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.     

Solubilization of native and derived forms of cellulose by cell-free microbial enzymes

1. Cell-free enzymes from Myrothecium verrucaria and Trichoderma koningii hydrolyse native undegraded cellulose, as found in cotton fibres, in a random manner to short insoluble fibres and to minor amounts of soluble products. 2. Enzyme preparations from M. verrucaria fail to attack the short fibres whereas preparations from T. koningii solubilize them completely to sugars at an optimum pH4.2-4.6. 3. The mode of hydrolysis of cotton cellulose by preparations from T. koningii involves from the earliest stages the formation of reducing sugars, followed closely by the appearance of short fibres, until the insoluble and soluble products each constitute about 40-50% of the weight of the initial substrate. After this stage the quantity of sugars increases at the expense of the insoluble short fibres. 4. Depending upon the method of preparation, derived forms of cellulose may be hydrolysed more slowly, much more rapidly, or at the same rate as cotton fibres by enzyme preparations from T. koningii.     

The purification and properties of the C1 component of Trichoderma koningii cellulase

1. The C(1) component that was isolated from a Trichoderma koningii cellulase preparation (Wood, 1968) by chromatography on DEAE-Sephadex with a salt gradient was still associated with a trace of CM-cellulase activity (determined by reducing-sugar and viscometric methods). 2. Further chromatography on DEAE-Sephadex, with a pH gradient instead of a salt gradient, provided a C(1) component that could still produce reducing sugars from a solution of CM-cellulose (to a very limited extent), but which could no longer decrease the viscosity (i.e. under the assay conditions employed). 3. No evidence for the non-identity of C(1) component and the trace of CM-cellulase activity could be found when electrofocusing was done in a stabilized pH gradient covering three pH units (pH3-6) or, alternatively, only 0.5 pH unit (pH3.72-4.25). 4. The two protein peaks that were separated by electrofocusing in carrier ampholytes covering only 0.5 pH unit (isoelectric pH values of 3.80 and 3.95) were shown to be isoenzymes of the C(1) component: they differed in the extent to which they were associated with carbohydrate (9% and 33%). 5. The purified C(1) component had little ability to attack CM-cellulose or highly ordered forms of cellulose, but degraded phosphoric acid-swollen cellulose readily: cellobiose was the principal product of the hydrolysis (97%). 6. Dewaxed cotton fibre was degraded to the extent of 15% when exposed to high concentrations of C(1) component over a prolonged period: cellobiose was again the principal sugar present in the supernatant (96%). 7. Cellotetraose and cellohexaose were hydrolysed almost exclusively to cellobiose. 8. Evidence indicates that the C(1) component is a beta-1,4-glucan cellobiosylhydrolase.     

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.     

Interactions between components of the cellulase complex of Trichoderma koningii on native substrates

Summary The cellulase complex of Trichoderma koningii has been separated into four apparently pure components namely cellobiase, a C₁-like component and two new components, one a CM-cellulase, the other named component C₂. All four are necessary for efficient solubilisation of native cellulose. 2. The two new components together constitute the composite CM-cellulase-short-fibre forming activity described by Halliwell and Riaz (1970). 3. Alone only components C₂ and C₁ have any action on the substrate, the former being somewhat more effective than the C₁ component which shows weak solubilising power. 4. Component C₂ degrades cellulose weakly to short-fibres but synergises extensively with the CM-cellulase in promoting this process. In contrast the CM-cellula se fails to react significantly with component C₁. 6. The interaction of all four components in contributing to the degradation of native cellulose is discussed.     

The cellulase of Fusarium solani. Resolution of the enzyme complex

1. Culture filtrates from Fusarium solani were fractionated by ion-exchange chromatography on DEAE-Sephadex, followed by gel chromatography on Sephadex G-100, into a C(1) component, a C(x) component (CM-cellulase) and a beta-glucosidase (cellobiase) component. 2. The individual components showed little capacity for the solubilization of cotton fibre (cellulase activity), but when recombined in their original proportions 81% of the original cellulase activity was recovered. 3. The C(1) components of F. solani and Trichoderma koningii were similar in their pH optima, heat stabilities over the pH range 5-8 and elution volumes on Sephadex G-100. 4. The C(1) component of F. solani synergized with the C(x) component of T. koningii and conversely. 5. The C(1) and the beta-glucosidase components of F. solani were devoid of the swelling-factor (S-factor) activity associated with the C(x) component.     

Catalytic decomposition of cellulose under biological conditions

1. The catalytic decomposition of undegraded cellulose in the form of cotton fibres is described with hydrogen peroxide at 0·4–0·04% (w/v) concentration in the presence of ferrous salts at pH3–5. 2. Complete solubilization of 5mg. of cotton fibres occurred in about 7 days in the presence of 0·4% hydrogen peroxide and 0·2mm-ferrous sulphate at the optimum pH4·2–4·3. 3. With 0·4% hydrogen peroxide the most rapid decomposition of cellulose was confined to ferrous sulphate concentrations of approx. 2–0·02mm. If the concentrations of the reagents were decreased in proportion extensive breakdown occurred but much more slowly. 4. In the primary stages of breakdown cotton fibres were disintegrated to very short fibres. These were subsequently solubilized, but there was little accumulation of soluble material. Organic matter was lost from solution as the reaction progressed. 5. Other naturally occurring cellulose-containing materials, such as grass, straw, hay and sawdust, were also disintegrated and solubilized by hydrogen peroxide and ferrous sulphate.     

Hydrolysis of fibrous cotton and reprecipitated cellulose by cellulolytic enzymes from soil micro-organisms

1. The catalytic decomposition of undegraded cellulose in the form of cotton fibres is described with hydrogen peroxide at 0·4–0·04% (w/v) concentration in the presence of ferrous salts at pH3–5. 2. Complete solubilization of 5mg. of cotton fibres occurred in about 7 days in the presence of 0·4% hydrogen peroxide and 0·2mm-ferrous sulphate at the optimum pH4·2–4·3. 3. With 0·4% hydrogen peroxide the most rapid decomposition of cellulose was confined to ferrous sulphate concentrations of approx. 2–0·02mm. If the concentrations of the reagents were decreased in proportion extensive breakdown occurred but much more slowly. 4. In the primary stages of breakdown cotton fibres were disintegrated to very short fibres. These were subsequently solubilized, but there was little accumulation of soluble material. Organic matter was lost from solution as the reaction progressed. 5. Other naturally occurring cellulose-containing materials, such as grass, straw, hay and sawdust, were also disintegrated and solubilized by hydrogen peroxide and ferrous sulphate.     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.     

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.     

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.     

Mechanism of cellulose synthesis in Agrobacterium tumefaciens.

Extracts of Agrobacterium tumefaciens incorporated UDP-[14C]glucose into cellulose. When the extracts were fractionated into membrane and soluble components, neither fraction was able to synthesize cellulose. A combination of the membrane and soluble fractions restored the activity found in the original extracts. Extracts of cellulose-minus mutants showed no significant incorporation of UDP-glucose into cellulose. When mixtures of the extracts were made, the mutants were found to fall into two groups: extracts of mutants from the first group could be combined with extracts of the second group to obtain cellulose synthesis. No synthesis was observed when extracts of mutants from the same group were mixed. The groups of mutants corresponded to the two operons identified in sequencing the cel genes (A. G. Matthysse, S. White, and R. Lightfoot. J. Bacteriol. 177:1069-1075, 1995). Extracts of mutants were fractionated into membrane and soluble components, and the fractions were mixed and assayed for the ability to synthesize cellulose. When the membrane fraction from mutants in the celDE operon was combined with the soluble fraction from mutants in the celABC operon, incorporation of UDP-glucose into cellulose was observed. In order to determine whether lipid-linked intermediates were involved in cellulose synthesis, permeablized cells were examined for the incorporation of UDP-[14C]glucose into material extractable with organic solvents. No radioactivity was found in the chloroform-methanol extract of mutants in the celDE operon, but radioactive material was recovered in the chloroform-methanol extract of mutants in the celABC operon. The saccharide component of these compounds was released after mild acid hydrolysis and was found to be mainly glucose for the celA insertion mutant and a mixture of cellobiose, cellotriose, and cellotetrose for the celB and celC insertion mutants. The radioactive compound extracted with chloroform-methanol form the celC insertion mutant was incorporated into cellulose by membrane preparations from celE mutants, which suggests that this compound is a lipid-linked intermediate in cellulose synthesis.     

Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85.

Two endoglucanases designated EG1 and EG2 were purified by column chromatography from the nonsedimentable extracellular culture fluid of Bacteroides succinogenes S85. They accounted for approximately 32 and 11%, respectively, of the total endoglucanase present in the nonsedimentable fraction. The most active enzyme (EG1) had a molecular weight of 65,000, pI of 4.8, and temperature and pH optima of 39 degrees C and 6.4, respectively. The Km for carboxymethyl cellulose was 3.6 mg/ml, and the Vmax was 84 U/mg. The major products of cellulose hydrolysis catalyzed by EG1 were cellotriose and cellobiose. EG2 was present as two components with molecular weights of 118,000 and 94,000. The two components had nearly identical cyanogen bromide peptide maps, thereby indicating that the 94,000-dalton component was a proteolytic degradation product of the 118,000-dalton enzyme. The larger component, which was more abundant in the culture fluid than the smaller form was, had a Km of 12.2 mg/ml and a Vmax of 10.4 U/mg. It was a basic protein with a pI of 9.4, a temperature optimum of 39 degrees C, and a pH optimum of 5.8. The major product of cellulose hydrolysis was cellotetraose. EG2 exhibited specific binding to acid-swollen cellulose, whereas EG1 did not, and neither of them had affinity for crystalline cellulose. Based on the substrate specificities and the affinities of the two enzymes for cellulose, we postulated that EG2 is involved in the early stages of cellulose hydrolysis and that EG1 is active primarily on the products arising from EG2.     

Treatment of cotton with an alkaline Bacillus spp cellulase: activity towards crystalline cellulose

We analysed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Although cellulases are commonly applied in detergent formulations due to their anti-redeposition and depilling benefits, determining the mechanism of action of alkaline cellulases on cotton fibres requires a deeper understanding of the morphology and structure of cotton fibres in terms of fibre cleaning. The accessibility of cellulose fibres was studied by evaluating the iodine sorption value and by fluorescent-labelled enzyme microscopy; the surface morphology of fabrics was analysed by scanning microscopy. The action of enzyme hydrolysis over short time periods can produce fibrillation on cotton fibre surface without any release of cellulosic material. The results indicate that several short consecutive treatments were more effective in increasing the fibre accessibility than one long treatment. In addition, no detectable hydrolytic activity, in terms of reducing sugar production, was found.     

Swelling and dissolution of cellulose. Part IV: Free floating cotton and wood fibres in ionic liquids

The objective of this paper is to investigate if the swelling and dissolution mechanisms found for aqueous solvents are valid for non-aqueous ones. Three different ionic liquids were used and the swelling and dissolution mechanisms were investigated by optical methods. Native and enzymatically treated cellulose fibres (cotton and wood fibres) are dipped into three ionic liquids (1-N-butyl-3-methylimidazolium chloride ([C4mim]+Cl−)/DMSO, allylmethylimidazolium bromide ([Amim]+Br−) and butenylmethylimidazolium bromide ([Bmim]+Br−). ([C4mim]+Cl−)/DMSO shows a swelling of cellulose by ballooning and then dissolution. ([Amim]+Br−) and ([Bmim]+Br−) show a homogeneous swelling but no dissolution. The swelling and dissolution mechanisms of cellulose in ionic liquids are similar to those observed in aqueous solvents. It indicates that the swelling and dissolution mechanisms are entirely due to the way cellulose fibres are structured, not depending on the type of solvent. The quality of the solvent is giving the type of mechanism.     

Growth and Cellulase Formation by Cellvibrio fulvus

S ummary : The aerobic cellulolytic bacterium Cellvibrio fulvus grew on several sugars and polysaccharides, but not on highly substituted cellulose derivatives, organic acids and alcohols. Whereas no growth was obtained on long cotton fibres, it occurred on such fibres cut into small pieces, and on filter paper and chromatography powders derived from cotton. Lignin free wood pulp was rapidly degraded. The organism grew best at pH 7–8 and utilized nitrate, ammonium and some amino acids as nitrogen sources. The bacteria have cell-bound cellulase but enzyme was also found in the culture medium. Glucose repressed cellulase formation and the enzyme activity of cultures grown on cellulose was much higher than on sugars. Reducing sugar was not detected in cellulose cultures. The pH optimum for hydrolysis of carboxymethylcellulose (CMC) was 7 and the enzyme was inhibited by mercuric acetate but not by p -chloromercuribenzoate or EDTA. Fractionation of cellulase preparations from cultures grown on partially hydrolysed filter paper gave many components of different molecular weights. The activities of these components against carboxymethylcellulose and microcrystalline cellulose differed.     

The cellulase of Trichoderma viride: Separation of the components involved in the solubilization of cotton

1. Culture filtrates from Trichoderma viride have been fractionated by gel filtration on Sephadex G-75 followed by ion-exchange chromatography on DEAE- and SE-Sephadex. 2. The components essential for attack on cotton are a carboxymethylcellulase, a cellobiase and a third (C₁) component which has no action on CM-cellulose, cellobiose or cotton. 3. These components, which together can completely convert cotton into water-soluble products, lose this ability when separated and regain it quantitatively when recombined in their original proportions.     

Cellulolytic Activity of Thermomonospora curvata: Optimal Assay Conditions, Partial Purification, and Product of the Cellulase

Thermomonospora curvata produces cellulases active against both cotton fibers (designated C(1) activity) and carboxymethylcellulose (C(x) activity). In reaction systems employing optimal substrate concentration, pH, and temperature, hydrolysis rates (measured by the release of soluble reducing sugars) were initially linear and decreased on prolonged incubation, although only a small amount of substrate (1 to 2%) had been hydrolyzed. Persistence of this lower rate, even after addition of fresh enzyme (in the C(1) assay system), indicated alteration of cellulose susceptibility to hydrolysis rather than enzyme inactivation. Partial purification by (NH(4))(2)SO(4) precipitation and exclusion chromatography resolved cellulase activity into two fractions. The sole product of purified cellulase activity on ground cotton fibers appears to be cellobiose.     

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.