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.     

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.     

The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size

Degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) was investigated by analyzing the insoluble cellulose fragments remaining after enzymatic hydrolysis. Changes in the molecular-size distribution of cellulose after attack by EGI, alone and in combination with CBHII, were determined by size exclusion chromatography of the tricarbanilate derivatives. Cotton cellulose incubated with EGI exhibited a single major peak, which with time shifted to progressively lower degrees of polymerization (DP; number of glucosyl residues per cellulose chain). In the later stages of degradation (8 days), this peak was eventually centered over a DP of 200 to 300 and was accompanied by a second peak (DP, (apprx=)15); a final weight loss of 34% was observed. Although CBHII solubilized approximately 40% of bacterial microcrystalline cellulose, the cellobiohydrolase did not depolymerize or significantly hydrolyze native cotton cellulose. Furthermore, molecular-size distributions of cellulose incubated with EGI together with CBHII did not differ from those attacked solely by EGI. However, a synergistic effect was observed in the reducing-sugar production by the cellulase mixture. From these results we conclude that EGI of T. reesei degrades cotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.     

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.     

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.     

Surface activation of dyed fabric for cellulase treatment

Surface activation of fabric made from cellulose fibres, such as viscose, lyocell, modal fibres and cotton, can be achieved by printing of a concentrated NaOH-containing paste. From the concentration of reducing sugars formed in solution, an increase in intensity of the cellulase hydrolysis by a factor of six to eight was observed, which was mainly concentrated at the activated parts of the fabric surface. This method of local activation is of particular interest for modification of materials that have been dyed with special processes to attain an uneven distribution of dyestuff within the yarn cross-section, e.g., indigo ring-dyed denim yarn for jeans production. Fabrics made from regenerated cellulose fibres were used as model substrate to express the effects of surface activation on indigo-dyed material. Wash-down experiments on indigo-dyed denim demonstrated significant colour removal from the activated surface at low overall weight loss of 4-5%. The method is of relevance for a more eco-friendly processing of jeans in the garment industry.     

The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase.

1. Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing. 2. All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack. 3. Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength. 4. All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem. J. 128, 1183-1192] of T. koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component. 5. The mode of action of the enzymes is discussed in relation to these synergistic effects. It is suggested that the results are compatible with the interpretation that the 'crystalline' areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains.     

Short-fibre formation during cellulose degradation by cellulolytic fungi

Summary All cell-free filtrates of 26 fungal strains containning cellulase activities degraded native cellulose to both reducing sugar and insoluble short fibres. Low-molecular components from the crude filtrates could also degrade native cellulose into short fibres, not accompanied with the production of reducing sugar. Short fibre formation played an important role in cellulose degradation to make the substrate more accessible to attack of cellulases.     

Methodological analysis for determination of enzymatic digestibility of cellulosic materials

Accurate measurement of enzymatic cellulose digestibility (X) is important in evaluating the efficiency of lignocellulose pretreatment technologies, assessing the performance of reconstituted cellulase mixtures, and conducting economic analysis for biorefinery processes. We analyzed the effect of sugars contained in enzymes solutions, usually added as a preservative, and random measurement errors on the accuracy of X calculated by various methods. The analysis suggests that exogenous sugars at levels measured in several commercial enzyme preparations significantly bias the results and that this error should be minimized by accounting for these sugars in the calculation of X. Additionally, a method of calculating X equating the ratio of the soluble glucose equivalent in the liquid phase after hydrolysis to the sum of the soluble glucose equivalent in the liquid phase and the insoluble glucose equivalent in the residual solid after hydrolysis was found to be the most accurate, particularly at high conversion levels (>ca. 50%).     

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.     

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.     

Incorporation of UDPGlucose into Cell Wall Glucans and Lipids by Intact Cotton Fibers

The [¹⁴C] moiety from [³H]UDP[¹⁴C]glucose was incorporated by intact cotton fibers into hot water soluble, acetic-nitric reagent soluble and insoluble components, and chloroform-methanol soluble lipids; the [³H] UDP moiety was not incorporated. The ³H-label can be exchanged rapidly with unlabeled substrate in a chase experiment. The cell wall apparent free space of cotton fibers was in the order of 30 picomoles per milligram of dry fibers; 25 picomoles per milligram easily exchanged and about 5 picomoles per milligram more tightly adsorbed. At 50 micromolar UDPglucose, 70% of the [¹⁴C]glucose was found in the lipid fraction after both a short labeling period and chase. The percent of [¹⁴C]glucose incorporated into total glucan increased slightly with chase, but the fraction of total glucans incorporated into insoluble acetic-nitric reagent (cellulose) did increase within a 30-minute chase period. The data supports the concept that glucan synthesis, including cellulose, as well as the synthesis of steryl glucosides, acetylated steryl glucosides, and glucosyl-phosphoryl-polyprenol from externally supplied UDPglucose occurs at the plasma membrane-cell wall interface. The synthase enzymes for such synthesis must be part of this interfacial membrane system.     

Unravelling carbon metabolism in anaerobic cellulolytic bacteria

Carbon metabolism in anaerobic cellulolytic bacteria has been investigated essentially in Clostridium thermocellum, Clostridium cellulolyticum, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. While cellulose depolymerization into soluble sugars by various cellulases is undoubtedly the first step in bacterial metabolisation of cellulose, it is not the only one to consider. Among anaerobic cellulolytic bacteria, C. cellulolyticum has been investigated metabolically the most in the past few years. Summarizing metabolic flux analyses in continuous culture using either cellobiose (a soluble cellodextrin resulting from cellulose hydrolysis) or cellulose (an insoluble biopolymer), this review aims to stress the importance of the insoluble nature of a carbon source on bacterial metabolism. Furthermore, some general and specific traits of anaerobic cellulolytic bacteria trends, namely, the importance and benefits of (i) cellodextrins with degree of polymerization higher than 2, (ii) intracellular phosphorolytic cleavage, (iii) glycogen cycling on cell bioenergetics, and (iv) carbon overflows in regulation of carbon metabolism, as well as detrimental effects of (i) soluble sugars and (ii) acidic environment on bacterial growth. Future directions for improving bacterial cellulose degradation are discussed.     

Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.     

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.     

Hydrolysis and transformation of cellulose with Aspergillus niger IBT-90 enzymes

Hydrolysis and transformation of Fibrenier cellulose (USA) with enzymes from Aspergillus niger IBT-90 was studied. The process was performed at 50°C and pH 4.8 for 24 h using an enzyme complex either as a properly diluted culture filtrate or as a mixture of isolated and purified enzymes from A.niger IBT-90. In the latter experiments, enzyme-substrate ratios expressed as units of activity per 1 g of cellulose were as follows: endoglucanase E₁ and E₂, 40; β-glucosidase, 40 and cellobio-hydrolase, 2. Cellulose concentration was 5%. It was proved that the crude celluloytic complex from A. niger IBT-90 exhibits higher efficiency in the decomposition of cellulose in comparison to the mixture of enzymes isolated from this complex, as was revealed in assays of reducing sugars and determinations of light transmission throughout cellulose fibres using a computer analysis of the microscopic image. Comparison of both the endoglucanases E₁ and E₂ showed that the first enzyme is more active against cellulose. It liberated more reducing sugars and caused more significant decomposition of fibres. The predominant effect of the endoglucanase E₂ was a smoothing of the fibre surface. The cellobiohydrolase split a cellulose fibre into many short fibres.     

Cellulolytic Activity of Thermomonospora curvata: Nutritional Requirements for Cellulase Production

The use of a minimal medium for cellulase (C(1) and C(x)) production by Thermomonospora curvata increased extracellular C(1) activity (measured by rate of cotton fiber hydrolysis) 11-fold compared with the previously used yeast extract medium. Ground cotton fibers supported the highest cellulase production when compared to other soluble and insoluble carbohydrate sources. Maximal cellulase production occurred at 45 C, slightly less at 55 C, and was insignificant at 65 C (the highest temperature at which cellulase activity appeared stable). At a temperature of 55 C, an optimal pH of 8.0, and a cotton fiber concentration of 8 mg/ml, shake cultures of T. curvata degraded about 75% of the cellulose during the 10-day period.     

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.     

Effects of adsorption properties and mechanical agitation of two detergent cellulases towards cotton cellulose

Abstract

The impacts of two hybrid cloned commercial cellulases designed for detergency on cotton fibres were compared. HiCel45 has a family 45 catalytic domain and a fungal cellulose binding module (CBM) from the fungus Humicola insolens. BaCel5 has a family 5 catalytic domain and a fungal CBM from Bacillus spp. BaCel5 bound irreversibly to cellulose under the buffer conditions tested while HiCel45 was found to bind reversibly to cellulose because it showed low adsorption. BaCel5 seems to yield more activity towards cotton than HiCel45 under mild stirring conditions, but under strong mechanical agitation both enzymes produce similar amount of sugars. HiCel45 had a more progressive production of residual reducing ends on the fabric than BaCel5. These studies seem to indicate that HiCel45 is a more cooperative enzyme with detergent processes where high mechanical agitation is needed.