Presence Of Cellulose Research Articles

Expression patterns of ten hemicellulase genes of the filamentous fungus Trichoderma reesei on various carbon sources

The filamentous fungus Trichoderma reesei produces a multitude of cellulolytic and hemicellulolytic enzymes. The expression of ten hemicellulase-encoding genes was studied by Northern analyses in various culture conditions including polymeric and di- and monomeric sugars. The study included genes encoding two β-xylanases ( xyn1 and xyn2) and a β-mannanase ( man1) which hydrolyze the main chain of hemicellulose polymers by endo fashion, and the acetyl xylan esterase ( axe1), the αr-glucuronidase ( glr1), the α- l-arabinofuranosidase ( abf1), and three α-galactosidases ( agi1, agl2 and agl3), which attack the side groups of hemicellulosic substrates. The genes encoding the oligosaccharide-hydrolysing enzymes β-glucosidase ( bgl1) and β-xylosidase ( bxl1) were also studied and as a reference the cellulase encoding gene cbh1. A general picture of their relative expression patterns and relative expression levels in particular conditions was obtained. Growth in the presence of cellulose, sophorose, xylobiose, xylan and l-arabitol gave rise to abundant expression of most genes studied, α-galactosidase I and II, β-xylosidase and α- l-arabinofuranosidase encoding genes were the ones most frequently expressed in the conditions studied, whereas expression of the mannanase and α-galactosidase III was rarely seen. Expression of the genes was not detected in the presence of glucose in the strain QM9414 but derepressed expression of most of them occurred after glucose depletion. Expression of at least xyn1, bxl1, agi1, agl2, agl3 and axe1 is under control of the glucose represser cre 1 as studied in the cre1-l mutant Rut-C30 and Rut-C30 transformed with the wild type cre 1 allele.

Cellular and Molecular Mechanisms Involved in the Interaction between Trichoderma harzianum and Pythium ultimum

The interaction between Trichoderma harzianum and the soilborne plant pathogen Pythium ultimum was studied by electron microscopy and further investigated by gold cytochemistry. Early contact between the two fungi was accompanied by the abnormal deposition of a cellulose-enriched material at sites of potential antagonist penetration. The antagonist displayed the ability to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, the presence of cellulose in the walls of severely damaged Pythium hyphae indicated that cellulolytic enzymes were not the only critical traits involved in the antagonistic process. The marked alteration of the (beta)-1,3-glucan component of the Pythium cell wall suggested that (beta)-1,3-glucanases played a key role in the process.

Cellulase Induction in Trichoderma reesei by Cellulose Requires Its Own Basal Expression

The induction of cellulases by cellulose, an insoluble polymer, in the filamentous fungus Trichoderma reesei is puzzling. We previously proposed a mechanism that is based on the presence of low levels of cellulase in the uninduced fungus; this basal cellulase activity would digest cellulose-releasing oligosaccharides that could enter the cell and trigger expression of cellulases. We now present experiments that lend further support to this model. We show here that transcripts of two members of the cellulase system, cbh1 and egl1, are present in uninduced T. reesei cells. These transcripts are induced at least 1100-fold in the presence of cellulose. We also show that a construct containing the hygromycin B resistance-encoding gene driven by the cbh1 promoter confers hygromycin B resistance to T. reesei cells grown in the absence of cellulose. Moreover, cellulose-induced production of the cbh1 transcript was suppressed when antisense RNA against three members of the cellulase system was expressed in vivo. Experiments are presented indicating that extracellular cellulase activity is the rate-limiting event in induction of synthesis of the cellulase transcripts by cellulose. The results reveal a critical requirement for basal expression of the cellulase system for induction of synthesis of its own transcripts by cellulose.

Comparative kinetics of the induced radical autocondensation of polyflavonoid tannins. III. Micellar reactions vs. cellulose surface catalysis

Cellulose surface catalysis of the induced autocondensation of tannins has been found to occur also for the radical mechanism of the reaction. Equally effective in accelerating pyran ring opening of polyflavonoids by radical mechanisms appears to be the presence of any substance leading to micellar structures in the tannin solution. Thus, soaps and synthetic and natural polymeric colloids have been found to accelerate the reaction. The micellar effect is distinct and different from the surface catalysis of the process induced by cellulose. Amorphous and crystalline cellulose appear to differ somewhat as regards the extent of catalytic activation. As for ionic mechanisms, also for catalyzed radical mechanisms, pro-cyanidins invert their favorite reaction of cleavage of the interflavonoid bond to favor, instead, pyran ring opening. This inversion of the favorite reaction is induced both by the presence of cellulose and by the level of the micellar state of the reaction. © 1996 John Wiley & Sons, Inc.

Comparative kinetics of the induced radical autocondensation of polyflavonoid tannins. III. Micellar reactions vs. cellulose surface catalysis

Cellulose surface catalysis of the induced autocondensation of tannins has been found to occur also for the radical mechanism of the reaction. Equally effective in accelerating pyran ring opening of polyflavonoids by radical mechanisms appears to be the presence of any substance leading to micellar structures in the tannin solution. Thus, soaps and synthetic and natural polymeric colloids have been found to accelerate the reaction. The micellar effect is distinct and different from the surface catalysis of the process induced by cellulose. Amorphous and crystalline cellulose appear to differ somewhat as regards the extent of catalytic activation. As for ionic mechanisms, also for catalyzed radical mechanisms, pro-cyanidins invert their favorite reaction of cleavage of the interflavonoid bond to favor, instead, pyran ring opening. This inversion of the favorite reaction is induced both by the presence of cellulose and by the level of the micellar state of the reaction. © 1996 John Wiley & Sons, Inc.

The cholesterol-lowering effect of oat bran cereals in rats: Influence of processing

In order to study the cholesterol-lowering properties of oat bran-based ready-to-eat cereal (RTE), rats (12/ group) were fed the following diets: basal semipurified (B), B plus 1% cholesterol (BC), BC plus 10% cellulose (fiber control), BC + 67% of an extruded RTE cereal; BC + 67% of a conventionally cooked RTE cereal; and BC + 67% of the raw ingredients of the latter. All the RTE cereal products were oat bran-based, and 1% cholesterol was included in the dietary formulations. At the end of a 3-week feeding period, serum total cholesterol was significantly increased from 2.02 ± 0.13 mmol/L to 3.57 ± 0.18 mmol/L due to cholesterol feeding alone. The presence of either cellulose or oat bran products in the diet negated the hypercholesterolemia. Inclusion of 1% cholesterol in the diet raised liver cholesterol almost 10 fold. All three of the oat bran-based products significantly inhibited the increase but cellulose was ineffective. Liver cholesterol content was due primarily to the accumulation of cholesteryl esters. Liver triglyceride was also increased by dietary cholesterol and the presence of cellulose or oat bran in the diet appeared to enhance this effect. The method of processing the RTE appears to affect the efficacy of oat bran.

The interaction between polyelectrolyte complexes made from poly(dimethyldiallylammonium chloride) (PDMDAAC) and poly(maleic acid-co-α-methylstyrene) (P(MS-α-MeSty)) and cellulosic materials

The interaction between the oppositely charged polyelectrolytes poly(dimethyldiallylammonium chloride) (PDMDAAC) and poly(maleic acid-co-α-methylstyrene) (P(MS-α-MeSty)) in the presence of cellulose or a mixture of cellulose and clay, respectively, has been investigated. The application of different methods allowed the characterization of flocculation and surface modification. It has been found for step-by-step addition that the flocculation behaviour of this two-component system (dual system) depends on the molar ratio of the two polymers used and also on the concentration of polycation. Only at a certain concentration of PDMDAAC does the two component system lead to a considerable improvement in the flocculation behaviour and in surface modification, as compared to a pure polycation. The addition of a premixed polyelectrolyte complex has also been found to be a successful method. However, the mechanism of flocculation is supposed to be different for complexes with different molar ratios.

Synthesis and characterization of cellulose/N‐methylolacrylamide/glycidyl methacrylate/methacrylic acid cation exchange resins

AbstractNew cation exchange resins based on cellulose/N‐methylolacrylamide (NMA)/glycidyl methacrylate (GMA)/methacrylic acid (MA) were prepared by polymerizing highly concentrated mixtures of NMA/GMA/MA in the presence of cellulose using a sodium persulfate/sodium sulfite redox system under different conditions including the concentrations of the redox system components, NMA and GMA as well as the polymerization temperature. Polymerization conditions were selected to synthesize three resins of different carboxy and nitrogen contents. Potentiometric titration showed that these resins were weakly basic cation exchangers of different strength. Water solubility and swellability as well as durability of these resins were examined.

Preparation and characterization of cellulose/glycidyl methacrylate/acrylic acid cation exchange composites

AbstractAcrylic acid (AA)/glycidyl methacrylate (GMA) mixtures were bulk polymerized in the presence of cellulose in order to prepare cellulose/GMA/AA cation exchange composites using sodium persulfate as an oxidant, alone or in conjunction with sodium sulfite, sodium thiosulfate, sodium metabisulfite or glucose as a reductant. The presence of the reductant has the effect of shortening the polymerization time, expressed as the solidification time (ST), with little improvement in the polymerization extent, expressed as percentage total conversion (%TC), as well as the percentage yield and the carboxyl content of the crude and the purified composites. Sodium sulfite was selected as the the reductant that can achieve best polymerization. The effect of the concentrations of “sodium persulfate/sodium sulfite, redox system, acrylic acid and glycidyl methacrylate” and temperature on the formation of the composite were investigated. Four cation exchange composites were prepared and characterized by investigating their potentiometric titration, water solubility and swellability as well as durability.

Transcriptional analysis of the Clostridium cellulovorans endoglucanase gene, engB.

An endoglucanase gene, which was shown to be identical to the previously sequenced engB gene [Attwood et al. (1993) Abstr. Ann. Meet. Am. Soc. Microbiol.], was isolated from a Clostridium cellulovorans genomic library. Because of the lack of transcriptional information concerning engB we examined its expression in C. cellulovorans and in the heterologous hosts Escherichia coli and C. acetobutylicum following transformation of engB. Northern analysis suggested that both E. coli and C. acetobutylicum produced several transcripts of various sizes. C. cellulovarans produced a single transcript of 1600 bp with the relative amount of engB mRNA from cellulose-grown cells being much greater than that from cellobiose-grown cells. Primer extensions showed that engB was transcribed from a single transcription initiation site in C. cellulovorans preceded by sequences similar to promoter sequences found in Gram-positive bacteria. Primer extensions from both E. coli and C. acetobutylicum strains containing the engB gene showed multiple transcription initiation sites, none of which corresponded to the site determined in C. cellulovorans. We conclude that transcriptional control of the engB gene is less stringent in heterologous backgrounds and postulate that expression of the engB gene in C. cellulovorans is increased in the presence of cellulose.

Solid-state NMR study of leaf cell walls of oil palm

Lignified and non-lignified cell walls from leaf laminae of oil palm Elaeis guineensis were separated by controlled homogenization and wet sieving. Their solid-state 13C NMR spectra were obtained at 75 MHz using cross-polarization and magic-angle spinning. The spectrum of the non-lignified cell walls was consistent with the presence of cellulose and xylans with little pectin, as in the Gramineae. The lignified cell walls appeared to contain a high-syringyl lignin, but both they and the non-lignified cell wall preparation contained substantial quantities of a cutin-like polymer accounting for at least some of the phenolic residues present.

The biological degradation of cellulose

Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the β-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.

The biological degradation of cellulose.

Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the beta-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.

Characterization of the Trichoderma reesei cbh2 promoter

A 613-bp fragment of the 5' upstream region of the Trichoderma reesei cbh2 gene (coding for the cellulolytic enzyme cellobiohydrolase II) has been isolated and sequenced. Fusion of this fragment to the E. coli uidA gene (coding for beta-glucuronidase) leads to--albeit low--expression of beta-glucuronidase activity in the presence of cellulose and upon the addition of low molecular weight inducers (sophorose, lactose) of cellobiohydrolase II. It also governed the formation of beta-glucuronidase activity during sporulation and its transport to the conidial surface. However, despite the presence of a signal peptide in the cbh2:uidA fusion, beta-glucuronidase was not secreted in T. reesei. Defined fragments of the 613-bp promoter region were isolated and used to identify areas involved in the regulation of cbh2 expression by protein-DNA binding assays. At least two binding areas--between -443/-363 and -363/-173, respectively--were identified. In both areas, the DNA-protein complex observed was appreciably larger when cell-free extracts from sophorose-induced mycelia were used. This suggests that at least one of the proteins regulating cbh2 transcription is itself induced by cellulose.

Observations of Gonapodya in Pure Culture: Growth, Development and Cell Wall Characterization

Gonapodya prolifera was isolated from a pond in Athens, Georgia, and its development in pure culture is reported for the first time. While the fungus grew well on a diversity of media, the morphology was closest to published descriptions when the fungus was grown on dogwood fruits with twigs held under reduced oxygen conditions. The distributions of chitin, cellulose, glycogen and N-acetyl-D-galactosamine residues in the cytoplasm, cell walls and pseudosepta of freeze substituted hyphae were studied with gold-complexed enzyme and lectin probes. The presence of cellulose in the cell walls of this member of the Chytridiomycetes was substantiated by compositional and methylation analyses of a cell wall extraction that was selective for cellulose. The observation that Helix pomatia agglutinin binds to glycogen in thin section is discussed, as is the first reported detection of cellulose in a member of the Chytridiomycetes.

Observations of Gonapodya in Pure Culture: Growth, Development and Cell Wall Characterization

Gonapodya prolifera was isolated from a pond in Athens, Georgia, and its development in pure culture is reported for the first time. While the fungus grew well on a diversity of media, the morphology was closest to published descriptions when the fungus was grown on dogwood fruits with twigs held under reduced oxygen conditions. The distributions of chitin, cellulose, glycogen and N-acetyl-D-galactosamine residues in the cytoplasm, cell walls and pseudosepta of freeze substituted hyphae were studied with gold-complexed enzyme and lectin probes. The presence of cellulose in the cell walls of this member of the Chytridiomycetes was substantiated by compositional and methylation analyses of a cell wall extraction that was selective for cellulose. The observation that Helix pomatia agglutinin binds to glycogen in thin section is discussed, as is the first reported detection of cellulose in a member of the Chytridiomycetes.

Regulatory aspects of endoglucanase production by the brown-rot fungus Gloeophyllum trabeum

Regulation of endoglucanase formation by the brown-rot fungus Gloeophyllum trabeum was investigated. This fungus produced endoglucanases in the presence of monosaccharides such as glucose or mannose as the sole carbon source, but the expression of these enzymes was four to five times higher in the presence of cellulose or cellobiose. In a lactose- or glucose-containing medium, endoglucanase production was induced by cellobiose. Glucose and glycerol did not repress enzyme production. We concluded that endoglucanase production by brown-rot fungi is inducible by cellulose and not subject to catabolite repression. Cellobiose is the most effective inducer of the system.

Endo-Cellulase of the Spruce Callus Culture (Picea abies (L.) Karst)

Summary Spruce callus culture ( Picea abies (L.) Karst) was cultivated on the hydrophilic surface of a gel prepared from crosslinked hydroxyethylcellulose as the only source of carbon in the growth medium. The gel became liquefied during the course of callus growth due to splitting of the β(1→4) linkages of the modified cellulose. Cellobiose and higher oligosaccharides as splitting products of cellulose were determined. Enzyme activity of the cellulase type was isolated from the culture medium; increased enzyme activity was found in the presence of cellulose, as well as its derivatives.

A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities.

A plant polysaccharide hydrolase cDNA, designated celD, was isolated from a cDNA library of the rumen fungus Neocallimastix patriciarum. The enzyme encoded by celD had endoglucanase, cellobiohydrolase and xylanase activities. Deletion analysis revealed that celD cDNA can be truncated to code for three catalytically active domains. Each domain had the same substrate specificity as the enzyme produced by the untruncated celD and also possessed cellulose-binding capacity. Substrate competition studies showed that carboxymethylcellulose and xylan appear to compete with methylumbelliferyl cellobioside for the same active site within each domain. Expression of celD transcript in the rumen fungus was constitutive and was not affected by the presence of cellulose in the culture medium.

Structure, organization, and transcription of a cellobiohydrolase gene cluster from Phanerochaete chrysosporium.

Restriction mapping and sequence analysis of cosmid clones revealed a cluster of three cellobiohydrolase genes in Phanerochaete chrysosporium. P. chrysosporium cbh1-1 and cbh1-2 are separated by only 750 bp and are located approximately 14 kb upstream from a cellulase gene previously cloned from P. chrysosporium (P. Sims, C. James, and P. Broda, Gene 74:411-422, 1988). Within a well-conserved region, the deduced amino acid sequences of P. chrysosporium cbh1-1 and cbh1-2 are, respectively, 80 and 69% homologous to that of the Trichoderma reesei cellobiohydrolase I gene. The conserved cellulose-binding domain typical of microbial cellulases is absent from cbh1-1. Transcript levels of the three P. chrysosporium genes varied substantially, depending on culture conditions. cbh1-1 and cbh1-2 were not induced in the presence of cellulose, nor did they appear to be subject to glucose repression. Therefore, aspects of the chromosomal organization, structure, and transcription of these genes are unlike those of any previously described cellulase genes.