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Cellulose degrading enzymes and their potential industrial applications
FEMS Microbiology Letters 50 (1988) 247-252 Published by Elsevier
247
FEM 03167
Breakdown of crystalline cellulose by synergistic action between cellulase components from Clostridium thermocellum and Trichoderma koningii L a u r a A. G o w a n d T h o m a s M. W o o d Rowett ResearchInstitute, Bucksburn, Aberdeen, U.K.
Received 17 December 1987 Revision received 15 January 1988 Accepted 20 January 1988 Key words: Cellulase; Synergism; Clostridium thermocellum," Trichoderma koningii; Cellobiohydrolase; (Mechanism of action)
1. S U M M A R Y Certain isolated components of fungal cellulases, which cannot effect the breakdown of highly ordered cellulose individually, interact together synergistically to do so when recombined. Surprisingly, 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 thermocellure cannot effect the extensive breakdown of highly ordered cellulose unless Ca z+ and dithiothreitol (DTT) are present. However, we now report that isolated cellobiohydrolase from Trichoderma koningii can combine with C. thermocellure cellulase to effect the breakdown of cellulose in the absence of Ca 2+ and DTT. Enhanced activity is observed if Ca z+ and D T T are present. This finding m a y have important applications in industry: it certainly has important implications
Correspondence to." Thomas M. Wood, Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, U.K.
for those interested in the basic mechanism of cellulase action in C. thermocellum.
2. I N T R O D U C T I O N There is now a world-wide interest in the use of cellulases for the industrial recycling of biomass to provide energy and food. With this development in mind, the extracellular cellulases of the anaerobe C. thermocellum [1-3] and the fungi Trichoderma reesei, T. koningii, Trichoderma oiride, Fusariurn solani and Penicillium pinophilum /funiculosum have been the subject of much research [4-6]. The interest in these cellulases, which are described in the literature as ' t r u e ' cellulases, is a result of their being able to solubilise hydrogen-bond-ordered (crystalline) cellulose to an extensive degree. Fractionation and other studies carried out on the 'true' cellulases of the fungi mentioned have established that the distinguishing feature of these cellulases is that they contain a cellobiohydrolase, in addition to the endoglucanases and fl-glucosidases that are found in the extracellular cellulases of most other cellulolytic microorganisms
0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies
248 [4]. When isolated and purified to homogeneity, these enzyme components are unable to effect the breakdown of crystalline cellulose, but when recombined act synergistically to render the cellulose soluble. Unfortunately, no such studies have been possible with the cellulase of C. thermocellum, which exists in solution as a multi-component enzyme complex, termed a cellulosome [1]. The integrity of this complex, which has been shown to contain a multiplicity of endoglucanase components, would appear to be essential for the manifestation of activity to crystalline cellulose [7]. So far, only partial disruption of the complex has been possible. Some data have been interpreted to indicate that a cellobiohydrolase may be involved in the complex, but the evidence cannot be regarded as definitive [8,9]. It seems also that a cellulose-binding factor is concerned in binding the enzyme to the substrate [1], and that Ca 2+ and D T T are required for activity to crystalline cellulose [2]. In view of the apparent differences between C. thermocellum cellulase and the fungal cellulases it is interesting to be able to report that the cellobiohydrolase of T. koningii can act synergistically with the cellulase of C. thermocellum to solubilise crystalline cellulose in the form of cotton fibre. This finding will clearly be of interest to those working on the basic mechanism of cellulase action in C. thermoeellum; the implications of the present observations are discussed in that context.
3. MATERIALS A N D M E T H O D S 3.1. Growth conditions C. thermocellum (NCIB10682) was grown without stirring at 6 0 ° C under N 2 gas in GS medium [10] except that cellulose (Solka Floc SW40; 10 g.1 -a) was used in place of cellobiose. Cultures (50 ml) were grown in Wheaton bottles (100 ml) and 500-ml cultures were grown in anaerobic bottles (1.0 1) adapted as described [11]. Cells were harvested after 3 - 4 days when about 10% of the cellulose remained undigested. T. koningii, cultured collection No. CMI73022, was obtained from the Commonwealth Mycological Institute, Kew, Surrey, U.K. Cultures were
grown in Mandels' medium [12] at 3 0 ° C in a 15-1 New Brunswick Microgen fermenter at an aeration rate of 0.5 vol./vol./min. Cultures were harvested after 7 days. 3.2. Assay of enzyme activities Cotton fibre-solubilising activity was measured using dewaxed fibre, as described [13]. Reaction mixtures were examined microscopically to check whether growth of microorganisms had occurred during the incubation. No such growth was observed. Endoglucanase activity was assayed in 50 m M MES, p H 6.0 by measuring either (a) reducing sugars produced [14]; or (b) the change in viscosity [15] of a 1% ( w / v ) solution of carboxymethylcelhilose (low viscosity). Cellobiohydrolase activity was assayed by measuring either the reducing sugars produced from a solution of phosphoric acid-swollen cellulose [16], or the decrease in absorbance of a solution of Avicel (3 mg in 10 ml) in a turbidity assay [2]. 3.3. Chemical determinations Reducing sugars were measured using the Somogyi-Nelson method [17,18] and protein by the method of Bradford [19]. 3.4. Isolation of enzymes C. thermocellum cultures were filtered through Whatman G F / C filters. The retentates were washed with water to remove cellulase that might still be attached to cells or cellulose. The combined water washings and filtrates were centrifuged (77000 × g; 20 min) to remove small fine particles of cellulose and ammonium sulphate was added to the supernatant to 80% saturation at 0 ° C . The precipitate obtained was centrifuged (77000 × g; 20 min) and redissolved in 50 mM MES buffer, p H 6.0, which was 10 m M with respect to DTT, and 7 m M with respect to CaC12Recovery of CM-cellulase was normally in excess of 95%. This partially purified preparation was used for assay of cotton fibre-sohibilising activity. T. koningii cellobiohydrolase was purified using ion exchange chromatography on DEAE-Sephadex as described in [20].
249 Table 2
4. R E S U L T S 4.1. Effect o f cellobiohydrolase on the breakdown o f cotton fibre by C. t h e r m o c e l l u m cellulase C. thermocellum cellulase has b e e n shown to b e m o s t active at t e m p e r a t u r e s in excess of 70 o C a n d at p H 5.7 [2]. T h e cellulase of the m e s o p h i l i c fungus 7". koningii, however, was m o s t active at 40 ° C a n d at p H 5 [12] a n d these c o n d i t i o n s were c h o s e n to m a x i m i s e a n y effect t h a t c e l l o b i o h y d r o lase might have w h e n acting in a s s o c i a t i o n w i t h the b a c t e r i a l cellulase. This c e l l o b i o h y d r o l a s e is i n a c t i v a t e d on p r o l o n g e d i n c u b a t i o n at 60 o C. A t 40 ° C a n d p H 5, C. thermocellum cellulase showed very little c a p a c i t y for solubilising crystalline cellulose in the f o r m of the c o t t o n fibre %) ( T a b l e 1). U n d e r the s a m e conditions, p u r i f i e d ceUobiohydrolase was u n a b l e to d e g r a d e the s a m e substrate. However, w h e n the b a c t e r i a l e n z y m e was c o m b i n e d with cellobiohydrolase, c o t t o n fibre was solubilised to the extent of 40%. T h e a d d i t i o n of C a 2+ or D T T resulted in further i m p r o v e m e n t s in the a p p a r e n t c o - o p e r a t i o n b e t w e e n the fungal a n d b a c t e r i a l enzymes. Thus, in the presence of C a 2+ the c o t t o n was solubilised to the extent of 20% b y the C. thermocellum enz y m e when acting alone, b u t s o l u b i l i s a t i o n was a r e m a r k a b l e 55% w h e n fungal c e l l o b i o h y d r o l a s e was also present. However, the highest activity
Table 1 Percentage hydrolysis of cotton by cellulases of C. thermocellurn and T. koningii acting alone and in combination Additions to C. therrnocetlum cellulase or T. koningii cellobiohydrolase None 10 mM DTT 7 mM CaCI 2 10 mM DTT + 7 mM CaC12
C. thermocetlum cellulase (A)
T. koningii cellobiohydrolase (B)
A+B
8
0
0 20
0 0
40 35 55
35
0
68
2 mg dewaxed cotton was incubated at 40 o C for 7 days in 50 mM sodium acetate buffer pH 5.0 with additions as indicated. The following quantities were used. C. therrnocellum cellulase, 0.37 IU; T. koningii cellobiohydrolase, 180 btg. Values are % hydrolysis.
Percentage of hydrolysis of Avicel by cellulase of C. thermocellure and T. koningii alone and in combination Avicel (3 mg) was incubated at 40°C for 18 h in sodium acetate buffer, pH 5.0 with additions as indicated. Activity was measured by following the decrease in turbidity at 660 nm. Enzyme additions were as in the legend to Table 1. Values are % hydrolysis. Additions to C. thermocellum cellulase or T. koningii cellobiohydrolase
C. thermocellum cellulase (A)
72. koningii cellobiohydrolase (B)
A+B
None 10 mM DTT 7 mM CaC12 10 mM DTI" + 7 mM CaC12
17 15 24
3 3 2
28 31 35
31
3
35
was o b t a i n e d w h e n b o t h C a 2+ a n d D T T were p r e s e n t (68%), d e s p i t e the fact that the a d d i t i o n of D T T a l o n e c o m p l e t e l y i n h i b i t e d the a c t i o n of C. thermocellum cellulase o n cotton, a n d slightly inh i b i t e d the c o m b i n e d a c t i o n of c e l l o b i o h y d r o l a s e a n d C. thermocellum cellulase (40% to 35%). N e i t h e r D T T n o r C a 2+ a p p e a r e d to affect cell o b i o h y d r o l a s e activity. This was d e t e r m i n e d using the s u b s t r a t e p h o s p h o r i c acid-swollen cellulose which is k n o w n to b e r e a d i l y h y d r o l y s e d b y the c e l l o b i o h y d r o l a s e [12]. T h e c o n c e n t r a t i o n s of C a z+ a n d D T T tested were those a l r e a d y s h o w n to b e o p t i m a l for the activity of C. thermocellum cellulase acting alone [9]. T h e p o s s i b i l i t y that these c o n d i t i o n s m a y n o t b e the b e s t for the c o m b i n e d a c t i o n of the cell o b i o h y d r o l a s e a n d the C. thermocellum cellulase has n o t y e t b e e n tested. Synergistic effects were also o b s e r v e d b e t w e e n C. thermocellum cellulase a n d T. koningii cell o b i o h y d r o l a s e using A v i c e l as substrate, b u t the c o - o p e r a t i o n was less m a r k e d ( T a b l e 2). 4.2. Effect o f Ca 2+ and D T T on C. t h e r m o c e l l u m cellulase A n o t h e r o b s e r v a t i o n w h i c h relates to the effect of D T T a n d C a 2÷ o n the activity of C. thermocellum cellulase r e c o r d e d in T a b l e 1 is w o r t h y of c o m m e n t . It has b e e n p r e v i o u s l y o b s e r v e d a n d
250
recorded that D T T is stimulatory [2] though low concentrations were later found to be inhibitory [9]. We now report that the C. thermocellum cellulase preparation used in the present study is completely inactivated in the presence of 10 mM DTT. It is also interesting to note that the presence of Ca 2÷ along with the D T T results in the highest cellulase activity (68%), and the presence of Ca 2÷ alone results in appreciable activity (20%). It seems that Ca 2+ protects against the inhibitory effect of DTT. Neither Ca 2+ nor D T T affected endoglucanase activity when the activity was measured using a solution of carboxymethylcellulose and the viscosity method (data not shown). 4. 3. Effect of temperature on synergism The optimum temperature for the activity of C. thermocellum cellulase has been reported to be 70 ° C when Avicel was the substrate and when the
incubation was carried out for only 18 h. However, as the cellobiohydrolase of T. koningii is inactivated at this high temperature on prolonged incubation [13], the study of the synergistic interaction of cellobiohydrolase and bacterial cellulase was done only in the temperature range 40-60 ° C. In the present investigation, C. thermocellum cellulase was found to be most active at 50 ° C in the prolonged incubation needed to solubilise the refractory cotton fibre (Table 3). The highest percentage of hydrolysis shown by the mixture of fungal and bacterial enzymes was found at 50 ° C in the presence of both Ca 2+ and D T T (Table 3). However, it was at 4 0 ° C that the synergistic activity between cellobiohydrolase and C. thermocellum cellulase was most pronounced. At 60 ° C the percentage hydrolysis fell even in the presence of D T T and Ca 2+, almost certainly as a result of the complete loss of activity of the cellobiohydrolase.
Table 3 Effect of temperature on C. thermocellum cellulase and its synergism with T. koningii cellobiohydrolase
5. DISCUSSION
2 m g dewaxed cotton was incubated at various temperatures for 7 days in 50 m M sodium acetate buffer, p H 5.0. Enzyme concentrations are as described in the legend to Table 1. Values are % hydrolysis.
To a large extent our ignorance of the mechanism of cellulase action of the C. thermocellum cellulase has been a consequence of the apparent inability of investigators to fractionate the cellulase complex into its component parts. That the cellulase consists of several endoglucanases is well established, but the presence of a cellobiohydrolase has not been demonstrated unequivocally. Because the activity of the complete cellulase system to crystalline cellulose has been shown to be affected by Ca 2+ and DTT, but the activity of the endoglucanases is not, it has been postulated that an unstable cellobiohydrolase may exist and that both cellobiohydrolase and endoglucanase activities are essential for cellulose breakdown [8,9]. Our finding that fungal cellobiohydrolases and C. thermocellum cellulase interact synergistically to hydrolyse crystalline cellulose in the absence of C a 2+ and D T T could support this hypothesis and give it more credence. The significance of the observed synergism between the eucaryotic and the procaryotic cellulases is highlighted by the studies that have already been carried out on the effectiveness of various
Enzyme
C. thermocellum cellulase alone
Temperature ( ° C) 40
50
60
8
12
4
C. thermocellum cellulase plus D T T (10 mM) and Ca 2+ (7 raM)
45
79
72
C. thermocellum cellulase plus T. koningii cellobiobydrolase
40
34
5
T. koningii cellobiohydrolase plus DTI? and Ca 2÷
0
0
0
C. thermocellurn cellulase plus T. koningii cellobiohydrolase plus D T T and Ca 2+
60
83
75
251
combinations of cellobiohydrolases and endoglucanases from different sources to solubilise crystalline cellulose. It has been observed that a high degree of synergistic activity is shown by any combination of cellobiohydrolase and endoglucanase of the cellulases of the fungi T. koningii, F. solani, and P. pinophilum, but that very little co-operation is shown between cellobiohydrolase of these fungi and the endoglucanases of the fungi, Myrothecium uerrucaria, Stachybotrys atra, and Memnoniella echinata [4]. Similarly, endoglucanases from the rumen bacteria Ruminococcus albus, Ruminococcus flavefaciens and Bacteroides succinogenes have shown no capacity for acting synergistically with the cellobiohydrolases from the fungi using cotton as substrates [21]. It has been suggested that these observations are highly significant in terms of the mechanism of cellulase action, and they have been rationalised to indicate that synergism between fungal cellobiohydrolase and endoglucanase originating in another microorganism occurs only in those situations where the endoglucanase has been isolated from a cellulase preparation that also contained a cellobiohydrolase, i.e., the cellulase preparation from which it was isolated was a 'true' cellulase [22]. Very few 'true' cellulases are known. The extracellular cellulases from the fungi M. verrucaria, S. atra and M. echinata which were unable to act synergistically with the fungal cellobiohydrolase are not 'true' cellulases; and the cellulases of the rumen bacteria, R. flavefaciens and B. succinogenes are the same in this respect. Given the special properties that are possessed by the cellulase of C. thermocellum, in particular the existence of a multi-component cellulase complex, and the fact that the integrity of this complex must be maintained for the activity to crystalline cellulose to be observed [7], it is indeed remarkable that the fungal cellobiohydrolase and the bacterial cellulase can act synergistically. As C. thermocellum cellulase and T. koningii cellobiohydrolase show activity in 10 mM D T T only when mixed, it is difficult to explain this result without assuming that some kind of complex is involved. Extrapolating further, it is intriguing to consider the possibility that the fungal cellobiohydrolase can (a) be integrated into the bacterial
cellulase complex in the correct orientation with respect to the endoglucanases and the substrate for effective hydrolysis of crystalline cellulose; and (b) has some affinity for the cellulose-binding factor which has been shown to exist in the C. thermocellum cellulase, and which is important in binding the enzyme to the substrate [1]. By using fungal cellobiohydrolase as a 'probe' it may now be possible to identify the cellulose-binding factor and other important enzymes of the cellulase complex, once the complex has been partially disrupted by dissociating agents. This new approach may therefore help to shed some new light on the complex interactions involved in the breakdown of cellulose by this commercially important cellulase: the direct approach of studying the component enzymes and their ability to act synergistically in a reconstituted mixture has not proved very fruitful so far [7].
ACKNOWLEDGEMENTS This work was supported by funds from the Commission for European Communities, Contract EN 3B-0084-UK (HI).
REFERENCES [1] Lamed, R., Setter, E. and Bayer, E.A. (1983) J. Bacteriol. 156, 828-936. [2] Johnson, E.A., Sakajoh, M., Halliwell, G., Madia, A. and Demain, A.L. (1982) Appl. Environ. Microbiol. 43, 1125-1132. [3] Beguin, P., Millet, J. and Aubert, J.-P. (1987) Microbiol. Sci. 4, 277-280. [4] Wood, T.M. and McCrae, S.I. (1979) Adv. Chem. Ser. 181, 181-209. [5] Wood, T.M. (1985) Biochem. Soc. Trans. 13, 407-410. [6] Mandels, M. (1982) in Annual Report on Fermentation Processes 5, 35-48. [7] Lamed, R. and Bayer, E.A. (1988) Adv. Appl. Microbiol., (in press). [8] Lamed, R., Kenig, R. and Setter, E. (1985) Enzyme Microbiol. Technol. 7, 37-41. [9] Johnson, E.A. and Demain, A.C. (1984) Arch. Microbiol. 137, 135-138. [10] Garcia-Martinez, D.v., Shinmyo, A., Madia, A. and Demain, A.L. (1980) Eur. J. Microbiol. Biotechnol. 9, 189-197.
252 [11] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R. and Wolfe, R.S. (1979) Microbiol. Rev. 43, 260-296. [12] Mandels, M. and Weber, J. (1969) Adv. Chem. Ser. 95, 391-414. [13] Wood, T.M. (1969) Biochem. J. 115, 457-464. [14] Wood, T.M. (1968) Biochem. J. 109, 217-227. [15] Wood, T.M. (1971) Biochem. J. 121, 353-362. [16] Wood, T.M. and McCrae, S.I. (1977) Carbohydr. Res., 57, 117-133. [17] Nelson, N. (1944) J. Biol. Chem. 153, 375-380.
[18] Somogyi, (1952) J. Biol. Chem. 195, 19-23. [19] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [20] Wood, T.M. and McCrae, S.I. (1972) Biochem. J. 128, 1183-1192. [21] Wood, T.M. (1983) in Biomass as a Source of Industrial Chemicals, (Heslot, H. and Villet, R., Eds.) pp. 55-80. Adeprina, Paris. [22] Wood, T.M. (1981) in Proceedings of The Ekman-Days International Symposium on Wood Pulping Chemistry 3, pp. 31-36. SPCI, Stockholm.
247
FEM 03167
Breakdown of crystalline cellulose by synergistic action between cellulase components from Clostridium thermocellum and Trichoderma koningii L a u r a A. G o w a n d T h o m a s M. W o o d Rowett ResearchInstitute, Bucksburn, Aberdeen, U.K.
Received 17 December 1987 Revision received 15 January 1988 Accepted 20 January 1988 Key words: Cellulase; Synergism; Clostridium thermocellum," Trichoderma koningii; Cellobiohydrolase; (Mechanism of action)
1. S U M M A R Y Certain isolated components of fungal cellulases, which cannot effect the breakdown of highly ordered cellulose individually, interact together synergistically to do so when recombined. Surprisingly, 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 thermocellure cannot effect the extensive breakdown of highly ordered cellulose unless Ca z+ and dithiothreitol (DTT) are present. However, we now report that isolated cellobiohydrolase from Trichoderma koningii can combine with C. thermocellure cellulase to effect the breakdown of cellulose in the absence of Ca 2+ and DTT. Enhanced activity is observed if Ca z+ and D T T are present. This finding m a y have important applications in industry: it certainly has important implications
Correspondence to." Thomas M. Wood, Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, U.K.
for those interested in the basic mechanism of cellulase action in C. thermocellum.
2. I N T R O D U C T I O N There is now a world-wide interest in the use of cellulases for the industrial recycling of biomass to provide energy and food. With this development in mind, the extracellular cellulases of the anaerobe C. thermocellum [1-3] and the fungi Trichoderma reesei, T. koningii, Trichoderma oiride, Fusariurn solani and Penicillium pinophilum /funiculosum have been the subject of much research [4-6]. The interest in these cellulases, which are described in the literature as ' t r u e ' cellulases, is a result of their being able to solubilise hydrogen-bond-ordered (crystalline) cellulose to an extensive degree. Fractionation and other studies carried out on the 'true' cellulases of the fungi mentioned have established that the distinguishing feature of these cellulases is that they contain a cellobiohydrolase, in addition to the endoglucanases and fl-glucosidases that are found in the extracellular cellulases of most other cellulolytic microorganisms
0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies
248 [4]. When isolated and purified to homogeneity, these enzyme components are unable to effect the breakdown of crystalline cellulose, but when recombined act synergistically to render the cellulose soluble. Unfortunately, no such studies have been possible with the cellulase of C. thermocellum, which exists in solution as a multi-component enzyme complex, termed a cellulosome [1]. The integrity of this complex, which has been shown to contain a multiplicity of endoglucanase components, would appear to be essential for the manifestation of activity to crystalline cellulose [7]. So far, only partial disruption of the complex has been possible. Some data have been interpreted to indicate that a cellobiohydrolase may be involved in the complex, but the evidence cannot be regarded as definitive [8,9]. It seems also that a cellulose-binding factor is concerned in binding the enzyme to the substrate [1], and that Ca 2+ and D T T are required for activity to crystalline cellulose [2]. In view of the apparent differences between C. thermocellum cellulase and the fungal cellulases it is interesting to be able to report that the cellobiohydrolase of T. koningii can act synergistically with the cellulase of C. thermocellum to solubilise crystalline cellulose in the form of cotton fibre. This finding will clearly be of interest to those working on the basic mechanism of cellulase action in C. thermoeellum; the implications of the present observations are discussed in that context.
3. MATERIALS A N D M E T H O D S 3.1. Growth conditions C. thermocellum (NCIB10682) was grown without stirring at 6 0 ° C under N 2 gas in GS medium [10] except that cellulose (Solka Floc SW40; 10 g.1 -a) was used in place of cellobiose. Cultures (50 ml) were grown in Wheaton bottles (100 ml) and 500-ml cultures were grown in anaerobic bottles (1.0 1) adapted as described [11]. Cells were harvested after 3 - 4 days when about 10% of the cellulose remained undigested. T. koningii, cultured collection No. CMI73022, was obtained from the Commonwealth Mycological Institute, Kew, Surrey, U.K. Cultures were
grown in Mandels' medium [12] at 3 0 ° C in a 15-1 New Brunswick Microgen fermenter at an aeration rate of 0.5 vol./vol./min. Cultures were harvested after 7 days. 3.2. Assay of enzyme activities Cotton fibre-solubilising activity was measured using dewaxed fibre, as described [13]. Reaction mixtures were examined microscopically to check whether growth of microorganisms had occurred during the incubation. No such growth was observed. Endoglucanase activity was assayed in 50 m M MES, p H 6.0 by measuring either (a) reducing sugars produced [14]; or (b) the change in viscosity [15] of a 1% ( w / v ) solution of carboxymethylcelhilose (low viscosity). Cellobiohydrolase activity was assayed by measuring either the reducing sugars produced from a solution of phosphoric acid-swollen cellulose [16], or the decrease in absorbance of a solution of Avicel (3 mg in 10 ml) in a turbidity assay [2]. 3.3. Chemical determinations Reducing sugars were measured using the Somogyi-Nelson method [17,18] and protein by the method of Bradford [19]. 3.4. Isolation of enzymes C. thermocellum cultures were filtered through Whatman G F / C filters. The retentates were washed with water to remove cellulase that might still be attached to cells or cellulose. The combined water washings and filtrates were centrifuged (77000 × g; 20 min) to remove small fine particles of cellulose and ammonium sulphate was added to the supernatant to 80% saturation at 0 ° C . The precipitate obtained was centrifuged (77000 × g; 20 min) and redissolved in 50 mM MES buffer, p H 6.0, which was 10 m M with respect to DTT, and 7 m M with respect to CaC12Recovery of CM-cellulase was normally in excess of 95%. This partially purified preparation was used for assay of cotton fibre-sohibilising activity. T. koningii cellobiohydrolase was purified using ion exchange chromatography on DEAE-Sephadex as described in [20].
249 Table 2
4. R E S U L T S 4.1. Effect o f cellobiohydrolase on the breakdown o f cotton fibre by C. t h e r m o c e l l u m cellulase C. thermocellum cellulase has b e e n shown to b e m o s t active at t e m p e r a t u r e s in excess of 70 o C a n d at p H 5.7 [2]. T h e cellulase of the m e s o p h i l i c fungus 7". koningii, however, was m o s t active at 40 ° C a n d at p H 5 [12] a n d these c o n d i t i o n s were c h o s e n to m a x i m i s e a n y effect t h a t c e l l o b i o h y d r o lase might have w h e n acting in a s s o c i a t i o n w i t h the b a c t e r i a l cellulase. This c e l l o b i o h y d r o l a s e is i n a c t i v a t e d on p r o l o n g e d i n c u b a t i o n at 60 o C. A t 40 ° C a n d p H 5, C. thermocellum cellulase showed very little c a p a c i t y for solubilising crystalline cellulose in the f o r m of the c o t t o n fibre %) ( T a b l e 1). U n d e r the s a m e conditions, p u r i f i e d ceUobiohydrolase was u n a b l e to d e g r a d e the s a m e substrate. However, w h e n the b a c t e r i a l e n z y m e was c o m b i n e d with cellobiohydrolase, c o t t o n fibre was solubilised to the extent of 40%. T h e a d d i t i o n of C a 2+ or D T T resulted in further i m p r o v e m e n t s in the a p p a r e n t c o - o p e r a t i o n b e t w e e n the fungal a n d b a c t e r i a l enzymes. Thus, in the presence of C a 2+ the c o t t o n was solubilised to the extent of 20% b y the C. thermocellum enz y m e when acting alone, b u t s o l u b i l i s a t i o n was a r e m a r k a b l e 55% w h e n fungal c e l l o b i o h y d r o l a s e was also present. However, the highest activity
Table 1 Percentage hydrolysis of cotton by cellulases of C. thermocellurn and T. koningii acting alone and in combination Additions to C. therrnocetlum cellulase or T. koningii cellobiohydrolase None 10 mM DTT 7 mM CaCI 2 10 mM DTT + 7 mM CaC12
C. thermocetlum cellulase (A)
T. koningii cellobiohydrolase (B)
A+B
8
0
0 20
0 0
40 35 55
35
0
68
2 mg dewaxed cotton was incubated at 40 o C for 7 days in 50 mM sodium acetate buffer pH 5.0 with additions as indicated. The following quantities were used. C. therrnocellum cellulase, 0.37 IU; T. koningii cellobiohydrolase, 180 btg. Values are % hydrolysis.
Percentage of hydrolysis of Avicel by cellulase of C. thermocellure and T. koningii alone and in combination Avicel (3 mg) was incubated at 40°C for 18 h in sodium acetate buffer, pH 5.0 with additions as indicated. Activity was measured by following the decrease in turbidity at 660 nm. Enzyme additions were as in the legend to Table 1. Values are % hydrolysis. Additions to C. thermocellum cellulase or T. koningii cellobiohydrolase
C. thermocellum cellulase (A)
72. koningii cellobiohydrolase (B)
A+B
None 10 mM DTT 7 mM CaC12 10 mM DTI" + 7 mM CaC12
17 15 24
3 3 2
28 31 35
31
3
35
was o b t a i n e d w h e n b o t h C a 2+ a n d D T T were p r e s e n t (68%), d e s p i t e the fact that the a d d i t i o n of D T T a l o n e c o m p l e t e l y i n h i b i t e d the a c t i o n of C. thermocellum cellulase o n cotton, a n d slightly inh i b i t e d the c o m b i n e d a c t i o n of c e l l o b i o h y d r o l a s e a n d C. thermocellum cellulase (40% to 35%). N e i t h e r D T T n o r C a 2+ a p p e a r e d to affect cell o b i o h y d r o l a s e activity. This was d e t e r m i n e d using the s u b s t r a t e p h o s p h o r i c acid-swollen cellulose which is k n o w n to b e r e a d i l y h y d r o l y s e d b y the c e l l o b i o h y d r o l a s e [12]. T h e c o n c e n t r a t i o n s of C a z+ a n d D T T tested were those a l r e a d y s h o w n to b e o p t i m a l for the activity of C. thermocellum cellulase acting alone [9]. T h e p o s s i b i l i t y that these c o n d i t i o n s m a y n o t b e the b e s t for the c o m b i n e d a c t i o n of the cell o b i o h y d r o l a s e a n d the C. thermocellum cellulase has n o t y e t b e e n tested. Synergistic effects were also o b s e r v e d b e t w e e n C. thermocellum cellulase a n d T. koningii cell o b i o h y d r o l a s e using A v i c e l as substrate, b u t the c o - o p e r a t i o n was less m a r k e d ( T a b l e 2). 4.2. Effect o f Ca 2+ and D T T on C. t h e r m o c e l l u m cellulase A n o t h e r o b s e r v a t i o n w h i c h relates to the effect of D T T a n d C a 2÷ o n the activity of C. thermocellum cellulase r e c o r d e d in T a b l e 1 is w o r t h y of c o m m e n t . It has b e e n p r e v i o u s l y o b s e r v e d a n d
250
recorded that D T T is stimulatory [2] though low concentrations were later found to be inhibitory [9]. We now report that the C. thermocellum cellulase preparation used in the present study is completely inactivated in the presence of 10 mM DTT. It is also interesting to note that the presence of Ca 2÷ along with the D T T results in the highest cellulase activity (68%), and the presence of Ca 2÷ alone results in appreciable activity (20%). It seems that Ca 2+ protects against the inhibitory effect of DTT. Neither Ca 2+ nor D T T affected endoglucanase activity when the activity was measured using a solution of carboxymethylcellulose and the viscosity method (data not shown). 4. 3. Effect of temperature on synergism The optimum temperature for the activity of C. thermocellum cellulase has been reported to be 70 ° C when Avicel was the substrate and when the
incubation was carried out for only 18 h. However, as the cellobiohydrolase of T. koningii is inactivated at this high temperature on prolonged incubation [13], the study of the synergistic interaction of cellobiohydrolase and bacterial cellulase was done only in the temperature range 40-60 ° C. In the present investigation, C. thermocellum cellulase was found to be most active at 50 ° C in the prolonged incubation needed to solubilise the refractory cotton fibre (Table 3). The highest percentage of hydrolysis shown by the mixture of fungal and bacterial enzymes was found at 50 ° C in the presence of both Ca 2+ and D T T (Table 3). However, it was at 4 0 ° C that the synergistic activity between cellobiohydrolase and C. thermocellum cellulase was most pronounced. At 60 ° C the percentage hydrolysis fell even in the presence of D T T and Ca 2+, almost certainly as a result of the complete loss of activity of the cellobiohydrolase.
Table 3 Effect of temperature on C. thermocellum cellulase and its synergism with T. koningii cellobiohydrolase
5. DISCUSSION
2 m g dewaxed cotton was incubated at various temperatures for 7 days in 50 m M sodium acetate buffer, p H 5.0. Enzyme concentrations are as described in the legend to Table 1. Values are % hydrolysis.
To a large extent our ignorance of the mechanism of cellulase action of the C. thermocellum cellulase has been a consequence of the apparent inability of investigators to fractionate the cellulase complex into its component parts. That the cellulase consists of several endoglucanases is well established, but the presence of a cellobiohydrolase has not been demonstrated unequivocally. Because the activity of the complete cellulase system to crystalline cellulose has been shown to be affected by Ca 2+ and DTT, but the activity of the endoglucanases is not, it has been postulated that an unstable cellobiohydrolase may exist and that both cellobiohydrolase and endoglucanase activities are essential for cellulose breakdown [8,9]. Our finding that fungal cellobiohydrolases and C. thermocellum cellulase interact synergistically to hydrolyse crystalline cellulose in the absence of C a 2+ and D T T could support this hypothesis and give it more credence. The significance of the observed synergism between the eucaryotic and the procaryotic cellulases is highlighted by the studies that have already been carried out on the effectiveness of various
Enzyme
C. thermocellum cellulase alone
Temperature ( ° C) 40
50
60
8
12
4
C. thermocellum cellulase plus D T T (10 mM) and Ca 2+ (7 raM)
45
79
72
C. thermocellum cellulase plus T. koningii cellobiobydrolase
40
34
5
T. koningii cellobiohydrolase plus DTI? and Ca 2÷
0
0
0
C. thermocellurn cellulase plus T. koningii cellobiohydrolase plus D T T and Ca 2+
60
83
75
251
combinations of cellobiohydrolases and endoglucanases from different sources to solubilise crystalline cellulose. It has been observed that a high degree of synergistic activity is shown by any combination of cellobiohydrolase and endoglucanase of the cellulases of the fungi T. koningii, F. solani, and P. pinophilum, but that very little co-operation is shown between cellobiohydrolase of these fungi and the endoglucanases of the fungi, Myrothecium uerrucaria, Stachybotrys atra, and Memnoniella echinata [4]. Similarly, endoglucanases from the rumen bacteria Ruminococcus albus, Ruminococcus flavefaciens and Bacteroides succinogenes have shown no capacity for acting synergistically with the cellobiohydrolases from the fungi using cotton as substrates [21]. It has been suggested that these observations are highly significant in terms of the mechanism of cellulase action, and they have been rationalised to indicate that synergism between fungal cellobiohydrolase and endoglucanase originating in another microorganism occurs only in those situations where the endoglucanase has been isolated from a cellulase preparation that also contained a cellobiohydrolase, i.e., the cellulase preparation from which it was isolated was a 'true' cellulase [22]. Very few 'true' cellulases are known. The extracellular cellulases from the fungi M. verrucaria, S. atra and M. echinata which were unable to act synergistically with the fungal cellobiohydrolase are not 'true' cellulases; and the cellulases of the rumen bacteria, R. flavefaciens and B. succinogenes are the same in this respect. Given the special properties that are possessed by the cellulase of C. thermocellum, in particular the existence of a multi-component cellulase complex, and the fact that the integrity of this complex must be maintained for the activity to crystalline cellulose to be observed [7], it is indeed remarkable that the fungal cellobiohydrolase and the bacterial cellulase can act synergistically. As C. thermocellum cellulase and T. koningii cellobiohydrolase show activity in 10 mM D T T only when mixed, it is difficult to explain this result without assuming that some kind of complex is involved. Extrapolating further, it is intriguing to consider the possibility that the fungal cellobiohydrolase can (a) be integrated into the bacterial
cellulase complex in the correct orientation with respect to the endoglucanases and the substrate for effective hydrolysis of crystalline cellulose; and (b) has some affinity for the cellulose-binding factor which has been shown to exist in the C. thermocellum cellulase, and which is important in binding the enzyme to the substrate [1]. By using fungal cellobiohydrolase as a 'probe' it may now be possible to identify the cellulose-binding factor and other important enzymes of the cellulase complex, once the complex has been partially disrupted by dissociating agents. This new approach may therefore help to shed some new light on the complex interactions involved in the breakdown of cellulose by this commercially important cellulase: the direct approach of studying the component enzymes and their ability to act synergistically in a reconstituted mixture has not proved very fruitful so far [7].
ACKNOWLEDGEMENTS This work was supported by funds from the Commission for European Communities, Contract EN 3B-0084-UK (HI).
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252 [11] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R. and Wolfe, R.S. (1979) Microbiol. Rev. 43, 260-296. [12] Mandels, M. and Weber, J. (1969) Adv. Chem. Ser. 95, 391-414. [13] Wood, T.M. (1969) Biochem. J. 115, 457-464. [14] Wood, T.M. (1968) Biochem. J. 109, 217-227. [15] Wood, T.M. (1971) Biochem. J. 121, 353-362. [16] Wood, T.M. and McCrae, S.I. (1977) Carbohydr. Res., 57, 117-133. [17] Nelson, N. (1944) J. Biol. Chem. 153, 375-380.
[18] Somogyi, (1952) J. Biol. Chem. 195, 19-23. [19] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [20] Wood, T.M. and McCrae, S.I. (1972) Biochem. J. 128, 1183-1192. [21] Wood, T.M. (1983) in Biomass as a Source of Industrial Chemicals, (Heslot, H. and Villet, R., Eds.) pp. 55-80. Adeprina, Paris. [22] Wood, T.M. (1981) in Proceedings of The Ekman-Days International Symposium on Wood Pulping Chemistry 3, pp. 31-36. SPCI, Stockholm.