The structure and histochemistry of sclerotia of Ophiocordyceps sinensis

The structure and histochemistry of sclerotia of Ophiocordyceps sinensis (synonym: Cordyceps sinensis) are described. The remains of the caterpillar epidermis and sometimes setae of the caterpillar were attached to the pigmented layer that is external to the rind of the sclerotium. The outer aerial hyphae and hyphae of the inner medulla were densely interwoven around the epidermis of the caterpillar; these eventually differentiated into the rind of the sclerotium. The medulla of the sclerotium consisted of three intergrading regions of hyphal density: high, low and a region of intermediate hyphal density. All hyphae of the medulla contained large quantities of protein, polysaccharide and polyphosphate; only the region of high hyphal density was rich in beta-1,3 glucans; the center of the sclerotium was almost devoid of hyphae and contained what are most likely the remains of caterpillar tissue. These features are compared with those of sclerotia of other fungi, and their possible significance is discussed.     

Scanning Electron Microscopy of Sclerotia of Sclerotinia trifoliorum and Related Species

The microanatomy of immature 'white', 'slightly pigmented' and mature, 1-month-old 'black' sclerotia of Sclerotinia trifoliorum , S. sclerotiorum , and S. minor were studied by scanning electron microscopy (SEM). A surface mycelial network was present over sclerotia at maturity. Also dried exudate on the superficial, sclerotial cells at maturity was observed. At this stage of morphogenesis an outer layer of the wall of medullary hyphae was synthesized. Two zones (i.e., rind and medulla) of hyphal tissue in sections of mature sclerotia were distinguished. The wall of rind cells was thick and one-layered, whereas the wall of medullary hyphae was thick and bi-layered.
No lacunac (intercellular spaces) in sclerotial rind were found but the sclerotial medulla appeared to be lacunate in all three species. At the SEM level the structural organization of sclerotia of S. trifoliorum was identical to that one of sclerotia of S. sclerotiorum and S. minor. Thus, in the conducted investigation of the sclerotial stromata, a unique, structural characteristic of taxonomic importance to distinguish S. trifoliorum from the other Sclerotinia species was not found. Observations on the sclerotial morphogenesis in S. trifoliorum and the related species agree with and supplement the light and transmission electron microscope studies of other researchers.     

Changes during development in the permeability of sclerotia ofSclerotinia minor to an apoplastic tracer

Summary Sclerotia ofSclerotinia minor produced in culture are permeable to the apoplastic tracer sulphorhodamine G (SR) in early stages of their development, but become impermeable as the rind differentiates at the onset of maturation. Reduction in permeability corresponds with deposition of the dark brown pigment in the rind cell walls rather than initiation of the rind as a distinct surface layer. Fluorochrome permeation into cut sclerotia indicates that, while the rind is the primary barrier, the walls and extracellular matrix of the cortex and medulla of mature sclerotia also impede SR movement. Some cells take up fluorochrome into the protoplast. This indicates enhanced proton pumping activity at the cell surface, which suppresses ionisation of the fluorochrome, allowing it to cross the plasma membrane and accumulate in the hyphae. In intact sclerotia such hyphae are very rare and were detected only at one stage of development. However, in cut sclerotia at the two earliest stages of development most of the hyphae near the cut surface accumulated SR and it is possible that this is due to proton pumping activity induced by wounding.Abbreviations ECM extracellular matrix - SR sulphorhodamine G     

Calcium ions and polyamines activate the plasma membrane-located 1,3-β-glucan synthase

Sucrose-density-gradient centrifugation and partitioning in a polyethylene glycol/dextran two-phase system were used to isolate plasmamembrane vesicles from microsomal preparations of soybean cell suspension cultures. Both methods resulted in the enrichment of the activity of a 1,3--glucan synthase which forms a polymer consisting of more than 99% of 1,3-linked glucose (callose). Digitonin increases the 1,3--glucan synthase activity in the various membrane fractions to a different degree, supporting the suggestion that this enzyme is vectorially arranged in the plasma membrane. The enzyme is greatly activated either by poly-l-ornithine or synergistically by Ca²⁺ and spermine, indicating that the same enzyme is affected and exhibits the regulatory properties necessary for callose synthesis.Abbreviations GSI glucan synthase I - 1,3--GS 1,3--glucan synthase (EC 2.4.1.34) - IDPase inosine 5-diphosphatase - poly-l-Orn poly-l-ornithine - PEG polyethyleneglycol - SGT sterol-glucosyl-transferase - UDPG uridine 5-diphosphoglucose Dedicated to W. Nultsch on the occasion of his 60th birthday     

Isolation and culture of anucleate protoplasts from cotton fiber; assessment of viability and analysis of regenerated wall polymers

Procedures were developed for the isolation and culture of an anucleate protoplast system from cotton fibers actively undergoing secondary wall synthesis. Because the fibers at this stage are elongated single cells (30 m × 1–2 cm), most of the cellular vesicles released in the process of isolation are anucleate. After purification, the protoplast population was nuclei-free. When transferred to culture medium, the anucleate protoplasts (cytoplasts) synthesized starch, hydrolyzed fluorescene diacetate for up to 9 days and formed cell wall material for at least 7 days. The composition of the regenerated cell walls was dependent upon the substrate supplied in the medium: -1,3-linked glucans were predominantly synthesized when 1 mM UDP[¹⁴C]glucose was supplied; -1,4-linked glucans were predominantly synthesized when 1 mM [¹⁴C]-glucose was supplied. Thus the composition of the regenerated cell walls formed by the anucleate protoplasts was similar to the secondary cell wall synthesized by intact cotton fibers under the same culture conditions.     

Morphological characteristics of sclerotia formed from hyphae of <Emphasis Type="Italic">Grifola umbellata</Emphasis> under artificial conditions

The morphological characteristics of sclerotia were induced in cultures of the fungus Grifola umbellata by introducing an unidentified companion fungus were studied by light microscope, scanning and transmission electron microscope (SEM and TEM). Light microscope and SEM investigations of developing sclerotia revealed that aerial mycelial hyphae diminished with age, and mature sclerotia had two tissue layers, the rind and medulla. The medulla was comprised of thin and thick-walled hyphae of varying diameter. The thick-walled cells always formed below the hyphal tips. Retraction of the cytoplasm was accompanied by the thickening of cell wall. There were crystalline initials in the newly formed sclerotium. Crystalline initials were always formed in the tip of medullary hyphae and were not of regular shape. A series of changes occurred in the cells in which the crystalline initials would be formed, such as enlargement of size, formation of one or several large vacuoles. Crystalline initials developed via amorphous materials in the cytoplasm deposited in the vacuoles. At last crystalline initials was released by degradation of the cell wall.     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses

Summary In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), -glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid -d-galactosidase, -d-N-acetylglucosaminidase, -d-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth.—After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET. When the drugs were applied to Zn-deficient pregnant rats, the alterations resembled those observed after drug treatment alone. In all cases of severe thymus degeneration, i.e. ET and DEXA treatment and Zn-deficiency, the number of macrophages and activities of lysosomal hydrolases in macrophages and reticular cells were increased; the lysosomal hydrolases were often homogeneously distributed over the cortex. Cell contacts between reticular cells and lymphocytes were reduced. Vacuoles occurred within the reticular cells.—The fetal thymus was reduced in size and the number of macrophages and the activities of their lysosomal enzymes were increased after Zn-deficiency, DEXA treatment and Zn-deficiency combined with ET administration.Supported by the Deutsche Forschungsgemeinschaft (Sfb 174)     

Structural aspects of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] tuberculate ectomycorrhizae

Summary Tubercles of Pseudotsuga menziesii consisted of clusters of ectomycorrhizae surrounded by a peridiumlike rind. Energy dispersive spectroscopy demonstrated that crystals found in the zone of loose hyphae extending from the inner rind to the mantle of each root probably contain calcium oxalate. Inner mantle and Hartig net hyphae showed a labyrinthine branching pattern and stored carbohydrates and protein. The Hartig net formed up to inner cortical cells which had thickened, darkly stained walls. Bacteria were located either along with hyphae within the rind or as colonies on the surface of the tubercle.     

Sclerotinia nivalis, sp. nov., the pathogen of snow mold of herbaceous dicots in Northern Japan

A newSclerotinia, previously reported asS. intermedia from Japan, is described asSclerotinia nivalis on the morphological basis of the sclerotial anamorph and teleomorph produced in culture. The characters assigning this species to the genusSclerotinia are the tuberoid sclerotia superficially produced on suscepts, the small sclerotia produced on aerial mycelium in culture, the interhyphal spaces in medullary tissue of sclerotia, and the globose cells constructing the ectal excipulum of apothecia. It is distinguishable fromS. sclerotiorum, S. minor, andS. trifoliorum by the intermediate sized sclerotia in culture, binucleate ascospores, the molecular mass of major proteins of sclerotia, and the patterns of esterase isozymes in sclerotial extracts. AlthoughS. nivalis causes snow mold of various dicots, it is a mesophile having an optimum temperature for mycelial growth of around 20°C. It attacks edible burdock(Arctium lappa), Chryhsanthemum morifolium, Ambrosia elatior, carrot(Daucus carota), Angelica acutiloba, Ajuga reptans, andPlantago lanceolata.     

Protoplast formation ofNeurospora crassa by an inducible enzyme system ofArthrobacter GJM-1

A novel method for preparation ofNeurospora crassa protoplasts has been developed based on treatment of hyphae with an inducible enzyme system ofArthrobacter GJM-1 that had been obtained by growing the bacterium onNeurospora cells as the sole carbon source. The lytic system was characterized for enzyme activities relevant to hydrolysis of major cell-wall components ofN. crassa, such as 1,3--d-glucanase, -mannanase, chitinase, proteinase, -amylase and -d-glucosidase. The optimal conditions for protoplast formation from young hyphae ofN. crassa (10 mg dry weight) were 180 min treatment by 40 mg/ml of freeze-dried lytic system. Protoplasts which were released from both hyphal tips and sides were found not to contain cell-wall material, as judged by electron microscopy and Calcofluor binding. When freed of the lytic system, 50% of protoplasts regenerated a mycelium.     

A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues

Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [¹⁴C]glucans using either guanosine 5-diphosphate (GDP)-D-[U-¹⁴C]glucose or uridine 5-diphosphate (UDP)-D-[U-¹⁴C]glucose as glycosyl donors. Although these glucans had -(13) and -(14) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed -(13) and -(14) glucan from GDP-D-[U-¹⁴C]glucose was changed to that of -(14) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-¹⁴C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K _m and V _max of the glucan synthase for GDP-D-glucose were 6.38 M and 5.08 M·min^-1, respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.Abbreviations GDP guanosine 5-diphosphate - GLC gasliquid chromatography - UDP trridine 5-diphosphate     

Structure and differentiation of the cell wall of Phytophthora palmivora: Cysts,hyphae and sporangia

Summary Walls from cysts, hyphae and sporangia of Phytophthora palmivora consist chiefly (ca. 90% dry wt) of -glucans with 1,3-, 1,4- and 1,6-links. The glucans are predominatly -1,3-linked but there are significant differences in the relative proportion of 1,3-, 1,6- and 1,4-linked glucosyl residues among the three wall types. There are also differences in protein content, susceptibility to degradation by various -glucanases, and surface texture. The isolated cyst wall consists solely of a thin fabric of long, tightly interwoven, randomly oriented microfibrils. Both inner and outer surfaces of the cyst wall are distinctly microfibrillar. The hyphal wall has two different textures; the internal surface is distinctly microfibrillar while the external surface is non-fibrillar. In a germinated cyst, there is a zone of demarcation where the microfibrils of the cyst wall disappear into the smooth outer texture of the germ tube wall. An exo--1,3-glucanase preferentially removed the amorphous material of the outer surface of the germ tube leaving exposed a continuous microfibrillar fabric from cyst to hyphal tube. Conceivably, the textural and structural differentiation of the cell wall may play a decisive role in cellular morphogenesis.     

A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca²⁺ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K _m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca²⁺, and in the presence of 2 mM Ca²⁺ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K _m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara l-arabinose - CHAPS 3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid - DAP diphenylamine-aniline-phosphoric acid stain - Gal d-galactose - Glc d-glucose - Man d-mannose - Mes 2-(N-morpholino)ethane sulphonic acid - Rha d-rhamnose - Rib d-ribose - TFA trifluoroacetic acid - UDPGlc uridine-diphosphate glucose - Xyl d-xylose This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project.     

Reserve substances inPaxillus involutus sclerotia

Summary The ectomycorrhizal fungus,Paxillus involutus, produces sclerotia in culture. These can be induced to form on agar medium by exposing mycelium grown at 25°C to various temperatures between6°C and 15°C. Sclerotia formed at 10°C and above were large and covered with drops of exudate, while those formed at 6°C or 8°C were very small and did not produce an exudate. Mature sclerotia were bounded by a compact rind and contained abundant storage reserves. Histochemistry of the larger sclerotia showed large quantities of protein stored as protein bodies in the cytoplasm, lipid present as small droplets, glycogen granules stored in the cytoplasm and polyphosphate present as small granules in the cytoplasm and in the protein bodies. Energy dispersive X-ray microanalysis confirmed the presence of phosphate in the granules and was used to map its distribution throughout the sclerotium. The smaller sclerotia induced at 8°C and below on the same medium had the same basic structure and composition, but lacked the complex phenolic cell network found in large sclerotia, and had abundant extracellular polysaccharides. The rind was not well developed and these small sclerotia are interpreted to have been arrested at an early stage of development.     

Glucomannan synthesis in pea epicotyls: The mannose and glucose transferases

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a -1,4-[su14C]mannan from GDP-d-[U-¹⁴C]-mannose, a mixed -1,3- and -1,4-[¹⁴C]glucan from GDP-d-[U-¹⁴C]-glucose and a -1,4-[¹⁴C]-glucomannan from both GDP-d-[U-¹⁴C]mannose and GDP-d-[U-¹⁴C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The -glucan synthase had different properties from other preparations which bring about the synthesis of -1,3-glucans (callose) and mixed -1,3- and -1,4-glucans and which use UDP-d-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-d-xylose in addition to GDP-d-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-d-glucose acted competitively in the presence of GDP-d-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-d-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-d-glucose and GDP-d-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-d-mannose and GDP-d-glucose to bring about the synthesis of the heteropolysaccharide.Abbreviations CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate - CHAPSO 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate - CHD 1,2-cyclohexanedione - CDP cytidine 5-diphosphate - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - GDP guanosine 5-diphosphate - NAI N-acetyl-imidazole - NEM N-ethylmaleimide - PGO phenylglyoxal This work has been made possible by grants of M.A.F. and M.U.R.S.T. 40% of Italy. Dr. A. Zuppa wishes to thank the C.N.R. of Italy for his research scolarship.     

Simple staining detects ultrastructural and biochemical differentiation of vegetative hyphae and fruit body initials in colonies of Pleurotus pulmonarius

AIMS: To know the ultrastructural and biochemical differences of vegetative hyphae and fruit body initials in colonies of Pleurotus pulmonarius. METHODS AND RESULTS: Feulgen reagent was used to detects differentiation of hyphae. The intracellular laccases, proteases and beta-1,3-glucanases activity, content of cytoplasmic protein, glycogen and glucans in the cell wall were evaluated in hyphae of fruit body initials and in vegetative hyphae. The thickness of hyphal walls of the vegetative hyphae was also evaluated. Substantial biochemical changes were observed in hyphae of different zones of the fruiting colony. Hyphae at the periphery had thinner walls than in the centre of the colony. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Staining correlated with the enzymatic activity, protein, glycogen and glucans, in mycelium and in fruit body initials. The implications are that hyphal maturity in P. pulmonarius involves storage of glucans, in part at least, in the form of a thickened hyphal wall.     

High molecular weight precursors of glucans inSaccharomyces cerevisiae

Nascent -1,3 glucan synthesized by mixed membrane fractions fromSaccharomyces cerevisiae was solubilized by extraction with hot SDS or urea. Nature of the material was analyzed by electrophoresis and gel filtration. As determined by gel filtration, Mr of synthesized glucans exceeded 1,500 kDa, but was below 20,000 kDa. This nascent material served as an acceptor for further glucose transfer reactions, giving rise to glucan molecules over 20,000 kDa. It is suggested that the high Mr precursor components represent protein-bound glucan molecules in transit to the cell surface.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Structural Characterization of <Emphasis Type="Italic">β</Emphasis>-glucans of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis> in Different Stages of Fruiting Body Maturity and their Use in Nutraceutical Products

β-Glucans of Agaricus brasiliensis fruiting bodies in different stages of maturity were isolated and characterized by FTIR and NMR. These fractions had greater amount of (1→6)-β-glucan and the (1→3)-β-glucan increased with fruiting bodies maturation. Yields of β-glucans increased from 42 mg β-glucans g⁻¹ fruiting bodies (dry wt) in immature stage to 43 mg g⁻¹ in mature stage with immature spores, and decreased to 40 mg g⁻¹ in mature stage with spore maturation. Mature fruiting bodies, which included these glucans, have potential therapeutical benefits for use in nutraceutical products.