Search Results (9)

Three novel endo-β-N-acetylglucosaminidases (AVUL01, BCAC01, and BFIN01) classified as members of the glucoside hydrolase (GH) family 18 were identified from human fecal samples and then cloned and characterized for their ability to hydrolyze two distinct classes of N-glycans. Endo-β-N-acetylglucosaminidases (ENGases) are known for the hydrolysis of chitin and the N,N′-diacetylchitobiose core of N-linked glycans, depending on the glycan architecture. N-glycans have shown bioactivity as substrates in the human gut microbiome for microbes that encode ENGases, thus demonstrating their ecological relevance in the gut. However, distinct types of N-glycan structures, for example, oligomannosidic or complex, have been shown to enrich different microbes within the human gut. Novel advances in food technology have commercialized animal-derived dietary proteins with oligomannosidic instead of traditionally complex N-glycans using precision fermentation. This indicates that there is an unmet need to identify the classes of N-glycans that gut-derived ENGases act upon to determine whether these novel proteins alter gut ecology. AVUL01, BCAC01, and BFIN01 all demonstrated activity on exclusively oligomannosidic N-glycans from RNase B and bovine lactoferrin; however, they failed to show activity on complex or α-1,3-core fucosylated high-mannose N-glycans derived from fetuin and horseradish peroxidase, respectively. These results suggest that α-1,3 core fucosylation and complex N-glycan architecture inhibit the activity of AVUL01, BCAC01, and BFIN01. Furthermore, BFIN01 performed significantly better than BCAC01, resulting in a greater amount of N-glycans, suggesting that certain ENGases may possess enhanced specificity and kinetics as an evolutionary strategy to compete for resources. Full article

Chitin, a polymer of β-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 °C with 1.0 mM isopropyl-β-d-thiogalactopyranoside induction for 6 h. Purified SkChi65 displayed high stability over the ranges of 30–50 °C and pH 5.5–8.0 with optima at 40 °C and pH 7.0. The kinetic parameters K_m, V_max, and k_cat of SkChi65 towards colloidal chitin were 27.2 μM, 299.2 μMs⁻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. Full article

N,N′-diacetylchitobiose, also known as GlcNAc₂, is a chitin oligosaccharide and is reported to possess antimicrobial activity against pathogenic bacteria. In this study, 1% (w/v) GlcNAc₂ solution was applied on ready-to-eat (RTE) shrimp and evaluated as an antimicrobial coating against Listeria monocytogenes during storage at 4 °C for 16 days. Texture properties, colour, TBARS values, moisture content and bacterial counts were monitored and analysed every four days. The results indicated that the GlcNAc₂ coating retarded the changes in texture properties, TBARS values and moisture content of the RTE shrimp during storage. The presence of GlcNAc₂ showed no significant changes in RTE shrimp colour in contrast to the control. However, the growth of L. monocytogenes inoculated on the GlcNAc₂-coated RTE shrimp was slower than that of the control sample with the highest log reduction of 0.5 log CFU/mL being observed. This study showed that the GlcNAc₂ used as an antimicrobial coating was able to inhibit the growth of L. monocytogenes, while maintaining the quality of the RTE shrimp during refrigerated storage. Full article

Chitin is a major source of energy and macroelements for many organisms. An important step in its degradation is the deacetylation of chitin or its fragments. Deacetylase from the extremophile Pyrococcus chitonophagus has been analyzed by X-ray crystallography, small-angle X-ray scattering, differential scanning calorimetry, isothermal titration calorimetry and NMR to determine its structure, thermodynamics and enzymatic properties. It is a hexameric, zinc-containing metalloenzyme that retains its structural integrity up to temperatures slightly exceeding 100 °C. It removes the acetyl group specifically from the non-reducing end of the sugar substrate. Its main substrate is N,N-diacetylchitobiose but it also active, at a reduced level, toward N-acetyl-d-glucosamine or a trimer of N-acetyl-d-glucosamine units. Crystallographic analysis includes the structure of the enzyme with its main substrate approaching the active site in a monodentate manner, replacing the single water molecule that is bound at the Zn²⁺ cation when the ligand is absent. The Zn²⁺ cation remains tetrahedrally coordinated, with three of its ligands provided by the protein’s conserved His-Asp-His triad. The crystal structures are consistent with the reaction mechanism proceeding via an anhydride intermediate. Hydrolysis as the first step cannot be ruled out in a hydrated environment but no defined ‘hydrolytic water’ site can be identified in the analyzed structures. Full article

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 α-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 α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45–50 °C and pH optima of 7.0–7.5. They are all stable at 40 °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 °C. When acting on crystalline α-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. Full article

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. Full article

ATP-binding cassette (ABC) transporters are a superfamily of proteins that transport nutrient substances and secondary metabolites through cell membranes. They also act as an uptake system for N,N′-diacetylchitobiose (GlcNAc)₂ in Streptomyces coelicolor. (GlcNAc)₂ is an important inducer of chitinase. However, whether the ABC transporter in Trichoderma spp. is also responsible for (GlcNAc)₂ uptake and chitinase induction has not yet been confirmed. In this study, we applied RNA interference and overexpression technologies to alter the expression level of the ABC-B transporter in order to detect changes in its transportation ability and the expression level of inducible endo-chitinase ECH42—an important biocontrol enzyme in Trichoderma asperellum. The results revealed that, after interference with the expression of the ABC-B transporter, T. asperellum T4 was only able to grow normally when glucose was the only carbon source. Compared with the wild-type, the efficiency of (GlcNAc)₂ by the overexpression strain evidently increased, along with the activity level of ECH42. In conclusion, one of the functions of the ABC-B transporter in T. asperellum is the uptake and transport of (GlcNAc)₂ into cells, and chitobiose is a strong inducer of ECH42 in T. asperellum T4. Full article

Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N'-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases. Full article

A quantitative determination method of N-acetyl-D-glucosamine (GlcNAc) and N,N'-diacetylchitobiose (GlcNAc)₂ is proposed using a proton nuclear magnetic resonance experiment. N-acetyl groups of GlcNAc and (GlcNAc)₂ are chosen as target signals, and the deconvolution technique is used to determine the concentration of the corresponding compound. Compared to the HPLC method, ¹H-NMR spectroscopy is simple and fast. The method can be used for the analysis of chitin hydrolyzed products with real-time analysis, and for quantifying the content of products using internal standards without calibration curves. This method can be used to quickly evaluate chitinase activity. The temperature dependence of ¹H-NMR spectra (VT-NMR) is studied to monitor the chemical shift variation of acetyl peak. The acetyl groups of products are involved in intramolecular H-bonding with the OH group on anomeric sites. The rotation of the acetyl group is closely related to the intramolecular hydrogen bonding pattern, as suggested by the theoretical data (molecular modeling). Full article