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The composition of the cell wall of Aspergillus niger Biochem
Abstract
1. The cell-wall composition of Aspergillus niger has been investigated. Analysis shows the presence of six sugars, glucose, galactose, mannose, arabinose, glucosamine and galactosamine, all in the d-configuration, except that a small amount of l-galactose may be present. Sixteen common amino acids are also present. 2. The wall consists chiefly of neutral carbohydrate (73-83%) and hexosamine (9-13%), with smaller amounts of lipid (2-7%), protein (0.5-2.5%) and phosphorus (less than 0.1%). The acetyl content (3.0-3.4%) corresponds to 1.0mole/mole of hexosamine nitrogen. 3. A fractionation of the cell-wall complex was achieved, with or without a preliminary phenol extraction, by using n-sodium hydroxide. Though this caused some degradation, 30-60% of the wall could be solubilized (depending on the preparation). Analyses on several fractions suggest that fractionation procedures bring about some separation of components although not in a clear-cut fashion. 4. Cell-wall preparations were shown to yield a fraction having [alpha](D) approx. +240 degrees (in n-sodium hydroxide) and consisting largely of glucose. This was separated into two subfractions, one of which had [alpha](D)+281 degrees (in n-sodium hydroxide) and had properties resembling the polysaccharide nigeran; the other had [alpha](D) +231 degrees (in n-sodium hydroxide). It is suggested that nigeran is a cell-wall component.
... There were several differences between the spectra of Chlorella sp. and A. niger, respectively, which are related to the different chemical composition of the two microorganisms. According to Johnson (1965), the main components building the mycelium of Aspergillus are carbohydrates (73-83%), with smaller portions of lipids (2-7%) and proteins (0.5-2.5%), while in the case of Chlorella sp., approximately 50% of the biomass is made up of proteins (Wild et al., 2019). Apart from proteins, Chlorella sp. ...
... This absorption may be assigned to amines, which were detected in various fungal species and strains (Stüttgen et al., 1978;Karimi et al., 2019). Johnson (1965) reported that the cell wall of A. niger contains approximately 9-13% hexosamines. Amine and carboxylate groups were identified as major functional groups for metal biosorption in the A. niger biomass (Cai et al., 2016). ...
... There were several differences between the spectra of Chlorella sp. and A. niger, respectively, which are related to the different chemical composition of the two microorganisms. According to Johnson (1965), the main components building the mycelium of Aspergillus are carbohydrates (73-83%), with smaller portions of lipids (2-7%) and proteins (0.5-2.5%), while in the case of Chlorella sp., approximately 50% of the biomass is made up of proteins (Wild et al., 2019). Apart from proteins, Chlorella sp. ...
... This absorption may be assigned to amines, which were detected in various fungal species and strains (Stüttgen et al., 1978;Karimi et al., 2019). Johnson (1965) reported that the cell wall of A. niger contains approximately 9-13% hexosamines. Amine and carboxylate groups were identified as major functional groups for metal biosorption in the A. niger biomass (Cai et al., 2016). ...
Despite the negative impact on the environment, incineration is one of the most commonly used methods for dealing with waste. Besides emissions, the production of ash, which usually shows several negative properties, such as a higher content of hazardous elements or strongly alkaline pH, is problematic from an environmental viewpoint as well. The subject of our paper was the assessment of biosorption of Ni from ash material by a microbial consortium of Chlorella sp. and Aspergillus niger. The solid substrate represented a fraction of particles of size <0.63 mm with a Ni content of 417 mg kg −1. We used a biomass consisting of two different organisms as the sorbent: a non-living algae culture of Chlorella sp. (an autotrophic organism) and the microscopic filamentous fungus A. niger (a heterotrophic organism) in the form of pellets. The experiments were conducted under static conditions as well as with the use of shaker (170 rpm) with different modifications: solid substrate, Chlorella sp. and pellets of A. niger; solid substrate and pellets of A. niger. The humidity-temperature conditions were also changed. Sorption took place under dry and also wet conditions (with distilled water in a volume of 30-50 ml), partially under laboratory conditions at a temperature of 25 • C as well as in the exterior. The determination of the Ni content was done using inductively coupled plasma optical emission spectrometry (ICP-OES). The removal of Ni ranged from 13.61% efficiency (Chlorella sp., A. niger with the addition of 30 ml of distilled water, outdoors under static conditions after 48 h of the experiment) to 46.28% (Chlorella sp., A. niger with the addition of 30 ml of distilled water, on a shaker under laboratory conditions after 48 h of the experiment). For the purpose of analyzing the representation of functional groups in the microbial biomass and studying their interaction with the ash material, we used Fourier-transform infrared Frontiers in Microbiology | www.frontiersin.org 1 December 2021 | Volume 12 | Article 792987 Šimonovičová et al. Treatment of Hazardous Ash Waste (FTIR) spectroscopy. We observed that the amount of Ni adsorbed positively correlates with absorbance in the spectral bands where we detect the vibrations of several organic functional groups. These groups include hydroxyl, aliphatic, carbonyl, carboxyl and amide structural units. The observed correlations indicate that, aside from polar and negatively charged groups, aliphatic or aromatic structures may also be involved in sorption processes due to electrostatic attraction. The correlation between absorbance and the Ni content reached a maximum in amide II band (r = 0.9; P < 0.001), where vibrations of the C=O, C-N, and N-H groups are detected. The presented results suggest that the simultaneous use of both microorganisms in biosorption represents an effective method for reducing Ni content in a solid substrate, which may be useful as a partial process for waste disposal.
... Generally, nonenzymatic antioxidants such as carotenoids, polysaccharides, chitosan, polyphenol, polyols, mannitol, glucose, and vitamins in fungi have oxygen radical scavenging effects [49][50][51][52]. It was well known that glucose and mannose exist in the cell wall of A. brasiliensis spores [53]. The filamentous fungus A. niger produces several different polyols, including glycerol, erythritol, and D-mannitol [54]. ...
... The PAW treatment clearly Generally, nonenzymatic antioxidants such as carotenoids, polysaccharides, chitosan, polyphenol, polyols, mannitol, glucose, and vitamins in fungi have oxygen radical scavenging effects [49][50][51][52]. It was well known that glucose and mannose exist in the cell wall of A. brasiliensis spores [53]. The filamentous fungus A. niger produces several different polyols, including glycerol, erythritol, and D-mannitol [54]. ...
Plasma-activated water (PAW) has emerged as a platform for sterilizing fungal pathogens. In this study, we investigated the influence of PAW on black melanized spores of Aspergillus brasiliensis to explore the mechanism of fungal spore inactivation. PAW was prepared by activating deionized water with a nonthermal atmospheric pressure air plasma jet (soft plasma jet). The concentrations of H 2 O 2 and NO x in the PAW treated by the soft plasma jet for 3 min were 50 µM and 1.8 mM, respectively, and the pH of the PAW was 3.10. The reactive oxygen and nitrogen species (RONS) in the PAW increased with longer plasma activation time. After being treated for 30 min in the PAW with a plasma activation time of 3 min, the spore viability dramatically dropped to 15%. The viabilities of 0.3% H 2 O 2-and 0.3% HNO 3-treated spores were 22% and 42%, respectively. The breakage of the spore cell wall by the PAW was revealed in scanning electron microscope images and flow cytometry measurements. Disruption of cell wall integrity provides a path for intracellular components to escape and RONS of the PAW can attack intracellular components directly. Degradation of high molecular genomic DNA was also observed by agarose gel electrophoresis. These results suggest that long-lived reactive species generated in the PAW play an important role in the inactivation of melanized fungal spores. Consequently, PAW produced by a soft plasma jet can be applied to sterilize bioprotective walled fungal spores in a relatively large volume.
... Analysis had shown the presence of six sugars: glucose, galactose, mannose, arabinose, glucosamine and galactosamine, all in the D-configuration, except that a small amount of L-galactose may be present. Sixteen common amino acids are also present [4]. At the same time, knowledge of filamentous fungi, as well as of A. niger, cell wall structure, mechanisms regulating cell wall biogenesis, and cell wall stress responses, is still limited in comparison with that of Saccharomyces cerevisiae. ...
Метою дослідження було проаналізувати інгібіторну дію на гриби, особливо на A. niger, складових сумішей, які складалися з ослаблених консервантів та ефірних олій. Було висунуто гіпотезу, що структура клітинної стінки A. niger може сприяти стійкості грибка до лікування, заснованого на комбінації ослаблених консервантів у поєднанні з ефірними оліями.
... Fungal agglutination by lectins is important to bind and eradicate the pathogens in marine invertebrates [22]. Presence of galactose sugars in the cell wall of Aspergillus niger and Tricoderma reesei could explain the agglutination by MytiLec-1 in this study [42][43][44]. Previously, both fungal strains were reported to be agglutinated by MTL, which affected their hyphal growth and spore germination as well [22]. ...
MytiLec-1, the recombinant form of a mussel lectin from Mytillus galloprovincialis, was purified by affinity chromatography and showed the maximum hemagglutination activity at a temperature range of 10°C to 40°C and at pH 7.0 to 9.0. Denaturants like urea and acidic-guanidine inhibited its hemagglutination activity significantly. MytiLec-1 was found to be metal-independent though Ca2+ slightly increased the activity of chelated MytiLec-1. The lectin suppressed 65% growth of Pseudomonas aeruginosa (ATCC 47085) at 200 μg/ml and reduced the formation of biofilm (15% at 200 μg/ml). Comparing to Shigella sonnei (ATCC 29930), Shigella boydii (ATCC 231903) and Shigella dysenteriae (ATCC 238135), Bacillus cereus (ATCC 14579) was slightly more sensitive to MytiLec-1. At a concentration of 200 μg/disc and 100 μg/ml, MytiLec-1 prevented the growth of Aspergillus niger and agglutinated the spores of Aspergillus niger and Trichoderma reesei, respectively. Amino acid sequences, physicochemical properties and antimicrobial activities of MytiLec-1 were compared with three other lectins (CGL, MTL and MCL from Crenomytilus grayanus, Mytilus trossulas and Mytilus californianus, respectively) from the mytilectin family of bivalve mollusks. It reconfirms the function of these lectins to recognize pathogens and perform important roles in innate immune response of mussels.
... To the best of our knowledge, a linear (1→2)-α-D-glucan has not yet been identified. The (1→3)-α-D-glucans are major components of the cell wall of filamentous fungi [55][56][57] and dimorphic yeasts [58][59][60][61] and are synthesized via the primer for (1→3)-α-D-glucans by intracellular amylases. The structural analysis of (1→3)-α-D-glucan was reported and it was mentioned that three crystalline forms I-III of (1→3)-α-D-glucan were detected and interconverted via dehydration and hydration reactions [32,62]. ...
Controlling the stereoselectivity of 1,2-cis glycosylation is one of the most challenging tasks in the chemical synthesis of glycans. There are various 1,2-cis glycosides in nature, such as α-glucoside and β-mannoside in glycoproteins, glycolipids, proteoglycans, microbial polysaccharides, and bioactive natural products. In the structure of polysaccharides such as α-glucan, 1,2-cis α-glucosides were found to be the major linkage between the glucopyranosides. Various regioisomeric linkages, 1→3, 1→4, and 1→6 for the backbone structure, and 1→2/3/4/6 for branching in the polysaccharide as well as in the oligosaccharides were identified. To achieve highly stereoselective 1,2-cis glycosylation, including α-glucosylation, a number of strategies using inter- and intra-molecular methodologies have been explored. Recently, Zn salt-mediated cis glycosylation has been developed and applied to the synthesis of various 1,2-cis linkages, such as α-glucoside and β-mannoside, via the 1,2-cis glycosylation pathway and β-galactoside 1,4/6-cis induction. Furthermore, the synthesis of various structures of α-glucans has been achieved using the recent progressive stereoselective 1,2-cis glycosylation reactions. In this review, recent advances in stereoselective 1,2-cis glycosylation, particularly focused on α-glucosylation, and their applications in the construction of linear and branched α-glucans are summarized.
... Polysaccharides are the main components of fungal cell walls [57], and α-1,3-glucan contributes to cell wall rigidity in Aspergillus spp., such as A. niger and A. fumigatus [58]. Subsequently, a feedback was induced, i.e., α-1,3-glucan (synthesized by α-1,3-glucan synthase) served to strengthen the fungal cell wall [59,60]. Therefore, the "repair" of cell wall structure through enhanced production of polysaccharides is an important strategy for A. niger to tolerate Pb stress. ...
The toxicity of metals to microorganisms is highly correlated with the type of metal used. However, the differences in the resistance mechanisms of filamentous fungi to multiple metals remain unclear. In this study, we investigated the responses of Aspergillus niger to three toxic metals, i.e., Pb2+, Cd2+, and Cu2+. Fungal growth and metabolism indices showed that A. niger had a higher tolerance to Pb2+ (>1000 mg L-1) than to Cu2+ (300 mg L-1) and Cd2+ (50 mg L-1). An appropriate Pb2+ concentration (<500 mg L-1) stimulated fungal growth and metabolic activity, whereas Cd2+ and Cu2+ stress showed continuously negative influences on fungal physiological parameters, such as biomass and secretion of oxalic acid. A. niger responded to Pb stress by constructing a new border layer around its cell wall. This pathway was also confirmed using RNA-seq analysis, i.e., the gene encoding cell wall α-1,3-glucan synthase was upregulated. This upregulation subsequently promoted the production of polysaccharides, which are the main components that support fungal cell walls. In contrast, the expression of genes encoding both AAA family ATPase and efflux pump antibiotic resistance proteins for Cd2+ and Cu2+ was significantly downregulated. Therefore, these findings elucidated the relatively complete fungal responses to different metal stresses.
... Significant residuals of CP, starch, and sugar were partly present in the substrates; however, they are rather no relevant nutrient suppliers on the scale of the entire ration. The mycelial cell walls of A. niger are mainly composed of neutral carbohydrates (glucose, mannose, arabinose, glucosamine, and galactosamine), hexosamines, and amino acids [23]. Their composition differs from that of plant fibers, but in the current analysis, they were recorded as detergent fibers as well. ...
Dietary fibers may have positive impact on health and wellbeing of pigs. The study examined physicochemical properties of two lignocelluloses (including and excluding bark), powdered cellulose, Aspergillus niger mycelium, lucerne chaff, soybean shells, wheat bran, and sugar beet pulp in relation to fermentability and digestibility using in vitro batch-culture incubation. Maize starch and a purified cellulose were used as standardized substrates for classification of the test substrates. The substrates covered a wide range regarding their physicochemical properties. Swelling capacity (SC) was 9–411%, water binding capacity (WBC) was 4.4–14.3 g/g dry matter (DM), and water holding capacity (WHC) was 4.1–10.6 g/g DM. Gas production and other fermentation parameters—namely post-incubation pH, CH4, NH3, and short chain fatty acids (SCFA) concentrations—revealed a significant fermentation of sugar beet pulp, soybean shells, lucerne chaff, wheat bran, A. niger mycelium, and powdered cellulose, whereas the lignocelluloses were not fermented. Significant correlations were found between the physicochemical properties and the fermentation parameters (p < 0.05). Enzymatic pre-digestion mostly reduced gas, NH3, and SCFA production. In vitro digestibility of DM (IVDMD) and organic matter (IVOMD) was mostly negligible after enzymatic pre-digestion. Fermentation alone led to only 0.10–0.15 IVDMD and 0.14–0.15 IVOMD in lignocelluloses and powdered cellulose, respectively, but 0.44–0.37 IVDMD and 0.46–0.38 IVOMD in the remainder of substrates (p < 0.05). In vitro digestibility was again correlated with the physicochemical properties of the substrates and the fermentation parameters (p < 0.05). The fiber preparations and fiber-rich byproducts were fermented to a relevant extent. In contrast, lignocelluloses were not fermented and can be used rather as bulk material.
... Nigerose is the constitutional unit of α-1,3-glucans produced in the cell wall of filamentous fungi [10,11] and fission yeast [12], which are sometimes used in the fermentation process. As a result of these fermentation processes, nigerose is also present in fermentation products in Japanese rice wine [13] and beer [14]. ...
3-O-Substituted reducing aldoses are commonly unstable under heat treatment at neutral and alkaline pH. In this study, to evaluate the decomposition products, nigerose (3-O-α-d-glucopyranosyl-d-glucose) and 3-O-methyl glucose were heated at 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Decomposition via β-elimination was observed that formed a mixture of 3-deoxy-arabino-hexonic acid and 3-deoxy-ribo-hexonic acid; upon further acid treatment, it was converted to their γ-lactones. Similarly, turanose (3-O-α-d-glucopyranosyl-d-fructose), a ketose isomer of nigerose, decomposed more rapidly than nigerose under the same conditions, forming the same products. These findings indicate that 3-O-substituted reducing glucose and fructose decompose via the same 1,2-enediol intermediate. The alkoxycarbonyl elimination of 3-O-substituted reducing glucose and fructose occurs readily if an O-glycosidic bond is located on the carbon adjacent to the 1,2-enediol intermediate. Following these experiments, we proposed a kinetic model for the3- decomposition of nigerose and turanose by heat treatment under neutral pH conditions. The proposed model showed a good fit with the experimental data collected in this study. The rate constant of the decomposition for nigerose was (1.2 ± 0.1) × 10⁻⁴ s⁻¹, whereas that for turanose [(2.6 ± 0.2) × 10⁻⁴ s⁻¹] was about 2.2 times higher.
... 4) glycosidic linkages. [3] As a part of the hyphal cell wall, [4,5] it can contribute up to 40% of the cell dry weight. [6] Nigeran is produced in a very limited number of species, that is, Aspergillus niger and Penicillium crustosum [7] and its commercial use is relatively low compared to other sugars (as for example, amylose). ...
Mycodextran—also known as nigeran—is an unbranched polysaccharide made of α‐d‐glucopyranose units alternatively connected by (1 → 3) and (1 → 4) glycosidic linkages produced intracellularly by Aspergillus niger and Penicillium crustosum. In this work we examine possible enforced conformational transitions in the glucopyranose rings in the nigeran oligosaccharide chains. In order to simulate such structural changes we used the Enforced Geometry Optimization (EGO) method.
... The proteome of JSC- 093350089 revealed differential levels of cell wall modulation proteins ( Table 1). Conidia of A. niger possess a relatively thick cell wall made of a network of carbohy- drates, including -glucans, chitin, -glucans, galactomannan, and galactosaminoga- lactan, with an outer cell wall layer consisting of complex melanin pigments (29). The polyketide synthase AlbA (An09g05730), which is required for the production of 1,8-dihydroxynaphthalene-melanin (DHN-melanin) in A. niger (24), was over 2-fold more enriched in JSC-093350089 than in ATCC 1015. ...
The initial characterization of the Aspergillus niger isolate JSC-093350089, collected from U.S. segment surfaces of the International Space Station (ISS), is reported, along with a comparison to the extensively studied strain ATCC 1015. Whole-genome sequencing of the ISS isolate enabled its phylogenetic placement within the A. niger / welwitschiae / lacticoffeatus clade and revealed that the genome of JSC-093350089 is within the observed genetic variance of other sequenced A. niger strains. The ISS isolate exhibited an increased rate of growth and pigment distribution compared to a terrestrial strain. Analysis of the isolate’s proteome revealed significant differences in the molecular phenotype of JSC-093350089, including increased abundance of proteins involved in the A. niger starvation response, oxidative stress resistance, cell wall modulation, and nutrient acquisition. Together, these data reveal the existence of a distinct strain of A. niger on board the ISS and provide insight into the characteristics of melanized fungal species inhabiting spacecraft environments.
IMPORTANCE A thorough understanding of how fungi respond and adapt to the various stimuli encountered during spaceflight presents many economic benefits and is imperative for the health of crew. As A. niger is a predominant ISS isolate frequently detected in built environments, studies of A. niger strains inhabiting closed systems may reveal information fundamental to the success of long-duration space missions. This investigation provides valuable insights into the adaptive mechanisms of fungi in extreme environments as well as countermeasures to eradicate unfavorable microbes. Further, it enhances understanding of host-microbe interactions in closed systems, which can help NASA’s Human Research Program maintain a habitat healthy for crew during long-term manned space missions.
... yeasts. The existence and importance of cell wall α-1,3-glucan were recognized well before the whole-genome analysis of some fungi [6,7]. Since α-1,3-glucan is accumulated in the cell wall in the early stage of Aspergillus nidulans culture and is degraded under carbon-limited conditions [8], it was thought to act as a storage polysaccharide in some fungi [8]. ...
Although α-1,3-glucan is a major cell wall polysaccharide in filamentous fungi, its biological functions remain unclear, except that it acts as a virulence factor in animal and plant pathogenic fungi: it conceals cell wall β-glucan on the fungal cell surface to circumvent recognition by hosts. However, cell wall α-1,3-glucan is also present in many of non-pathogenic fungi. Recently, the universal function of α-1,3-glucan as an aggregation factor has been demonstrated. Applications of fungi with modified cell wall α-1,3-glucan in the fermentation industry and of in vitro enzymatically-synthesized α-1,3-glucan in bio-plastics have been developed. This review focuses on the recent progress in our understanding of the biological functions and biosynthetic mechanism of cell wall α-1,3-glucan in fungi. We briefly consider the history of studies on α-1,3-glucan, overview its biological functions and biosynthesis, and finally consider the industrial applications of fungi deficient in α-1,3-glucan.
... Interestingly, strain AR6 showed strong antagonistic activity against plant pathogenic fungi A. niger (Fig. 4). In general, cell wall of A. niger majorly consists of carbohydrate (73-83%) hexosamine (9-13%), lipid (2-7%), protein (0.5-2.5%) and phosphorus (less than 0.1%) (Johnston, 1965). Thus, rhizobacterial strain AR6 with protease, lipase and amylase producing ability can easily hydrolyze the cell wall of A. niger. ...
... Na podstawie uzyskanych wyników dowodzi siê, ¿e testowane glukany skrê-caj¹ w prawo p³aszczyznê oewiat³a spolaryzowanego, a otrzymane wartooeci mieoeci³y siê w przedziale od +216°do +300°. Johnson (24), izoluj¹c a-(1®3)-glukan z Aspergillus niger NRRL 326, okreoeli³ jego skrêcalnooeae optyczn¹ na poziomie +233°( 1M NaOH), natomiast Hasegawa i wsp. (15) dla glukanu z tego samego gatunku grzyba otrzymali skrêcalnooeae o wartooeci +257°. ...
a-(1®3)-Glucans from cell wall of Laetiporus sulphureus (Bull.:Fr.) Murrill – isolation, characteristics and application for induction of mutanase synthesis S u m m a r y Five different methods described in the literature were used for the isolation of a-(1®3)-glucans from the cell wall of fruiting bodies of Laetiporus sulphureus (Bull.:Fr.) Murrill, and their comparative analysis was performed. The separated fungal biopolymers were well-characterized in respect of their structure and some physicochemical properties. Structural analyses, i.e., Fou-rier-transform infra-red (FT-IR) spectroscopy, 1 H nuclear magnetic resonance (NMR) spectroscopy and specific rotation, revealed that the alkali-soluble wall fraction from this basidiomycetous fungus contained about 56% of (1®3)-linked a-glucans. Four out of five a-(1®3)-glucans isolated by different methods from the mycelium of the polypore fungus L. sulphureus induced higher activity of fungal and bacterial mutanase than those obtained on mutan. Therefore, the a-(1®3)-glucans from fruiting bodies of L. sulphureus can be used as a new alternative to streptococcal mutan, which so far has been known as the best inducer of mutanase production.
... Suppl. Bardalaye and Nordin (1976) 0.04 Nigeran (Johnston, 1965;Damveld et al., 2005) 0.25 ...
Supplementary Information
... Total hydrolysis of the S. cerevisiae cell wall gives glucose and N-acetylglucosamine, which come from polysaccharides, and mannose from mannoproteins that represent 30-50% of the yeast cell wall [2]. The cell wall of Aspergillus niger, one of the most biotechnologically relevant fungi, contains not only derivatives of glucose, mannose and N-acetylglucosamine (normally found in the S. cerevisiae cell wall), but also D-galactose, Darabinose, D-galactosamine, and small amounts of L-galactose [3]. The cell wall of the plant pathogen Fusarium oxysporum, apart from glucose and N-acetyl-glucosamine, also contains mannose, galactose, and uronic acid, which presumably originate from cell wall glycoproteins [4]. ...
In this chapter we compare the cell wall structure of filamentous fungi and the yeast Saccharomyces cerevisiae. The genetic engineering of the cell wall of Trichoderma, Aspergillus, Neurospora, S. cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha, Kluyveromyces and others is discussed and the consequences of direct changes in the synthesis of cell wall components, especially those affecting protein production and secretion by the mutants and their sensitivity to antifungal agents are highlighted. Furthermore, we discuss the close correlation between the cell wall structure and the activity of enzymes engaged in protein glycosylation and in the biosynthesis of glycosylphosphatidy linositol anchors. We also show problems with stability of the generated changes in the cell wall structure, which are detected and often overcome by cell wall compensatory mechanisms.
... Na podstawie uzyskanych wyników dowodzi siê, ¿e testowane glukany skrê-caj¹ w prawo p³aszczyznê oewiat³a spolaryzowanego, a otrzymane wartooeci mieoeci³y siê w przedziale od +216°do +300°. Johnson (24), izoluj¹c a-(1®3)-glukan z Aspergillus niger NRRL 326, okreoeli³ jego skrêcalnooeae optyczn¹ na poziomie +233°( 1M NaOH), natomiast Hasegawa i wsp. (15) dla glukanu z tego samego gatunku grzyba otrzymali skrêcalnooeae o wartooeci +257°. ...
Five different methods described in the literature were used for the isolation of α-(1→3)-glucans from the cell wall of fruiting bodies of Laetiporus sulphureus (Bull.:Fr.) Murrill, and their comparative analysis was performed. The separated fungal biopolymers were well-characterized in respect of their structure and some physicochemical properties. Structural analyses, i.e., Fourier-transform infra-red (FT-IR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy and specific rotation, revealed that the alkali-soluble wall fraction from this basidiomycetous fungus contained about 56% of (1→3)-linked α-glucans. Four out of five α-(1→3)- glucans isolated by different methods from the mycelium of the polypore fungus L. sulphureus induced higher activity of fungal and bacterial mutanase than those obtained on mutan. Therefore, the α-(1→3)-glucans from fruiting bodies of L. sulphureus can be used as a new alternative to streptococcal mutan, which so far has been known as the best inducer of mutanase production.
... Therefore the carbohydrate monomer composition of cell walls was analyzed at seven time points during exponential growth and autolysis. At the end of the exponential growth phase, the total carbohydrates in wild-type strain N402 cell walls were composed of 13% glucosamine, 8% galactose, 73% glucose and 6% mannose (Fig. 4A), consistent with the previously reported composition of A. niger cell walls [69]. The glucosamine may arise from either N-acetyl-glucosamine or glucosamine, present in the cell wall in chitin or chitosan since the used acid hydrolysis removes the acetyl group from carbohydrates. ...
Background
The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis.
Results
In this study, we used developmental mutants (ΔflbA and ΔbrlA) which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced.
Conclusion
The combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall modification, which contributes to understanding cell wall remodeling mechanisms during development.
... (ii) Glucans (I) <x-Glucans.-These consisted of glycogen (Peat, Whelan, and Edwards 1955) and nigeran (Johnston 1965). ...
Infrared spectroscopy is assessed as a technique for identifying polymers
derived from fungal cell walls, both as isolated materials and in mixtures with one
another. The technique is then applied to a study of the composition of fungal cell
walls and the conclusion reached that infrared spectra provide a rapid and valuable
indication of the major components of such walls. They can also be used to follow
the effect of chemical treatments designed to separate major wall components.
Nigeran (α-1,3-alt-α-1,4-glucan) is a linear glucan with alternating α-1,3- and α-1,4-glycosidic linkages. It is extracted from the cell walls of certain species in the genera Aspergillus and Penicillium. Nigeran can be esterified and used as a film, but its strength is only approximately 4–25 MPa. However, in the present study, high-strength nigeran ester films with tensile strengths of 100 MPa were successfully prepared by thermally stretching and annealing the melt-quenched films. Two-dimensional wide-angle X-ray diffraction (2D-WAXD) analysis revealed that the highly oriented films of nigeran butyrate (NGBu), nigeran valerate (NGVa), and nigeran hexanoate (NGHx) studied herein had two-fold helix symmetry along the molecular axis. Assuming the crystal systems to be orthorhombic, the unit lattice of each ester was calculated as NGBu (a=28.6Å, b=9.07Å, c=16.5Å), NGVa (a=31.5Å, b=9.07Å, c=16.2Å), and NGHx (a=36.7Å, b=9.07Å, c=16.2Å). In the calculated unit lattice parameters, only the a-axis was extended as the number of ester carbons increased.
Polysaccharides are the most abundant polymers in nature, and polysaccharide esters, represented by cellulose acetate, exhibit unique properties as thermoplastic resins, depending on the backbone structures (linkage positions and types of glucose units) and ester side chain length and structures (linear or branched). For example, pullulan esters show high transparency and zero birefringence, paramylon esters show fine thermoplasticity and form melt‐spun fibers, and the unnatural polysaccharide α‐1,3‐glucan can be enzymatically polymerized and its esters show outstanding thermal stability comparable to super engineering plastics. The esterification methods are divided into two broad categories: one is chemical esterification and the other is enzymatic esterification. The latter method seems to be not only an environmentally benign process without using strong acids but also has high substrate specificity, enabling well‐designed structures and functionalities of polysaccharide esters.
Nigeran is a linear α-d-glucan with alternating α-1,3- and α-1,4-glycosidic linkages. Nigeran (α-1,3-alt-α-1,4-glucan) esters with different ester chain lengths were synthesized from high molecular weight nigeran extracted from mycelium of Aspergillus oryzae over-expressing a nigeran synthase gene derived from Aspergillus luchuensis. All prepared nigeran esters were crystalline polymers with high melting point, including those with ester groups longer than octanoate. This unique crystallization behavior is not reported in other polysaccharide esters. Both solvent-cast and melt-quenched films were self-standing and high transparency. Solvent-cast films were stretched up to four times the initial length and showed well-oriented X-ray fiber diagrams. Nigeran propionate had twofold screw symmetry along the molecular axis (same as neat nigeran), suggesting that the molecular conformation remained unchanged despite esterification. This result indicates that the molecular conformation of nigeran with its α-1,3-alt-α-1,4-glycosidic linkage is very stable despite chemical modification and retains high crystallinity.
Fungal pathogens of humans cause severe and life-threatening infections worldwide. The fungal cell wall is the first point of contact between the invading pathogen and the host innate immune system. It plays a crucial role in the interaction between host and pathogen cells, mediating processes essential to infection, such as adhesion and phagocytosis. If the fungus successfully invades the host it might then be destroyed by the host immune cells but if the fungus is able to disseminate hematogenously there is a high risk of life-threatening systemic infection. This article focusses on fungal cell wall biosynthesis and the mechanisms by which it interacts with host cell receptors.
To establish a sustainable material production system and preserve the Earth’s environment, “biomass plastics” that are made from renewable biomass instead of petroleum and “biodegradable plastics” that are completely degraded into carbon dioxide and water by enzymes secreted by microorganisms in the environment are desirable products. This miniature review describes a series of studies on microbial polyesters and polysaccharide ester derivatives, including the synthesis of novel polymers, development of new processing techniques for high-performance films and fibers, elucidation of the relationship between structure and properties using synchrotron radiation, and control of the rate of enzymatic degradation.
Endo-1,4-β-mannosidases catalyze the cleavage of the β-d-1,4-mannopyranosyl linkage in mannan and have potential biotechnological applications in biofuel production, manno-oligosaccharide production, cleansing, and food and feed industries. In this study, an endo-1,4-β-mannosidase gene (TaMan3A) from wheat (Triticum aestivum) was successfully expressed in Pichia pastoris, and the antifungal effectiveness and manno-oligosaccharide production of the enzyme were evaluated. The purified TaMan3A exhibited a molecular weight of approximately 43 kDa, and its highest enzyme activity at pH 4.0 and 40 °C. Comparisons with other β-mannanases showed that TaMan3A had a conserved mannan-binding V-shaped groove, catalytic acid/base residue (E179), and nucleophilic residue (E297). Antifungal assays for TaMan3A against seven fungi commonly associated with wheat kernels showed that this enzyme had higher inhibitory effects on hyphal growth of the field fungi Fusarium graminearum and Alternaria sp. in comparison with that of storage fungi. TaMan3A could hydrolyze mannan polymers of galactomannan and Konjac glucomannan to mannobiose, mannotriose, and mannotetraose as the main products. Overall, these results showed the potential of TaMan3A for enhancing host resistance against fungal pathogens in wheat and manno-oligosaccharide production in the feed and food industries.
Filamentous fungi are covered by a cell wall consisting mainly of chitin and glucan. The synthesis of chitin, a β-1,4-linked homopolymer of N-acetylglucosamine, is essential for hyphal morphogenesis. Fungal chitin synthases are integral membrane proteins that have been classified into seven classes. ChsB, a class III chitin synthase, is known to play a key role in hyphal tip growth and has been used here as a model to understand the cell biology of cell wall biosynthesis in Aspergillus nidulans. Chitin synthases are transported on secretory vesicles to the plasma membrane for new cell wall synthesis. Super-resolution localization imaging as a powerful biophysical approach indicated dynamics of the Spitzenkörper where spatiotemporally regulated exocytosis and cell extension, whereas high-speed pulse-chase imaging has revealed ChsB transport mechanism mediated by kinesin-1 and myosin-5. In addition, live imaging analysis showed correlations among intracellular Ca²⁺ levels, actin assembly, and exocytosis in growing hyphal tips. This suggests that pulsed Ca²⁺ influxes coordinate the temporal control of actin assembly and exocytosis, which results in stepwise cell extension. It is getting clear that turgor pressure and cell wall pressure are involved in the activation of Ca²⁺ channels for Ca²⁺ oscillation and cell extension. Here the cell wall synthesis and tip growth meet again.
Nigeran, a hot-water-extractable glucan found in the hyphal walls of certain Aspergillus and Penicillium species has been utilized as a biochemical marker to study their phylogenetic relationships. The presence or absence of nigeran in members of certain previously assigned taxonomic groups suggests that some of these taxa may not be natural. The amount of nigeran deposited in the walls of competent species can vary over a 100-fold range under the experimental conditions employed. Of all species examined which contain nigeran, only Penicillium melinii is capable of degrading its endogenous polysaccharide.
Aspergillus is a fungal genus comprising several hundred species, many of which can damage the health of plants, animals and humans by direct infection and/or due to the production of toxic secondary metabolites known as mycotoxins. Aspergillus-specific antibodies have been generated against polypeptides, polysaccharides and secondary metabolites found in the cell wall or secretions, and these can be used to detect and monitor infections or to quantify mycotoxin contamination in food and feed. However, most Aspergillus-specific antibodies are generated against heterogeneous antigen preparations and the specific target remains unknown. Target identification is important because this can help to characterize fungal morphology, confirm host penetration by opportunistic pathogens, detect specific disease-related biomarkers, identify new candidate targets for antifungal drug design, and qualify antibodies for diagnostic and therapeutic applications. In this review, we discuss how antibodies are raised against heterogeneous Aspergillus antigen preparations and how they can be characterized, focusing on strategies to identify their specific antigens and epitopes. We also discuss the therapeutic, diagnostic and biotechnological applications of Aspergillus-specific antibodies.
We investigated the crystal structure and molecular arrangement of the linear (1 → 3)-α-D-glucan synthesized by glucosyltransferase GtfJ cloned from Streptococcus salivarius using sucrose as a substrate. The synthetic products had two morphologies: wavy fibril-like crystals as major and thin lamellae as minor products. Their structures were analyzed using electron microdiffraction, synchrotron X-ray powder diffraction, and solid-state ¹³C NMR spectroscopy. The fibrils and lamellae had the same allomorphic form but different molecular arrangements. The wet crystals were in a hydrated form, which converted into an anhydrous form with a significant decrease in crystallinity on drying. The hydrated and anhydrous forms had an extended-chain conformation with 2/1 helix, and the hydrated form was estimated to contain one water molecule per glucose residue. The long glucan chains were folded in the fibril crystals, while the short, extended chains were arranged perpendicular to the base plane of the lamellae.
Bio-based polymer is considered as one of potentially renewable materials to reduce the consumption of petroleum resources. We report herein on the one-pot synthesis and development of unnatural-type bio-based polysaccharide, α-1,3-glucan. The synthesis can be achieved by in vitro enzymatic polymerization with GtfJ enzyme, one type of glucosyltransferase, cloned from Streptococcus salivarius ATCC 25975 utilizing sucrose, a renewable feedstock, as a glucose monomer source, via environmentally friendly one-pot water-based reaction. The structure of α-1,3-glucan is completely linear without branches with weight-average molecular weight (Mw) of 700 kDa. Furthermore, acetate and propionate esters of α-1,3-glucan were synthesized and characterized. Interestingly, α-1,3-glucan acetate showed a comparatively high melting temperature at 339 °C, higher than that of commercially available thermoplastics such as PET (265 °C) and Nylon 6 (220 °C). Thus, the discovery of crystalline α-1,3-glucan esters without branches with high thermal stability and melting temperature opens the gate for further researches in the application of thermoplastic materials.
The fungal wall performs many functions, giving the cell its shape, serving a protective role and regulating the flow of molecules, including digestive enzymes, out of the cell (Chang and Trevithick, 1974; Pugh and Cawson, 1977). Protection is of particular importance with fungal pathogens. It has been postulated that primary pathogens differ from opportunistic pathogens such as Aspergillus in the greater resistance of the former to microbicidal agents produced by the phagocytic cells of an invaded host (Diamond, 1991).
Exocellular antigenic (EP) material excreted into the culture medium and composed of proteins and polysaccharides was studied in order to characterize isolates of various species of the Aspergillus fumigatus group (Aspergillus sect. Fumigati).
The EP produced by forty five strains of the A. fumigatus group (including teleomorphs of the genus Neosartorya) were analysed. The polysaccharide moiety of the extracellular fraction varies from 10 to 65% of the total lyophilized product. Galactose, mannose, and glucose are the main constituents of the EP of all strains studied. Their proportions vary considerably from one strain to another (galactose 4 to 58%, mannose 11 to 61%, glucose 4 to 68%). In spite of these variations, pathogenic and non-pathogenic strains A. fumigatus sensu stricto or teleomorphs and anamorphs cannot be separated on the basis of monosaccharidic composition. All isolates tested reacted positively with one monoclonal antibody directed against A. fumigatus galactomannan indicating the presence of galactofuran in all EP studied. The Elisa results with the rabbit antiserum indicated that A. brevipes, A. duricaulis, A. unilateralis are different from A. fumigatus and suggested the presence of specific sugar linkages in the EP of these species.
Most of the work done on Aspergillus cell wall has centered on the analysis of polysaccharidic structures. The major components have been identified as glucans and chitin with galactomannan as a minor constituent (Azuma et al.,1969). The protein content of the cell wall of those Aspergilli which have been studied is relatively low (Johnston, 1965; Bull, 1970 and Hearn and Mackenzie, 1979; Latgé et al., chapter 11). Pyridine extraction of a galactomannan-protein (APSK-66) from Aspergillus fumigatus mycelium has been reported (Azuma et al.,1968). The protein moiety was responsible for eliciting a delayed-type skin reactivity in both sensitized guinea pigs and rabbits while the carbohydrate moiety elicited an Arthus-type reaction (Suzuki and Hayashi, 1975). Molecules in situ on germ tube and mature hyphal surfaces have been used in indirect immunofluorescence tests as antigens to detect specific binding to anti-Aspergillus antibodies (Drouhet et al., 1972; Gordon et al.,1977; Schoenheyder et al., 1982). In similar tests, a germling surface antigen (Ag 5) has been used and identified as a non-ConA-binding, heat labile, protein molecule of apparent molecular mass (mol.mass.) of 34 kDa (Taylor and Longbottom, 1988). Recently, antibodies from patients with allergic bronchopulmonary aspergillosis (ABPA) were shown to bind specifically to antigens expressed on the surface of A. fumigatus spores and mycelium (Kurup et al., 1990). However, the chemical composition of these reactive surface antigens remains essentially undefined. The situation is further complicated by the known effects of age on the nature and distribution of cell wall components (Hunsley and Burnett, 1970; Barkai-Golan et al.,1978). Antigenic moieties of spore, germ-tube and hyphal structures have all been monitored and while common entities are found, marked differences have also been noted (Hearn, 1984.; John et al.,1984; Kauffman et al., 1984, Piechura et al., 1987). Recent EM studies have shown that most of the antigens recognized by antibodies from patients are localized in the mycelial cell wall (Fig.1) (Hearn et al., 1991). This result would reinforce the interest in studying cell wall glycoproteins.
Glucans were isolated from the cell wall of the yeast (Y) and mycelial (M) forms of Paracoccidioides brasiliensis. The alkali-soluble glucan of the Y form had properties of alpha-1, 3-glucan. The alkali-insoluble glucan of the M form was identified as a beta-glucan which contains a, beta-(1 -> 3)-glycosidic linkage by infrared absorption spectrum, by effect of beta-1,3-glucanase, and by partial acid hydrolysis. The alkalisoluble glucans of the M form were a mixture of alpha-and beta-glucans and the ratio of alpha-to beta-glucan was variable, depending on the preparations.
Galactomannans (GM) are widely distributed polysaccharides amongst plant and microorganisms. In plants, mainly legumes, they are reserve polysaccharides composed of linear β(1–4) mannopyranosyl residues with single stubs of α galactopyranosyl groups joined by (1–6) linkages along the chain (Dey, 1978). In contrast to plants, GMs associated with vertebrate mycopathogens are known to stimulate host defense reaction (Reiss, 1986). Their fundamental structure is different from the plant GMs. In fungi, most GMs contain (1–6) mannans with a great variety of side chain structures (Gander, 1974; Barreto-Bergter and Gorin, 1983). Man α substituted at C-2, Galp substituted at C-4 and Galf substituted at C-5 and Galf non-reducing end units are the groups most commonly found in the side chains of different fungi. Galactofuranosyl groups have been studied more thoroughly because they are presently considered to be immunodominant amongst vertebrate fungi (Bennett et al., 1985; Notermans et al., 1988). However, the length, branching point and phosphorylation of the galactose chain vary with the fungus (Barreto-Bergter and Gorin, 1983). For example, 8–10 residues are found per galactofuran side chain of Penicillium charlesii (Gander et al.,1974) whereas only single non reducing end group of galactofuranose joined by a (1–2) linkage to the mannan backbone have been detected amongst Dermatophytes (Bishop et al., 1965, 1966). The GM and peptido-GM of Cladosporium and Penicillium contain phosphorus whereas GM of Aspergillus are not phosphorylated (Lloyd, 1970; Gander et al., 1974; Barreto-Bergter et al., 1981).
The dependency between antimicrobial activity and lipophilicity (logP) of monooxygenated alklyphenols (MOAPs) was studied for a greater number of bacterial and fungal species from literature data., which was found to be significant for all species examined. The antimicrobial activity is explained with unspecific cytoplasmic membrane damaging effects that is in optimum at a lipophilicity (logP0) of 4 (±0.5) for gram-negative bacteria, at logPo of 5 for fungi and at logPo of 6 (±0.5) for gram-positive bacteria. Antimicrobials with reported mechanism of action, e.g. MOAPs with complex carbon skeleton or azole antifungal drugs, separated clearly from the data points of unspecifically acting compounds in regression analysis. It was therefore concluded that the regression lines calculated for unspecifically acting MOAPs are useful as standard curves in the analysis of microbiological inhibitory data. This method allows a systematical and computer-aided selection of potential mechanism-based inhibitors. In addition, three types of microbial inhibitors are classified: membrane damaging compounds (≤logPo), compounds desorbing from the cytoplasmic membrane (logPmax. ≤7.5 to 8), and compounds selectively acting in the cytoplasmic membrane or cell wall (logP >8).
Glucans constitute important structural or skeletal components of the cell envelope of yeasts and filamentous fungi. Isolation of individual glucan components and elucidation of their chemical structures have presented enormous difficulties. Early investigators generally subjected whole cells of baker’s or brewer’s yeast to more or less drastic treatments with alkali and acid to obtain cell wall glucan residues. It is now recognized that some glucans are extracted by these treatments and their contribution to the cell wall structure was overlooked. In addition, chemical degradation of some polysaccharides occurred as a result of heating with alkali and acids. Thirdly, as will be discussed later on, it was not recognized until the last decade that the alkali-insoluble glucan of baker’s yeast actually consists of two different polysaccharides that are difficult to separate. As a consequence, the value of the early structural investigations on yeast “glucan” was greatly diminished.
Chitosan is an antimicrobial substance used to protect against a wide range of target organisms. At present, chitosan nanoparticles are being applied for many uses but there are very few reports of their antifungal activity in seeds. This research aimed to study the effect of chitosan polymer (M.W. 200 kDa) and chitosan nanoparticles on mycelial growth of four fungal species and on chilli seed quality. Potato dextrose agar (PDA) incorporating chitosan polymer and chitosan nanoparticles at a concentration of 0.6% w/v significantly delayed mycelial growth of Rhizopus sp. Colletotrichum capsici, C. gloeosporioides, and Aspergillus niger when compared with 0.15% w/v captan, 0.2% w/v chitosan nanoparticles, and the control (PDA). Chitosan polymer solution dissolved with 0.5% v/v acetic acid was applied as a coating substance on chilli seeds. The coating procedure was done by spraying chitosan at 20 and 100 ppm on chilli seeds using top-spray fluidized bed coating equipment. Another two samples were sprayed with distilled water and 0.5% v/v acetic acid. The treated samples were then monitored for infection and compared with the non-treated control samples. Coating the seeds with chitosan at 20 and 100 ppm by this technique resulted in lower seed infection compared with the other treatments. Seed coating with chitosan at 20 and 100 ppm, however, did not significantly affect moisture content, germination, germination after accelerated ageing, germination index, and mean germination time when compared with the control samples. Therefore, chitosan appears to preserve seed quality, and further investigations into longer periods of storage are being carried out.
This chapter discusses the chemical structures of the polysaccharides of fungi and lichens investigated from 1967 to the middle of 1980. It is convenient to group the polysaccharides in terms of their chemical structures, according to the nature of the component sugars, the predominant linkage and configuration, and, in the case of heteropolymers, the nature of the main chain. The cell walls of Fusicoccum amygdali are stained blue with iodine and attacked by alpha amylase. Extracts of Sporothrix schenckii and Ceratocystis stenoceras contain 4-O-substituted D-glucopyranosyl units and the solutions give a blue color with iodine. Glycogens have been isolated from Candida albicans, Blastocladiella emersonii, Neurospora crassa, Allomyces macrogynus, Rhizophydium sphaerotheca, and Monoblepharella elongata. They have β-amylolysis values of 45–45% and average chain-length (x) values of 11–14 D-glucosyl units. This chapter briefly discusses about glucans including pseudonigeran, pullulan, cellulose etc; mannans including linear mannans etc; galactans; and heteropolysaccharides based on D-mannan main-chains and galactan main-chains.
The ability of live and dead biomass of indigenous Aspergillus niger was examined for the removal of uranium (VI) from dilute aqueous solutions. The influences of different experimental parameters such as initial solution pH, initial uranium concentration, biomass concentration, and contact time were investigated. The results indicated that the maximum sorption of biomass was observed at pH 5 and pH 4, and biosorption equilibrium was established in 120 and 60 min, for live and dead biomass, respectively. Optimal biosorption was observed at concentrations of 0.2 and 0.3 g dry biomass per 100 mL for live and dead biomass, respectively. Langmuir and Freundlich models were well able to explain the sorption-equilibrium data with satisfactory correlation coefficients higher than 0.9 for both sorbents. Moreover, the Langmuir isotherm in linear form fit better for both live and dead biomass of A. niger. The kinetic study showed that the pseudosecond-order model was appropriate to describe the sorption process.
The common saprophyte Aspergillus niger may experience carbon starvation in nature as well as during industrial fermentations. Starvation survival strategies, such as conidiation or the formation of exploratory hyphae, require energy and building blocks, which may be supplied by autolysis. Glycoside hydrolases are key effectors of autolytic degradation of fungal cell walls, but knowledge on their identity and functionality is still limited. We recently identified agnB and cfcA as two genes encoding carbohydrate-active enzymes that had notably increased transcription during carbon starvation in A. niger. Here, we report the biochemical and functional characterization of these enzymes. AgnB is an α-1,3-glucanase that releases glucose from α-1,3-glucan substrates with a minimum degree of polymerization of 4. CfcA is a chitinase that releases dimers from the nonreducing end of chitin. These enzymes thus attack polymers that are found in the fungal cell wall and may have a role in autolytic fungal cell wall degradation in A. niger. Indeed, cell wall degradation during carbon starvation was reduced in the double deletion mutant ΔcfcA ΔagnB compared to the wild-type strain. Furthermore, the cell walls of the carbon-starved mycelium of the mutant contained a higher fraction of chitin or chitosan. The function of at least one of these enzymes, CfcA, therefore appears to be in the recycling of cell wall carbohydrates under carbon limiting conditions. CfcA thus may be a candidate effector for on demand cell lysis, which could be employed in industrial processes for recovery of intracellular products.
Crude mannans extracted from Candida albicans and Saccharomyces cerevisiae by autoclaving yeast cells in citrate buffer (pH 7.0) according to Peat's method, were fractionated repeatedly by column chromatography on DEAE-Sephadex, acetate form, yielding neutral and acidic mannans. The former fraction showed a single peak by boundary electrophoresis and ultracentrifugal analysis, while the latter contained small amounts of phosphorus and protein. Using purified mannans as controls, various serological experiments were carried out with mannan antigens extracted from C. albicans with 45% phenol water and with 3% NaOH. No remarkable differences were observed in the antigenic activity of 4 mannan antigens from C. albicans, and the purified mannan exhibited very high antigenic activity. It was found that the mannan of S. cerevisiae was antigenically less specific than that of C. albicans mannan. The difference in serological specificity between mannans of both species may reflect not only differences in mannopyranose linkages but differences in the structure of the macromolecules.
The polysaccharides obtained by alkaline extraction of hyphae of Aspergillus niger 2022 contain d-mannosyl, d-galactosyl, and d--glucosyl units, the proportion of d-galactose decreasing and that of d-mannose increasing with age of culture. The polysaccharide of 5-day cultures contains d-gaIacto-d-mannans with a range of chemical structures, Fehling, precipitation providing polysaccharides of sugar composition which varied from one preparation to the other. This step removed most of the α-d-glucan present and analysis of the d-galacto-d-mannan-rich polysaccharide showed it to contain a core with a main chain of (1→6)-Iinked α-d-mannopyranosyl units substituted in the 2-position with side chains of α-d-mannopyranosyl, O-α-d-mannopyranosyl-(1→2)-O-α-d-mannopyranosyl, and O-α-d-mannopyranosyl-(1→2)-O-α-d-mannopyranosyl-(1→2)-O-α-d-mannopyranosyl units. The methylation and 13C-n.m.r. data indicated side chains of (1→5)-Iinked β-d-galactofuranosyl residues having an average length of ∼4 units and linked to the mannan core at OH-6.Polysaccharides of Aspergillus fumigatus, Aspergillus terreus, Aspergillus flavus, and Aspergillus nidulans, prepared by alkaline extraction of hyphae, and precipitated by Cetavlon in the presence of borate have interrelated chemical structures.
Cell-wall components of mycelia and conidia of Aspergillus fumigatus contain alkali-soluble polysaccharides comprised of d-galacto-d-mannans which coprecipitated with small proportions of a d-glucan, tentatively identified as glycogen. The fine structures of the d-galacto-d-mannans varied as a function of the cell type. In a 5-day-old mycelium, the polysaccharide consisted of a main chain of (1→6)-linked α-d-mannopyranose residues substituted at O-2 by 1 to 3 α-d-mannopyranosyl units that are (1→2)-interlinked. β-d-Galactofuranosyl units are (1→6)-linked to the d-mannan core, being components of side-chains of average length of ∼6 units, which are (1→5)-interlinked. The 10-day-old mycelium had a similar d-galacto-d-mannan, but the proportion of glycogen was smaller. Conidia contain polysaccharides of different structure, as shown by the 13C-n.m.r. spectrum and by methylation analysis. Side chains composed of a single unit of β-d-galactofuranosyl linked (1→6) to adjacent d-mannopyranosyl units were identified with a minor proportion of 6-O-substituted d-galactofuranosyl units. Also present were nonreducing d-galacto-pyranosyl end-groups and 2-amino-2-deoxyglycosyl units. The glucan component was not glycogen. Conidial walls have much less protein than mycelial walls. Predominant amino acids in the latter were aspartic and glutamic acids, tyrosine, alanine, and glycine. Fatty acids C16, C18, C18:1, and C18:2 were present in the mycelial and conidial walls, C18:2 was present in minor amounts in the mycelial wall, but was a major component of the lipid fraction from whole cells.
Bartnicki-Garcia, S. (Rutgers, The State University, New Brunswick, N. J.) and Walter J. Nickerson. Induction of yeastlike development in Mucor by carbon dioxide. J. Bacteriol.
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829–840. 1962—Vegetative development of Mucor rouxii may follow either one of two patterns of morphogenesis (mold-yeast dimorphism), depending on the atmosphere of incubation. Under air or N2, a filamentous (moldlike) growth developed, commonly followed by fragmentation of hyphae into spherical cells (arthrospores). Introduction of CO2 into an anaerobic atmosphere induced development of spherical, budding yeastlike cells. Anaerobically, a pCO2 of 0.3 atm or higher produced a purely yeastlike development. Presence of oxygen annulled the effect of CO2 On germination, spores gave rise directly to either type of vegetative development, depending on the atmosphere of incubation. Induction of yeastlike development by CO2 occurred in five strains of M. rouxii, and in most species of Mucor tested. M. subtilissimus, however, did not require CO2; it developed in the yeastlike form under anaerobic conditions. Strains of Rhizopus grew under CO2, but developed only filamentous mycelium. Members of other genera of Mucorales were unable to grow under an atmosphere of CO2.
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Chemically intact cell walls of filamentous and yeast-like forms of Mucor rouxii were isolated. Comparative studies were made on their composition and structure to explore possible morphogenetic implications. Both types of cell walls exhibited a complex chemical composition consisting of polysaccharides (glucosamine, galactose, mannose and fucose), phosphate, proteins (at least 13 common amino acids), lipids (readily extracted and bound), purines and pyrimidines (RNA type), Mg2+ and Ca2+. Chitosan was the most abundant component of both types of cell walls. Chitin was present in smaller quantities. No qualitative differences were found between the two types of cell walls. Major quantitative differences were found in protein, purine-pyrimidine, and especially mannose content, all of which were higher in the yeast walls. Electron microscopy of ultrathin section of whole cells showed pronounced differences in thickness and fine structure of the walls. Whereas yeast walls were seemingly composed of two layers, no distinct layering was apparent in filamentous walls, which were only one tenth as thick as yeast walls.
1. A highly dextrorotatory polysaccharide (alpha(D)+232 degrees in n-sodium hydroxide), previously isolated as a fragment of Aspergillus niger cell walls, was prepared from whole mycelium and subjected to partial acid hydrolysis. 2. Fractionation of the hydrolysate on a charcoal column with a linear gradient of ethanol yielded a series of oligosaccharides. The disaccharide member was shown to be nigerose (3-O-alpha-d-glucopyranosyl-alpha-d-glucopyranose), although a small proportion of the disaccharide peak (10%) was present as maltose despite the fact that all the nigeran had been removed from the starting material. 3. The oligosaccharides forming the main peaks from the column were shown to be members of a polymer-homologous series (nigerodextrins) by (a) the relationship between the logarithm of their chromatographic mobility and degree of polymerization, (b) obeying the Freudenberg relationship, and (c) partial acid hydrolysis.
A method is described which permits separation, identification, and determination of glucosamine and galactosamine by paper chromatography. Amounts as small as 2–5 μg. of one of the amino sugars can be detected in the presence of more than 100 μg. of the other.
A polysaccharide isolated from cultures of Nocardia asteroides contained arabinose and galactose in a molar ratio of 1.7:1. The two monosaccharides were unequivocally identified as D-arabinose and D-galactose by isolating them in crystalline form. Partial hydrolysis showed that some of the D-arabinose units in the polysaccharide were in the furanoside ring form while the D-galactose units possessed the pyranoside structure. Methylation studies showed that the polysaccharide was a branched structure of D-arabinose and D-galactose units with some of the arabinose forming non-reducing, terminal residues. The findings reported here are regarded as further evidence of the close, taxonomic relationship between Nocardia asteroides and Mycobacterium tuberculosis, members of the same order.
Recently, the study of alkaline degradation has been re-opened, with the objective of using it as a means of determining the structures of oligo- and polysaccharides and of oxidized polysaccharides. That interest has indeed been rekindled is attested by the recent reviews on saccharinic acids: on the modification of monosaccharides in alkaline solution, and on the hot alkali stability of chemically-treated, cellulose fibers. Ether derivatives of monosaccharides, oligosaccharides, and alkali-sensitive glycosides' have been used as models in determining the effect of alkalis on oxygen-free solutions of polysaccharides and oxidized polysaccharides. Alkaline degradation of polysaccharides begins, in general, at the reducing end of the molecule and proceeds in a stepwise manner through the anhydroglycose chain. Consequently, an understanding of the effects of aqueous, alkaline solutions on the reducing end-group (or on modifications of it) is essential to comprehension of the mechanism of alkaline degradation. Reducing glycose units of polysaccharide chains will be transformed, in part, to their C2-epimers in alkaline solution. The classical transformation of Lobry de Bruyn and Alberda van Ekenstein is a base-catalyzed enolization giving an enediol, which may either revert to the starting aldose or be converted to epimers of the original aldose; they showed that the main product is the ketose. However, the ions may also be in equilibrium by prototropy, without transition through an enediol.
Incubation of mycelium ofNeurospora crassa with inorganic polyphosphate brought about adsorption of the polyphosphate to receptor substances on the surface of the hyphae. The binding sites appear to be identical with those previously found to bind intracellular polyphosphate in cell-fractionation studies.Binding of polyphosphate to isolated cell walls led to the formation of two complexes. In an acid environment, an acid-dissociable complex was formed due to interaction of the polyphosphate with protein components of the cell wall. A second complex, stable to cold acid, which formed even in neutral solution, resulted from interaction of the polyphosphate with free amino groups of a polysaccharide. The latter was isolated and shown to be a polymer of galactosamine.
The glucose in c.p. galactose (I) amounting to about 2.5% can be removed by enzymic oxidation of glucose to gluconic acid with glucose oxidase-catalase system (II). When untreated I is acted on by excess II, and O2 uptake is plotted vs. time, a triphasic curve is obtained. The initial rapid O2 uptake due to the oxidation of β-d-glucose is followed by an intermediate less rapid O2 uptake in which the limiting factor is the rate of mutarotation of α-d-glucose to its β isomer. The third phase represents the slow oxidation of the galactose component by II. The glucose content is calculated by extrapolating the third phase to zero time, the intercept representing the glucose present. When enzymepurified I is treated with II, O2 uptake vs. time gives a straight line passing through the origin. This line is parallel to the third phase of the triphasic curve obtained with I.
Purified preparations of cell walls have been made from Penicillium chrysogenum. The sugar constituents of the preparations were identified by chromatography as mannose, galactose, glucosamine, glucose, xylose, and rhamnose and were found in the approximate molar ratios of 1:3:4.5:9:0.5:0.5. Fractionation with alkali and acid indicated that the walls have at least two chemically distinct layers. An explanation for some of the contradictions of earlier work is advanced.
A low-sporing isolate of Pithomyces chartarum was grown in submerged liquid culture in defined medium, and a hyphal wall fraction isolated by mechanical disruption. Itcontains about 20% protein, 40% bound hexoses, 10% bound glucosamine, 10% lipid and 5% ash.
Spore coats of Aspergillus oryzae have been mechanically isolated. They exhibited a complex chemical composition consisting of polysaccharide (mannose, glucose, galactose and glucosamine), phosphate, protein and nucleic acid. Spore coats were partially hydrolyzed by the lytic enzyme from Bacillus circulans, and glucose, laminaribiose and other unknown sugars were detected in the hydrolyzate. No qualitative differences were found between the cell walls and the spore coats. The major quantitative difference was in protein content, which was higher in the spore coats.
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