Search

Chitin is a highly abundant N-acetylglucosamine polysaccharide that has been linked to immune responses in the context of fungal infections and allergic asthma, especially to T helper 2 immune responses. Unfortunately, due to the frequent use of crude chitin preparations of unknown purity and degree of polymerization, there is still great uncertainty about how chitin activates different parts of the human immune system. We recently identified chitin oligomers of 6 N-acetylglucosamine units as the smallest immunologically active chitin motif and the innate immune receptor TLR2 as a primary chitin sensor on human and murine myeloid cells, but the response of further immune cells (e.g. lymphoid cells) to oligomeric chitin has not been investigated. Our analysis of primary human immune cells now shows that chitin oligomers activate immune responses of both innate and adaptive lymphocytes: notably, chitin oligomers activated natural killer cells but not B lymphocytes. Moreover, chitin oligomers induced maturation of dendritic cells and enabled potent CD8+ T-cell recall responses. Our results suggest that chitin oligomers not only trigger immediate innate responses in a limited range of myeloid cells but also exert critical activities across the entire human immune system. This highlights chitin oligomer immune activation as an interesting and broadly applicable potential target for both adjuvant development and therapeutic interference in chitin-mediated pathologies., Competing Interests: Conflict of interest statement. K.F., Y.C.G., C.G., and/or A.N.R.W. were listed as inventors on 2 German patents, which have been discontinued since 2020. There are no further conflicts of interest to declare., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chitin, the second richest polymer in nature, is composed of the monomer N-acetylglucosamine (GlcNAc), which has numerous functions and is widely applied in the medical, food, and chemical industries. However, due to the highly crystalline configuration and low accessibility in water of the chitin resources, such as shrimp and crab shells, the chitin is difficult utilize, and the traditional chemical method causes serious environment pollution and a waste of resources. In the present study, three genes encoding chitinolytic enzymes, including the N-acetylglucosaminidase from Ostrinia furnacalis ( OfHex1 ), endo-chitinase from Trichoderma viride ( TvChi1 ), and multifunctional chitinase from Chitinolyticbacter meiyuanensis ( CmChi1 ), were expressed in the Pichia pastoris system, and the positive transformants with multiple copies were isolated by the PTVA (post-transformational vector amplification) method, respectively. The three recombinants OfHex1, TvChi1, and CmChi1 were induced by methanol and purified by the chitin affinity adsorption method. The purified recombinants OfHex1 and TvChi1 were characterized, and they were further used together for degrading chitin from shrimp and crab shells to produce GlcNAc through liquid-assisted grinding (LAG) under a water-less condition. The substrate chitin concentration reached up to 300 g/L, and the highest yield of the product GlcNAc reached up to 61.3 g/L using the mechano-enzymatic method. A yield rate of up to 102.2 g GlcNAc per 1 g enzyme was obtained.

Direct conversion of crystalline chitin to N-acetylglucosamine and the related chitooligomers through enzymatic approaches is gaining importance owing to their various biological applications. Here we report the crystalline chitin degradation ability of chitinolytic cocktail produced by the isolate Paenibacillus sp. LS1. Growth studies of the isolate in presence of different chitin substrates revealed preference for β-chitin and colloidal chitin. FE-SEM micrographs showed formation of visible perforations on the crystalline chitin particles by the isolate. Further, zymogram analysis revealed the presence of six potential chitinase isozymes. The enzyme-cocktail produced by the isolate was optimally active at 50 °C in 50 mM sodium acetate, pH-4.0. Time-course hydrolysis of crystalline α- and β-chitin with the Paenibacillus sp. LS1 enzyme-cocktail produced N-acetylglucosamine and N,N'-diacetylchitobiose as the predominant products. Taken together, our results confirm that the Paenibacillus sp. LS1 enzyme-cocktail would have potential application in eco-friendly enzymatic approaches for efficient saccharification of crystalline chitin., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chitin represents the second most abundant biomass after lignocelluloses in the biosphere. It can be depolymerized into either N-acetylglucosamine (GlcNAc) or glucosamine (GlcN) and acetate by different degradation strategies. However, these chitin-derived carbon sources have been scarcely compared for lipid production. Here, GlcNAc was found superior to GlcN or acetate for lipid accumulation by Cutaneotrichosporon oleaginosum. The lipid accumulation potential of these carbon sources was calculated based on a small scale metabolic model of C. oleaginosum. Co-fermentation of GlcN and acetate under phosphate limitation rendered improved lipid production. GlcN and acetate were assimilated simultaneously. The highest lipid titer and yield of 10.1 g/L and 0.25 g/g, respectively, was reached when GlcNAc was used under phosphate limitation. The fatty acids composition of the lipid samples showed similarities to vegetable oils, demonstrating the suitability in biodiesel industry. This study provides profitable guidance for the design of chitin-to-lipids routes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chitin, abundant biomass found in crab shells and other marine life, has wide applications in the production of food, pharmaceuticals, and cosmetics. Our recent studies have focused on the development of new functional materials by derivatizing chitin oligosaccharides and monosaccharides. For example, we have prepared various derivatives by chemoenzymatic synthesis using N-acetylglucosamine (GlcNAc) or chitin oligosaccharide prepared from chitin as starting materials. First, we have achieved the total synthesis of two secondary metabolites (furanodictine A and B) with neuronal differentiation-inducing activity on PC12 cells by using a simple heatinduced structural transformation of GlcNAc and esterification reaction. Second, we synthesized both a novel inhibitor that has facilitated a re-examination of the reaction mechanism of hen egg-white lysozyme, and a new substrate for assaying lysozyme activity by using chitin oligosaccharides as raw materials. Thus, the development of new materials by simple derivatization of chitin mono- or oligo-saccharides is paving the way for effective use of chitin., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Bacillus subtilis PdaC ( Bs PdaC) is a membrane-bound, multidomain peptidoglycan N- deacetylase acting on N -acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. Bs PdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, Bs PdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the Bs PdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc 3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that Bs PdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, Bs PdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase Sp PgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of Bs PdaC and compared them with those of Sp PgdA and Bs PdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. Bs PdaC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. Bs PdaC contains short loops similar to those in Sp PgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases., (© 2019 Grifoll-Romero et al.)

Chitin is massively produced by freshwater plankton species as a structural element of their exoskeleton or cell wall. At the same time, chitin does not accumulate in the predominantly anoxic sediments, underlining its importance as carbon and nitrogen sources for sedimentary microorganisms. We studied chitin degradation in littoral sediment of Lake Constance, Central Europe's third largest lake. Turnover of the chitin analog methyl-umbelliferyl- N , N -diacetylchitobioside (MUF-DC) was highest in the upper oxic sediment layer, with 5.4 nmol MUF-DC h -1 (g sediment [dry weight]) -1 In the underlying anoxic sediment layers, chitin hydrolysis decreased with depth from 1.1 to 0.08 nmol MUF-DC h -1 (g sediment [dry weight]) -1 Bacteria involved in chitin degradation were identified by 16S rRNA (gene) amplicon sequencing of anoxic microcosms incubated in the presence of chitin compared to microcosms amended either with N -acetylglucosamine as the monomer of chitin or no substrate. Chitin degradation was driven by a succession of bacteria responding specifically to chitin only. The early phase (0 to 9 days) was dominated by Chitinivibrio spp. ( Fibrobacteres ). The intermediate phase (9 to 21 days) was characterized by a higher diversity of chitin responders, including, besides Chitinivibrio spp., also members of the phyla Bacteroidetes , Proteobacteria , Spirochaetes , and Chloroflexi In the late phase (21 to 43 days), the Chitinivibrio populations broke down with a parallel strong increase of Ruminiclostridium spp. (formerly Clostridium cluster III, Firmicutes ), which became the dominating chitin responders. Our study provides quantitative insights into anaerobic chitin degradation in lake sediments and linked this to a model of microbial succession associated with this activity. IMPORTANCE Chitin is the most abundant biopolymer in aquatic environments, with a direct impact on the carbon and nitrogen cycles. Despite its massive production as a structural element of crustaceans, insects, or algae, it does not accumulate in sediments. Little is known about its turnover in predominantly anoxic freshwater sediments and the responsible microorganisms. We proved that chitin is readily degraded under anoxic conditions and linked this to a succession of the members of the responsible microbial community over a 43-day period. While Fibrobacteres and Firmicutes members were driving the early and late phases of chitin degradation, respectively, a more diverse community was involved in chitin degradation in the intermediate phase. Entirely different microorganisms responded toward the chitin monomer N -acetylglucosamine, which underscores that soluble monomers are poor and misleading substrates to study polymer-utilizing microorganisms. Our study provides quantitative insights into the microbial ecology driving anaerobic chitin degradation in freshwater sediments., (Copyright © 2019 Wörner and Pester.)

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable β-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc) 2-5 , p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc) 1-3 -pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc) 2 . MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

This study aimed to investigate the ability of chitin and heat-treated shrimp shells to bind aflatoxin M1 (AFM1) in liquid matrix. Several concentrations of chitin or shrimp shells (grinded and ungrinded) were incubated in AFM1-contaminated phosphate-buffered saline (PBS) at different incubation times. The stability of the formed adsorbent-AFM1 complex was also tested in milk at different incubation times and temperatures. The unbound AFM1 was quantified by HPLC. Thereby, the percentages of the initial bounded AFM1 varied between 14.29 and 94.74%. Interestingly, in milk, an increase in incubation time coupled with a decrease in temperature affected positively the amount of bounded AFM1 to chitin and negatively those bounded to ungrinded shells. Results also revealed a partial reversibility in the binding of AFM1 to these adsorbents. These findings provided strong evidence on ability of chitin or shrimp shells by-product to bind AFM1 in milk and in PBS.

N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chitin and β-glucan are conserved throughout evolution in the fungal cell wall and are the most common polysaccharides in fungal species. Together, these two polysaccharides form a structural scaffold that is essential for the survival of the fungus. In the present study, we demonstrated that Aspergillus fumigatus alkali-insoluble cell wall fragments (AIF), composed of chitin linked covalently to β-glucan, induced enhanced immune responses when compared with individual cell wall polysaccharides. Intranasal administration of AIF induced eosinophil and neutrophil recruitment, chitinase activity, TNF-α and TSLP production in mice lungs. Selective destruction of chitin or β-glucan from AIF significantly reduced eosinophil and neutrophil recruitment as well as chitinase activity and cytokine expression by macrophages, indicating the synergistic effect of the cell wall polysaccharides when presented together as a composite PAMP. We also showed that these cell wall polysaccharides induced chitin-specific IgM in mouse serum. Our in vivo and in vitro data indicate that chitin and β-glucan play important roles in activating innate immunity when presented as composite cell wall PAMPs., (Copyright © 2013. Published by Elsevier GmbH.)

N-Acetylglucosamine (GlcNAc) is a monosaccharide that usually polymerizes linearly through (1,4)-β-linkages. GlcNAc is the monomeric unit of the polymer chitin, the second most abundant carbohydrate after cellulose. In addition to serving as a component of this homogeneous polysaccharide, GlcNAc is also a basic component of hyaluronic acid and keratin sulfate on the cell surface. In this review, we discuss the industrial production of GlcNAc, using chitin as a substrate, by chemical, enzymatic and biotransformation methods. Also, newly developed methods to obtain GlcNAc using glucose as a substrate in genetically modified microorganisms are introduced. Moreover, GlcNAc has generated interest not only as an underutilized resource but also as a new functional material with high potential in various fields. Here we also take a closer look at the current applications of GlcNAc, and several new and cutting edge approaches in this fascinating area are thoroughly discussed.

Peatlands are invaluable but threatened ecosystems that store huge amounts of organic carbon globally and emit the greenhouse gasses carbon dioxide (CO2) and methane (CH4). Trophic interactions of microbial groups essential for methanogenesis are poorly understood in such systems, despite their importance. Thus, the present study aimed at unraveling trophic interactions between fermenters and methanogens in a nitrogen-limited, subarctic, pH-neutral fen. In situ CH4 emission measurements indicated that the fen is a source of CH4, and that CH4 emissions were higher in plots supplemented with ammonium compared to unsupplemented plots. The amino sugar N-acetylglucosamine was chosen as model substrate for peat fermenters since it can serve as organic carbon and nitrogen source and is a monomer of chitin and peptidoglycan, two abundant biopolymers in the fen. Supplemental N-acetylglucosamine was fermented to acetate, ethanol, formate, and CO2 during the initial incubation of anoxic peat soil microcosms without preincubation. Subsequently, ethanol and formate were converted to acetate and CH4. When methanogenesis was inhibited by bromoethanesulfonate, acetate and propionate accumulated. Long-term preincubation considerably increased CH4 production in unsupplemented microcosms and microcosms supplemented with methanogenic substrates. Supplemental H2-CO2 and formate stimulated methanogenesis the most, whereas acetate had an intermediary and methanol a minor stimulatory effect on methane production in preincubated microcosms. Activity of acetogens was suggested by net acetate production in microcosms supplemented with H2-CO2, formate, and methanol. Microbial community analysis of field fresh soil indicated the presence of many physiologically unresolved bacterial taxa, but also known primary and secondary fermenters, acetogens, iron reducers, sulfate reducers, and hydrogenotrophic methanogens (predominately Methanocellaceae and Methanoregulaceae). Aceticlastic methanogens were either not abundant (Methanosarcinaceae) or could not be detected due to limited coverage of the used primers (Methanotrichaceae). The collective results indicate a complex interplay of synergy and competition between fermenters, methanogens, acetogens, and potentially iron as well as sulfate reducers. While acetate derived from fermentation or acetogenesis in this pH-neutral fen likely plays a crucial role as carbon source for the predominant hydrogenotrophic methanogens, it remains to be resolved whether acetate is also converted to CH4 via aceticlastic methanogenesis and/or syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis. [ABSTRACT FROM AUTHOR]

Featured Application: The developed indirect sandwich ELLA offers a practical solution for the routine screening and detection of insects in food processing facilities, and holds promise for implementation by inspection authorities. Edible insects have been officially classified as food in the European Union since 2015. Currently, four insect species are approved for food use. However, no official method for detecting insects in food has been recognized to date. To establish a simple analytical method for insect detection in food, we developed an indirect sandwich (enzyme-linked lectin sorbent assay) ELLA specific for N-acetylglucosamine in chitin and chitosan polymers. The validation of the method demonstrated that the ELLA developed in this study is reliable for insect detection. The limit of detection (LOD) and quantification (LOQ) were 0.006 and 0.028 mg/mL, respectively. Intra-day precision ranged from 2.45% to 30.29%, and inter-day precision from 0.36% to 12.87%. Significant differences in the total amount of chitin and chitosan were observed among the insect products, processed insect products, and samples without any insect addition (p < 0.05). [ABSTRACT FROM AUTHOR]

Introduction: Chitin, abundant in marine environments, presents significant challenges in terms of transformation and utilization. A strain, T22.7.1T, with notable chitin deacetylation capabilities, was isolated from the rhizosphere of Acanthus ebracteatus in the North Sea of China. Comparative 16S rDNA sequence analysis showed that the new isolate had the highest sequence similarity (99.79%) with Rhodococcus indonesiensis CSLK01-03T, followed by R. ruber DSM 43338T, R. electrodiphilus JC435T, and R. aetherivorans 10bc312T (98.97%, 98.81%, and 98.83%, respectively). Subsequent genome sequencing and phylogenetic analysis confirmed that strain T22.7.1T belongs to the R. indonesiensis species. However, additional taxonomic characterization identified strain T22.7.1T as a novel type strain of R. indonesiensis distinct from CSLK01-03T. Methods: This study refines the taxonomic description of R. indonesiensis and investigates its application in converting chitin into chitosan. The chitin deacetylase (RiCDA) activity of strain T22.7.1T was optimized, and the enzyme was isolated and purified from the fermentation products. Results: Through optimization, the RiCDA activity of strain T22.7.1T reached 287.02 U/mL, which is 34.88 times greater than the original enzyme’s activity (8.0 U/mL). The natural CDA enzyme was purified with a purification factor of 31.83, and the specific activity of the enzyme solution reached 1200.33 U/mg. RiCDA exhibited good pH and temperature adaptability and stability, along with a wide range of substrate adaptabilities, effectively deacetylating chitin, chitooligosaccharides, N-acetylglucosamine, and other substrates. Discussion: Product analysis revealed that RiCDA treatment increased the deacetylation degree (DD) of natural chitin to 83%, surpassing that of commercial chitosan. Therefore, RiCDA demonstrates significant potential as an efficient deacetylation tool for natural chitin and chitooligosaccharides, highlighting its applicability in the biorefining of natural polysaccharides. [ABSTRACT FROM AUTHOR]

N‐acetylglucosamine (GlcNAc) is an important structural component of the cell wall chitin, N‐glycans, glycolipids, and GPI‐anchors in eukaryotes. GlcNAc kinase phosphorylates GlcNAc into GlcNAc‐6‐phosphate, a precursor of uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc) that serves as a substrate for glycan synthesis. Although GlcNAc kinase is found widely in organisms ranging from microorganisms to mammals, it has never been found in the model yeast Saccharomyces cerevisiae. Here, we demonstrate the presence of GlcNAc metabolism for UDP‐GlcNAc biosynthesis in S. cerevisiae through Ngk1, a GlcNAc kinase we discovered previously. The overexpression or deletion of Ngk1 in the presence of GlcNAc affected the amount of both UDP‐GlcNAc and chitin, suggesting that GlcNAc metabolism via Ngk1 promotes UDP‐GlcNAc synthesis. Our data suggest that the Ngk1‐mediated GlcNAc metabolism compensates for the hexosamine pathway, a known pathway for UDP‐GlcNAc synthesis. [ABSTRACT FROM AUTHOR]

Edible insects have been officially classified as food in the European Union since 2015. Currently, four insect species are approved for food use. However, no official method for detecting insects in food has been recognized to date. To establish a simple analytical method for insect detection in food, we developed an indirect sandwich (enzyme-linked lectin sorbent assay) ELLA specific for N-acetylglucosamine in chitin and chitosan polymers. The validation of the method demonstrated that the ELLA developed in this study is reliable for insect detection. The limit of detection (LOD) and quantification (LOQ) were 0.006 and 0.028 mg/mL, respectively. Intra-day precision ranged from 2.45% to 30.29%, and inter-day precision from 0.36% to 12.87%. Significant differences in the total amount of chitin and chitosan were observed among the insect products, processed insect products, and samples without any insect addition (p < 0.05).

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite −2 and −1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite −2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite −1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs. Chitin deacetylases are a valuable tool for producing custom chitosans with non-random N-acetylglucosamine and glucosamine units. Analysis of a chitin deacetylase active site reveals residues crucial for substrate binding, allowing engineering to drastically change the acetylation pattern of a chitosan product. [ABSTRACT FROM AUTHOR]

3‐Acetamido‐5‐acetylfuran (3A5AF) is a potential platform compound for the production of nitrogen‐containing pharmaceuticals and chemicals. 3A5AF can be obtained by dehydration of chitin or its monomer, N‐acetylglucosamine (NAG). Here, we examined the use of solid catalysts for the dehydration of NAG to 3A5AF to achieve a more economical process that uses a recyclable catalyst. NAG was dehydrated using various solid catalysts in the presence of NaCl and N,N‐dimethyl acetamide as solvent at 433 K. The yield of 3A5AF with the solid catalysts decreased in the following order: Al‐exchanged montmorillonite>H‐ZSM‐5 (SiO2/Al2O3=40)>H‐montmorillonite (K‐10)>Amberlyst15>H‐ZSM‐5 (SiO2/Al2O3=300)>TiO2>γ‐Al2O3>ZrO2>SiO2 ⋅ MgO>Na‐montmorillonite. The highest yield of 3A5AF (14 %) was obtained with the Al‐exchanged montmorillonite. The montmorillonite catalysts were characterized by using inductively coupled plasma optical emission spectroscopy, energy‐dispersive X‐ray spectroscopy, N2 adsorption, Fourier‐transformed infrared spectroscopy, X‐ray diffraction, and 27Al magic‐angle spinning nuclear magnetic resonance spectroscopy (MAS‐NMR). In addition, a combined catalyst of Al‐exchanged montmorillonite and Cl− from synthetic hydrotalcite was found to be an active and recyclable solid catalyst for NAG dehydration to 3A5AF. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine (GlcNAc) is an amino monosaccharide that has a variety of bioactivities and is widely used in pharmaceutical and food industries. Production of GlcNAc by chitin hydrolysis is limited by the supply of raw materials and encounters the risk of shellfish protein contamination. For efficient biosynthesis of GlcNAc, one challenge is to balance the carbon distribution between growth and production. Here, we applied the strategy of synergistic carbon utilization, in which glycerol supports cell growth and provides the acetyl group of GlcNAc while glucose serves as the precursor to glucosamine. The efficiency of GlcNAc production was stepwise improved by blocking the product re-uptake and degradation, strengthening the biosynthetic pathway and synergistically utilizing two carbon sources. With these efforts, the final strain produced 41.5 g/L GlcNAc with a yield of 0.49 g/g of total carbon sources. In addition, we also explored the feasibility of using acetate as a cheap carbon source to partly replace glycerol. This study provides a promising alternative strategy for sustainable and efficient production of GlcNAc. [ABSTRACT FROM AUTHOR]

The conversion of chitin enables the utilisation of naturally-fixed nitrogen in addition to carbon toward establishing a sustainable carbon and nitrogen cycle. Chitin is an abundant biomass, 100 Gt per year, but most chitin-containing waste is discarded due to its recalcitrant properties. This feature article summarises the challenges and our work on chitin conversion to N-acetylglucosamine and oligomers with fascinating applications. Afterwards, we introduce recent progress on the chemical transformation of N-acetylglucosamine, followed by a discussion of future perspectives based on current status and findings. [ABSTRACT FROM AUTHOR]

Background: Filamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications. Results: A novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%. Conclusions: This PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications. [ABSTRACT FROM AUTHOR]

Earlier studies have shown that the outer layers of the conidial and mycelial cell walls of Aspergillus fumigatus are different. In this work, we analyzed the polysaccharidome of the resting conidial cell wall and observed major differences within the mycelium cell wall. Mainly, the conidia cell wall was characterized by (i) a smaller amount of α-(1,3)-glucan and chitin; (ii) a larger amount of β-(1,3)-glucan, which was divided into alkali-insoluble and water-soluble fractions, and (iii) the existence of a specific mannan with side chains containing galactopyranose, glucose, and N-acetylglucosamine residues. An analysis of A. fumigatus cell wall gene mutants suggested that members of the fungal GH-72 transglycosylase family play a crucial role in the conidia cell wall β-(1,3)-glucan organization and that α-(1,6)-mannosyltransferases of GT-32 and GT-62 families are essential to the polymerization of the conidium-associated cell wall mannan. This specific mannan and the well-known galactomannan follow two independent biosynthetic pathways. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine (GlcNAc), a chitin monomer, can be used as a natural preservative to ensure food quality and safety. Combining natural preservatives with low storage temperature offers physical hurdles to bacterial growth in food. This study aimed to produce chitin hydrolysate containing GlcNAc using Serratia marcescens PT6 crude chitinase and investigate its effect on bacterial growth rate as a function of temperature. Crude chitinase from partial purification was used to hydrolyze 1.3% colloidal chitin. The optimal enzymatic conditions were pH 6 and 45°C for 120 min, at an enzyme:substrate ratio of 1:1, yielding a 65.6 μg/mL GlcNAc. Inhibitory activity of hydrolysate containing 2.5-7.5 ppm GlcNAc on Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus was measured at 4, 15, and 30oC in nutrient broth. Bacterial growth was measured using of optical density for each combination of GlcNAc concentration and temperature. Growth curves fitted by the Baranyi and Roberts model were developed using DMFit software. The growth rate was converted to the square root and then modeled as a function of temperature using the Ratkowsky square root model. Incubation temperature exerted a pronounced effect on the inhibition of all bacterial species (P<0.0001), with the greatest effect observed for E. coli at 30°C (P<0.0001), and the least effect for V. parahaemolyticus (P=0.0878). The inhibitory effect of GlcNAc in chitin hydrolysate was only significant for E. coli (P<0.0001) and S. aureus (P=0.0041). This study revealed that the effect of temperature in growth inhibition was more significant than GlcNAc addition. However, a reduction in bacterial growth with the addition of GlcNAc at 30°C was observed, which may be effective for food encountered thermal abuse conditions. Further investigation of the effect of GlcNAc on bacteria structure and metabolism is required to elucidate the mechanism of GlcNAc as a food preservative. [ABSTRACT FROM AUTHOR]

Chitin is the second most abundant biopolymer in nature after cellulose and is composed of N -acetylglucosamine (GlcNAc) connected via β(1 → 4)-glycosidic bonds. Despite its prominence in nature and diverse roles in pharmaceutical and food technological applications, there is still a need to develop methods to study structure and function of chitin and its corresponding oligomers. Efforts have been made to analyse chitin oligomers by NMR spectroscopy, but spectral overlap has prevented any differentiation between the interior residues. In this study, chitin oligomers up to hexaose with natural abundance of 15N were analysed with NMR spectroscopy in aqueous solution. Different 1H,15N-HSQC pulse sequences were evaluated to obtain the best resolution and sensitivity. Interior residues were resolved in the 15N dimension and detailed chemical shifts of amide proton and nitrogen are reported for the first time. Additionally, all oligomers were analysed for the presence of the amide cis form and its corresponding chemical shifts were assigned. This study exploits the information that can be obtained from chitin oligomers with NMR spectroscopy and depicts methods for detailed analysis of glycans containing oligomers of N -acetylglucosamine. [Display omitted] [ABSTRACT FROM AUTHOR]

N-acetylglucosamine (GlcNAc) is widely used in nutritional supplement and is generally produced from chitin using chitinases. While most GlcNAc is produced from colloidal chitin, it is essential that chitinases be acidic enzymes. Herein, we characterized an acidic, highly salinity tolerance and thermostable chitinase AfChiJ, identified from the marine fungus Aspergillus fumigatus df673. Using AlphaFold2 structural prediction, a truncated Δ30AfChiJ was heterologously expressed in E. coli and successfully purified. It was also found that it is active in colloidal chitin, with an optimal temperature of 45°C, an optimal pH of 4.0, and an optimal salt concentration of 3% NaCl. Below 45°C, it was sound over a wide pH range of 2.0–6.0 and maintained high activity (≥97.96%) in 1–7% NaCl. A notable increase in chitinase activity was observed of Δ30AfChiJ by the addition of Mg2+, Ba2+, urea, and chloroform. AfChiJ first decomposed colloidal chitin to generate mainly N-acetyl chitobioase, which was successively converted to its monomer GlcNAc. This indicated that AfChiJ is a bifunctional enzyme, composed of chitobiosidase and β-N-acetylglucosaminidase. Our result suggested that AfChiJ likely has the potential to convert chitin-containing biomass into high-value added GlcNAc.

Background: N-acetylglucosaminidase (NAGase) could liberate N-acetylglucosamine (GlcNAc) from GlcNAc-containing oligosaccharides. Trichoderma spp. is an important source of chitinase, particularly NAGase for industrial use. nag1 and nag2 genes encoding NAGase, are found in the genome in Trichoderma spp. The deduced Nag1 and Nag2 shares ~ 55% homology in Trichoderma virens. Most studies were focus on Nag1 and nag1 previously. Results: The native NAGase (TvmNAG2) was purified to homogeneity with molecular mass of ~ 68 kDa on SDS-PAGE analysis, and identified as Nag2 by MALDI/MS analysis from an isolate T. virens strain mango. RT-PCR analyses revealed that only nag2 gene was expressed in liquid culture of T. virens, while both of nag1 and nag2 were expressed in T. virens cultured on the plates. TvmNAG2 was thermally stable up to 60 °C for 2 h, and the optimal pH and temperature were 5.0 and 60–65 °C, respectively, using p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) as substrate. The hydrolytic product of colloidal chitin by TvmNAG2 was suggested to be GlcNAc based on TLC analyses. Moreover, TvmNAG2 possesses antifungal activity, inhibiting the mycelium growth of Sclerotium rolfsii. And it was resistant to the proteolysis by papain and trypsin. Conclusions: The native Nag2, TvmNAG2 was purified and identified from T. virens strain mango, as well as enzymatic properties. To our knowledge, it is the first report with the properties of native Trichoderma Nag2. [ABSTRACT FROM AUTHOR]

ABSTRACT Communication between plants and microorganisms is vital because it influences their growth, development, defense, propagation, and metabolism in achieving maximal fitness. N-acetylglucosamine (N-GlcNAc), the building block of bacterial and fungal cell walls, was first reported to promote tomato plant growth via stimulation of microorganisms typically known to dominate the tomato root rhizosphere, such as members of Proteobacteria and Actinobacteria. Using KEGG pathway analysis of the rhizosphere microbial operational taxonomic units, the streptomycin biosynthesis pathway was enriched in the presence of N-GlcNAc. The biosynthesis of 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol, two foremost types of plant growth promotion-related volatile organic compounds, were activated in both Bacillus subtilis and Streptomyces thermocarboxydus strains when they were cocultured with N-GlcNAc. In addition, the application of N-GlcNAc increased indole-3-acetic acid production in a dose-dependent manner in strains of Bacillus cereus, Proteus mirabilis, Pseudomonas putida, and S. thermocarboxydus that were isolated from an N-GlcNAc-treated tomato rhizosphere. Overall, this study found that N-GlcNAc could function as microbial signaling molecules to shape the community structure and metabolism of the rhizosphere microbiome, thereby regulating plant growth and development and preventing plant disease through complementary plant–microbe interactions. IMPORTANCE While the benefits of using plant growth-promoting rhizobacteria (PGPRs) to enhance crop production have been recognized and studied extensively under laboratory conditions, the success of their application in the field varies immensely. More fundamentally explicit processes of positive, plant–PGPRs interactions are needed. The utilization of organic amendments, such as chitin and its derivatives, is one of the most economical and practical options for improving soil and substrate quality as well as plant growth and resilience. In this study, we observed that the chitin monomer N-GlcNAc, a key microbial signaling molecule produced through interactions between chitin, soil microbes, and the plants, positively shaped the community structure and metabolism of the rhizosphere microbiome of tomatoes. Our findings also provide a new direction for enhancing the benefits and stability of PGPRs in the field.

Biowaste originating from aquaculture sector represents a potential feedstock to produce value-added substances and materials like chitin and chitosan. They are the long chain polymers of N-acetylglucosamine polymers with huge industrial and biomedical significance. Chitin has long been recognized as a useful biomaterial for drug delivery and neurological therapy. Similarly, chitosan oligosaccharide, a short chain polymer derived from chitin/chitosan, has been identified for its potential biomedical applications, including antibacterial, anti-inflammatory, and anti-Alzheimer action. Chitosan nanoparticles are also used extensively in biomedical applications. Here we have critically summarized various methods for the extraction of chitin, chitosan, and chitooligossacharides and chitosan nanoparticle preparation and their diverse biomedical applications. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In pursuit of a more green and sustainable society, the preparation of versatile chemicals from renewable biomass is essential. Nitrogen-containing chemicals, as compared with hydrocarbon fuels or oxygen-containing compounds, usually have higher values and play key roles in modern industry. The industrial synthesis of nitrogen-containing chemicals relies heavily on the Haber–Bosch process, a high-energy-consumption and environment-polluting process. Therefore, the production of these organonitrogen chemicals from biomass has attracted increasing interest in recent years. Chitin is the most abundant nitrogen-containing biopolymer on earth, and its monomer N-acetylglucosamine (GlcNAc) not only has great application values in multiple areas, but also is a key intermediate for the conversion of chitin to other organonitrogen compounds. This review provides a comprehensive summary and perspective of the latest cutting-edge innovations on biomass conversion to GlcNAc. Biological methods, chemical methods as well as combined methods for GlcNAc preparation are systematically elaborated, and their respective advantages and disadvantages in terms of product yield, process safety, economy, efficiency, and environmental friendliness are discussed. To achieve an economic, environmentally friendly, and efficient production of GlcNAc from renewable biomass, the development of combined methods that involve multiple actions in a synergistic way under mild and green conditions should be the focus of future research. [ABSTRACT FROM AUTHOR]

The study was undertaken to size-fractionate the bacterial population associated with Microcystis blooms of an eutrophic lake. Two bacterial cultures were isolated from the particulate-bound fraction, and biochemical tests, phylogenetic analysis and homology of 16S rRNA gene sequences revealed their high similarity with Aeromonas veronii and Shigella flexneri. Another isolate with characteristics resembling Escherichia fergusonii was obtained in phytoplankton-free water samples. Since these three cultures were enriched on chitin-selective medium, their capability to utilize chitin as a sole C source was examined. The in vivo chitinase activity (as µg N-acetyglucosamine produced min--1 mL--1 culture) was 82.1, 28.5 and 18.5 for A. veronii, S. flexneri and E. fergusonii, respectively. The corresponding N-acetylglucosamine accumulation in medium was 421, 288 and 122 µg mL--1. There was also a gradation in growth indices in the three bacteria, which corresponded to their chitin utilization ability. Notwithstanding differences in chitinase activity, the three strains utilized almost equally the exogenous N-acetylglucosamine. We propose that chitinase activity may have a role in affinity of the particular bacterial cell to phytoplankton extracellular polymeric substances and therefore, adherence ability. [ABSTRACT FROM AUTHOR]

Research background. Shrimp shells contain chitin that can be further processed into N-acetylglucosamine, which has been extensively used to treat joint damage. Providencia stuartii has a strong chitinolytic activity and may be utilized in the form of immobilized cells in repeated fermentation. Pumice is a porous and rigid stone that offers superior mechanical strength, making it suitable for immobilization. Experimental approach. In the research submerged fermentation with different pumice stone sizes and pumice stone/growth medium ratios (m/V) was carried out for 4 days at 37 °C and pH=7.0. The optimum pumice stone size and pumice stone/growth medium ratio (m/V) were used to determine the optimum fermentation cycle for the production of N-acetylglucosamine using immobilized P. stuartii. Results and conclusions. Pumice stones of 1.0 cm×1.0 cm×1.0 cm and pumice stone/growth medium ratio of 1:5 were found to be the optimum conditions for successful immobilization of (90.0±1.6) % cells and production of (331.4±7.3) g/L N-acetylglucosamine. The highest N-acetylglucosamine concentration of (323.0±2.5) g/L was obtained in the first fermentation cycle, which then decreased and remained stable throughout the last three cycles. Novelty and scientific contribution. P. stuartii, a strong chitinolytic bacterium previously isolated from rotten shrimp shells, was used for the first time in immobilized form to produce N-acetylglucosamine. The findings in this research showed the potential use of P. stuartii cells immobilized in pumice stone for continuous production of N-acetylglucosamine in repeated fermentation. [ABSTRACT FROM AUTHOR]

Background: Lacto-N-triose II (LNT II), an important backbone for the synthesis of different human milk oligosaccharides, such as lacto-N-neotetraose and lacto-N-tetraose, has recently received significant attention. The production of LNT II from renewable carbon sources has attracted worldwide attention from the perspective of sustainable development and green environmental protection. Results: In this study, we first constructed an engineered E. coli cell factory for producing LNT II from N-acetylglucosamine (GlcNAc) feedstock, a monomer of chitin, by introducing heterologous β-1,3-acetylglucosaminyltransferase, resulting in a LNT II titer of 0.12 g L−1. Then, lacZ (lactose hydrolysis) and nanE (GlcNAc-6-P epimerization to ManNAc-6-P) were inactivated to further strengthen the synthesis of LNT II, and the titer of LNT II was increased to 0.41 g L−1. To increase the supply of UDP-GlcNAc, a precursor of LNT II, related pathway enzymes including GlcNAc-6-P deacetylase, glucosamine synthase, and UDP-N-acetylglucosamine pyrophosphorylase, were overexpressed in combination, optimized, and modulated. Finally, a maximum titer of 15.8 g L−1 of LNT II was obtained in a 3-L bioreactor with optimal enzyme expression levels and β-lactose and GlcNAc feeding strategy. Conclusions: Metabolic engineering of E. coli is an effective strategy for LNT II production from GlcNAc feedstock. The titer of LNT II could be significantly increased by modulating the gene expression strength and blocking the bypass pathway, providing a new utilization for GlcNAc to produce high value-added products. [ABSTRACT FROM AUTHOR]

Using commercial API-ZYM screening kits, highly active α-glucosidase, β- glucosidase, and β-N-acetylglucosaminidase were found in Grifola frondosa, having potential for carbohydrate utilization. Of these, β-N- acetylglucosaminidase, which converts chitin to N-acetylglucosamine, was purified and characterized. The recovery was 24.5%, and the purified enzyme had a specific activity 0.67 U/mg protein. Chitinase activity was confirmed by zymogram analysis. The enzyme was also shown to be β-N- acetylglucosaminidase, as N-acetylglucosamine was the main hydrolysis product from colloidal chitin. Thus, the molecule was named NAG38, to indicate β-N-acetylglucosaminidase activity and a molecular weight of 38 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity was optimal at pH 7.0 and 50 °C, with Km and Vmax values of 0.112 mM and 0.570 μmol/min/mg protein against p-nitrophenyl N-acetyl-β-D-glucosaminide. The bioactivity was inhibited by Hg2+, Ag+, Mg2+, Zn2+, Ca2+, and Mn2+, with residual enzyme bioactivity only 11.1% after incubation in Hg2+, but was not substantially inhibited by Ba2+, K+, and Na+. [ABSTRACT FROM AUTHOR]

Abstract Arbuscular mycorrhizal (AM) symbiosis is heavily and positively implicated in phosphorus (P) acquisition from soil to plants, including many important agricultural crops. Its role in plant nitrogen (N) nutrition is generally not as prominent or beneficial, with exception of some situations when N is available predominantly in organic forms. Yet the AM fungi (AMF) are, due to their poor exo-enzymatic repertoire, unlikely to degrade organic compounds on their own, therefore they possibly depend on other microorganisms to liberate nutrients contained in those materials. Here, we review current knowledge on the roles played by the AMF in plant N nutrition in general and uptake of N from organic compounds in particular, with a specific reference to microbes and processes involved in liberation and AM fungal utilization of N from organic compounds. Future research needs and directions are outlined, as is the agronomic and societal context of such research.

N-acetylglucosamine (NAG) is the monomer of chitin which is the most abundant nitrogen-containing biomass. Thus, NAG is a potential nitrogen-rich feedstock for the production of renewable organonitrogen chemicals. In this work, ethanolamine ionic liquids were synthesized easily from inexpensive raw materials and subsequently used as catalysts for NAG conversion to produce 3-acetamido-5-acetylfuran (3A5AF). Using triethanolamine hydrochloride ([TEA]Cl) ionic liquid as the catalyst, a high 3A5AF yield of 62.0% was obtained at the optimum reaction conditions of 170°Cand 20 min. Moreover, this ionic liquid exhibited good reusability and still had good catalytic activity after recycling five times. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis provided the relevant basis for rationalizing the reaction mechanism. Therefore, our study presents an intriguing and sustainable protocol for the direct conversion of NAG. [ABSTRACT FROM AUTHOR]

Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc2, chitin deacetylase to convert GlcNAc2 into GlcN2 and acetate, and glucosaminidase to cleave GlcN2 into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a β-1,4-glucosaminidase degraded chitin to GlcNAc2 or GlcN2 to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin. Key Points • A bacterial consortium was developed to use chitin as feedstock for the bioeconomy. • Substrate converter and producer strain use different chitin hydrolysis products. • Substrate converter and producer strain are mutually dependent on each other. [ABSTRACT FROM AUTHOR]

This study assessed the effects of diets supplemented with different types of chitin hydrolysate on larval growth of the Japanese eel Anguilla japonica. Larvae were reared for 26 days on artificial diets containing 2% of purified monomeric N-acetylglucosamine (NAG1), a mixture of mono-, di- and trimers (NAG3), a mixture of mono-, di-, tri-, tetra-, penta- and hexamers (NAG6), or a control diet. The larvae fed NAG3 and NAG6 grew significantly more rapidly than the control larvae (p < 0.05). However, the survival rate of the NAG6-fed larvae was low (8%) for unknown reasons, whereas the survival rates of the NAG1 and NAG3-fed larvae were similar to that of control larvae, about 46% (p > 0.9). These results indicate that NAG3 is the most effective supplement for eel larvae. A dose–response analysis showed that the optimal level of NAG3 in the diet was around 1%. Although the biological activity of chitin hydrolysates in eel larvae remains unclear, part of these hydrolysates are likely utilized by eel larvae as components of glycosaminoglycan, a major constituent of their bodies. Alternatively, the hydrolysates may act as a bioactive substance, influencing fish health, or in the effective utilization of energy in eel larvae, enhancing their growth. [ABSTRACT FROM AUTHOR]

Chitin, which is the polymer of N-acetylglucosamine (GlcNAc) linked by β1,4 glycoside bonds, has been reported as a soil amendment to mitigate plant soil diseases, increasing the population of chitindegrading bacteria, and chitinolytic enzymatic activity in the soil. In some chitin-degrading bacteria, whose chitinolytic systems have been intensively studied, the chitin degradation product N,N'-diacetylchitobiose {(GlcNAc)2} induces expression of genes for chitinases whereas GlcNAc does not. To evaluate the effects of these mono- and disaccharides on the population and activity of chitinolytic bacteria in soil, we investigated the chitinolytic enzyme activity and bacterial community structure in an incubated upland soil supplemented with GlcNAc or (GlcNAc)2. The added GlcNAc and (GlcNAc)2 (2 mg g-1) were consumed within 1 d when incubated at 25°C. Chitinase activity was induced by (GlcNAc)2 and chitin after 1-d and 7-d incubation, respectively, but not by GlcNAc. N-acetylglucosaminidase (GlcNAcase) activity was induced by GlcNAc but was lower than those by (GlcNAc)2 and chitin. Amplicon sequencing analysis targeting 16S rRNA genes demonstrated that both GlcNAc and (GlcNAc)2 significantly increased the rate of the order Bacillales, but the compositions of Bacillales differed from each other: the family Planococcaceae significantly increased in either GlcNAc- or (GlcNAc)2-added soil, but the genera Bacillus and Paenibacillus were increased mainly by GlcNAc and (GlcNAc)2, respectively. The family Streptomycetaceae of the order Actinomycetales was significantly increased by (GlcNAc)2 and chitin, but GlcNAc did not. Thus, GlcNAc and (GlcNAc)2, which were promptly consumed in the incubated soil, indicated partly similar but distinctive effects on chitinolytic enzyme activity and bacterial communities. Both aminosugars increased GlcNAcase activity and the population size of Planococcaceae. GlcNAc increased Bacillus. Chitinase activity and the populations of Paenibacillus and Streptomycetaceae, a number of strains of which are known as potent chitin-degraders, were increased by (GlcNAc)2, but not by GlcNAc. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the chitin degradation potential and whole-genome sequence of Streptomyces diastaticus strain CS1801, which had been screened out in our previous work. The results of fermentation revealed that CS1801 can convert the chitin derived from crab shells, colloidal chitin and N-acetylglucosamine to chitooligosaccharide. Additional genome-wide analysis of CS1801 was also performed to explore the genomic basis for chitin degradation. The results showed that CS1801 possesses a chromosome with 5,611,479 bp (73% GC) and a plasmid with 1,388,284 bp (73% GC). The CS1801 genome consists of 7584 protein-coding genes, 90 tRNA and 21 rRNA operons. In addition, the results of genomic CAZyme analysis indicated that CS1801 comprises 103 glycoside hydrolase family genes, which could regulate the glycoside hydrolases that contribute to chitin degradation. The whole-genome information of CS1801 could highlight the mechanism underlying the chitin degradation activity of CS1801, strongly indicating that CS1801 is characterized by a substantial number of genes encoding chitinases and the complete metabolic pathway of chitin, conferring CS1801 with promising potential applicability in chitooligosaccharide production. [ABSTRACT FROM AUTHOR]

Chitinase is an enzyme that degrades chitin on β-1,4-acetamido-2-deoxy-D-glycoside bound to produce Chitin oligosaccharide or its soluble monomer, N-acetylglucosamine (NAG). Chitinase are usually produced naturally by chitinolytic microorganism that are found in soil or water environment. Chitinase have been widely used for biocontrol agents of plant pests. The product from chitinase hydrolysis can be used as antiinflammatory agent and anti hyperpigmentation. This research was aimed to screen and evaluate the chitinolytic ability of 9 isolate. The best isolate was the one which produced high chitinolytic activity and hydrolyzed chitin into NAG. 9 isolate were examined and BPPTCC-2 was the best isolate which produced highest level of chitinolytic activity 0.1780 U/ml. The chitinase was able to hydrolyzed chitin resulting high yields of NAG 11.5% as the only product.

ABSTRACT Soil microorganisms play fundamental roles in cycling of soil carbon, nitrogen, and other nutrients, yet we have a poor understanding of how soil microbiomes are shaped by their nutritional and physical environment. In this study, we investigated the successional dynamics of a soil microbiome during 21 weeks of enrichment on chitin and its monomer, N-acetylglucosamine. We examined succession of the soil communities in a physically heterogeneous soil matrix as well as a homogeneous liquid medium. The guiding hypothesis was that the initial species richness would influence the tendency for the selected consortia to stabilize and maintain a relatively constant community structure over time. We also hypothesized that long-term, substrate-driven growth would result in consortia with reduced species richness compared to the parent microbiome and that this process would be deterministic with relatively little variation between replicates. We found that the initial species richness does influence the long-term community stability in both liquid media and soil and that lower initial richness results in a more rapid convergence to stability. Despite use of the same soil inoculum and access to the same major substrate, the resulting community composition differed greatly in soil from that in liquid medium. Hence, distinct selective pressures in soils relative to homogenous liquid media exist and can control community succession dynamics. This difference is likely related to the fact that soil microbiomes are more likely to thrive, with fewer compositional changes, in a soil matrix than in liquid environments. IMPORTANCE The soil microbiome carries out important ecosystem functions, but interactions between soil microbial communities have been difficult to study due to the high microbial diversity and complexity of the soil habitat. In this study, we successfully obtained stable consortia with reduced complexity that contained species found in the original source soil. These consortia and the methods used to obtain them can be a valuable resource for exploration of specific mechanisms underlying soil microbial community ecology. The results of this study also provide new experimental context to better inform how soil microbial communities are shaped by new environments and how a combination of initial taxonomic structure and physical environment influences stability. Author Video: An author video summary of this article is available.

Chitin is not only the most abundant nitrogen‐containing biopolymer on the planet, but also a renewable feedstock that is often treated as a waste. Current chemical methods to break down chitin typically employ harsh conditions, large volumes of solvent, and generate a mixture of products. Although enzymatic methods have been reported, they require a harsh chemical pretreatment of the chitinous substrate and rely on dilute solution conditions that are remote from the natural environment of microbial chitinase enzymes, which typically consists of surfaces exposed to air and moisture. We report an innovative and efficient mechanoenzymatic method to hydrolyze chitin to the N‐acetylglucosamine monomer by using chitinases under the recently developed reactive aging (RAging) methodology, based on repeating cycles of brief ball‐milling followed by aging, in the absence of bulk solvent. Our results demonstrate that the activity of chitinases increases several times by switching from traditional solution‐based conditions of enzymatic catalysis to solventless RAging, which operates on moist solid substrates. Importantly, RAging is also highly efficient for the production of N‐acetylglucosamine directly from shrimp and crab shell biomass without any other processing except for a gentle wash with aqueous acetic acid. [ABSTRACT FROM AUTHOR]

Background: Serratia marcescens is a chitinolytic bacterium that can potentially be used for consolidated bioprocessing to convert chitin to value-added chemicals. Currently, S. marcescens is poorly characterized and studies on intracellular metabolic and regulatory mechanisms would expedite development of bioprocessing applications. Results: In this study, our goal was to characterize the metabolic profile of S. marcescens to provide insight for metabolic engineering applications and fundamental biological studies. Hereby, we constructed a constraint-based genome-scale metabolic model (iSR929) including 929 genes, 1185 reactions and 1164 metabolites based on genomic annotation of S. marcescens Db11. The model was tested by comparing model predictions with experimental data and analyzed to identify essential aspects of the metabolic network (e.g. 138 essential genes predicted). The model iSR929 was refined by integrating RNAseq data of S. marcescens growth on three different carbon sources (glucose, N-acetylglucosamine, and glycerol). Significant differences in TCA cycle utilization were found for growth on the different carbon substrates, For example, for growth on N-acetylglucosamine, S. marcescens exhibits high pentose phosphate pathway activity and nucleotide synthesis but low activity of the TCA cycle. Conclusions: Our results show that S. marcescens model iSR929 can provide reasonable predictions and can be constrained to fit with experimental values. Thus, our model may be used to guide strain designs for metabolic engineering to produce chemicals such as 2,3-butanediol, N-acetylneuraminic acid, and n-butanol using S. marcescens. [ABSTRACT FROM AUTHOR]

Insect meals (IM) are being pointed out as a promising sustainable protein source. However, chitin in IM may negatively interfere with fish performance and nutrient availability. Given that carnivorous fish have a low or inexistent ability to digest chitin, novel strategies are needed to overcome chitin-imposed bottlenecks. Carbohydrolytic sporeforming probiotics offer a competitive industrial approach, being able to resist the harsh feed manufacturing process, transport, storage, and animal's gastrointestinal tract, while being able to increase the digestibility of otherwise indigestible components. Aiming to increase IM chitin digestibility, two chitinolytic sporeforming fish isolates (Fish isolates 645 (FI645) and 658 (FI658)), closely related to Bacillus licheniformis , were isolated from European sea bass (ESB) gastrointestinal tract, and incorporated (individually or as a mixture) in ESB diets with high defatted Hermetia illucens larvae meal (HM) levels (30% inclusion). FI were evaluated regarding their effects on ESB growth performance and digestibility. The spores were able to maintain their viability throughout the 180 days of diet storage at room temperature. Dietary inclusion of FI645 led to higher chitin digestibility. Concomitantly, the digestibility of dry matter, protein, and energy increased, overall leading to higher feed efficiency and protein efficiency ratio. Dietary inclusion of FI645 led to increased plasma N -acetylglucosamine (GlcNAc) levels. This is the first evidence for diet-mediated modulation of plasma GlcNAc concentrations. Dietary inclusion of FI645 also increased the expressions of the genes coding for N -acetylglucosamine kinase (nagk) and GlcNAc phosphomutase (pgm3) , key enzymes from the GlcNAc salvage pathway and the hexosamine biosynthetic pathway (HBP). On the other hand, no differences were found for the expression of N -acetyl-D-glucosamine-6-phosphate deacetylase (amdhd2) , a precursor enzyme for GlcNAc redirection to glycolysis. These results hint that free GlcNAc resulting from increased chitin digestibility might be preferentially redirected to protein O-GlcNAcylation processes through HBP in fish cells. Additionally, when challenged with Vibrio anguillarum , and after being feed for one month with the same diets, D645 also increased ESB survival from 52.5% to 77.5%, when compared to the control. Together, these results establish the potential of using FI645 as a probiotic for enhancing chitin digestion and acting as a prophylactic agent for ESB protection against V. anguillarum infection. This strategy opens novel possibilities for using commodities, including high chitin levels in aquafeeds, and reducing the use of antibiotics in aquaculture. • Fish isolate 645 increases chitin, protein, and energy digestibility and feed efficiency. • Increase in chitin digestibility increased N -acetylglucosamine in plasma. • The hexosamine biosynthetic pathway is potentially modulated by N -acetylglucosamine. • Fish isolate 645 increases European sea bass survivability to Vibrio anguillarum. [ABSTRACT FROM AUTHOR]

[Display omitted] • C.curvatus concurrently assimilated GlcNAc and glucose for lipid production. • GlcNAc has the capacity to supply both carbon and nitrogen sources. • The co-fermentation of rice hydrolysates and chitin hydrolysate was effective. • Scaling-up experiments in a 5-L bioreactor resulted in lipid content of 60.1 %. • The fatty acid compositions of lipids were similar to that for vegetable oils. Microbial lipids were produced through a two-stage process with Cryptococcus curvatus by co-fermenting rice and shrimp shells hydrolysates. In the first stage, biomass production of glucose and N -acetylglucosamine was optimized by response surface methodology with the maximum biomass yield (17.60 g/L) under optimum conditions (43.2 g/L mixed sugar concentration, pH 5.8, 200 rpm, and 28 °C). In the second stage, according to a single-factor optimization setting (43.2 g/L sugar mixture solutions, pH 5.5, and shift time of 36 h), lipid titer of 10.08 g/L with content of 55.30 % was achieved. Scaling up to a 5-L bioreactor increased lipid content to 60.07 % with 0.233 g/g yield. When Cryptococcus curvatus was cultured in the blends of rice hydrolysates and shrimp shells hydrolysate, lipid content and yield were 52.25 % and 0.204 g/g. The fatty acid compositions of lipid were similar to those of typical vegetable oils. [ABSTRACT FROM AUTHOR]

Density functional theory (DFT) studies have contributed to analyze the interaction between chitosan and chitin with Hg(I), Hg(II) and Pb(I), Pb(II) ions in the absence or in the presence of one to three water molecules. All calculations were performed at the M06-2X/LanL2DZ level of theory using Glucosamine and N-acetylglucosamine monomers as models of chitosan and chitin biopolymers, respectively. Geometries of all complexes were optimized, enthalpies and Gibbs free energies of the interactions were calculated. The obtained results suggest that Hg(I) and Pb(I) cations tend to bind to glucosamine at the O4/N5 position with the best stability, while these cations prefer the O3/O4 position in coordinating to N-acetylglucosamine. The most favorable binding positions of these two molecules with Hg(II) and Pb(II) cations are all at the O3/O4 ones. The presence of water molecules slightly affects the most favorable binding configuration, and strongly stabilizes the metallic complexes by forming several hydrogen bonds. Additionally, the Pb(I) complexes are more stable than the Hg(I) ones, whereas the Pb(II) complexes are less stable than the Hg(II) ones. This observation is quite coherent with reported experimental data. Finally, natural bond orbital (NBO) analysis shows that all the metallic cations play as electron acceptors in receiving lone pairs of electron from the heteroatoms of the ligands to their vacant orbitals. [ABSTRACT FROM AUTHOR]