in sera. Our results indicate that there are varieties of free oligosaccharides in sera and the mechanism responsible for their formation is more complicated than currently envisaged.

Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat.[Pubmed:34884628]

Int J Mol Sci. 2021 Nov 26;22(23):12822.

Four chitinases were cloned and characterized from three strains isolated from a mudflat: Aeromonas sp. SK10, Aeromonas sp. SK15, and Chitinibacter sp. SK16. In SK10, three genes, Chi18A, Pro2K, and Chi19B, were found as a cluster. Chi18A and Chi19B were chitinases, and Pro2K was a metalloprotease. With combinatorial amplification of the genes and analysis of the hydrolysis patterns of substrates, Chi18A and Chi19B were found to be an endochitinase and exochitinase, respectively. Chi18A and Chi19B belonged to the glycosyl hydrolase family 18 (GH18) and GH19, with 869 and 659 amino acids, respectively. Chi18C from SK15 belonged to GH18 with 864 amino acids, and Chi18D from SK16 belonged to GH18 with 664 amino acids. These four chitinases had signal peptides and high molecular masses with one or two chitin-binding domains and, interestingly, preferred alkaline conditions. In the activity staining, their sizes were determined to be 96, 74, 95, and 73 kDa, respectively, corresponding to their expected sizes. Purified Chi18C and Chi18D after pET expression produced N,N'-diacetylchitobiose as the main product in hydrolyzing chitooligosaccharides and colloidal chitin. These results suggest that Chi18A, Chi18C, and Chi18D are endochitinases, that Chi19B is an exochitinase, and that these chitinases can be effectively used for hydrolyzing natural chitinous sources.

Structural characterization of two solute-binding proteins for N,N'-diacetylchitobiose/N,N',N''-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7.[Pubmed:34195603]

J Struct Biol X. 2021 Jun 10;5:100049.

The chitinolytic bacterium Paenibacillus sp. str. FPU-7 efficiently degrades chitin into oligosaccharides such as N-acetyl-D-glucosamine (GlcNAc) and disaccharides (GlcNAc)(2) through multiple secretory chitinases. Transport of these oligosaccharides by P. str. FPU-7 has not yet been clarified. In this study, we identified nagB1, predicted to encode a sugar solute-binding protein (SBP), which is a component of the ABC transport system. However, the genes next to nagB1 were predicted to encode two-component regulatory system proteins rather than transmembrane domains (TMDs). We also identified nagB2, which is highly homologous to nagB1. Adjacent to nagB2, two genes were predicted to encode TMDs. Binding experiments of the recombinant NagB1 and NagB2 to several oligosaccharides using differential scanning fluorimetry and surface plasmon resonance confirmed that both proteins are SBPs of (GlcNAc)(2) and (GlcNAc)(3). We determined their crystal structures complexed with and without chitin oligosaccharides at a resolution of 1.2 to 2.0 A. The structures shared typical SBP structural folds and were classified as subcluster D-I. Large domain motions were observed in the structures, suggesting that they were induced by ligand binding via the "Venus flytrap" mechanism. These structures also revealed chitin oligosaccharide recognition mechanisms. In conclusion, our study provides insight into the recognition and transport of chitin oligosaccharides in bacteria.

Genome analysis of Streptomyces sp. UH6 revealed the presence of potential chitinolytic machinery crucial for chitosan production.[Pubmed:34192819]

Environ Microbiol Rep. 2022 Jun;14(3):431-442.

Chitosan and its derivatives have numerous applications in wastewater treatment as bio-coagulants, flocculants and bio-adsorbents against both particulate and dissolved pollutants. Chitinolytic bacteria secrete an array of enzymes, which play crucial role in chitin to chitosan conversion. Consequently, there is a growing demand for identification and characterization of novel bacterial isolates with potential implications in chitosan production. We describe genomic features of the new isolate Streptomyces sp. UH6. Analysis of the 6.51 Mb genome revealed the GC content as 71.95% and presence of 6990 coding sequences of which 63% were functionally annotated. Further, we identified two possible chitin-utilization pathways, which employ secreted enzymes like lytic polysaccharide monooxygenases and family-18 glycoside hydrolases (GHs). More importantly, the genome has six family-4 polysaccharide deacetylases with probable role in chitin to chitosan conversion, as well as two chitosanases belonging to GH46 and GH75 families. In addition, the gene clusters, dasABC and ngcEFG coding for transporters, which mediate the uptake of N,N'-diacetylchitobiose and N-acetyl-d-glucosamine were identified. Several genes responsible for hydrolysis of other polysaccharides and fermentation of sugars were also identified. Taken together, the phylogenetic and genomic analyses suggest that the isolate Streptomyces sp. UH6 secretes potential chitin-active enzymes responsible for chitin to chitosan conversion.