Structural investigation of a water-soluble glucan from an edible mushroom, Astraeus hygrometricus

A water-soluble glucan, Fraction I, was isolated from the aqueous extract of the fruit bodies of the mushroom Astraeus hygrometricus. On the basis of total hydrolysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, 2D-COSY, TOCSY, NOESY, and HSQC), the structure of the repeating unit of the glucan is determined as:This glucan shows strong splenocyte activation.     

饲料中灵芝提取物对异育银鲫生长、饲料利用、免疫应答及抗病的影响

通过生长实验探求饲料中添加灵芝提取物饲料对异育银鲫（6.720.11） g生长、饲料利用及免疫应答的影响,以及停止灵芝提取物饲料后,使用效果的持续性。在实验进行的第21天（D21）、第46天（D46）、第65天（D65）进行取样,结果表明所有的灵芝提取物组的特定生长率在饲喂65d后与对照组无显著差异（P0.05）。摄食率随着灵芝提取物在饲料中含量的增加而增加（P0.05）,饲料效率则有着相反的趋势（P0.05）。在D46和D65时,随着灵芝提取物的含量增高,吞噬活力略有增高,但无显著差异（P0.05）。在D21、D46和D65时,呼吸暴发活力随着灵芝提取物的含量增高而增高（P0.05）,在D21时,（0.6%-21d、0%-44d）处理组和（3%-21d、0%-44d）处理组其呼吸暴发活力高于对照组（P0.05）。在D65时,（0.6%-21d、0%-44d）处理组和（3%-21d、0%-44d）处理组其呼吸暴发活力仍高于对照组（P0.05）,但差异不显著。D21、D46和D65时,替代途径补体溶血活力随着灵芝提取物的增高而降低（P0.05）。所有处理组溶菌酶活力在D21时没有产生显著差异。在D46时,对照组溶菌酶活力比最高添加组低但高于低添加组（P0.05）,（3%-1d、0%-44d）处理组也仍高于对照组（P0.05）。在D65时,所有添加组的溶菌酶活力均低于对照组（P0.05）。在D65时,髓过氧化物酶活力随着灵芝提取物的添加剂量升高而升高（P0.05）,（0.6%-21d、0%-44d）处理组仍高于对照组（P0.05）。灵芝提取物的能提高白细胞呼吸暴发活力,且在在高剂量摄入21d后（D46时）,仍可以保持较高的活力,说明灵芝提取物对免疫应答有一定时间延续效应。随着灵芝提取物添加量的升高,异育银鲫在经爱德华氏菌攻毒后的存活率显著提高。最高存活率组为3%添加组,显著高于对照组（P0.05）。实验周期为65d时,饲料中灵芝提取物推荐添加剂量为3%。       

Chemical analysis of a new (1-->3)-, (1-->6)-branched glucan from an edible mushroom, Pleurotus florida

A glucan that was soluble in aqueous sodium chloride was isolated from the aqueous extract of the fruiting bodies of Pleurotus florida. On the basis of total hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies (1H, 13C, TOCSY, DQF-COSY, NOESY, and HSQC), the structure of the repeating unit of the polysaccharide is established as: This glucan stimulates the phagocytic activity of macrophages.     

Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase

Dextransucrase S (DSRS) is the only active glucansucrase that has been found in Leuconostoc mesenteroides NRRL B-512F strain. Native DSRS produces mainly 6-linked glucopyranosyl residue (Glcp), while Escherichia coli recombinant DSRS was observed to produce a glucan consisting of 70% 6-linked Glcp and 15% 3,6-Glcp. Lys residues were introduced at the N-terminal end of the core domain by site-directed mutagenesis. In glucans produced by the one-point mutants T350K and S455K, the amount of 6-linked Glcp was increased to about 85% of the total glucan produced, more similar in structure to native B-512F dextran. The double mutant T350K/S455K produced adhesive, water-insoluble glucan with 77% 6-linked Glcp, 8% 3,6-linked Glcp and 4% 2,6-linked Glcp. The T350K/S455K mutant exhibited a 10-fold increase in glucosyltransferase activity over those of the parental DSRS-His(6) and its T350K and S455K mutants. This is the first report demonstrating a change in the properties of a dextransucrase or a related glucosyltransferase through simple site-directed mutagenesis to create 2,6-linked Glcp.     

The structure and conformation of a water-insoluble (1-->3)-,(1-->6)-beta-d-glucan from the fruiting bodies of Pleurotus florida

A water-insoluble glucan, PFPSIN, has been isolated from the aqueous extract of an edible mushroom Pleurotus florida. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and (13)C NMR experiments, the repeating unit of the polysaccharide was established as Conformational analysis revealed the triple helical conformation of this glucan.     

A glucanase-driven fractionation allows redefinition of Schizosaccharomyces pombe cell wall composition and structure: assignment of diglucan

Purified endoglucanases have been used to determine the composition of Schizosaccharomyces pombe cell wall. This structure has been traditionally studied after isolating its components (mannoproteins, alpha1,3-glucan, beta1,3-glucan, and a branched beta-glucan) with hot alkali. Instead, we sequentially removed the polysaccharides by digesting with endo-beta1,3-glucanase and with a novel endo-alpha1,3-glucanase (mutanase). After this gentle isolation we observed that a branched beta1,3-beta1,6-glucan is much more abundant than previously described. By scaling-up the new protocol we prepared large amounts of the highly branched glucan and determined its structural features. We have named this highly branched beta-glucan diglucan, reflecting its two types of beta linkages. We have also identified an insoluble endoglucanase-resistant type of 1,3-linked glucan present in S. pombe cell walls. We redefined the wall composition of S. pombe vegetative cells by this new method. Finally, to demonstrate its application, we determined the cell wall composition of known mutant strains.     

Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module

The crystal structures of a carbohydrate-binding module (CBM) family 28 domain of endoglucanase Cel5A from Clostridium josui have been determined in ligand-free and complex forms with cellobiose, cellotetraose, and cellopentaose as the first complex structures of this family. In the cleft of a β-sandwich fold, the ligands are recognized by stacking interactions and hydrogen bonds. Conformations of the bound cellooligosaccharides are similar to those in crystals and solution but clearly different from the cellulose structure. Interestingly, the glucan chain bound on CBM28 is in the opposite direction of that bound to CBM17, although these families share significant structural similarity.     

Arrangement of mixed-linkage glucan and glucuronoarabinoxylan in the cell walls of growing maize roots

Background and Aims

Plant cell enlargement is unambiguously coupled to changes in cell wall architecture, and as such various studies have examined the modification of the proportions and structures of glucuronoarabinoxylan and mixed-linkage glucan in the course of cell elongation in grasses. However, there is still no clear understanding of the mutual arrangement of these matrix polymers with cellulose microfibrils and of the modification of this architecture during cell growth. This study aimed to determine the correspondence between the fine structure of grass cell walls and the course of the elongation process in roots of maize (Zea mays).

Methods

Enzymatic hydrolysis followed by biochemical analysis of derivatives was coupled with immunohistochemical detection of cell wall epitopes at different stages of cell development in a series of maize root zones.

Key Results

Two xylan-directed antibodies (LM11 and ABX) have distinct patterns of primary cell wall labelling in cross-sections of growing maize roots. The LM11 epitopes were masked by mixed-linkage glucan and were revealed only after lichenase treatment. They could be removed from the section by xylanase treatment. Accessibility of ABX epitopes was not affected by the lichenase treatment. Xylanase treatment released only part of the cell wall glucuronoarabinoxylan and produced two types of products: high-substituted (released in polymeric form) and low-substituted (released as low-molecular-mass fragments). The amount of the latter was highly correlated with the amount of mixed-linkage glucan.

Conclusions

Three domains of glucuronoarabinoxylan were determined: one separating cellulose microfibrils, one interacting with them and a middle domain between the two, which links them. The middle domain is masked by the mixed-linkage glucan. A model is proposed in which the mixed-linkage glucan serves as a gel-like filler of the space between the separating domain of the glucuronoarabinoxylan and the cellulose microfibrils. Space for glucan is provided along the middle domain, the proportion of which increases during cell elongation.     

Comparison of beta-glucan structures in a cell wall mutant of Saccharomyces cerevisiae and the wild type

A mutant of Saccharomyces cerevisiae defective in the cell wall beta-glucan structure was obtained. The mutant cells are extremely sensitive to (beta 1-3)-glucanase digestion and mild alkali treatment. Structural analysis revealed that the alkali-insoluble, skeletal glucan from wild type cells contains two components, a (beta 1-3) linked glucan with a laminated structure, and a highly branched glucan containing predominantly (beta 1-6) linkages. The mutant cells lack the latter component.     

Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings

Switchgrass (Panicum virgatum) was subjected to hydrothermolysis pretreatment and then used to study the effect of enzyme loading and temperature in a simultaneous saccharification and fermentation (SSF) with the thermotolerant yeast strain Kluyveromyces marxianus IMB3 at 8% solid loading. Various loadings of Accellerase 1500 between 0.1 and 1.1 mL g(-1) glucan were tested in SSF at 45 °C (activity of enzyme was 82.2 FPU mL(-1)). The optimum enzyme loading was 0.7 mL g(-1) glucan based on the six different enzyme loadings tested. SSFs were performed at 37, 41 and 45 °C with an enzyme loading of 0.7 mL g(-1) glucan. The highest ethanol concentration of 22.5 g L(-1) was obtained after 168 h with SSF at 45 °C, which was equivalent to 86% yield. Four different batch and fed-batch strategies were evaluated using a total solid loading of 12% (dry basis). About 32 g L(-1) ethanol was produced with the four strategies, which was equivalent to 82% yield.     

Structural characterization of beta-D-(1-->3)-glucans from different growth phases of the marine diatoms Chaetoceros mülleri and Thalassiosira weissflogii

We have investigated the content and structure of the chrysolaminarans isolated from the two marine diatoms Chaetoceros mülleri and Thalassiosira weissflogii. Samples were taken from different phases of growth, and the structure of the chrysolaminaran was seen in relation to the specific growth rate of the diatoms. The structure determined for the glucan from C. mülleri was found not to vary with different specific growth rates. T. weissflogii showed some variance in the structure, both throughout the different stages of growth and between samples taken from the stationary phase. C. mülleri was found to have a chrysolaminaran with a degree of polymerization (DP) of 22-24 and a degree of beta-(1-->6) branching of 0.006-0.009. These results corresponded well with previous results obtained in our laboratories. The chrysolaminaran isolated from T. weissflogii was found to have a DP of 5-13 and no beta-(1-->6) branching. This is to our knowledge the first characterization of the chrysolaminaran from T. weissflogii.     

Structure of a -D-glucan from the mycelial wall of Basidiomycete QM 806

A water-soluble β-D-glucan has been isolated from the mycelial wall of Basidiomycete QM 806. The structure of this glucan was investigated by methylation, periodate, and enzymic studies. Hydrolysis of the methylated glucan gave 2,3,4,6-tetra-, 2,4,6-, 2,3,4- and 2,3,6-tri-, and 2,4-di-O-methyl-D-glucose in the following molar proportions: 1.0:1.0:O.8:1.2:1.0. Periodate oxidation of the glucan followed by reduction and mild acid hydrolysis gave glycerol, erythritol, and D-glucose in the molar proportions, 2.1, 1.0, and 2.0, respectively. The glucan was degraded to the extent of 38% by an exo-β-(1→3)-glucanase isolated from the same organism, though the branch points (joined through O-1, O-3, and O-6) appeared to be resistant to the enzyme whereas the (1→4) linkages were not. On the basis of these findings, the structure of the glucan and the possible role of the glucanase are discussed.     

Immune-enhancing effect of water-soluble beta-glucan derived from enzymatic hydrolysis of yeast glucan

Immunostimulants play an important role in the treatment of immunodeficiency. Macrophages are the first line in our immune defense system and play a critical role in the immune response. Therefore, finding new and better substances to induce an immune response by activating macrophages is an attractive research topic, especially in the fields of immunopharmacology and cancer prevention. Keratinocytes actively crosstalk with immune cells during wound repair, so enhancing the function of keratinocytes is also an important part of improving immunity. Beta-glucans are naturally occurring polysaccharides, consisting of d-glucose monomers linked by beta-glycosidic bonds. Several studies have investigated the immunomodulatory effects of beta-glucan, such as its anti-inflammatory and antibacterial properties. However, the use of yeast cell wall glucan has been limited because it is not soluble in water. In this study, we produced low-molecular-weight water-soluble yeast glucan (WSY glucan) and confirmed various aspects of its immune-enhancing effect. The structure of the beta-(1→3) and (1→6) bonds of WSY glucan were confirmed by nuclear magnetic resonance spectroscopy (¹H-NMR) analysis. Our results showed that treatment with WSY glucan significantly and dose-dependently induced the production of inflammatory mediators (prostaglandin E₂ (PGE₂) and nitric oxide (NO)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) in macrophages. In addition, WSY glucan treatment showed changes in the morphological structure of the macrophages and promoted phagocytic activity of the macrophages and wound healing in keratinocytes. Based on these results, WSY glucan is considered as a potential candidate for the treatment of diseases related to the weakening of the immune system without the limitation of insolubility.     

长白山树舌水溶性色素多糖CF_1的分离纯化与结构研究

 从长白山树舌子实体中分离色素多糖,其均一性检查用Sepharose CL-4B柱层析、高压玻璃纤维纸电泳、醋酸纤维素薄膜电泳、超离心分析等方法,用凝胶层析测定的分子量为18.5万。 均一的脱色多糖(CF_1a)分子量为14万。用红外光谱,G.C,~1H-N.M.R,~(13)CN.M.R,高碘酸氧化与Smith降解,甲基化分析等确定其结构为葡聚糖,其基本结构可能如下式: 总色素用次氯酸氧化法测定为24％。在色素多糖中的色素可能是聚合形式。色 素经薄层层析、紫外吸收性质检查等表明可能是新黄酮类化合物。     

Cell wall metabolism during fruiting of the basidiomycete Schizophyllum commune

During the development of fruit bodies of the basidiomycete Schizophyllum commune, the alkali-insoluble (R glucan) and alkali-soluble (S glucan) cell wall fractions are synthesized during the entire course of morphogenesis. The water soluble glucan (WSG) is not synthesized after an early stage. There is also a relative increase in the proportion of S glucan during development which appears related to a change in the proportion of the components synthesized. Data are also presented to show that several fruiting mutants also have specific cell wall differences, and that there is a significant contribution to cell wall structure by genes which do not cause a macroscopically observable change in phenotype.     

Morphology of yeast cell wall as affected by genetic manipulation of beta(1 --> 6) glycosidic linkage

The morphology of yeast cells as it is affected by the glycosidic linkages of constituent glucan was studied. Four different strains of Saccharomyces cerevisiae were studied. A cell wall matrix particle representing the intact original morphology and composed entirely of beta-glucan was prepared. Using prepared cell wall glucan particles, the morphology and cell wall matrix structure were examined. Genetic modification of the cell wall structure during growth results in the alteration of the shape and hydrodnamic volume of the intact cell wall particles. The shape and hydrodynamic volume of the cell wall particles can also be modified by in vitro chemical and enzymatic treatment. The shape factor and hydrodynamic volume of the whole glucan cell wall matrix particles were evaluated quantitatively using a rheological analysis. An increased degree of beta(1 --> 6) cross-linking in the cell wall matrix induces a nearly 2-fold increase in the shape factor and a 10-fold increase in the compression modulus of the glucan particles. The disruption of beta(1 --> 6) glycosidic cross-linking causes the particles to swell by up to 18% of their original volume. This was used as a strategy to isolate a yeast mutant with a high beta(1 --> 6) glycosidic content in the cell wall glucan.     

Crystal structure of glucansucrase from the dental caries pathogen Streptococcus mutans

Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosyl-enzyme intermediate in subsite − 1. On the other hand, novel structural features were revealed in subsites + 1 and + 2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite + 1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1-3) glycosidic linkages or soluble glucan with α(1-6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI.     

An alpha-glucan elicitor from the cell wall of a biocontrol binucleate Rhizoctonia isolate

Binucleate Rhizoctonia (BNR) isolate (232-C6) is an effective biocontrol agent for protection of potato from Rhizoctonia canker, a disease caused by Rhizoctonia solani. Production of hydrolytic enzymes is one of the best known inducible defense responses following microbial infection. We isolated and characterized a cell wall alpha-glucan from BNR, which induces beta-1,3 glucanase activities in potato sprouts, the primary site of infection by R. solani. An autoclaving method, previously reported for isolation of oligosaccharide elicitors was used, and the glucan purified by chromatographic techniques. Maximal induction of beta-1,3 glucanase activity in potato sprouts was obtained with 250 microg of the alpha-glucan elicitor after 6 days from inoculation time. Both, BNR mycelium and the alpha-glucan produced a similar kinetic response of beta-1,3 glucanase. However, the alpha-glucan did not induce phytoalexin accumulation, previously correlated with the defense response. Uronic acids (approximately 10% with respect to total neutral sugars) were determined and identified as glucuronic acid by high-pH anion-exchange chromatography. Methylation analysis showed that the glucan consists of (1-->3) and (1-->4)-linked glucose units with preponderance of the first ones. Some of the (1-->4) linkages were branched at position 6. The glucan was partially degraded with amyloglucosidase. This, together with the NMR spectra data and the high optical rotation of the original (+195 degrees ) and degraded glucans (+175 degrees ) proved the alpha configuration. Further methylation of the amyloglucosidase degraded glucans indicated that they consist of (1-->3)-linked glucoses. The present study is the first report on the isolation and characterization of an alpha-glucan from Rhizoctonia, that may be important as a biocontrol factor.     

Analysis of alkali-soluble glucan produced by<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> wild-type and mutants

The alkali-soluble glucan of the yeast cell wall contains beta-(1,3)- and (1,6)-D-linkages and systemically enhances the immune system. To isolate Saccharomyces cerevisiae mutants producing glucan with a high degree of beta-(1,6)-D-glycosidic bonds, a wild-type strain was mutagenized with ultraviolet light. The mutants were then selected by treatment with 1.0 mg laminarinase, endo-beta-(1,3)-D-glucanase/ml. The alkali-soluble glucan was extracted by modified alkalysis followed by the Cetavlon method and concanavalin-A chromatography. The prepared alkali-soluble glucans from the wild-type and the mutants were compared with respect to yield and polymer structure using gas chromatography, 13C-NMR spectrometry, high performance liquid, and multi-angle laser light scattering and refractive index detectors. The results indicated that the S. cerevisiae mutants had ten-fold more alkali-soluble glucan than the wild-type. Structural analysis revealed that the alkali-soluble glucan from the mutants also had a higher degree of beta-(1,6)-D-linkage than that from the wild-type.