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1 FEMS Microbiology Letters 147 (1997) 239^243 Synergistic interaction of the Clostridium stercorarium cellulases Avicelase I (CelZ) and Avicelase II (CelY) in the degradation of microcrystalline cellulose Abstract Kathrin Riedel, Johannes Ritter, Karin Bronnenmeier * Lehrstuhl fuër Mikrobiologie, Technische Universitaët Muënchen, Muënchen, Germany Received 28 November 1996; revised 18 December 1996; accepted 20 December 1996 Avicelase I and Avicelase II purified from the cellulolytic thermophile Clostridium stercorarium acted in synergism to hydrolyze microcrystalline cellulose. The degree of synergism proved to be dependent on the ratio of the two enzymes and on the type of the cellulosic substrate. The activity of the combined enzymes towards Avicel was about double the sum of the individual activities. No synergism was found with amorphous cellulose preparations. It is shown that the simultaneous concerted action of both Avicelases is required to observe synergism. We suggest that synergism results from an exo-exo type cooperativity and present a mechanistic model explaining the synergistic interaction between Avicelase I and Avicelase II. Keywords: Clostridium; Synergism; Avicelase; Cellulase; Endoglucanase; Exoglucanase 1. Introduction * Corresponding author. Fax: +49 (89) ; bronnen@biol.chemie.tu-muenchen.de The enzymatic sacchari cation of crystalline cellulose by fungal and bacterial enzyme systems or complexes requires the action of several enzymes including endo-1,4-l-glucanases, exo-1,4-l-glucanases (cellobiohydrolases), and L-glucosidases. One of the most intriguing phenomena in cellulose hydrolysis is the synergistic interaction between the individual components. The molecular basis for synergism is not well understood and the published data are inconclusive or even contradictory. Traditionally, the observed synergism is attributed to sequential endoexo cooperativity: in an initial random attack on the amorphous regions of the substrate endoglucanases would provide new chain ends for the action of cellobiohydrolases which would then proceed with the degradation of the crystalline regions. The thermophilic, anaerobic bacterium Clostridium stercorarium produces a `low-complexity' cellulase system consisting only of two cellulolytic enzymes designated Avicelase I and Avicelase II. Avicelase I has been characterized as an unusual cellulolytic enzyme combining both endoglucanase and exoglucanase activities [1]. Avicelase II represents a novel type of exoglucanase exhibiting cellodextrinohydrolase activity [2]. In this paper we report the synergistic e ects observed during reconstitution experiments performed with puri ed Avicelase I and Avicelase II / 97 / $17.00 Copyright ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S (96)

2 240 K. Riedel et al. / FEMS Microbiology Letters 147 (1997) 239^ Materials and methods Avicelase I and Avicelase II were puri ed from culture supernatants of C. stercorarium as described in previous papers [1,2]. The properties of the enzymes are reported therein. Avicel PH 101, Avicel PH 102, Avicel PH 105, cellulose HBS, cellulose MN 300 and cellulose HL were purchased from Serva, cellulose CF 1 and CF 11 were from Whatman. Hydrochloric-acid swollen Avicel was prepared by incubating Avicel in concentrated hydrochloric acid with continuous agitation for 2 h. Enzyme assays were performed at 68³C in 0.75 ml reaction mixtures containing 1% (mass/vol.) suspensions of the substrates in 50 mm Mes/10 mm CaCl 2, ph 6.0. After removal of solids by centrifugation, aliquots (0.5 ml) of the supernatants were further incubated at 37³C for 3 h with 5 U almond L-glucosidase (Sigma) in order to achieve complete conversion of the soluble hydrolysis products into glucose. Glucose was determined as reducing sugar with the 3,5-dinitrosalicylic acid reagent [3]. For accurate determination of concentrations below 50 Wg, an internal standard of 100 Wg glucose was added to the 0.5 ml reaction mixtures. The degree of synergistic e ect (DSE) was de ned according to Beldman et al. [4] as the ratio of the observed activity of the combined enzymes to the sum of the observed individual activities. HPLC analysis of Avicel degradation products was performed at 85³C on an Aminex HPX 42A column (Bio-Rad) with water as eluent. 3. Results and discussion According to their ability to degrade the microcrystalline cellulose substrate Avicel, the cellulolytic enzymes puri ed from C. stercorarium have been designated Avicelase I and Avicelase II. Meanwhile, the genes coding for both cellulases have been cloned and identical substrate speci cities have been determined for the recombinant enzymes produced in E. coli [5,6]. The activity of Avicelase I towards Avicel proved to be higher by an order of magnitude than that determined for Avicelase II [1,2]. When both Avicelases were used together to hydrolyze Avicel Fig. 1. Hydrolysis of Avicel by C. stercorarium cellulases Avicelase I and Avicelase II, alone and in combination. Avicel PH 105 was incubated with 13.3 Wg of puri ed Avicelase I and 40 Wg of puri ed Avicelase II in 5 ml reaction mixtures under standard assay conditions. At the times indicated, aliquots (0.5 ml) were withdrawn for the determination of soluble saccharides as described in Section 2. The numbers in parentheses represent the degree of synergism calculated as described in Section 2. Representative results for one of at least 5 experiments are shown. and other cellulosic substrates, synergistic interaction was observed. With Avicel, the activity of the combined enzymes was almost twice as high as the sum of the activities of the single components (Fig. 1). The degree of synergism proved to be dependent upon the ratio of the cellulolytic enzymes (Fig. 2). Maximal synergistic degradation of Avicel was observed between a 2:1 to 5:1 ratio of Avicelase II to Avicelase I. An in uence of the ratios of cellulase components on the magnitude of the synergistic effect has also been described by several authors for fungal cellulase systems [7^9]. Synergism between the C. stercorarium cellulases depended strongly on the type of cellulosic substrate. Table 1 shows that synergism occurred exclusively when crystalline celluloses were used as substrates. The degree of synergism observed with cellulose MN 300 was approximately twice as high as the values obtained with the di erent Avicel preparations. Although both individual enzymes exhibited signi cantly higher activities towards amorphous cellulose preparations like hydrochloric-acid pretreated Avicel [1,2], no synergism was

3 K. Riedel et al. / FEMS Microbiology Letters 147 (1997) 239^ Table 1 Synergism observed with di erent cellulosic substrates Cellulosic substrate DSE Avicel PH 105 (particle size 0.02 mm) 1.8 Avicel PH 105, hydrochloric acid swollen 0.7 Avicel PH 101 (particle size 0.05 mm) 1.4 Avicel PH 102 (particle size 0.10 mm) 2.1 Cellulose CF 11 ( bres medium) 2.0 Cellulose CF 1 ( bres long) 2.5 Cellulose HL 2.1 Cellulose HBS 2.7 Cellulose MN 300 (particle size 0.002^0.02 mm) 3.8 Standard reaction mixtures containing 1% (mass/vol.) suspensions of the substrates, 4 Wg of puri ed Avicelase I and 20 Wg of puri ed Avicelase II were incubated for 32 h. The DSE (degree of synergistic e ect) was determined as described in Section 2. The table summarizes representative results for one of three independent experiments. The individual values are means of triplicate assays. Fig. 2. Degree of synergism obtained after combining constant amounts of Avicelase I with increasing amounts of Avicelase II. Standard reaction mixtures (0.75 ml) contained 1 Wg of puri ed Avicelase I and corresponding amounts of puri ed Avicelase II. Avicel PH 105 was used as substrate. Representative results for one of three independent experiments are shown. The individual values are means of triplicate assays. found with such a more easily accessible substrate. A comparable dependency between the synergistic action and the nature of the cellulosic substrate has been described for cellulases of Penicillium pinophilum [8], Trichoderma reesei [10] and Thermomonospora fusca [11]. Most remarkably, a simultaneous action of both Avicelases was required to observe synergism (Table 2). This is hard to reconcile with the traditional explanation for synergism, sequential endo-exo cooperativity (reviewed in [12]). Since Avicelase I and Avicelase II had previously been characterized as endoand exoglucanase, respectively, this result was somewhat unexpected. On the other hand, our previous work has demonstrated the ability of Avicelase I to attack crystalline rather than amorphous or soluble substrates in an exoglucanolytic mode of action [1]. Since the results described above for native Avicelase I and Avicelase II puri ed from C. stercorarium could be veri ed with the recombinant enzymes produced in E. coli (data not shown), it can be ruled out that trace amounts of a contaminating endoglucanase are responsible for the synergistic e ect. Therefore, the observed synergy between the two C. stercorarium Avicelases seems to result from exo-exo cooperativity. Exo-exo synergism has been demonstrated between cellobiohydrolases from fungi [7,8,13] and cellulases from T. fusca [11]. For T. reesei this kind of synergism has been attributed to the formation of a `loose complex' between the two cellobiohydrolases CBH I and CBH II prior to adsorption to microcrystalline Avicel [14]. Non-denaturing PAGE and interaction Western blotting [15] were used to examine whether the synergistic e ect described in the present study is brought about by a physical association of C. stercorarium Avicelase I and Avicelase II. We have, however, not found any evidence for such an interaction in free solution, but complex formation might be induced by binding to the surface of the crystalline substrate. In any case, formation of such a hypothetical complex does not a ect the binding characteristics of the enzymes. Both enzymes were shown to adsorb strongly onto microcrystalline Avicel [16]. The dissociation constants calculated from equilibrium binding data (Scatchard analysis) and the amounts maximally adsorbed were almost identical for Avicelase I and Avicelase II. In reconstituted mixtures, the presence of either enzyme had no e ect on the adsorption of the other (data not shown). Recently, an alternative explanation for exo-exo synergy has been presented: Based on their speci cities towards labeled cellooligosaccharides, enzymes from T. reesei and T. fusca were divided into two functional classes of exoglucanases attacking cellulose chains from either the reducing or the non-reducing ends [17]. This new con-

4 242 K. Riedel et al. / FEMS Microbiology Letters 147 (1997) 239^243 Fig. 3. HPLC analysis of degradation products released from Avicel. Standard reaction mixtures were incubated with Avicelase I (A), Avicelase II (B) and a 1:2 mixture of Avicelase I and Avicelase II (C) for 48 h at 68³C. G1, glucose; G2, cellobiose; G3, cellotriose. cept is consistent with the nding that T. reesei cellobiohydrolases can act synergistically even when added sequentially, but it does not explain the absolute requirement of simultaneous attack observed for the C. stercorarium enzymes. We suggest that exo-exo synergism between the C. stercorarium cellulases results from the enhancement of Avicelase II activity following preparation of a more easily accessible substrate by the action of Avicelase I. Since both individual enzymes are able to attack a crystalline substrate, Avicelase I as well as Avicelase II are supposed to possess a `structure-disrupting' or `amorphase' activity. In the case of a synergistic cooperation, the task of substrate pretreatment seems to be taken over by Avicelase I, the partner exhibiting signi cantly higher activity towards the crystalline substrate. Although amorphous substrates are more readily hydrolyzed by Avicelase II, the activity is again lower than that of Avicelase I. Therefore, Avicelase II must be present in excess in order to be able to exploit the `amorphase' activity of Avicelase I. Since a simultaneous action of both enzyme is required to observe synergism, disruption of the crystalline cellulose structure is supposed to be a reversible process depending on the presence of the enzyme exhibiting `amorphase' activity. An analysis of the degradation products formed by Avicelase I and Avicelase II action on cellulosic substrates supports the concept presented above (Fig. 3). From our previous work [1] we know that Avicelase I releases preponderantly cellotetraose (G4) and minor amounts of cellotriose (G3) as primary degradation products from Avicel. While G3 accumulates, G4 is further degraded to G3, cellobiose (G2) and glucose (G1). Thus, the products found after prolonged incubation are G3, G2 and Table 2 Synergism during sequential and simultaneous attack of Avicelase I and Avicelase II on microcrystalline cellulose Enzyme Soluble saccharides (Wg) DSE Avicelase I 137 ^ Avicelase II 57 ^ Avicelase I+Avicelase II, simultaneously Avicelase I+Avicelase II, preincubation with Avicelase I Avicelase I+Avicelase II, preincubation with Avicelase II Incubations were carried out for 72 h with 1.5 Wg of puri ed Avicelase I and 7.5 Wg of puri ed Avicelase II. Avicel PH 105 was used as substrate. Preincubations were terminated by boiling for 30 min. The DSE (degree of synergistic e ect) was determined as described in Section 2. The table summarizes representative results for one of three independent experiments. The individual values are means of triplicate assays.

5 K. Riedel et al. / FEMS MicrobiologyLetters 147 (1997) 239^ G1 (Fig. 3A). Avicelase II produces G2 and G3 at a constant molar ratio of 4:1 from Avicel (Fig. 3B). G4 is cleaved into two molecules of G2. Formation of G1 could never be detected for Avicelase II. The combined enzymes released approx. 60% more G2, 70% less G3 and 50% less G1 than expected by summation. Based on the reduced formation of G1 and G3, we have to attribute the enhanced release of G2 to Avicelase II action. The increase in G2 formation can be explained either by preferential hydrolysis of the initially released G4 or by higher activity of Avicelase II towards the polymeric substrate. Since no signi cant di erence in G4 degradation could be observed between Avicelase I and II, the second explanation seems to prove true. A prerequisite for the postulated increase in Avicelase II activity is a disruption of the cellulose structure, a capability which we ascribed above to Avicelase I. Acknowledgments This work was supported by grants from the Deutsche Forschungsgemeinschaft (Br 1472/2) and the Volkswagen-Stiftung. References [1] Bronnenmeier, K. and Staudenbauer, W.L. (1990) Cellulose hydrolysis by a highly thermostable endo-1,4-l-glucanase (Avicelase I) from Clostridium stercorarium. Enzyme Microb. Technol. 12, 431^436. [2] Bronnenmeier, K., Ruëcknagel, K.P. and Staudenbauer, W.L. (1991) Puri cation and properties of a novel type of exo-1,4-lglucanase (Avicelase II) from the cellulolytic thermophile Clostridium stercorarium. Eur. J. Biochem. 200, 379^385. [3] Wood, T.M. and Bhat, K.M. (1988) Methods for measuring cellulase activities. Methods Enzymol. 160, 87^112. [4] Beldman, G., Voragen, A.G.J., Rombouts, F.M. and Pilnik, W. (1988) Synergism in cellulose hydrolysis by endoglucanases and exoglucanases puri ed from Trichoderma viride. Biotechnol. Bioeng. 31, 173^178. [5] Jauris, S., Ruëcknagel, K.P., Schwarz, W.H., Kratzsch, P., Bronnenmeier, K. and Staudenbauer, W.L. (1990) Sequence analysis of the Clostridium stercorarium celz gene encoding a thermostable cellulase (Avicelase I): identi cation of catalytic and cellulose-binding domains. Mol. Gen. Genet. 223, 258^ 267. [6] Bronnenmeier, K., Kundt, K., Riedel, K., Schwarz, W.H. and Staudenbauer, W.L. (1997) Structure of the Clostridium stercorarium gene cely encoding the exo-l-1,4-glucanase Avicelase II. Microbiology, in press. [7] Henrissat, B., Driguez, H., Viet, C. and Schuëlein, M. (1985) Synergism of cellulases from Trichoderma reesei in the degradation of cellulose. Bio/Technology 3, 722^726. [8] Wood, T.M. and McCrae, S.I. (1986) The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellobiohydrolases. Biochem. J. 234, 93^99. [9] Nidetzky, B., Steiner, W., Hayn, M. and Claeyssens, M. (1994) Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction. Biochem. J. 298, 705^710. [10] Nidetzky, B., Hayn, M., Macarron, R. and Steiner, W. (1993) Synergism of Trichoderma reesei cellulases while degrading di erent celluloses. Biotechnol. Lett. 15, 71^76. [11] Irwin, D.C., Spezio, M., Walker, L.P. and Wilson, D.B. (1993) Activity studies of eight puri ed cellulases: speci city, synergism, and binding domain e ects. Biotechnol. Bioeng. 42, 1002^1013. [12] Beèguin, P. and Aubert, J.-P. (1994) The biological degradation of cellulose. FEMS Microbiol. Rev. 13, 25^58. [13] Faëgerstam, L.G. and Pettersson, L.G. (1980) The 1,4-L-glucan cellobiohydrolases of Trichoderma reesei QM9414. A new type of cellulolytic synergism. FEBS Lett. 119, 97^100. [14] Tomme, P., Heriban, V. and Claeyssens, M. (1990) Adsorption of two cellobiohydrolases from Trichoderma reesei to Avicel: evidence for `exo-exo' synergism and possible `loose complex' formation. Biotechnol. Lett. 12, 525^530. [15] Takagi, M., Hashida, S., Goldstein, M.A. and Doi, R.H. (1993) The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has speci c interactions with endoglucanases. J. Bacteriol. 175, 7119^7122. [16] Bronnenmeier, K., Adelsberger, H., Lottspeich, F. and Staudenbauer, W.L. (1996) A nity puri cation of cellulose-binding enzymes of Clostridium stercorarium. Bioseparation 6, 41^ 45. [17] Barr, B.K., Hsieh, Y.-L., Ganem, B. and Wilson, D.B. (1996) Identi cation of two functionally di erent classes of exocellulases. Biochemistry 35, 586^592.