Engineering of a chitin deacetylase to generate tailor-made chitosan polymers.

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.

Nasal delivery of polymeric nanoDisc mobilizes a synergy of central and peripheral amyloid-ß clearance to treat Alzheimer's disease.

The disequilibrium of amyloid ß-peptide (Aß) between the central and peripheral pools has been claimed as an initiating event in Alzheimer's disease (AD). In this study, we employ discoidal high-density lipoproteins (HDL-Disc) mimicking Aß antibody for directional flux of Aß from central to peripheral catabolism, with desirable safety and translation potential. Structurally, HDL-Disc assembly (polyDisc) is prepared with aid of chitosan derivative polymerization. After intranasal administration and response to slightly acidic nasal microenvironment, polyDisc depolymerizes into carrier-free HDL-Disc with chitosan derivatives that adhere to the mucosal layer to reversibly open tight junctions, helping HDL-Disc penetrate the olfactory pathway into brain. Thereafter, HDL-Disc captures Aß into microglia for central clearance or ferries Aß out of the brain for liver-mediated compensatory catabolism. For synergy therapy, intranasal administration of polyDisc can effectively reduce intracerebral Aß burden by 97.3% and vascular Aß burden by 73.5%, ameliorate neurologic damage, and rescue memory deficits in APPswe/PS1dE9 transgenic AD mice with improved safety, especially vascular safety. Collectively, this design provides a proof of concept for developing Aß antibody mimics to mobilize a synergy of central and peripheral Aß clearance for AD treatment.

The LysM Receptor-Like Kinase SlLYK10 Controls Lipochitooligosaccharide Signaling in Inner Cell Layers of Tomato Roots.

Establishment of arbuscular mycorrhiza relies on a plant signaling pathway that can be activated by fungal chitinic signals such as short-chain chitooligosaccharides and lipo-chitooligosaccharides (LCOs). The tomato LysM receptor-like kinase SlLYK10 has high affinity for LCOs and is involved in root colonization by arbuscular mycorrhizal fungi (AMF); however, its role in LCO responses has not yet been studied. Here, we show that SlLYK10 proteins produced by the Sllyk10-1 and Sllyk10-2 mutant alleles, which both cause decreases in AMF colonization and carry mutations in LysM1 and 2, respectively, have similar LCO-binding affinities compared to the WT SlLYK10. However, the mutant forms were no longer able to induce cell death in Nicotiana benthamiana when co-expressed with MtLYK3, a Medicago truncatula LCO co-receptor, while they physically interacted with MtLYK3 in co-purification experiments. This suggests that the LysM mutations affect the ability of SlLYK10 to trigger signaling through a potential co-receptor rather than its ability to bind LCOs. Interestingly, tomato lines that contain a calcium (Ca2+) concentration reporter [genetically encoded Ca2+ indicators (GECO)], showed Ca2+ spiking in response to LCO applications, but this occurred only in inner cell layers of the roots, while short-chain chitooligosaccharides also induced Ca2+ spiking in the epidermis. Moreover, LCO-induced Ca2+ spiking was decreased in Sllyk10-1*GECO plants, suggesting that the decrease in AMF colonization in Sllyk10-1 is due to abnormal LCO signaling.

Interactions between copper homeostasis and the fungal cell wall affect copper stress resistance.

Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1Δ mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin / chitosan deposition and exposure. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein could be involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake.

LysM receptor-like kinases involved in immunity perceive lipo-chitooligosaccharides in mycotrophic plants.

Symbiotic microorganisms such as arbuscular mycorrhizal fungi (AMF) produce both conserved microbial molecules that activate plant defense and lipo-chitooligosaccharides (LCOs) that modulate plant defense. Beside a well-established role of LCOs in the activation of a signaling pathway required for AMF penetration in roots, LCO perception and defense modulation during arbuscular mycorrhiza is not well understood. Here we show that members of the LYRIIIA phylogenetic group from the multigenic Lysin Motif Receptor-Like Kinase family have a conserved role in dicotyledons as modulators of plant defense and regulate AMF colonization in the Solanaceae species Nicotiana benthamiana. Interestingly, these proteins have a high-affinity for LCOs in plant species able to form a symbiosis with AMF but have lost this property in species that have lost this ability. Our data support the hypothesis that LYRIIIA proteins modulate plant defense upon LCO perception to facilitate AMF colonization in mycotrophic plant species and that only their role in plant defense, but not their ability to be regulated by LCOs, has been conserved in non-mycotrophic plants.

Continuous production of chitooligosaccharides in a column reactor by the PUF-immobilized whole cell enzymes of Mucor circinelloides IBT-83.

BACKGROUND: Chitosan oligosaccharides (COS) have great potential for applications in several fields, including agriculture, food industry or medicine. Nevertheless, the large-scale use of COS requires the development of cost-effective technologies for their production. The main objective of our investigation was to develop an effective method of enzymatic degradation of chitosan in a column reactor using Mucor circinelloides IBT-83 cells, immobilized in a polyurethane foam (PUF). These cells serve as a source of chitosanolytic enzymes. RESULTS: The study revealed that the process of freeze-drying of immobilized mycelium increases the stability of the associated enzymes during chitosan hydrolysis. The use of stabilized preparations as an active reactor bed enables the production of COS at a constant level for 16 reactor cycles (384 h in total), i.e. 216 h longer compared to non-stabilized mycelium. In the hydrolysate, oligomers ranging in structure from dimer to hexamer as well as D-glucosamine were detected. The potential application of the obtained product in agriculture has been verified. The results of phytotests have demonstrated that the introduction of COS into the soil at a concentration of 0.01 or 0.05% w/w resulted in an increase in the growth of Lepidium sativum stem and root, respectively (extensions by 38 and 44% compared to the control sample). CONCLUSIONS: The research has verified that the PUF-immobilized M. circinelloides IBT-83 mycelium, which has been stabilized through freeze-drying, is a promising biocatalyst for the environmentally friendly and efficient generation of COS. This biocatalyst has the potential to be used in fertilizers.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Antimicrobial activity and nanoremediation of heavy metals using biosynthesized CS/GO/ZnO nanocomposite by Bacillus subtilis ATCC 6633 alone or immobilized in a macroporous cryogel.

BACKGROUND: The world society is still suffering greatly from waterborne infections, with developing countries bearing most of the morbidity and death burden, especially concerning young children. Moreover, microbial resistance is one of the most prevalent global problems that extends the need for self-medication and the healing period, or it may be linked to treatment failure that results in further hospitalization, higher healthcare expenses, and higher mortality rates. Thus, innovative synthesis of new antimicrobial materials is required to preserve the environment and enhance human health. RESULTS: The present study highlighted a simple and cost-effective approach to biosynthesize a chitosan/graphene oxide/zinc oxide nanocomposite (CS/GO/ZnO) alone and immobilized in a macroporous cryogel as a new antimicrobial agent. Bacillus subtilis ATCC 6633 was used as a safe and efficient bio-nano-factory during biosynthesis. The formation of CS/GO/ZnO was confirmed and characterized using different analyses including ultraviolet-visible spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), selective area diffraction pattern (SADP), Zeta analyses, scanning electron microscope (SEM) and transmission electron microscopy (TEM). GO combined with ZnO NPs successfully and displayed an adsorption peak at 358 nm. The XRD results showed the crystalline composition of the loaded ZnO NPs on GO sheets. FTIR spectrum confirmed the presence of proteins during the synthesis which act as stabilizing and capping agents. The nanocomposite has a high negative surface charge (-32.8 ± 5.7 mV) which increases its stability. SEM and TEM showing the size of biosynthesized ZnO-NPs was in the range of 40-50 nm. The CS/GO/ZnO alone or immobilized in cryogel revealed good antimicrobial activities against B. cereus ATCC 14,579, Escherichia coli ATCC 25,922, and Candida albicans ATCC 10,231 in a dose-dependent manner. The CS/GO/ZnO cryogel revealed higher antimicrobial activity than GO/ZnO nanocomposite and standard antibiotics (amoxicillin and miconazole) with inhibition zones averages of 24.33 ± 0.12, 15.67 ± 0.03, and 17.5 ± 0.49 mm, respectively. The MIC values of the prepared nanocomposite against B. cereus, E. coli, and C. albicans were 80, 80, and 90 µg/ml compared to standard drugs (90, 120 and 150 µg/ml, respectively). According to the TEM ultrastructure studies of nanocomposite-treated microbes, treated cells had severe deformities and morphological alterations compared to the untreated cells including cell wall distortion, the separation between the cell wall and plasma membrane, vacuoles formation moreover complete cell lyses were also noted. In the cytotoxicity test of CS/GO/ZnO alone and its cryogel, there was a significant reduction (p˂0.05) in cell viability of WI-38 normal lung cell line after the concentration of 209 and 164 µg/ml, respectively. It showed the low toxic effect of the nanocomposite and its cryogel on the WI-38 line which implies its safety. In addition, water treatment with the CS/GO/ZnO cryogel decreased turbidity (0.58 NTU), total coliform (2 CFU/100 ml), fecal coliform (1 CFU/100 ml), fecal Streptococcus (2 CFU/100 ml), and heterotrophic plate counts (53 CFU/1 ml) not only in comparison with the chlorine-treated samples (1.69 NTU, 4 CFU/100 ml, 6 CFU/100 ml, 57 CFU/100 ml, and 140 CFU/1 ml, respectively) but also with the raw water samples (6.9 NTU, 10800 CFU/100 ml, 660 CFU/100 ml, 800 CFU/100 ml, and 4400 CFU/1 ml, respectively). Moreover, cryogel significantly decreased the concentration of different heavy metals, especially cobalt compared to chlorine (0.004 ppm, 0.002 ppm, and 0.001 ppm for raw water, chlorine-treated, and cryogel-treated groups, respectively) which helped in the reduction of their toxic effects. CONCLUSION: This study provides an effective, promising, safe, and alternative nanocomposite to treat different human and animal pathogenic microbes that might be used in different environmental, industrial, and medical applications.

Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation.

BACKGROUND: In the inflammatory milieu of diabetic chronic wounds, macrophages undergo substantial metabolic reprogramming and play a pivotal role in orchestrating immune responses. Itaconic acid, primarily synthesized by inflammatory macrophages as a byproduct in the tricarboxylic acid cycle, has recently gained increasing attention as an immunomodulator. This study aims to assess the immunomodulatory capacity of an itaconic acid derivative, 4-Octyl itaconate (OI), which was covalently conjugated to electrospun nanofibers and investigated through in vitro studies and a full-thickness wound model of diabetic mice. RESULTS: OI was feasibly conjugated onto chitosan (CS), which was then grafted to electrospun polycaprolactone/gelatin (PG) nanofibers to obtain P/G-CS-OI membranes. The P/G-CS-OI membrane exhibited good mechanical strength, compliance, and biocompatibility. In addition, the sustained OI release endowed the nanofiber membrane with great antioxidative and anti-inflammatory activities as revealed in in vitro and in vivo studies. Specifically, the P/G-CS-OI membrane activated nuclear factor-erythroid-2-related factor 2 (NRF2) by alkylating Kelch-like ECH-associated protein 1 (KEAP1). This antioxidative response modulates macrophage polarization, leading to mitigated inflammatory responses, enhanced angiogenesis, and recovered re-epithelization, finally contributing to improved healing of mouse diabetic wounds. CONCLUSIONS: The P/G-CS-OI nanofiber membrane shows good capacity in macrophage modulation and might be promising for diabetic chronic wound treatment.

Pharmacodynamic study of chitosan in combination with salvianolic acid B in the treatment of CC14 -induced liver fibrosis in mice.

Liver fibrosis is a chronic liver lesion caused by excessive deposition of the extracellular matrix after liver damage, resulting in fibrous scarring of liver tissue. The progression of liver fibrosis is partially influenced by the gut microbiota. Chitosan can play a therapeutic role in liver fibrosis by regulating the gut microbiota based on the 'gut-liver axis' theory. Salvianolic acid B can inhibit the development of liver fibrosis by inhibiting the activation of hepatic stellate cells and reducing the production of extracellular matrix. In this study, the therapeutic effect of chitosan in combination with salvianolic acid B on liver fibrosis was investigated in a mouse liver fibrosis model. The results showed that the combination of chitosan and salvianolic acid B was better than the drug alone, improving AST/ALT levels and reducing the expression of α-SAM, COL I, IL-6 and other related genes. It improved the structure of gut microbiota and increased the abundance of beneficial bacteria such as Lactobacillus. The above results could provide new ideas for the clinical treatment of liver fibrosis.

Batch-mode degradation of high-strength phenolic pollutants by Pseudomonas aeruginosa strain STV1713 immobilized on single and hybrid matrices.

Controlled environments are pivotal in all bioconversion processes, influencing the efficacy of biocatalysts. In this study, we designed a batch bioreactor system with a packed immobilization column and a decontamination chamber to enhance phenol and 2,4-dichlorophenol degradation using the hyper-tolerant bacterium Pseudomonas aeruginosa STV1713. When free cells were employed to degrade phenol and 2,4-DCP at a concentration of 1000 mg/L, the cells completely removed the pollutants within 28 h and 66 h, respectively. Simultaneous reductions in chemical oxygen demand and biological oxygen demand were observed (phenol: 30.21 mg/L/h and 16.92 mg/L/h, respectively; 2,4-dichlorophenol: 12.85 mg/L/h and 7.21 mg/L/h, respectively). After assessing the degradation capabilities, the bacterium was immobilized on various matrices (sodium alginate, alginate-chitosan-alginate and polyvinyl alcohol-alginate) to enhance pollutant removal. Hybrid immobilized cells exhibited greater tolerance and degradation capabilities than those immobilized in a single matrix. Among them, polyvinyl alcohol-alginate immobilized cells displayed the highest degradation capacities (up to 2000 mg/L for phenol and 2500 mg/L for 2,4-dichlorophenol). Morphological analysis of the immobilized cells revealed enhanced cell preservation in hybrid matrices. Furthermore, the elucidation of the metabolic pathway through the catechol dioxygenase enzyme assay indicated higher activity of the catechol 1,2-dioxygenase enzyme, suggesting that the bacterium employed an ortho-degradation mechanism for pollutant removal. Additionally, enzyme zymography confirmed the presence of catechol 1,2-dioxygenase, with the molecular weight of the enzyme determined as 245 kDa.

Exploring rumen fermentation and microbial populations in Dhofari goats fed a chitosan-added diet.

The use of chitosan (CHI) in ruminant diets is a promising natural modifier for rumen fermentation, capable of modulating both the rumen pattern and microbial activities. The objective of this study was to explore the rumen fermentation and microbial populations in Dhofari goats fed a diet supplemented with CHI. A total of 24 Dhofari lactating goats (body weight, 27.32 ± 1.80 kg) were assigned randomly into three experimental groups (n = 8 ewes/group). Goats were fed a basal diet with either 0 (control), 180 (low), or 360 (high) mg CHI/kg of dietary dry matter (DM) for 45 days. Feeding high CHI linearly increased (p < 0.05) the propionate level and reduced the acetate, butyrate, and total protozoa count (p < 0.05). Ruminal ammonia nitrogen (NH3-N) concentrations and the acetate:propionate ratio decreased linearly when goats were fed CHI (p < 0.05). The abundances of both Spirochetes and Fibrobacteres phyla were reduced (p < 0.05) with both CHI doses relative to the control. Both low and high CHI reduced (p < 0.05) the relative abundances of Butyrivibrio hungatei, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, Selenomonas ruminantium and Neocallimastix californiae populations. Adding CHI significantly decreased (p < 0.05) the abundances of Ascomycota, Basidiomycota, and Bacillariophyta phyla compared to the control. Adding CHI to the diet reduces the abundance of fibrolytic-degrading bacteria, however, it increases the amylolytic-degrading bacteria. Application of 360 mg of CHI/kg DM modified the relative populations of ruminal microbes, which could enhance the rumen fermentation patterns in Dhofari goats.

Effectiveness of chitosan nanohydrogel mediated encapsulation of EcR dsRNA against the whitefly, Bemisia tabaci Asia-I (Gennedius) (Hemiptera: Aleyordidae).

Bemisia tabaci is a global invasive pest causing substantial loss on several economically important crops and has developed a very high level of resistance to insecticides making current management practices ineffective. Thus, the novel pest management strategy like RNA interference (RNAi) has emerged as a potential molecular tool in the management of insect pests particularly B. tabaci. The present study investigated RNAi mediated silencing of the Ecdysone Receptor (EcR) gene in B. tabaci Asia-I using biodegradable Chitosan Nanoparticles (CNPs) hydrogel containing EcR dsRNA. The formation of nanohydrogel and dsRNA loading were characterized by gel retardation assay, scanning electron microscopy (SEM); transmission electron microscopy (TEM) and Fourier transform infrared microscopy (FTIR). The stability of CNPs/dsRNA was assessed by exposure to direct sunlight and UV light for different time periods. The CNPs/dsRNA exhibited increased stability over the untreated control and further confirmed by bioassay studies which yielded mortality over 80% and effectively down regulated the expression of the EcR gene as confirmed by qRT-PCR analysis. These investigations provide potential avenues for advancing innovative pest management strategies using biopolymer CNPs hydrogel, which can enhance the efficiency of dsRNA as a safe and targeted solution in the management of whiteflies.

Hijacking 5-Fluorouracil Chemoresistance in Triple Negative Breast Cancer via microRNAs-Loaded Chitosan Nanoparticles.

Chemotherapy is still the mainstay of treatment for triple-negative breast cancer (TNBC) patients. Yet only 20% of TNBC patients show a pathologic complete response (pCR) after neoadjuvant chemotherapy. 5-Fluorouracil (5-FU) is a stable cornerstone in all recommended chemotherapeutic protocols for TNBC patients. However, TNBC patients' innate or acquired chemoresistance rate for 5-FU is steeply escalating. This study aims to unravel the mechanism behind the chemoresistance of 5-FU in the aggressive TNBC cell line, MDA-MB-231 cells, to explore further the role of the tumor suppressor microRNAs (miRNAs), miR-1275, miR-615-5p, and Let-7i, in relieving the 5-FU chemoresistance in TNBC, and to finally provide a translational therapeutic approach to co-deliver 5-FU and the respective miRNA oligonucleotides using chitosan-based nanoparticles (CsNPs). In this regard, cellular viability and proliferation were investigated using MTT and BrdU assays, respectively. 5-FU was found to induce JAK/STAT and PI3K/Akt/mTOR pathways in MDA-MB-231 cells with contaminant repression of their upstream regulators miR-1275, miR-615-5p, and Let-7i. Moreover, CsNPs prepared using the ionic gelation method were chosen and studied as nanovectors of 5-FU and a combination of miRNA oligonucleotides targeting TNBC. The average particle sizes, surface charges, and morphologies of the different CsNPs were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. In addition, the encapsulation efficiency (EE%), drug loading capacity (DLC%), and release manner at two different pH values were assessed. In conclusion, the novel CsNPs co-loaded with 5-FU and the combination of the three miRNA oligonucleotides demonstrated synergistic activity and remarkable repression in cellular viability and proliferation of TNBC cells through alleviating the chemoresistance to 5-FU.

Partial nitrification-denitrification and enrichment of paracoccus induced by iron-chitosan beads addition in an intermittently-aerated activated sludge system.

Insufficient carbon source has become the main limiting factor for efficient nitrogen removal in wastewater treatment. In this study, an intermittently-aerated activated sludge system with iron-chitosan (Fe-CS) beads addition was proposed for nitrogen removal from low C/N wastewater. By adding Fe-CS beads, partial nitrification-denitrification (PND) process and significant enrichment of Paracoccus (with ability of iron reduction/ammonium oxidation/aerobic denitrification) were observed in the reactor. The accumulation rate of NO2--N reached 81.9 %, and the total nitrogen removal efficiency was improved to 93.9 % by shortening the aeration time. The higher activity of ammonium oxidizing bacteria and inhibited activity of nitrite-oxidizing bacteria in Fe-CS assisted system mediated the occurrence of PND. In contrast, the traditional nitrification and denitrification process occurred in the control group. The high-throughput sequencing analysis and metagenomic results confirmed that the addition of Fe-CS induced 77.8 % and 54.9 % enrichment of Paracoccus in sludge and Fe-CS beads, respectively, while almost no enrichment was observed in control group. Furthermore, with the addition of Fe-CS beads, the expression of genes related to outer membrane porin, cytochrome c, and TCA was strengthened, thereby enhancing the electron transport of Fe(Ⅱ) (electron donor) and Fe(Ⅲ) (electron acceptor) with pollutants in the periplasm. This study provides new insights into the direct enrichment of iron-reducing bacteria and its PND performance induced by the Fe-CS bead addition. It therefore offers an appealing strategy for low C/N wastewater treatment.

Chitinase induction in Trichoderma harzianum: a solid-state fermentation approach using shrimp waste and wheat bran/commercial chitin for chitooligosaccharides synthesis.

This study innovatively employed solid-state fermentation (SSF) to evaluate chitinase induction in Trichoderma harzianum. Solid-state fermentation minimizes water usage, a crucial global resource, and was applied using shrimp waste chitin and a mixture of commercial chitin with wheat bran as substrates. Shrimp waste and wheat bran were pretreated and characterized for SSF, and the fungus's utilization of the substrates was assessed using spectrophotometric and microscopic methods. The resulting enzymes' ability to produce chitooligosaccharides (COS) mixtures was studied. Wheat bran/commercial chitin demonstrated superior performance, with a 1.8-fold increase in chitinase activity (76.3 U/mg protein) compared to shrimp waste chitin (41.8 U/mg protein). Additionally, the COS mixture obtained from wheat bran/commercial chitin showed a higher concentration of reducing sugars, reaching 87.85 mM, compared to shrimp waste chitin (14.87 mM). The COS profile from wheat bran/commercial chitin included monomers to heptamers, while the profile from shrimp waste chitin was predominantly composed of monomers. These results highlight the advantages of SSF for chitinase induction and COS production in T. harzianum, offering potential applications as dietary fiber, antioxidants, and antimicrobial agents. The findings contribute to by-product valorization, waste reduction, and the sustainable generation of valuable products through SSF-based enzyme production.

Tailored chitosan integration in diatomaceous earth particles as a scaffold for fructosyltransferase immobilization in fructo-oligosaccharide production.

BACKGROUND: Fructo-oligosaccharide (FOS) belongs to the group of short inulin-type fructans and is one of the most important non-digestible bifid-oligosaccharides capable of biotransforming sucrose using fructosyltransferase (FTase). However, there are no immobilized FTase products that can be successfully used industrially. In this study, diatomite was subjected to extrusion, sintering and granulation to form diatomaceous earth particles that were further modified via chitosan aminomethylation for modification. FTase derived from Aspergillus oryzae was successfully immobilized on the modified support via covalent binding. RESULTS: The immobilized enzyme activity was 503 IU g-1 at an enzyme concentration of 0.6 mg mL-1, immobilization pH of 7.0 and contact time of 3 h. Additionally, the immobilization yield was 56.91%. Notably, the immobilized enzyme was more stable under acidic conditions. Moreover, the half-life of the immobilized enzyme was 20.80 and 10.96 times as long as that of the free enzyme at 45 and 60 °C, respectively. The results show good reusability, as evidenced by the 84.77% retention of original enzyme activity after eight cycles. Additionally, the column transit time of the substrate was 35.56 min when the immobilized enzyme was applied in a packed-bed reactor. Furthermore, a consistently high FOS production yield of 60.68% was achieved and maintained over the 15-day monitoring period. CONCLUSIONS: Our results suggest that immobilized FTase is a viable candidate for continuous FOS production on an industrial scale. © 2024 Society of Chemical Industry.

Single-channel characterization of the chitooligosaccharide transporter chitoporin (SmChiP) from the opportunistic pathogen Serratia marcescens.

Serratia marcescens is an opportunistic pathogen that can utilize chitin as a carbon source, through its ability to produce chitin-degrading enzymes to digest chitin and membrane transporters to transport the degradation products (chitooligosaccharides) into the cells. Further characterization of these proteins is important to understand details of chitin metabolism. Here, we investigate the properties and function of the S. marcescens chitoporin, namely SmChiP, a chitooligosaccharide transporter. We show that SmChiP is a monomeric porin that forms a stable channel in artificial phospholipid membranes, with an average single-channel conductance of 0.5 ± 0.02 nS in 1 M KCl electrolyte. Additionally, we demonstrated that SmChiP allowed the passage of small molecules with a size exclusion limit of <300 Da and exhibited substrate specificity toward chitooligosaccharides, both in membrane and detergent-solubilized forms. We found that SmChiP interacted strongly with chitopentaose (Kd = 23 ± 2.0 µM) and chitohexaose (Kd = 17 ± 0.6 µM) but did not recognize nonchitose oligosaccharides (maltohexaose and cellohexaose). Given that S. marcescens can use chitin as a primary energy source, SmChiP may serve as a target for further development of nutrient-based antimicrobial therapies directed against multidrug antibiotic-resistant S. marcescens infections.

Swimming and L-arginine loaded chitosan nanoparticles ameliorates aging-induced neuron atrophy, autophagy marker LC3, GABA and BDNF-TrkB pathway in the spinal cord of rats.

Aging is associated with muscle atrophy, and erosion and destruction of neuronal pathways in the spinal cord. The study aim was to assess the effect of swimming training (Sw) and L-arginine loaded chitosan nanoparticles (LA-CNPs) on the sensory and motor neuron population, autophagy marker LC3, total oxidant status/total antioxidant capacity, behavioural test, GABA and BDNF-TrkB pathway in the spinal cord of aging rats. The rats were randomized to five groups: young (8-weeks) control (n = 7), old control (n = 7), old Sw (n = 7), old LA-CNPs (n = 7) and old Sw + LA-CNPs (n = 7). Groups under LA-CNPs supplementation received 500 mg/kg/day. Sw groups performed a swimming exercise programme 5 days per week for 6 weeks. Upon the completion of the interventions the rats were euthanized and the spinal cord was fixed and frozen for histological assessment, IHC, and gene expression analysis. The old group had more atrophy in the spinal cord with higher changes in LC3 as an indicator of autophagy in the spinal cord compared to the young group (p < 0.0001). The old Sw + LA-CNPs group increased (improved) spinal cord GABA (p = 0.0187), BDNF (p = 0.0003), TrkB (p < 0.0001) gene expression, decreased autophagy marker LC3 protein (p < 0.0001), nerve atrophy and jumping/licking latency (p < 0.0001), improved sciatic functional index score and total oxidant status/total antioxidant capacity compared to the old group (p < 0.0001). In conclusion, swimming and LA-CNPs seems to ameliorate aging-induced neuron atrophy, autophagy marker LC3, oxidant-antioxidant status, functional restoration, GABA and BDNF-TrkB pathway in the spinal cord of aging rats. Our study provides experimental evidence for a possible positive role of swimming and L-arginine loaded chitosan nanoparticles to decrease complications of aging.

Achieving Single-Molecule Tracking of Subcellular Regulation in Bacteria during Real-Time Environmental Perturbations.

Bacteria rely on protein systems for regulation in response to external environmental signals. Single-molecule fluorescence imaging and tracking has elucidated the complex mechanism of these protein systems in a variety of bacteria. We recently investigated Vibrio cholerae, the Gram-negative bacterium responsible for the human cholera disease, and its regulation of the production of toxins and virulence factors through the membrane-localized transcription factors TcpP and ToxR. These experiments determined that TcpP and ToxR work cooperatively under steady-state conditions, but measurements of how these dynamical interactions change over the course of environmental perturbations were precluded by the traditional preparation of bacterial cells confined on agarose pads. Here, we address this gap in technology and access single-molecule dynamics during real-time changes by implementing two alternative sample preparations: microfluidic devices and chitosan-coated coverslips. We report the first demonstration of single-molecule tracking within live bacterial cells in a microfluidic device. Additionally, using the chitosan-coated coverslips, we show that real-time environmental changes impact TcpP-PAmCherry dynamics, activating a virulence condition in the bacteria about 45 min after dropping to pH 6 and about 20 min after inducing ToxR expression. These new technology advances open our ability for new experiments studying a variety of bacteria with single-molecule imaging and tracking during real-time environmental perturbations.