The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellobiohydrolases.

Two immunologically unrelated cellobiohydrolases (I and II), isolated from the extracellular cellulase system elaborated by the fungus Penicillum pinophilum, acted in synergism to solubilize the microcrystalline cellulose Avicel; the ratio of the two enzymes for maximum rate of attack was approx. 1:1. A hypothesis to explain the phenomenon of synergism between two endwise-acting cellobiohydrolases is presented. It is suggested that the cellobiohydrolases may be two stereospecific enzymes concerned with the hydrolysis of the two different configurations of non-reducing end groups that would exist in cellulose. Only one type of cellobiohydrolase has been isolated so far from the cellulases of the fungi Fusarium solani and Trichoderma koningii. Only cellobiohydrolase II of P. pinophilum acted synergistically with the cellobiohydrolase of the fungi T. koningii or F. solani to solubilize Avicel. Cellobiohydrolase II showed no capacity for co-operating with the endo-1,4-beta-glucanase of T. koningii or F. solani to solubilize crystalline cellulose, but cellobiohydrolase I did. These results are discussed in the context of the hypothesis presented.     

Synergism between components of the cellulase system of the anaerobic rumen fungus Neocallimastix frontalis and those of the aerobic fungi Penicillium pinophilum and Trichoderma koningii in degrading crystalline cellulose

The cellulase system of Neocallimastix frontalis was separated by differential affinity on cellulose into an adsorbed fraction that could solubilize crystalline cellulose (crystalline-cellulose-solubilizing fraction, CCSF), and a non-adsorbed fraction that contained endoglucanase and -glucosidase activities (non-adsorbed endoglucanase/ -glucosidas, NAE/-G) but which showed no activity to crystalline cellulose. Both fractions were tested for their capacity to act synergistically with the cellobiohydrolase (CBH) components of aerobic fungi in degrading crystalline cellulose. The CCSF acted synergistically with CBH I components of both Penicillium pinophilum and Trichoderma koningii but not with CBH II. The NAE/-G fraction also acted synergistically with the CBH components of P. pinophilum but, remarkably, only when both CBH I and CBH II were present in the reaction mixture. By comparison with previously published studies on the mechanism of action of P. pinophilum cellulase it is speculated that the CCSF of N. frontalis may contain CBH I- and CBH II-type enzymes.     

Multiple forms of endo-1,4-beta-D-glucanase in the extracellular cellulase of Penicillium pinophilum

The influence of the composition of the growth medium on the production of endo-1,4-beta-D-glucanase (CM-cellulase) activity by P. pinophilum was studied in shake flask cultures using Avicel PH101 as the carbon source. It was observed that the culture conditions had a profound effect on the level of endoglucanase (CM-cellulase) produced by P. pinophilum. However, isoelectric focusing of the endoglucanase activity obtained from shake flask and fermenter cultures using the same growth medium revealed that the enzyme system found in both cultures was identical qualitatively, and contained seven or eight different endoglucanase components. All the endoglucanase components appeared simultaneously in the early stages of culture and prolonged incubation resulted only in an increase in the concentration of these enzymes. Protease levels were found to be low in both types of culture but were particularly so in the growth medium which contained corn steep liquor. The proteases were unable to release low molecular weight peptides when P. pinophilum cellulase protein was used as a substrate. The results were interpreted to indicate that the multiplicity of endoglucanase components found in cultures of P. pinophilum is most likely the result of expression of a number of specific genes rather than by post-secretional modification of one or more endoglucanase(s) synthesized by the fungus.     

Studies on the capacity of the cellulase of the anaerobic rumen fungus Piromonas communis P to degrade hydrogen bond-ordered cellulose

The anaerobic rumen fungus Piromonas communis, when cultured on cotton fibre as the carbon source, produces an extracellular cellulase that is capable of solubilizing crystalline hydrogen-bond-ordered cellulose, in the form of the cotton fibre, at a rate that is greater than that of any other cellulases reported in the literature hitherto. The cell-free culture fluid is also very rich in xylan-degrading enzymes. The activity towards crystalline cellulose resides in a high-molecular-mass (approximately 700–1000 kDa) component (so-called crystalline-cellulose-solubilizing component, CCSC) that comprises endo (1 4)--D-gluconase (carboxymethylcellulase), -D-glucosidase and another enzyme that appears to be important for the breakdown of hydrogen-bond-ordered cellulose. The CCSC is associated with only a small amount of the endo-(1 4)--D-glucanase (1.9%), -D-glucosidase (0.7%) and protein (0.5%) found in the crude cell-free cellulase preparation. The CCSC, unlike the bulk of the endo-(1 4)--D-glucanase and -D-glucosidase, is very strongly absorbed on the microcrystalline cellulose, Avicel.     

Breakdown of crystalline cellulose by synergistic action between cellulase components from Clostridium thermocellum and Trichoderma koningii

Abstract Certain isolated components of fungal cellulases, which cannot effect the breakdown of highly ordered cellulose individually, interact together synergistically to do so when recombined. Suprisingly, not all fungal cellulase components exhibit this property, and no such synergism has been observed so far between fungal and bacterial cellulases.
The cellulase complex of Clostridium thermocellum cannot effect the extensive breakdown of highly ordered cellulose unless Ca²⁺ and dithiothreitol (DTT) are present. However, we now report that isolated cellobiohydrolase from Trichoderma koningii can combine with C. thermocellum cellulase to effect the breakdown of cellulose in the absence of Ca²⁺ and DTT. enhanced activity is observed if Ca²⁺ and DTT are present.
This finding may have important applications in industry: it certainly has important implications for those interested in the basic mechanism of cellulase action in C. thermocellum .     

Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei.

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1----4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.     

Effect of pretreatment on the hydrolysis of cellulose by Penicillium funiculosum cellulase and recovery of enzyme

Penicillium funiculosum produces a complete cellulase which brings about 97% hydrolysis of cotton and has high beta-glucosidase, xylanase, laminarinase, and lichenase activities. This article deals with the effect of different pretreatments on the hydrolysis of sugarcane bagasse by P. funiculosum enzymes and the recovery of enzyme from the insoluble residues. Enzymic saccharification of bagasse pretreated with hot 1N NaOH followed by neutralization with HCI and steam treated under pressure (7 kg/cm(2)) gave 63 and 59% saccharification, respectively, in 48 h. Hemicellulose is not lost in these pretreatments. With a 30% slurry of steam-treated bagasse, a semisolid mass containing 14% sugar was obtained. A 90% recovery of CMCase, beta-glucosidase, and filter paper activity from the hydrolysates was obtained under the following conditions: (1) maintaining the ratio of enzyme to substrate high by stepwise addition of substrate, (2) brief grinding of the residual substrate with glass powder, and (3) addition of 0.4% Tween-80 to the eluting buffer. The high recovery of cellulolytic enzymes indicates that the adsorption of these enzymes on cellulose is not irreversible.     

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 mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence that the hydrogen transfer mechanism involves a second intermediate hydrogen carrier in addition to the cofactor

Ethanolamine ammonia-lyase catalyzes the adenosylcobalamin (AdoCbl)-dependent conversion of ethanolamine to acetaldehyde and ammonia. During this reaction, a hydrogen atom migrates from the carbinol carbon of ethanolamine to the methyl carbon of acetaldehyde. Previous studies have shown that this migrating hydrogen equilibrates with the hydrogens on the 5'-(cobalt-linked) carbon of the cofactor. On the basis of those studies, a two-step mechanism for hydrogen transfer has been postulated in which the migrating hydrogen is first transferred from the substrate to the cofactor, then in a subsequent step is returned from the cofactor to the product. We now show that this migrating hydrogen is transferred not only to the cofactor, but also to a second acceptor at the active site. Hydrogens on this acceptor do not exchange with water during the course of the reaction, but are released to water when the enzyme is denatured. The catalytic significance of this second hydrogen acceptor was demonstrated by the findings that the transfer of hydrogen to this acceptor required both AdoCbl and active enzyme and that hydrogen at the second acceptor site could be washed out by unlabeled ethanolamine. On the basis of these results, we propose an expanded hydrogen transfer mechanism in which AdoCbl and the second acceptor site serve as alternative intermediate hydrogen carriers during the course of ethanolamine deamination.     

The action pattern of Penicillium lilacinum dextranase.

The product distributions resulting from the action of Penicillium lilacinum dextranase on end-labelled oligosaccharides of the isomaltose series have been determined. The initial rates of formation of labelled products were measured for isomaltotriose up to isomalto-octaose, and the molar proportions and radioactivity of the final products from isomaltotriose up to isomaltohexaose were determined. D-Glucose was released only from isomaltotriose and isomaltotetraose, by hydrolysis of the first linkage from the reducing end (linkage 1); the terminal bonds of higher members of the series were not attacked. All oligosaccharides except isomaltotriose were hydrolyzed at more than one linkage. The main points of attack on isomaltotetraose up to isomalto-octaose were at linkage 2, and at the third linkage from the non-reducing end; these two positions coincide for isomaltopentaose. The degradation of isomaltotriose up to isomalto-octaose was entirely hydrolytic. The enzyme also catalyzed an extremely slow, concentration-dependent degradation of isomaltose, and this may have occurred via a condensation to isomaltotetraose, followed by hydrolysis of linkage 1 to give D-glucose and isomaltotriose.     

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.     

On the mechanism of enzyme action. LIII. The amphoteric properties of trypsin

Several methods were employed for the preparation of salt-free trypsin samples which were used to determine the electrometric titration curves of the enzyme. These curves point to a maximum acid-binding capacity below pH 2. Stoichiometric analysis indicates the presence of 6 carboxyl groups per 10,000 g. of proteins, 1 imidazole group, and 13 hydroxyl-binding groups. Calcium has a specific effect on the titration curves by increasing the acidity of the carboxyl groups in the pH range 3.5 – 5. This effect is not shown by potassium, sodium, or even the bivalent magnesium ion. It is attributed to the formation of a specific complex between the enzyme and the calcium ions, involving the carboxyl groups of the protein. The equally specific protective effect of calcium on the self-digestion of trypsin can therefore be explained by assuming the formation of a complex which stabilizes the enzyme.     

The fungal cell wall and the mechanism of action of anidulafungin

The fungal cell wall is a structure with a high plasticity that protects the cell from different types of environmental stresses including changes in osmotic pressure. In addition to that, the cell wall allows the fungal cell to interact with its environment, since some of its proteins are adhesins and receptors. Some of its components are highly immunogenic. The structure of the fungal cell wall is unique to the fungi, and it is composed of glucan, chitin and glycoproteins. Since humans lack the components present in the cell walls of fungi, this structure is an excellent target for the development of antifungal drugs. Anidulafungin, like the rest of echinocandins acts on beta-1,3-D-glucan synthase inhibiting the formation of beta-1,3-D-glucan and causing, depending on the type of fungus, a fungicidal or either a fungistatic effect.     

Competitive adsorption of cellulase components and its significance in a synergistic mechanism

Some studies on the adsorption of cellulase on cellulose revealed part of the mechanisms involved in the enzymatic hydrolysis of cellulose and provided some clues to the synergistic mechanism of cellulase complex. The adsorption of cellulase was significantly affected by the reaction conditions and physical chemical characteristics of cellulose. Endoglucanase consisted of adsorbable and nonadsorbable components. Cellobiohydrolase had the strongest adsorption affinity. Each cellulase component is postulated to have distinctly different adsorption sites on cellulose, corresponding to the active sites in the hydrolysis reaction. Competitive adsorption kinetics between cellulase components were also observed during the adsorption process. The degree of competitive adsorption was most remarkable when the composition of cellulase components was nearly the same as that in the crude cellulase complex. This seems to show the optimal relative composition of cellulase components. The synergism between cellobiohydrolase and endoglucananse could be elucidated more clearly by this competitive adsorption model of the reaction mechanism.