Catriona A. Wilson

The anaerobic fungus Neocallimastix frontalis: isolation and properties of a cellulosome-type enzyme fraction with the capacity to solubilize hydrogen-bond-ordered cellulose

SummaryA minor component isolated from the extra-cellular cellulase of the anaerobic rumen fungus Neocallimastix frontalis by adsorption on cellulose had a remarkable capacity to degrade crystalline hydrogen-bond-ordered cellulose. When produced in a semi-defined medium the component comprised normally less than 4% of the total protein and only 0.3% of the protein in cultures containing rumen fluid. The minor component showed endoglucanase (carboxymethylcellulase) and β-glucosidase activity and effected the extensive hydrolysis of “crystalline” cellulose in the form of the cotton fibre when acting alone. Glucose was the sole product of hydrolysis. The specific activity of the crystalline cellulose solubilizing factor (CCSF) in degrading cotton fibre was much higher than any other cellulase or cellulase component reported so far. The activity of the CSSF to crystalline hydrogen-bond-ordered cellulose resides in a high molecular mass complex of 670 kDa, that comprised a number of subunits ranging in size from 68 to 135 kDa.

Studies on the cellulase of the rumen anaerobic fungus Neocallimastix frontalis, with special reference to the capacity of the enzyme to degrade crystalline cellulose

Abstract By using cotton fiber, carboxymethylcellulose (CM-cellulose), and o -nitrophenyl-β- d -glucoside as substrates, it was possible to demonstrate that there were at least three different types of enzyme present in culture filtrates of Neocallimastix frontalis RK21. The activity to crystalline cellulose (cotton fiber) resided in a high-molecular-weight complex that comprised endoglucanase activity, β-glucosidase activity, and another enzyme. However, synergism between the components in the high-molecular-weight complex and between the complex and low-molecular-weight endoglucanases and β-glucosidases was also apparent in the solubilization of crystalline cellulose. The composition of the complex varied according to the growth conditions: it was, however, between 750 and 1000 kDa in size. Cultures containing rumen fluid contained only small amounts of the high-molecular-weight complex. Cultures grown on defined medium were rich in high-molecular-weight complex, but only when the concentration of the carbon source was less than 1.0%. Treatment of the crude culture filtrates with chitinase under conditions that had little effect on the activity of the enzyme to endoglucanase (CM-cellulose) or β-glucosidase completely destroyed the activity to crystalline cellulose. It is tentatively suggested that an enzyme crucial for the activity to crystalline cellulose may be cell wall-bound and may be dependent on its association with the cell wall for the maintenance of its conformation for attacking crystalline cellulose. The enzyme involved in degrading crystalline cellulose is much more thermolabile than the CM-cellulase or the β-glucosidase: activity is optimal at pH 6.0 and 40°C.

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)-β-D-gluconase (carboxymethylcellulase), β-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)-β-D-glucanase (1.9%), β-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)-β-D-glucanase and β-D-glucosidase, is very strongly absorbed on the microcrystalline cellulose, Avicel.

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 β-glucosidase activities (non-adsorbed endoglucanase/ β-glucosidas, NAE/β-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/β-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.

Characterisation of a β-d-glucosidase from the anaerobic rumen fungus Neocallimastix frontalis with particular reference to attack on cello-oligosaccharides

Abstract A β- d -glucosidase was purified by a series of chromatographic procedures involving gel filtration, ion exchange, adsorption/desorption on hydroxyapatite and hydrophobic interaction. The enzyme had an isoelectric pH of 3.7 and an apparent molecular mass, determined by gel filtration, of 153 kDa. The enzyme exhibited activity on cello-oligosaccharides up to degree of polymerisation 6. Activity on cellotriose was slightly greater than on cellobiose, but thereafter the rate of attack fell as the d.p. of the substrate decreased: Km values for attack on cello-oligosaccharides, d.p. 2–6, were 31, 46, 30 and 26 μM, respectively. Analysis of the products of hydrolysis by anion exchange chromatography on a Dionex PA1 column and pulsed amperometric detection indicated an exo-action and the successive removal of d -glucose from each cello-oligosaccharide substrate. A feature of the action of the enzyme on each cello-oligosaccharide was the synthesis of a cello-oligosaccharide one glucose unit longer than the substrate. This transferase activity was dependent on substrate concentration. Apart from o- and p-nitrophenyl β- d -glucosides, all other aryl and alkyl glucosides tested were poor substrates. However, p-nitrophenyl β- d -fucoside was readily hydrolysed: the β- l -, α- d - and α- l -forms were not substrates.

α-(4-O-Methyl)-d-glucuronidase activity produced by the rumen anaerobic fungusPiromonas communis: A study of selected properties

The rumen anaerobic fungusPiromonas communis, unlike the rumen anaerobic fungiNeocallimastix frontalis andNeocallimastix patriciarum, produced extracellular α-(4-O-methyl)-d-glucuronidase when grown in cultures containing filter-paper, barley straw, birchwood xylan or birchwood sawdust as carbon source. The highest concentration of enzyme was produced in cultures containing birchwood sawdust. The aldobiouronic acidO-α-(4-O-methyl-d-glucopyran-osyluronic acid)-(1 → 2)-d-xylopyranose (MeGlcAXyl) was the best substrate of those tested: the aldotriouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid (1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl2) and the aldotetraouronic acidO-α-(4-O-methyl-d-glucopyranosyluronic acid)-(1 → 2)-O-\-d-xylopyranosyl-(1 → 4)-O-\-d-xylopyranosyl-(1 → 4)-d-xylopyranose (MeGlcAXyl3) were also attacked but the rate fell as the degree of polymerisation increased. When the same substituted xylooligosaccharides were reduced to the corresponding alditols the enzyme activity disappeared. Similarly,p-nitrophenyl-α-d-glucuronide was not a substrate. Remarkably, the relative rates of attack shown by the α-(4-O-methyl)-d-glucuronidase on the aldouronic acids and on xylans extracted from birchwood, oat spelts and oat straw differed according to the carbon source used to produce the enzyme. The α-(4-O-methyl)-d-glucuronidase had a pH optimum of 5.5 and a temperature optimum of 50°C. On gel filtration the enzyme was shown to be associated with proteins covering the range 100–300 kDa, but a major peak of activity in the column effluent appeared to have a molecular mass of 103 kDa.

The cellulase system of the anaerobic rumen fungus Neocallimastix frontalis: studies on the properties of fractions rich in endo-(1→4)-β-D-glucanase activity

Abstract Seven fractions rich in endoglucanase activity were separated from the extracellular cellulase system of the anaerobic rumen fungus Neocallimastix frontalis. The fractions (ES1, ES3, ES2U1, ES2U2, ES2U4, ES2U3C1 and ES2U3C2) were separated from each other and from a fraction that could solubilize crystalline cellulose (the so-called crystalline-cellulose-solubilizing component, CCSC) by the sequential use of differential adsorption on the microcrystalline cellulose Avicel, gel filtration and affinity chromatography on concanavalin-A–Sepharose. The molecular masses of the endoglucanase fractions, when determined by gel filtration, were 64, 30, 61, 113, 17, 38 and 93 kDa respectively. Each enzyme degraded carboxymethylcellulose and was rich in activity to cellulose swollen in phosphoric acid to break the hydrogen bonding: cellobiose, cellotriose and cellotetraose were released in differing proportions. Each fraction showed a characteristic gradient when the capacity of each enzyme to increase the fluidity of a solution of carboxymethylcellulose was plotted against the increase in reducing power of the solution. Although neither endoglucanase fraction, acting in isolation, could degrade crystalline cellulose, three of the fractions (ES1, ES3 and ES2U1) could act synergistically with the CCSC fraction in this regard. Remarkably, the same three fractions also acted in synergism with the cellobiohydrolases (CBH I and CBH II) of the aerobic fungus Penicillium pinophilum in degrading crystalline cellulose, but only when both cellobiohydrolase enzymes were present in the solution along with any one of the three endoglucanases. These observations support the conclusion that the mechanism of action of the cellulase system of N. frontalis in degrading crystalline cellulose may be similar to that operating in the aerobic fungi.

Observations on the complex interactions involved in the enzymatic hydrolysis of cellulose

Although fractionation studies performed on the cellulases of the fungiPencillium funiculosum, Trichoderma koningii, andFusarium solani have shown that the solubilization of high ordered crystalline cellulose can be effected by mixtures of endo-1,4-Β-glucanase, cellobiohydrolase, and Β-glucosidase, factors that affect the interaction of these enzymes are not well understood. Sequential action between endo-1,4-Β-glucanase and cellobiohydrolase is almost certainly a feature of these cellulase systems, but experimental observations would suggest that it is not possible to discuss the mechanism purely in these terms. Some of the steric problems confronting the enzymes may explain, in part, many of the anomalous observations recorded.The cellulase ofP. funiculosum is of special interest in that, in addition to four endo-1,4-Β-glucanases and two Β-glucosidases, it contains two cellobiohydrolases and a glucohydrolase. A detailed study of all these enzymes has not yet been carried out, but several properties of the glucohydrolase and cellobiohydrolase are worthy of note.The glucohydrolase, in being strongly inhibited by glucono-1,5-lactone, in possessing transferase activity and in exhibiting activity on all other Β-linked, glucose disaccharides, had several properties normally associated with Β-glucosidases. It could be distinguished from the Β-glucosidases, however, in retaining anomeric configuration during hydrolysis and in being able to attack long glucan chains. The glucohydrolase was unable to cooperate with the endo- 1,4-Β-glucanase in solubilizing cotton cellulose and this contrasts with the high degree of cooperation shown by the cellobiohydrolase and endo-l,4-Β-glucanase in effecting the extensive solubilization of this substrate. The observation that an enzyme that removes two glucose units (cellobiohydrolase) from the end of the cellulose chain can act synergistically with the endo-l,4-Β-glucanase, but an enzyme that removes only one (glucohydrolase) cannot, must be connected in some way with steric rigidity of the anhydroglucose unit in the cellulose crystallite and the fact that cellobiose is the repeating unit.Recently, studies have been extended to the two cellobiohydrolases ofP. funiculosum. Not surprisingly, it was found that the cellobiohydrolase enzymes had no capacity for cooperating to solubilize cotton cellulose. However, it was unexpected to find that they acted synergistically in degrading the microcrystalline cellulose, Avicel. Two immunologically unrelated cellobiohydrolases have been found recently in cultures of the fungusTrichoderma reesei. Experiments are now in progress to determine whether the two cellobiohydrolases ofP. funiculosum are also immunologically different. However, even if the cellobiohydrolases prove to be merely isoenzymes, the observation with Avicel is an unusual one.It has already been observed that the cellobiohydrolase of one fungus can act synergistically with the endo-1,4-Β-glucanase of another in solubilizing highly ordered cellulose. However, when fungal cellobiohydrolases were added to the endo-1,4-Β-glucanase of the rumen bacteriaRuminococcus albus,Ruminococcus flavefaciens, andBacteroides succinogenes, no synergism was observed. One interpretation of this would be that these bacteria use mechanisms of cellulase action different from that used in the fungi.