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1.
Melanocarpus albomyces, a thermophilic fungus isolated from compost by enrichment culture in a liquid medium containing sugarcane bagasse, produced
cellulase-free xylanase in culture medium. The fungus was unusual in that xylanase activity was inducible not only by hemicellulosic
material but also by the monomeric pentosan unit of xylan but not by glucose. Concentration of bagasse-grown culture filtrate
protein followed by size-exclusion and anion-exchange chromatography separated four xylanase activities. Under identical conditions
of protein purification, xylanase I was absent in the xylose-grown culture filtrate. Two xylanase activities, a minor xylanase
IA and a major xylanase IIIA, were purified to apparent homogeneity from bagasse-grown cultures. Both xylanases were specific
for β-1,4 xylose-rich polymer, optimally active, respectively, at pH 6.6 and 5.6, and at 65°C. The xylanases were stable between
pH 5 to 10 at 50°C for 24 h. Xylanases released xylobiose, xylotriose and higher oligomers from xylans from different sources.
Xylanase IA had a M r of 38 kDa and contained 7% carbohydrate whereas xylanase IIIA had a M r of 24 kDa and no detectable carbohydrate. The K m for larchwood xylan (mg ml −1) and V max (μmol xylose min −1 mg −1 protein) of xylanase IA were 0.33 and 311, and of xylanase IIIA 1.69 and 500, respectively. Xylanases IA, II and IIIA showed
no synergism in the hydrolysis of larchwood glucuronoxylan or oat spelt and sugarcane bagasse arabinoxylans. They had different
reactivity on untreated and delignified bagasse. The xylanases were more reactive than cellulase on delignified bagasse. Simultaneous
treatment of delignified bagasse by xylanase and cellulase released more sugar than individual enzyme treatments. By contrast,
the primary cell walls of a plant, particularly from the region of elongation, were more susceptible to the action of cellulase
than xylanase. The effects of xylanase and cellulase on plant cell walls were consistent with the view that hemicellulose
surrounds cellulose in plant cell walls. 相似文献
2.
In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance.Fractionation of CSE yielded two fractions: FI and FII, of which only FI exhibited inhibitory activity. Chemical analysis of CSE and its two fractions revealed that CSE contains over 70% of proteins, most of which were found in the non-active fraction. In addition, 3% of amino-sugars were found in the FI active fraction. Lipids were also detected in the unfractionated CSE and in both fractions.Experiments to further characterize the component(s) in the CSE inhibiting the attachment of C. albicans are in progress in our laboratory. 相似文献
3.
The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram-positive bacteria were studied using the anti-sheep red blood cell (SRBC) or anti-trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS-responder), C3H/HeJ (LPS-non-responder), (CBA/N × Balb/c) F1 male with an X-linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA-ability of synthetic peptidoglycan, muramyl dipeptide ( N-acetylmuramyl-L -alanyl-D -isoglutamine, MDP), was also examined, and a similar activity was observed in MDP. 相似文献
4.
The present work reports the results of a study on the isolation and characterization of matrix polysaccharides in the cell walls of galls formed by an aphid ( Neothoracaphis yanonis) on Distylium racemosum leaves. Cell walls were isolated from both healthy Distylium leaf and gall tissues and then extracted sequentially with cyclohexane‐ trans‐1,2‐diaminetetra‐acetate (CDTA), Na 2CO 3, 1 m KOH, and 4 m KOH. The amount of pectin solubilized from gall cell walls was approximately 2.6‐fold higher than the pectin solubilized from leaf cell walls, whereas the amount of hemicellulose solubilized from gall cell walls was 1.4‐fold higher than that from normal leaf cell walls. When the polysaccharides were fractionated by anion‐exchange chromatography, considerable increases in arabinose and galactose were observed in CDTA‐soluble pectic polymer (fraction PI‐1) from gall cell walls, whereas the gall cell walls had less xylose in 1 m KOH‐soluble hemicellulosic polymers (fractions HI‐2, HI‐3, and HI‐4) than did the cell walls from the healthy leaf. The hemicellulosic polymers of the gall cell walls exhibited distinctly different patterns of molecular mass, compared with the healthy leaf cell walls. These results suggest that an extensive change occurs in the matrix polysaccharide structure of the cell walls of Distylium galls formed by an aphid. In addition, many glycosylhydrolase activities were detected in the protein fraction solubilized with strong saline solution from the gall cell walls, and the activities of β‐galactosidase, β‐xylosidase and α‐l ‐arabinofuranosidase were considerably increased under gall formation. 相似文献
5.
Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields. 相似文献
6.
Xylans, the major portion of the hemicellulose of plant cell walls and grasses, are heteropolymers consisting principally of xylose and arabinose. Microbial xylanases with different multiplicities and properties are reported. Most studies on the mode of action of these xylanases have been carried out with fungi and there is very little information available on bacterial xylanases. Fungal xylanases have three or more substrate binding sites: for example Aspergillus niger, Ceratocytis paradoxa, Cryptococcus albidus and Chainia sp. endoxylanases have four to seven subsites with the catalytic site located at the centre of these sub-sites. The analysis of these sub-sites is either by kinetic or end-product analysis studies. Kinetic studies are used for exo-type enzymes while the end-product analysis studies are more convenient for endo-type enzymes. This review covers microbial xylanases with special emphasis on studies of sub-site mapping. The industrial applications of the microbial xylanases are also discussed. 相似文献
7.
Fusarium oxysporum f. sp. albedinis, the bayoud disease agent of date palm, grows on a mineral medium containing the cell walls of date palm roots as a sole carbohydrate source. The growth and development of pathogen under these conditions was related to the production of extracellular cell wall-degrading enzymes (CWDE): cellulases, polygalacturonases, polygalacturonate transeliminases, and pectinmethylesterases. The mycelial growth and the sporulation of pathogen were higher in the presence of cell walls of susceptible cultivars (BFG, JHL, BSK) than in the presence of those of resistant cultivars (IKL, SLY, BSTN). After 8 d of fungal culture, the activity of CWDE was equal whatever is the origin of the cell walls (resistant or susceptible cultivars). After 16 d of culture, the activity of these CWDE was higher when the parasite was cultivated on the cell walls of the susceptible cultivars than on those of the resistant cultivars. A positive correlation was observed between CWDE activities and the growth and the sporulation of F. oxysporum after 16 d of culture. These results clearly show a relation between the susceptibility/resistance of the cell walls of the roots of the date palm to the parasitic CWDE and the susceptibility/resistance of the cultivars. 相似文献
8.
Botrytis cinerea is a phytopathogenic fungus infecting a number of crops (tomatoes, grapes and strawberries), which has been adopted as a model system in molecular phytopathology. B. cinerea uses a wide variety of infection strategies, which are mediated by a set of genes/proteins called pathogenicity/virulence factors. Many of these factors have been described as secreted proteins, and thus the study of this sub‐proteome, the secretome, under changing circumstances can help us to understand the roles of these factors, possibly revealing new loci for the fight against the pathogen. A 2‐DE, MALDI TOF/TOF‐based approach has been developed to establish the proteins secreted to culture media supplemented with different carbon sources and plant‐based elicitors (in this study: glucose, cellulose, starch, pectin and tomato cell walls). Secreted proteins were obtained from the culture media by deoxycholate‐trichloroacetic acid/phenol extraction, and 76 spots were identified, yielding 95 positive hits that correspond to 56 unique proteins, including several known virulence factors ( i.e. pectin methyl esterases, xylanases and proteases). The observed increases in secretion of proteins with established virulence‐related functions indicate that this in vitro‐induction/proteome‐mining approach is a promising strategy for discovering new pathogenicity factors and dissecting infection mechanisms in a discrete fashion. 相似文献
9.
Three-day-cultured cells of Vinca rosea L. (in the cell division phase) and 5-day-cultured cells (in the cell expansion phase) prelabelled with d -[U- 14C] glucose were incubated in a medium containing unlabelled glucose. After various periods of chase, extra-cellular polysaccharides (ECP) and cell walls were isolated, and cell walls were fractionated into pectic substances, hemicellulose, and cellulose fractions. After acid hydrolysis, the radioactive constituents in the pectic substances and hemicellulose fractions were analyzed. Active turnover was observed in arabinose and galactose in the hemicellulose fraction of cell walls, while the constituents of the pectic substances, and xylose and glucose in the hemicellulose fraction did not undergo active turnover. The proportion of radioactivities of arabinose and galactose in total radioactivity of ECP increased markedly after chasing. These results indicate that arabinogalactan was synthesized, deposited in the cell wall, degraded rapidly, and made soluble in the medium as a part of ECP. 相似文献
10.
The cell wall components cellulose, xylan and pectin in different tissues of noninoculated healthy and Fusarium culmorum (W. G. Smith) Sacc-infected wheat spikes were localized by means of enzyme-gold and immuno-gold labelling techniques. The cell walls in the ovary, lemma and rachis of the healthy wheat spike showed labellings in different patterns and densities with cellulase-gold and xylanase-gold probes, as well as with the antipectin monoclonal antibody JIM7. The inter- and intracellular growth of the pathogen in the ovary, lemma and rachis of the infected wheat spike, not only caused pronounced alterations of cell walls and middle lamella matrices, but also led to marked modifications of cell wall components. The enzyme-gold and immuno-gold labellings in the infected host tissues revealed that the labelling densities for cellulose, xylan and pectin were significantly reduced in the cell walls of infected ovary, lemma and rachis as compared with corresponding healthy host tissues. The host cell walls in contact with or close to hyphae of the pathogen showed more marked morphological changes and much greater reduction of the labelling density than those in distance from the hyphae. These results provide evidence that F. culmorum may produce cell-wall-degrading enzymes such as cellulases, xylanases and pectinases during infection and colonization of wheat spikes tissues. Furthermore, at the early stage of infection (e.g. 3 days after inoculation), the degradation of pectin was greater than that of cellulose and xylan in the cell walls of the same infected host tissues, indirectly suggesting that the pectinases may be secreted earlier or exert higher activities than cellulases and xylanases. 相似文献
11.
It was shown that Botrytis cinerea, an isolate infecting apples, secreted in vivo and in culture a variety of glycosidic hydrolases with substrate specificity towards the polysaccharides of cell walls. The following enzymes were partially separated by column chromatography on DEAE-Sepharose CL-6B: two cellulases, three xylanases, one arabinanase, polygalacturonase, β-glucosidase, β-xylosidase, β-galactosidase, β-mannosidase and α-galactosidase. The activity of glycosidic hydrolases tested was strongly inactivated by NBS and weaker by PCMB, tetranitromethan, dibromoacetophenon and Fe 3+, The results indicate synergistic action of the obtained cellulase, xylanase, polygalacturonase and arabinanase in apple cell wall degradation. 相似文献
12.
The growth of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici race 2 (FOL 2) was observed in dual culture with two soil fungi as biocontrol agents, Trametes versicolor and Pleurotus eryngii. In both cases, an interaction zone with the pathogen was found with the Fusarium’s hyphae becoming free of cytoplasmic content. The enzymatic complex of fungi, studied as biocontrol agents, showed β-(1,3)-glucanase
activity, and no other important glucanase activities were noted in all of the media studied. As the principal components
of F. oxysporum cell walls are glucans, the results of the positive attack on the cell walls of FOL 2 by the T. versicolor and P. eryngii enzymatic complex demonstrated the contribution of glucanases in the degradation of the hyphal cell walls of F. oxysporum. The lack of cellulase and xylanase activities (acting on plant cell wall polysaccharides) in T. versicolor makes this species a better alternative for the potential control of diseases caused by Fusarium spp. 相似文献
13.
Summary Sonic oscillation was used for the purpose of obtaining clean, chemically intact cell walls. The rate of disruption was determined
for cells of Hanseniaspora uvarum and Saccharomyces cerevisiae.
The carbohydrate fractions of cell walls of Hanseniaspora uvarum, H. valbyensis, Kloeckera apiculata, Saccharomycodes ludwigii and Saccharmyces cerevisiae were shown to be similar. Chromatography of cell wall hydrolysates of all these species demonstrated that glucose and mannose
were the only sugars present (in about equal amounts) besides traces of glucosamine.
The cell walls of H. uvarum contained 78.1 per cent carbohydrates, 7 per cent protein and approximately 0.05 per cent of chitin. Fractionation of the
polysaccharides lead to a recovery of 83.3 per cent of the carbohydrates present (30.4 per cent glucan and 34.9 per cent mannan).
Saccharomyces cerevisiae cell walls were found to have a carbohydrate content of 82.8 per cent, 6.5 per cent protein and a trace of chitin (0.04 per
cent).
Nadsonia elongata contained a relatively large amount of chitin ( ca. 5 per cent) and lacked mannan in its cell walls.
It was concluded that Hanseniaspora and Saccharomycodes are closely related to the Saccharomyceteae but they have little in common with species of Nadsonia. 相似文献
14.
Isolated wheat ( Triticum aestivum var. Potam) aleurone layers have a high capacity to acidify their environment, and secrete hydrolytic enzymes (endoxylanase, glucanase, α-amylase, proteases, etc.) under the control of GA 3. Acidic pH and xylanases are found to be essential for cell wall relaxation in growing tissues, but aleurone is a non-growing, non-dividing tissue. In this tissue, we studied the effect of these loosening factors on aleurone cell walls.Exposure to pH 3.0 caused the release of carbohydrates and calcium ions from the pericarp, and a small amount of carbohydrates, mainly polysaccharides, from aleurone layers from which pericarp tissue had been removed. 50 percnt; of the total sugars released into the incubation medium by these isolated aleurone tissue was arabinose, but no xylose, calcium ions, or phenolic compounds were found. Acid preincubation decreased by 30 percnt; the susceptibility of aleurone cell walls to degradation by exogenously-applied endoxylanase, and also modified the architecture of cell wall as observed by autofluorescence of phenolic groups. These findings suggest that acid treatment and endoxylanase action, rather than having a loosening effect on aleurone cell wall, can have an opposite effect, increasing the resistance of aleurone cell walls to loosening. 相似文献
15.
Activities of phosphatases were found in the fractions whichwere solubilized from cell walls of both pea and cowpea seedlingswith 0.5 M NaCl. These phosphatases hydrolyzed triphosphonucleotidesin the order: UTP=CTP>GTP>ATP; and UTP=GTP>CTP=ATP,respectively. The activities of a pyrophosphatase and a p-nitrophenylphosphatasewere also detected in these fractions. The suppressor in thespore germination fluid of a pea pathogen, Mycosphaerella pinodes,inhibited all of these phosphatase activities in the fractionsolubilized from pea cell walls, but it rather enhanced onlythe activity of the ATPase among those phosphatases from thecowpea cell wall. Hydrolysis of ATP by a cell wall fractionof pea was also markedly inhibited by the suppressor, whilehydrolysis of ATP by similar fractions from cowpea, kidney beanand soybean were rather enhanced by the suppressor, as wellas by the elicitor. Thus, the cell wall-bound ATPases respondedto the suppressor species-specifically. These cell wall-boundATPases seemed to be different from the plasma membrane ATPasesin several respects. The results suggest that plants recognizethe fungal signals not only on their plasma membranes but alsoon their cell walls and, moreover that putative receptors forthe fungal signals might be located close to cell wall-boundATPases or might even be these ATPases themselves. (Received November 16, 1994; Accepted April 20, 1995) 相似文献
16.
Phytopathogenic fungi devastate agricultural crops worldwide. The biological agents, such as Trichoderma spp., antagonize phytopathogenic fungi by secreting various cell wall-degrading enzymes, for example, endochitinase and β-1,3-glucanase that target glycosidic linkages in β-glucan and chitin polymers of fungal cell walls, thus inhibiting pathogen growth. In this study, two antifungal genes endochitinase and β-1,3-glucanase cloned from local Trichoderma spp. were ligated in pET28a+ expression vector individually to generate two recombinant vectors. The vectors were mobilized into Escherichia coli host strain Rosetta-gami 2 for protein expression, and the 6xHis-tagged recombinant proteins were purified through Ni-NTA affinity chromatography. The purified proteins were individually confronted in vitro with pure cultures of Phytophthora parasitica (destructive pathogen affecting several hundred plant species worldwide) for analyzing their effect on pathogen growth. In vitro confrontation assay revealed P. parasitica growth inhibition by purified β-1,3-glucanase. The pathogen growth inhibition was due to hyphal morphological distortions, such as breakages, swelling, and holes evinced through electron micrography confirming direct role of β-1,3-glucanase in pathogen structural degradation. 相似文献
17.
The external envelope of wheat grain ( Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants ( Kd) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls. 相似文献
18.
Fusariosis, caused by the fungus Fusarium subglutinans f. sp. ananas (Syn. F. guttiforme), is one of the main phytosanitary threats to pineapple ( Ananas comosus var. comosus). Identification of plant cell responses to pathogens is important in understanding the plant–pathogen relationship and establishing
strategies to improve and select resistant cultivars. Studies of the structural properties and phenolic content of cell walls
in resistant (Vitoria) and susceptible (Perola) pineapple cultivars, related to resistance to the fungus, were performed.
The non-chlorophyll base of physiologically mature leaves was inoculated with a conidia suspension. Analyses were performed
post-inoculation by light, atomic force, scanning and transmission electron microscopy, and measurement of cell wall-bound
phenolic compounds. Non-inoculated leaves were used as controls to define the constitutive tissue characteristics. Analyses
indicated that morphological differences, such as cell wall thickness, cicatrization process and lignification, were related
to resistance to the pathogen. Atomic force microscopy indicated a considerable difference in the mechanical properties of
the resistant and susceptible cultivars, with more structural integrity, associated with higher levels of cell wall-bound
phenolics, found in the resistant cultivar. p-Coumaric and ferulic acids were shown to be the major phenolics bound to the cell walls and were found in higher amounts
in the resistant cultivar. Leaves of the resistant cultivar had reduced fungal penetration and a faster and more effective
cicatrization response compared to the susceptible cultivar. 相似文献
19.
Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U- 14C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U- 14C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions. 相似文献
20.
It was shown for Cochlearia anglica roots in comparison with Phaseolus vulgaris, roots that: (a) the cell walls of the halophyte are heavy; (b) they have a high Ca 2+ content; (c) they may be a convenient means of transfer for Na +.In Aster tripolium as well as in Atriplex littoralis NaCl alters the composition of the various pectic fractions and maintains a low pectinemethylesterase activity and a great cellulase activity. This effect may be responsible for the great plasticity of the cell walls which allows an increased cell growth and leads to the formation of succulent organs, therefore to the dilution of salt within the cells. 相似文献
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