Enhancing nutritional and potential antimicrobial properties of poultry feed through encapsulation of metagenome-derived multi-enzymes.

BACKGROUND: The encapsulation of metagenome-derived multi-enzymes presents a novel approach to improving poultry feed by enhancing nutrient availability and reducing anti-nutritional factors. By integrating and encapsulated enzymes such as carbohydrate-hydrolyzing enzymes, protease, lipase, and laccase into feed formulations, this method not only improves feed digestibility but also potentially contributes to animal health and productivity through antimicrobial properties. RESULTS: This study investigates the encapsulation of metagenome-derived enzymes, including carbohydrate-hydrolyzing enzymes, protease, lipase, and laccase, using Arabic and Guar gums as encapsulating agents. The encapsulated multi-enzymes exhibited significant antimicrobial activity, achieving a 92.54% inhibition rate against Escherichia coli at a concentration of 6 U/mL. Fluorescence tracking with FITC-labeled enzymes confirmed efficient encapsulation and distribution, while physical characterization, including moisture content and solubility assessments, along with Atomic Force Microscopy (AFM) imaging, validated successful encapsulation. The encapsulated enzymes also effectively hydrolyzed poultry feed, leading to an increase in phenolic content and antioxidant activity, as confirmed by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. CONCLUSIONS: The encapsulated multi-enzymes improved the overall feed quality by increasing reducing sugars and enhancing physical properties such as solubility and water-holding capacity. The encapsulated multi-enzymes improved the overall feed quality by increasing reducing sugars, antioxidant activity and enhancing physical properties such as solubility and water-holding capacity. Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FTIR) analyses confirmed the enzymatic breakdown of the feed structure. These results suggest that supplementing poultry feed with encapsulated multi-enzymes can enhance its physical, nutritional, and functional properties, leading to improved digestibility and overall feed quality.

Date nawah powder as a promising waste for ß-mannanase production from a new isolate Aspergillus niger MSSFW, statistically improving production and enzymatic characterization.

ß-Mannanase producing fungus was isolated from coffee powder waste and identified as Aspergillus niger MSSFW (Gen Bank accession number OR668928). Dates nawah powder as industrial and agricultural waste was the most inducer of ß-mannanase production. The Plackett-Burman and Central Composite designs were used to improve ß-mannanase titer. Optimization studies enhanced the enzyme yield with approximate 13.50-times. ß-Mannanase was purified by Sephadex G-150 gel filtration column and the molecular weight was estimated to be 60 kDa by SDS-PAGE. Crude and purified ß-mannanase displayed maximum activity at temperature 60 °C and 50 °C, respectively. Crude ß-mannanase showed an activation energy value 2.35-times higher than the purified enzyme. Activation energy for thermal denaturation of the purified ß-mannanase was 1.08-times higher than that of the crude enzyme. Purified ß-mannanase exhibited higher deactivation rate constant (Kd) and lower half-life (t0.5) and decimal reduction time (D-value) compared with the crude enzyme. Thermodynamic parameters of enthalpy, entropy, and free energy values for crude and purified ß-mannanase were calculated. Substrate kinetic parameters suggested that the purified ß-mannanase had a strong affinity toward locust bean gum by showing 3.44-times lower Km and 1.99-times higher Vmax compared to the crude enzyme.

The sunflower jacalin Helja: biological and structural insights of its antifungal activity against Candida albicans.

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies.

FimH-mannose noncovalent bonds survive minutes to hours under force.

The adhesin FimH is expressed by commensal Escherichia coli and is implicated in urinary tract infections, where it mediates adhesion to mannosylated glycoproteins on urinary and intestinal epithelial cells in the presence of a high-shear fluid environment. The FimH-mannose bond exhibits catch behavior in which bond lifetime increases with force, because tensile force induces a transition in FimH from a compact native to an elongated activated conformation with a higher affinity to mannose. However, the lifetime of the activated state of FimH has not been measured under force. Here we apply multiplexed magnetic tweezers to apply a preload force to activate FimH bonds with yeast mannan, then we measure the lifetime of these activated bonds under a wide range of forces above and below the preload force. A higher fraction of FimH-mannan bonds were activated above than below a critical preload force, confirming the FimH catch bond behavior. Once activated, FimH detached from mannose with multi-state kinetics, suggesting the existence of two bound states with a 20-fold difference in dissociation rates. The average lifetime of activated FimH-mannose bonds was 1000 to 10,000 s at forces of 30-70 pN. Structural explanations of the two bound states and the high force resistance provide insights into structural mechanisms for long-lived, force-resistant biomolecular interactions.

Investigating functional mechanisms in the Co-biodegradation of lignite and guar gum under the influence of salinity.

The biodegradation of guar gum by microorganisms sourced from coalbeds can result in low-temperature gel breaking, thereby reducing reservoir damage. However, limited attention has been given to the influence of salinity on the synergistic biodegradation of coal and guar gum. In this study, biodegradation experiments of guar gum and lignite were conducted under varying salinity conditions. The primary objective was to investigate the controlling effects and mechanisms of salinity on the synergistic biodegradation of lignite and guar gum. The findings revealed that salinity had an inhibitory effect on the biomethane production from the co-degradation of lignite and guar gum. The biomethane production declined with increasing salinity levels, decreasing from 120.9 mL to 47.3 mL. Even under 20 g/L salt stress conditions, bacteria in coalbeds could effectively break the gel and the viscosity decreased to levels below 5 mPa s. As salinity increased, the removal rate of soluble chemical oxygen demand (SCOD) decreased from 55.63% to 31.17%, and volatile fatty acids (VFAs) accumulated in the digestion system. High salt environment reduces the intensity of each fluorescence peak. Alterations in salinity led to changes in microbial community structure and diversity. Under salt stress, there was an increased relative abundance of Proteiniphilum and Methanobacterium, ensuring the continuity of anaerobic digestion. Hydrogentrophic methanogens exhibited higher salt tolerance compared to acetoclastic methanogens. These findings provide experimental evidence supporting the use of guar gum fracturing fluid in coalbeds with varying salinity levels.

A novel lectin with a distinct Gal_Lectin and CUB domain mediates haemocyte phagocytosis in oyster Crassostrea gigas.

Invertebrate lectins exhibit structural diversity and play crucial roles in the innate immune responses by recognizing and eliminating pathogens. In the present study, a novel lectin containing a Gal_Lectin, a CUB and a transmembrane domain was identified from the Pacific oyster Crassostrea gigas (defined as CgGal-CUB). CgGal-CUB mRNA was detectable in all the examined tissues with the highest expression in adductor muscle (11.00-fold of that in haemocytes, p < 0.05). The expression level of CgGal-CUB mRNA in haemocytes was significantly up-regulated at 3, 24, 48 and 72 h (8.37-fold, 12.13-fold, 4.28-fold and 10.14-fold of that in the control group, respectively) after Vibrio splendidus stimulation. The recombinant CgGal-CUB (rCgGal-CUB) displayed binding capability to Mannan (MAN), peptidoglycan (PGN), D-(+)-Galactose and L-Rhamnose monohydrate, as well as Gram-negative bacteria (Escherichia coli, V. splendidus and Vibrio anguillarum), Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, and Bacillus sybtilis) and fungus (Pichia pastoris). rCgGal-CUB was also able to agglutinate V. splendidus, and inhibit V. splendidus growth. Furthermore, rCgGal-CUB exhibited the activities of enhancing the haemocyte phagocytosis towards V. splendidus, and the phagocytosis rate of haemocytes was descended in blockage assay with CgGal-CUB antibody. These results suggested that CgGal-CUB served as a pattern recognition receptor to bind various PAMPs and bacteria, and enhanced the haemocyte phagocytosis towards V. splendidus.

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth.

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.

Ancient Origin of Acetyltransferases Catalyzing O-acetylation of Plant Cell Wall Polysaccharides.

Members of the domain of unknown function 231/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases catalyzing the acetylation of plant cell wall polysaccharides, including pectins, mannan, xyloglucan and xylan. However, little is known about the origin and evolution of plant cell wall polysaccharide acetyltransferases. Here, we investigated the biochemical functions of TBL homologs from Klebsormidium nitens, a representative of an early divergent class of charophyte green algae that are considered to be the closest living relatives of land plants, and Marchantia polymorpha, a liverwort that is an extant representative of an ancient lineage of land plants. The genomes of K. nitens and Marchantia polymorpha harbor two and six TBL homologs, respectively. Biochemical characterization of their recombinant proteins expressed in human embryonic kidney 293 cells demonstrated that the two K. nitens TBLs exhibited acetyltransferase activities acetylating the pectin homogalacturonan (HG) and hence were named KnPOAT1 and KnPOAT2. Among the six M. polymorpha TBLs, five (MpPOAT1 to 5) possessed acetyltransferase activities toward pectins and the remaining one (MpMOAT1) catalyzed 2-O- and 3-O-acetylation of mannan. While MpPOAT1,2 specifically acetylated HG, MpPOAT3,4,5 could acetylate both HG and rhamnogalacturonan-I. Consistent with the acetyltransferase activities of these TBLs, pectins isolated from K. nitens and both pectins and mannan from M. polymorpha were shown to be acetylated. These findings indicate that the TBL genes were recruited as cell wall polysaccharide O-acetyltransferases as early as in charophyte green algae with activities toward pectins and they underwent expansion and functional diversification to acetylate various cell wall polysaccharides during evolution of land plants.

Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies.

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

Functional studies on tandem carbohydrate-binding modules of a multimodular enzyme possessing two catalytic domains.

Although functional studies on carbohydrate-binding module (CBM) have been carried out extensively, the role of tandem CBMs in the enzyme containing multiple catalytic domains (CDs) is unclear. Here, we identified a multidomain enzyme (Lc25986) with a novel modular structure from lignocellulolytic bacterial consortium. It consists of a mannanase domain, two CBM65 domains (LcCBM65-1/LcCBM65-2), and an esterase domain. To investigate CBM function and domain interactions, full-length Lc25986 and its variants were constructed and used for enzymatic activity, binding, and bioinformatic analyses. The results showed that LcCBM65-1 and LcCBM65-2 both bind mannan and xyloglucan but not cellulose or ß-1,3-1,4-glucan, which differs from the ligand specificity of reported CBM65s. Compared to LcCBM65-2, LcCBM65-1 showed a stronger ligand affinity and a preference for acetylation sites. Both CBM65s stimulated the enzymatic activities of their respective neighboring CDs against acetylated mannan, but did not contribute to the activities of the distal CDs. The time course of mannan hydrolysis indicated that the full-length Lc25986 was more effective in the complete degradation of mixed acetyl/non-acetyl substrates than the mixture of single-CD mutants. When acting on complex substrates, LcCBM65-1 not only improved the enzymatic activity of the mannanase domain, but also directed the esterase domain to the acetylated polysaccharides. LcCBM65-2 adopted a low affinity to reduce interference with the catalysis of the mannanase domain. These results demonstrate the importance of CBMs for the synergism between the two CDs of a multidomain enzyme and suggest that they contribute to the adequate degradation of complex substrates such as plant cell walls. IMPORTANCE: Lignocellulolytic enzymes, particularly those of bacterial origin, often harbor multiple carbohydrate-binding modules (CBMs). However, the function of CBM multivalency remains poorly understood. This is especially true for enzymes that contain more than one catalytic domain (CD), as the interactions between CDs, CBMs, and CDs and CBMs can be complex. Our research demonstrates that homogeneous CBMs can have distinct functions in a multimodular enzyme. The tandem CBMs coordinate the CDs in catalytic conflict through their differences in binding affinity, ligand preference, and arrangement within the full-length enzyme. Additionally, although the synergism between mannanase and esterase is widely acknowledged, our study highlights the benefits of integrating the two enzymes into a single entity for the degradation of complex substrates. In summary, these findings enhance our understanding of the intra-synergism of a multimodular enzyme and emphasize the significance of multiple CBMs in this context.

Optimized production and characterization of endo-ß-mannanase by Aspergillus niger for generation of prebiotic mannooligosaccharides from guar gum.

Optimized production of Aspergillus niger ATCC 26011 endo-ß-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.

Metabolism mechanisms of biogenic methane production by synergistic biodegradation of lignite and guar gum.

Coalbed methane (CBM) presents a promising energy source for addressing global energy shortages. Nonetheless, challenges such as low gas production from individual wells and difficulties in breaking gels at low temperatures during extraction hinder its efficient utilization. Addressing this, we explored native microorganisms within coal seams to degrade guar gum, thereby enhancing CBM production. However, the underlying mechanisms of biogenic methane production by synergistic biodegradation of lignite and guar gum remain unclear. Research results showed that the combined effect of lignite and guar gum enhanced the production, yield rate and concentration of biomethane. When the added guar gum content was 0.8 % (w/w), methane production of lignite and guar gum reached its maximum at 561.9 mL, which was 11.8 times that of single lignite (47.3 mL). Additionally, guar gum addition provided aromatic and tryptophan proteins and promoted the effective utilization of CC/CH and OCO groups on the coal surface. Moreover, the cooperation of lignite and guar gum accelerated the transformation of volatile fatty acids into methane and mitigated volatile fatty acid inhibition. Dominant bacteria such as Sphaerochaeta, Macellibacteroides and Petrimonas improved the efficiency of hydrolysis and acidification. Electroactive microorganisms such as Sphaerochaeta and Methanobacterium have been selectively enriched, enabling the establishment of direct interspecies electron transfer pathways. This study offers valuable insights for increasing the production of biogenic CBM and advancing the engineering application of microbial degradation of guar gum fracturing fluid. Future research will focus on exploring the methanogenic capabilities of lignite and guar gum in in-situ environments, as well as elucidating the specific metabolic pathways involved in their co-degradation.

A glucomannan produced by Bacillus velezensis HY23 and its growth promoting effect on soybeans under salt stress.

The Bacillus genus is widely distributed in nature, has bacteriostatic and growth-promoting activities, and has broad application potential in agriculture. An exopolysaccharide (EPS) was extracted and purified from Bacillus velezensis HY23. Structural characterisation of the EPS was performed by chemical and spectroscopic analyses. Methylation analysis showed that the EPS of HY23 was composed of mannose and glucose at a ratio of 82:18 and was identified as glucomannan. Combined with the nuclear magnetic resonance (NMR) analysis, EPS from HY23 had a backbone of →2)-α-D-Manp-(1 â†’ and →2,6)-α-D-Manp-(1 â†’ branched at C-6 with terminal α-(3-O-Me)-D-Manp-(1 â†’ and →6)-α-D-Manp-(1 â†’ residues as the side chain. A certain amount of ß-D-Glcp residues were also present in backbone. Moreover, EPS significantly improved the nitrogen-fixing activity and salt resistance of soybean seedlings by regulating the antioxidant pool and expression of ion transporters. These findings indicate that EPS from B. velezensis HY23 is a potential biostimulant for enhancing plant resistance to salt stress.

Understanding the dynamic evolution of hemicellulose during Pinus taeda L. growth.

Pinus taeda L. is a fast-growing softwood with significant commercial value. Understanding structural changes in hemicellulose during growth is essential to understanding the biosynthesis processes occurring in the cell walls of this tree. In this study, alkaline extraction is applied to isolate hemicellulose from Pinus taeda L. stem segments of different ages (1, 2, 3, and 4 years old). The results show that the extracted hemicellulose is mainly comprised of O-acetylgalactoglucomannan (GGM) and 4-O-methylglucuronoarabinoxylan (GAX), with the molecular weights and ratios (i.e., GGM:GAX) of GGM and GAX increasing alongside Pinus taeda L. age. Mature Pinus taeda L. hemicellulose is mainly composed of GGM, and the ratio of (mannose:glucose) in the GGM main chain gradually increases from 2.45 to 3.60 with growth, while the galactose substitution of GGM decreases gradually from 21.36% to 14.65%. The acetylation of GGM gradually increases from 0.33 to 0.45 with the acetyl groups mainly substituting into the O-3 position in the mannan. Furthermore, the contents of arabinose and glucuronic acid in GAX gradually decrease with growth. This study can provide useful information to the research in genetic breeding and high-value utilization of Pinus taeda L.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a cellular receptor for delta inulin adjuvant.

Delta inulin, or Advax, is a polysaccharide vaccine adjuvant that significantly enhances vaccine-mediated immune responses against multiple pathogens and was recently licensed for use in the coronavirus disease 2019 (COVID-19) vaccine SpikoGen. Although Advax has proven effective as an immune adjuvant, its specific binding targets have not been characterized. In this report, we identify a cellular receptor for Advax recognition. In vitro uptake of Advax particles by macrophage cell lines was substantially greater than that of latex beads of comparable size, suggesting an active uptake mechanism by phagocytic cells. Using a lectin array, Advax particles were recognized by lectins specific for various carbohydrate structures including mannosyl, N-acetylgalactosamine and galactose moieties. Expression in nonphagocytic cells of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a C-type lectin receptor, resulted in enhanced uptake of fluorescent Advax particles compared with mock-transfected cells. Advax uptake was reduced with the addition of ethylenediaminetetraacetic acid and mannan to cells, which are known inhibitors of DC-SIGN function. Finally, a specific blockade of DC-SIGN using a neutralizing antibody abrogated Advax uptake in DC-SIGN-expressing cells. Together, these results identify DC-SIGN as a putative receptor for Advax. Given the known immunomodulatory role of DC-SIGN, the findings described here have implications for the use of Advax adjuvants in humans and inform future mechanistic studies.

Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Discovery of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in Aspergillus fumigatus mycelium.

The cellular surface of the pathogenic filamentous fungus Aspergillus fumigatus is enveloped in a mannose layer, featuring well-established fungal-type galactomannan and O-mannose-type galactomannan. This study reports the discovery of cell wall component in A. fumigatus mycelium, which resembles N-glycan outer chains found in yeast. The glycosyltransferases involved in its biosynthesis in A. fumigatus were identified, with a focus on two key α-(1→2)-mannosyltransferases, Mnn2 and Mnn5, and two α-(1→6)-mannosyltransferases, Mnn9 and Van1. In vitro examination revealed the roles of recombinant Mnn2 and Mnn5 in transferring α-(1→2)-mannosyl residues. Proton nuclear magnetic resonance (1H-NMR) analysis of cell wall extracts from the ∆mnn2∆mnn5 strain indicated the existence of an α-(1→6)-linked mannan backbone in the A. fumigatus mycelium, with Mnn2 and Mnn5 adding α-(1→2)-mannosyl residues to this backbone. The α-(1→6)-linked mannan backbone was absent in strains where mnn9 or van1 was disrupted in the parental ∆mnn2∆mnn5 strain in A. fumigatus. Mnn9 and Van1 functioned as α-(1→6)-linked mannan polymerases in heterodimers when co-expressed in Escherichia coli, indicating their crucial role in biosynthesizing the α-(1→6)-linked mannan backbone. Disruptions of these mannosyltransferases did not affect fungal-type galactomannan biosynthesis. This study provides insights into the complexity of fungal cell wall architecture and a better understanding of mannan biosynthesis in A. fumigatus. IMPORTANCE: This study unravels the complexities of mannan biosynthesis in A. fumigatus, a key area for antifungal drug discovery. It reveals the presence of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in A. fumigatus mycelium, offering fresh insights into the fungal cell wall's design. Key enzymes, Mnn2, Mnn5, Mnn9, and Van1, are instrumental in this process, with Mnn2 and Mnn5 adding specific mannose residues and Mnn9 and Van1 assembling the α-(1→6)-linked mannan structures. Although fungal-type galactomannan's presence in the cell wall is known, the existence of an α-(1→6)-linked mannan adds a new dimension to our understanding. This intricate web of mannan biosynthesis opens avenues for further exploration and enhances our understanding of fungal cell wall dynamics, paving the way for targeted drug development.

Antibacterial activity of lysozyme after association with carboxymethyl konjac glucomannan.

The unique high isoelectric point of lysozyme (LYZ) restricts its application in composite antibacterial coating due to the unfavorable liability to electrostatic interaction with other components. In this work, the antibacterial activity of a dispersible LYZ-carboxymethyl konjac glucomannan (CMKGM) polyelectrolyte complex was evaluated. Kinetic analysis revealed that, compared with free LYZ, the complexed enzyme exhibited decreased affinity (Km) but markedly increased Vmax against Micrococcus lysodeikticus, and QCM and dynamic light scattering analysis confirmed that the complex could bind with the substrate but in a much lower ratio. The complexation with CMKGM did not alter the antibacterial spectrum of LYZ, and the complex exerted antibacterial function by delaying the logarithmic growth phase and impairing the cell integrity of Staphylococcus aureus. Since the LYZ-CMKGM complex is dispersible in water and could be assembled easily, it has great potential as an edible coating in food preservation.

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

The immunogenicity of p24 protein from HIV-1 virus is strongly supported and modulated by coupling with liposomes and mannan.

Anti-viral and anti-tumor vaccines aim to induce cytotoxic CD8+ T cells (CTL) and antibodies. Conserved protein antigens, such as p24 from human immunodeficiency virus, represent promising component for elicitation CTLs, nevertheless with suboptimal immunogenicity, if formulated as recombinant protein. To enhance immunogenicity and CTL response, recombinant proteins may be targeted to dendritic cells (DC) for cross presentation on MHCI, where mannose receptor and/or other lectin receptors could play an important role. Here, we constructed liposomal carrier-based vaccine composed of recombinant p24 antigen bound by metallochelating linkage onto surface of nanoliposomes with surface mannans coupled by aminooxy ligation. Generated mannosylated proteonanoliposomes were analyzed by dynamic light scattering, isothermal titration, and electron microscopy. Using murine DC line MutuDC and murine bone marrow derived DC (BMDC) we evaluated their immunogenicity and immunomodulatory activity. We show that p24 mannosylated proteonanoliposomes activate DC for enhanced MHCI, MHCII and CD40, CD80, and CD86 surface expression both on MutuDC and BMDC. p24 mannosylated liposomes were internalized by MutuDC with p24 intracellular localization within 1 to 3 h. The combination of metallochelating and aminooxy ligation could be used simultaneously to generate nanoliposomal adjuvanted recombinant protein-based vaccines versatile for combination of recombinant antigens relevant for antibody and CTL elicitation.