Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1.

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A homologue of EGL1 encoding endo-1,4-beta-glucanase in elongating pea stems

A gene (EGL2) encoding an endo-1,4-beta-glucanase in peas has been cloned as a homologue of EGL1. EGL2 encodes a polypeptide of 506 amino acids, including a 24-mer putative signal polypeptide. The gene product contains a domain conserved in endo-1,4-beta-glucanase (family 9) showing 60% amino acid identity to EGL1. EGL2 mRNA was accumulated only in the elongating regions of pea stems, although EGL1 mRNA was abundant in both elongating and non-elongating tissues. However, the level of EGL2 mRNA was not increased by the treatment with sucrose and auxin in pea segments. These results suggest that the expression of EGL2 either requires the presence of other factors related to the auxin effect or occurs independent of auxin in the elongating pea stems.     

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium

Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, 1,4-beta-glucosidase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase and 1,4-beta-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-beta-glucanase, 1,4-beta-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-beta-mannosidase in P. ostreatus.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cullulose. Functional characterization of five endo-1,4-beta-glucanases and one exo-1,4-beta-glucanase.

A strong synergistic response was observed between the five endo-1,4-beta-glucanases and the exo-1,4-beta-glucanase obtained from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum), when these enzymes were allowed to degrade de-waxed cotton and Avicel. No synergism was observed if Walseth cellulose, an acid-swollen cullulose, was used. If de-waxed cotton was pretreated with endo-1,4-beta-glucanases, the exo-1,4-beta-glucanase enzyme released much more degradation products than from an untreated cotton...     

Towards the mechanism of cellulose synthesis

Recent research has provided insights into how plants make cellulose - the major structural material of their cell walls and the basis of the cotton and wood fibre industries. Arabidopsis thaliana mutants impaired in cellulose production are defective in genes encoding membrane-bound glycosyltransferases, an endo-1,4-beta-glucanase and several enzymes involved in the N-glycosylation and quality-control pathways of the endoplasmic reticulum. The glycosyltransferases form the rosette terminal complexes seen in plasma membranes making cellulose. Synthesis might start by making lipoglucans, which, in turn, might form the substrate for the endo-1,4-beta-glucanase, before being elongated to form the long, crystalline microfibrils that assemble in the cell wall.     

A Sensitive Method Using 4-Methylumbelliferyl-beta-Cellobiose as a Substrate To Measure (1,4)-beta-Glucanase Activity in Sediments

A sensitive method to measure (1,4)-beta-glucanase activity in organic matter-rich sediments, using 4-methyl-umbelliferyl-beta-cellobiose as a substrate, is described. beta-Glucosidases, which were also able to hydrolyze this substrate, were inhibited with d-glucono-delta-lactone. The produced 4-methylumbelliferone was recovered quantitatively out of the sediment by an extraction with 80% ethanol. An inhibition experiment with known substrates or inhibitors suggested that at least 59% of the measured activity could be explained by enzymes of the exo-(1,4)-beta-glucanase type and that the contribution of endo-(1,4)-beta-glucanases was minor. Results of the inhibition experiment also suggested that the measured activity was of bacterial origin in the sediment used. First results of field measurements are given for the sediment from the reed bed of Lake Gooimeer.     

Formation and Location of 1,4-beta-Glucanases and 1,4-beta-Glucosidases from Penicillium janthinellum

Formation and location of 1,4-beta-glucanases and 1,4-beta-glucosidases were studied in cultures of Penicillium janthinellum grown on Avicel, sodium carboxymethyl cellulose, cellobiose, glucose, mannose, and maltose. Endo-1,4-beta-glucanases were found to be cell free, and their formation was induced by cellobiose. 1,4-beta-Glucosidases, on the other hand, were formed constitutively and were primarily cell free, but with a small amount strongly associated with the cell wall. Low 1,4-beta-glucosidase activities of periplasmic or intracellular origin were also found. A rotational viscosimetric method was developed to measure the total endo-1,4-beta-glucanase activity of the culture (broth and solids). By this method, it was possible to determine the endo-1,4-beta-glucanase activity not only in the supernatant of the culture but also on the surface of the mycelium or absorbed on residual Avicel. During a 70-liter batch cultivation of P. janthinellum, the adsorption of endo-1,4-beta-glucanases by residual and newly added 10% Avicel was measured. The adsorption of soluble protein and endo-1,4-beta-glucanases by Avicel was found to be largely independent of the pH value but dependent on temperature.     

Altered pectin composition in primary cell walls of korrigan, a dwarf mutant of Arabidopsis deficient in a membrane-bound endo-1,4-β-glucanase

Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.     

Role of endo-1,4-beta-glucanases from neisseria sicca SB in synergistic degradation of cellulose acetate

An enzyme hydrolyzing beta-1,4 bonds in cellulose acetate was purified 10.5-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which assimilate cellulose acetate as the sole carbon and energy source. The enzyme was an endo-1,4-beta-glucanase, to judge from the substrate specificity and hydrolysis products of cellooligosaccharides, we named it endo-1,4-beta-glucanase I (EG I). Its molecular mass was 50 kDa, 9 kDa larger than EG II from this strain, and its isoelectric point was 5.0. Results of N-terminal and inner-peptide sequences of both enzymes, and a similarity search, suggested that EG I contained a carbohydrate-binding module at the N-terminus and that EG II lacked this module. The pH and temperature optima of EG I were 5.0-6.0 and 45 degrees C. It hydrolyzed water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for these compounds were 0.296% and 1.29 micromol min(-1) mg(-1), and 0.448% and 13.6 micromol min(-1) mg(-1), respectively. Both glucanases and cellulose acetate esterase from this strain degraded water-insoluble cellulose acetate synergistically.     

Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56 kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1,4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012 bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194 bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.     

Involvement of a conidial endoglucanase and a plasma-membrane-bound beta-glucosidase in the induction of endoglucanase synthesis by cellulose in Trichoderma reesei

The induction of endo-1,4-beta-glucanase synthesis by Trichoderma reesei QM 9414 was investigated in conidia, mycelia and protoplasts. Cellulose induced endoglucanase synthesis only in conidia, but not in glucose-grown mycelia or protoplasts. Cellooligosaccharides and sophorose induced endoglucanase synthesis in mycelia, conidia and protoplasts. Only conidia exhibited detectable basal endoglucanase levels, whereas beta-glucosidase activity was found in conidia, mycelia and protoplasts. The beta-glucosidase was inhibited in vitro by nojirimycin and glucono-delta-lactone. Addition of either of these inhibitors to the induction medium blocked de noro synthesis of endo-1,4-beta-glucanase with cellulose (conidia) or cellooligosaccharides (protoplasts and mycelia) as inducer, whereas induction by sophorose remained unaffected. The results are consistent with the assumption that basal constitutive levels of endoglucanase and beta-glucosidase are involved in the induction of cellulase synthesis by cellulose in T. reesei.     

Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda

The formation and location of endo-1,4-beta-glucanases and beta-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-beta-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-beta-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. beta-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. beta-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and beta-glucosidases and the end products of cellulose degradation were determined.     

Influence of mineral and organic fertilization on soil fungi,enzyme activities and humic substances in a long-term field experiment

Changes in microfungal communities, fungal activities and humic substances (HS) in agricultural soils kept under different fertilization regimes were observed and their causal relationships were investigated in a long-term field experiment. Fertilization did not change the abundance of HS-utilizing microfungi and, except for organic amendment alone, total culturable microfungi were also unaffected by this factor. Organic fertilization increased activities of manganese peroxidase (MnP) and proteinase, but decreased endo-1,4-beta-glucanase activity compared to the corresponding control without organic fertilization. In soils treated with mineral fertilizers, the activities of MnP, endo-1,4-beta-glucanase and proteinase were higher than in control without any mineral treatment. Both the aromaticity of fulvic acid and the molar mass of humic acid was lower in soil with organic fertilization, which may be a result of oxidative degradation mediated by higher MnP activity observed in treatments with organic fertilization.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 1. Separation, purification and physico-chemical characterization of five endo-1,4-beta-glucanases.

Five endo-1,4-beta-glucanases (EC 3.2.1.4) have been separated from culture solutions OF THE ROT FUNGUS Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source. They have been extensively purified and characterized with regard to some physicochemical properties. The purifications have been carried out on a quantitative basis, the purity of the enzymes being tested in several ways. After purification they all showed one single protein band in analytical polyacrylamide electrophoresis, on dodecyl-sulphate gels and in analytical isoelectric focusing on flat-bed polyacrylamide gels. One exo-1,4-beta-glucanase has also been identified in the culture solution and separated from the endo-1,4-beta-glucanases. From the data obtained during the quantitative purification it has been possible to calculate that the ratio of activity between the five endoglucanases T1, T2a, T2b, T3a, and T3b in the culture solutions is 4:1:1:1:1. It has also been calculated that the weight ratio endoglucanase protein to exoglucanase protein is approximately 1:1. Flat-bed isoelectric focusing has been used for the identification of the individual endoglucanases and a new zymogram technique, useful for studies of carbohydrases in general, has been developed. The molecular weights, determined by ultracentrifugation, and calculated on the basis of a knowledge of the amino acid composition and carbohydrate content vary between 28200 and 37500. Small but significant differences in the amino acid compositions of the different endoglucanases have been found. The carbohydrate content varies between 0 and 10.5%, all but one of the enzymes being glycoproteins. For two of these the exact carbohydrate composition has been determined. Enzyme T1 contains 2 glucose and 19 mannose units per enzyme molecule while enzyme T2b contains 5 mannose, 7 galactose, 1 glucose and 1 arabinose unit per molecule.     

Cloning and expression of an endo-1,3-1,4-beta-glucanase gene from Bacillus macerans in Lactobacillus reuteri.

Strains of the gastrointestinal species Lactobacillus reuteri were electrotransformed with plasmid constructs containing the endo-1,3-1,4-beta-glucanase gene (bglM) of Bacillus macerans. The enzyme was expressed and secreted by the lactobacilli. A plasmid construct containing the bglM gene lacking its promoter was derived and was demonstrated to be useful as a promoter probe vector.     

Evidence that endo-1,4-beta-glucanases act on cellulose in suspension-cultured poplar cells

Suspension-cultured poplar (Populus alba) cells produce two distinct endo-1,4-beta-glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wall-bound endo-1, 4-beta-glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo-1,4-beta-glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello-oligosaccharides. Cello-oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello-oligosaccharides by the extracellular enzyme by 90% and that by the wall-bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo-1,4-beta-glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension-cultured poplar cells.