The formation of short fibres from native cellulose by components of Trichoderma koningii cellulase

Cellulolytic enzyme components of culture filtrates of Trichoderma koningii were fractionated on ionic and non-ionic forms of Sephadex and on cellulose powder (Whatman) and examined for their ability to hydrolyse soluble carboxymethyl-cellulose, and to saccharify, solubilize and form short fibres from native undegraded cellulose of the type found in cotton. DEAE-Sephadex provided two CM-cellulase components and a C(1) component; the C(1) component acted weakly and solely on cotton, forming soluble products but not short fibres. The ability to form short fibres was confined almost wholly to one of the CM-cellulase components which completely degraded cotton, minimally to soluble products and extensively to short fibres. The latter action was unaffected by the presence of the other two components. The two CM-cellulase components solubilized cellulose synergistically whereas the short-fibre-forming component and C(1) component were inhibitory.     

Cellulase production with Penicillium iriense (n. sp.)

Summary A new cellulase producing species of penicillium, named Penicillium iriense, has been isolated. Cultures of this fungus in liquid media containing cellulose as carbon source. excrete into the medium an enzyme complex able to degrade both soluble and insoluble forms of cellulose. This complex has been separated into five protein fractions. Three of them are endowed with CM-cellulase activity, one contains a cellobiase and one contains a C₁-like factor. These fractions show a moderate synergism in the attack of cotton fibres.     

Comparison of cell-walls of Lolium multiflorum with cotton cellulose in relation to their digestion with enzymes associated with cellulolysis

Activities of several enzymes associated with cellulolysis were compared using as substrates cell-walls of Lolium multiflorum and cotton cellulose. Purified enzymes C₁ (see Ref. 1 for definition), C._x (CM-cellulase) and β-glucosidase were employed as well as culture filtrates containing C_x. Activities were determined by ability to digest the substrates and to release H₂O-soluble phenolic compounds from the grass cell-walls. The culture filtrates most active on cotton cellulose were obtained using the fungi Trichoderma viride and Fusarium solani; with grass cell-walls the most active were from T. viride, Gliocladium roseum, a species of Basidiomycetes, and one strain of Myrothecium verrucaria (IMI Strain 25 291). For the crude enzyme preparations tested, there were highly significant correlations between the digestibility of grass cell-walls and the UV-absorption of the filtrate at λ_max 290 nm and at λ_max 324 nm but there was no significant correlation between the digestibility of grass cell-walls and that of cotton cellulose. Partially purified C₁ and C_x from two different fungal sources showed activity on both substrates. Differences in MW of the H₂O-soluble phenolic compounds obtained by treatment of grass cell-walls with C₁ and C_x components suggest that these enzymes could have different modes of action. Synergism between C₁ and C_x from T. koningii occurred with both substrates but with C₁ and C_x from F. solani synergism only occurred with cotton cellulose.     

Characterization and purification of hydrolytic enzymes in Sinorhizobium fredii CCRC15769

Rhizobial symbiosis provides the nitrogen for the leguminous plant through fixation of the gaseous nitrogen component of air. For the bacteria in the plant root-hair wall, carboxymethylcellulase (CM-cellulase, EC 3.2.1.4) may be the key enzyme in this symbiotic process, with polygalacturonase (pectinase, EC 3.2.1.15) another critical enzyme involved early in the mechanism of nitrogen supply. The precise cytosolic location, function and expression of CM-cellulase are still uncertain, however. To detect the relevant enzyme activity in Sinorhizobium fredii CCRC15769, various assay methods were used including double-layer plate assay and quantitation of reducing sugar products. After sonication of the cell pellet, ammonium sulphate precipitation, gel filtration, and ion-exchange chromatography are the preferred methods for derivation of the purified protein, with CM-cellulase characterized as follows: purification ratio, 33.35; recovery, 10.8%; and specific activity, 0.053 U mg^–1. The endoglucanase in the purified samples was resolved using native and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis; it was then assayed with an ultrathin CM-cellulose overlay stained with Congo Red. Two CM-cellulase isozymes were determined by native activity stain assay, with gel-filtration revealing molecular weights of approximately 196 and 30 kD; the SDS-PAGE activity gel resolved four enzyme subunits of 94, 67, 37, and 30 kD. It is suggested that the CM-cellulase in S.fredii CCRC15769 is a two-isozyme form, one a trimer of 196 kD (94, 67 and 37 kD), and the other a 30 kD monomer.     

Cellulolytic enzyme system of Trichoderma koningii. Separation of components attacking native cotton

1. Cell-free culture filtrates from Trichoderma koningii were concentrated by precipitation with ammonium sulphate between the limits of 20% and 80% saturation. 2. Removal of a low-molecular-weight carboxymethylcellulase (CM-cellulase) component by chromatography on Sephadex G-75 had no effect on the ability of the enzyme complex to solubilize cotton. 3. Further chromatography on DEAE-Sephadex separated a component (C(1)) from the C(x) (CM-cellulase) and beta-glucosidase activities. Separately these components had little ability to produce soluble sugars from cotton, but when recombined in their original proportions this capacity was almost completely recovered. 4. The C(x) component was further fractionated on SE-Sephadex into a fraction containing only CM-cellulase and a fraction showing CM-cellulase and beta-glucosidase activities: the latter two components could be separated by heat treatment. 5. The C(1) component had no swelling factor (S-factor) activity (Marsh, Merola & Simpson, 1953; Reese & Gilligan, 1954) on its own, but it had a synergistic effect on the S-factor activity associated with the CM-cellulase and beta-glucosidase components.     

The purification and properties of microsomal palmitoyl-coenzyme A synthetase

1. Cell-free culture filtrates of the fungus Fusarium solani were examined for homogeneity with respect to beta-d-glucosidase and C(x) activities. 2. o-Nitrophenyl beta-d-glucoside and cellobiose were both used as substrates for beta-d-glucosidase activity. 3. No evidence for the non-identity of nitrophenyl beta-d-glucosidase and cellobiase activities could be found, either by heat treatment, gel filtration on Sephadex G-100 or by isoelectric focusing. 4. The beta-d-glucosidase component was also a feeble exo-beta-glucanase: it had a molecular weight of approx. 400000. 5. The fall in viscosity of a solution of CM-cellulose, the formation of reducing sugars in a solution of CM-cellulose and the solubilization of phosphoric acid-swollen cellulose (Walseth cellulose), were all used for the measurement of C(x) activity. 6. The ratio of the two types of CM-cellulase activity was not changed after gel filtration on Sephadex G-100 or after chromatography on DEAE-Sephadex. 7. Three peaks of C(x) activity were obtained after electrofocusing, but all three possessed the same ratio of the two types of CM-cellulase activity as well as the same CM-cellulase/Walseth activity ratio, as the unfractionated enzyme; all three isoenzymes (isoelectric points, 4.75, 4.80-4.85 and 5.15) acted in synergism with a mixture of the C(1) and the beta-d-glucosidase components to the same extent in the solubilization of cotton fibre. 8. The molecular weight of the C(x) component was approx. 37000.     

Cellulolytic Enzyme System in Culture Filtrates of Aspergillus sydowi

During growth in liquid culture medium, that contained single soluble or insoluble cellulosic carbon source, Aspergillus sydowi (Bain. & Start.) Thom & Church released cellulolytic enzymes into the medium. The enzymes were separated by gel filtration followed by ion exchange chromatography into three components, all of high molecular weight. One of the components (Ac) has the character of a C₁ cellulase enzyme. In the assay for hydrolysis of insoluble cellulose, the combined fractions, especially whenever the fraction under test contained the component Ac, released more glucose than when each component was employed alone.     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

Synergism of isoenzymes of cellobiohydrolase I and II of trichoderma reesei in hydrolysis of amorphous cellulose

Decompositions of amorphous cellulose induced by cellulases of Trichoderma reesei were evaluated from gradients at zero time of exponential functions which were fitted to nephelometrically measured values of turbidty of incubated solutions of cellulose [turbidity = A × exp (B × t)+ C [A, B, C = constants, t = time]]. Synergistic enhancements of decomposition of amorphous cellulose resulted in the range of 300 p.c. whenever of the two isoenzymes of cellobiohydrolase I of Trichoderma reesei (CBH I, being an exo-glucanase) one was incubated together with one of the isoenzymes of CBH II (being really an endo-glucanase). Accessibility of amorphous cellulose to enzymatic decomposition being calculated from the fitted function by the term (A/(A + C)) × 100 [p.c.] resulted for the CBH I isoenzymes and for the CBH II/1 in the range of 27 to 38 p.c. of the total substrate. Incubations of CBH II/1 in with CBH I/1 and CBH I/2 were followed by increases of accessibility to 85 and 87 p.c., respectively. CBH II/2 by itself caused a substrate accessibility in the range of 80 p.c., which increased to 96 p.c. when it was incubated together with CBH I/1 or CBH I/2. Amorphous cellulose dispersing activity (ACD activity) being evaluated from the fitted function by the term (A + C)/(A_c + C_c) × 100 [p.c.] (A_c + C_c × control turbidity at zero time) was not increased when a CBH I isoenzyme was incubated together with a CBH II isoenzyme. EG I, a convetional endo-glucanase from Tr. reesei proved not to act synergistically in any case when incubated together with one of the CBH isoenzymes. On the contrary, EG I turned out to act antagonistically to CBH II/1 and CBH II/2. Results can be interpreted as an exo-endo-synergism taking place between C₁-specific exo- and endo-glucanases.     

Shifting dominance from native C4 to non‐native C3 grasses: relationships to community diversity

Many field studies have examined how site fertility, soil differences and site history influence the diversity of a plant community. However, only a few studies have examined how the identity of the dominant species influences the diversity in grasslands. Plant species differ widely in phenology, growth form and resource uses; thus, communities dominated by different species are also likely to strongly differ in the environment that they create and in which the subdominant species exist. We examined the correlation between the four most dominant species and community diversity in 2100 plots, located in 21 abandoned agricultural fields in central Minnesota over a 23‐year period. The four most common species were two non‐native C₃ cool season species, Poa pratensis and Agropyron repens, and two native C₄ warm season species, Schizachyrium scoparium and Andropogon gerardii. We found that the differences in the dominants explained up to 27% of the community diversity. Thus, the identity of the dominant species can have a strong influence on community diversity and studies examining factors that influence plant community diversity need to incorporate the effect of the dominants. Secondly, we found that the non‐native C₃ grass dominated communities had lower overall and lower native species richness relative to the native C₄ grass dominated communities. Therefore, a shift in dominants from C₄ to C₃ may lead to a large community diversity decline. We found that Poa pratensis, the most abundant non‐native C₃ grass increased in abundance over the 23 years; thus, the negative influence of non‐natives on the community diversity is not decreasing over time and active management is required to restore native grassland plant communities.     

Microbial decomposition of carboxymethyl cellulose continuously added to the soil

If heterocontinuous flow-cultivation method was used to study the degradation of soluble carboxymethyl cellulose (CMC) in soil, neither the potential CM-cellulase activity of the soil nor the total degree of CMC mineralization significantly differed under aerobic condition and in a nitrogen atmosphere. In contrast, the end products of the enzymatic hydrolysis and their mutual proportions were different: under anaerobic conditions, the formation of reducing sugars was increased at the expense of CO₂ production and organic acids were detectable in the extract. The composition of soil microflora also differed. Addition of ammonium ions affected the maximum CM-cellulase activity in the soil, the degree of substrate mineralization, the proportion of CO₂ and reducing sugars that are formed and the concentration of the present soil microflora.     

Effect of cultural conditions on the lipid profile of Thiobacillus ferroxidas

The intensitive investigations on the lipid profile of Thiobacillus ferrooxidans at various culture ages suggest some correlations of the lipid constitutents with the membrane-bound iron oxidation system. Phosphatidic acid, phosphatidyl serine and phosphatidyl ethanolamine were the major polar components; hydrocarbon, triglyceride and diglyceride were the main neutral components. Major fatty acids were C_16:0, C_16:1, C_16:3, C_18:1, C_18:3, C_22:1 while C_20:1, C_20:2, C_12:0, C_14:2, C_18:0, C_18:2, C_20:0, C_22:0 were found in trace amounts which also depended upon the phase of the growth. One lipoamino acid was identified as ornithine lipid in the polar fraction. Each and every component varied to some extent at different growth phasesindicating relationship of these lipids to the iron oxidation system of the strain.     

Kinetic behavior of penicillin acylase immobilized on acrylic carrier

The usefulness of Lilly's kinetic equation to describe penicillin G hydrolysis performed by immobilized penicillin acylase onto the acrylic carrier has been shown. Based on the experimental results characteristic kinetic constants have been estimated. The effect of noncompetitive inhibition of 6-amino penicillanic acid has not been found. Five components of reaction resistance have been defined. These components were also estimated for the reaction of the native enzyme as well as the Boehringer preparation.List of Symbols C _E g/m³ enzyme concentration - C _P,C _Q mol/m³ product concentrations - C _S mol/m³ substrate concentration - C _SO mol/m³ initial substrate concentration - K _A mol/m³ constant which defines the affinity of a substrate to the enzyme - K _iS mol/m³ substrate inhibitory constant - K _iP mol/m³ PhAA inhibitory constant - K _iQ mol/m³ 6-APA inhibitory constant - k ₃ mol/g/min constant rate of dissociation of the active complex - R(1) concentrational component of reaction resistance - R(2) resistance component derived from substrate affinity - R(3) resistance component due to the inhibition of the enzyme by substrate - R(4) resistance component due to the inhibition of the enzyme by PhAA - R(5) resistance component due to inhibition of the enzyme by 6-APA - r = dC_s/dt mol/m³ min rate of reaction - t min reaction time - (i) relative resistance of reaction     

Biochemical and Immunological Relationships Between Endo-β-1,4-Glucanases from Cockroaches

The cellulase from Geoscapheus dilatatus consisted of two major and four minor endo-β-1,4-glucanase components. Two major and one minor component were purified to homogeneity. The major endo-β-1,4-glucanase components, named GD1 and GD2, were similar to EG1 and EG2 from Panesthia cribrata in terms of M_r and kinetic properties. The purified minor component, named GD3, was distinct from GD1 and GD2 because of a lower M_r and a lower specific activity. Polyclonal antibodies raised against the two major endo-β-1,4-glucanase components, EG1 and EG2, of the cellulase from P. cribrata cross-reacted with each other and with pure GD1 and GD2 from the foregut and midgut of the related cockroach G. dilatatus but did not cross-react with GD3. Endo-β-1,4-glucanase components were partially purified from the foregut and midgut of four other cockroaches. These comprised three other Australian cockroaches also from the superfamily Blaberoidea and one American cockroach, Cryptocercus punctulatus, from the superfamily Blattoidea. The endogenous cellulases from all cockroaches examined consisted of either two or three major endo-β-1,4-glucanase components. The amino acid sequence of the N-terminus region of the two major endo-β-1,4-glucanase components from P. cribrata were determined and are homologous with those belonging to glycosyl hydrolase family 9 (cellulase family E).     

Polyacrylamide Gel Separation and Molecular Weight Determination of the Components of Cucumber Mosaic Virus RNA

The six principal components of cucumber mosaic virus RNA were eluted from polyacrylamide gels. After reaction with formaldehyde, their molecular weights were determined by means of sedimentation velocity ultracentrifugation. The molecular weights found were 0.91 million Daltons (mixture of components C₂ and C₁); 0.68 million Daltons (component B); 0.33 million Daltons (component A); 0.11 million Daltons (component 0); and 0.01 million Daltons (component 00). Individual molecular weights of components C₂ and C₁ (determined by polyacrylamide electrophoresis) were 1.01 million and 0.89 million Daltons respectively.     

Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export

FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N‐terminal transmembrane domain (FlhA_TM) and a C‐terminal cytoplasmic domain (FlhA_C). FlhA_C is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhA_C at 2.8 Å resolution. FlhA_C consists of four subdomains (A_CD1, A_CD2, A_CD3 and A_CD4) and a linker connecting FlhA_C to FlhA_TM. The sites of temperature‐sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation suggested that FlhA_C changes its conformation for its function. Molecular dynamics simulation demonstrated an open‐close motion with a 5–10 ns oscillation in the distance between A_CD2 and A_CD4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhA_C is essential for protein export.     

Carbohydrates and carbohydrate esters of ferulic acid released from cell walls of Lolium multiflorum by treatment with cellulolytic enzymes

41% of the cell walls from mature leaf blades of Lolium multiflorum were digested by treatment during 14 days with C₁ enzyme (cellulase) which had been purified by gel filtration and ion-exchange chromatography. Cellobiose was the main sugar released from the walls, together with some glucose and higher oligosaccharides. Considerable amounts of carbohydrate esters of ferulic and p-coumaric acids were also released. When the C₁ enzyme was further purified by isoelectric focusing, only 8% of the cell walls were digested. Purified C_x (CM-cellulase) containing β-glucosidase digested 51% of the cell walls in 16 hours: the major component detected in the soluble products was glucose together with some β (1 → 4)-xylobiose, xylose and arabinose. Higher oligosaccharides and carbohydrate esters of ferulic and p-coumaric acids were also present. It was shown that these acids were present in the cell walls mainly in the trans-configuration.     

Cellulase from Fusarium solani: Purification and properties of the C1 component

The C₁ component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H₃PO₄-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O-(carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (C_x enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H₃PO₄-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (C_x, CM-cellulases), indicate that C₁ is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting C_x enzyme when solubilizing cotton fibre.     

Characterization of the Glycolipid Associated with Alzheimer Paired Helical Filaments

Abstract: In the present study, analytical techniques including gas chromatography/mass spectrometry (GC/MS)-assisted carbohydrate linkage-analysis, one- and two-dimensional NMR, and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-MS) have been used to characterize the structure of the glycolipid associated with the paired helical filaments (PHF) isolated from the neurofibrillary tangles of Alzheimer's diseased brain. The ¹H NMR spectrum of acid-hydrolyzed protein-resistant core PHF (prcPHF) displays resonances that can be assigned to fatty acid and glucose. There are no resonances present that would indicate the presence of protein, amino acids, or a sphingosine base. Using two-dimensional homonuclear correlated spectroscopy, homonuclear Hartmann-Hahn, and heteronuclear multiple quantum coherence experiments, resonances in the ¹H and ¹³C NMR spectrum of native PHF were assigned to a nonreducing terminal α-1,6-glycosidically linked glucose, an internal α-1,6-linked glucose, and an α-1,2,6-linked glucose. The narrow line-widths observed for these residues suggest that they arise from glucose residues undergoing rapid segmental motion. The carbohydrate portion of the PHF-associated glycolipid was analyzed using GC/MS linkage analysis and confirmed the presence of terminal and internal α-1,6-linked glucose and α-1,2,6-linked glucose in a molar ratio of 2:1:1. Three components of the PHF-associated glycolipid fraction having masses 2,416, 2,325, and 2,237 Da were observed using MALDI-MS. The least abundant, heavier mass component (2,416 Da) was best fit to a structure with a tridecamer of glucose having a single esterified C₂₀ fatty acid (Glc₁₃ + C₂₀ or Glc₁₃ + C_20:1), whereas the more abundant, lower mass components were best fit to noncovalently associated glycolipid dimers, each with a glucose pentamer or hexamer having two C₁₄, C₁₆, or C₁₈ esterified fatty acids {D[(Glc₅ + C₁₈) + (Glc₆ + C₁₆)] or D[(Glc₅ + C₁₄) + (Glc₆ + C₁₄)]}. The ratio of glucose to fatty acid calculated from these best-fit structures of the more abundant mass components (5.5 ± 1.1:1.0) is in reasonable agreement with the same ratio calculated from peak integrations in the NMR spectra of acid-hydrolyzed prcPHF (6.2 ± 1.6). Structural similarities between PHF-associated glycolipid and other glycolipid amphiphiles known to form PHF-like filaments indirectly suggest that this unique glycolipid may be an integral component of the PHF suprastructure.     

Biosynthèse de la cellulose bacterienne deutériée: étude par R. M. N. du taux d'incorporation et de la localisation du deutérium

The use of the NMR spectra (250 MHz) of cellulose triacetate allows the determination of the percentage of deuterium bonded to each of the six carbon atoms of the monomer residue (except for H_?1 and one of the protons bonded to C₆ where the signals overlap). Deuterated derivatives of D -glucose and/or deuterated water were used for the biosynthesis of bacterial cellulose by Acetobacter xylinum. Analysis of NMR spectra of acetylated samples gives the following results. About 90% of the protons linked to C₁ and C₆ come from the D -glucose used in the nutrition medium, whereas 10% are exchanged with other sources of protons. Over 40% of the protons linked to C₂, C₃, C₄, and C₅ arise from the water of the nutrition medium. Discrepancies between results of biosynthesis from deuterated water and from deuterated D -glucose can only be explained if more than one enzymatic process is involved in the biosynthesis of bacterial cellulose.