Does cellobiohydrolase II core protein from Trichoderma reesei disperse cellulose macrofibrils?

Does cellobiohydrolase II core protein from Trichoderma reesei disperse cellulose macrofibrils? Jonathan Woodward, Kathleen A. Affholter, Kandy K. Noles, Nancie T. Troy and Sherebanu F. Gaslightwala Chemical Technology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee

Papain digestion of a Trichoderma ressei cellulase resulted in the generation of a protein, tentatively identified as cellobiohydrolase H core protein (CBH Ilcp), that could be separated from the rest of the enzyme by gel filtration. It apparently possessed the ability to disperse cellulose macrofibrils based on our findings that: (1) the amount of CBH Ilcp that bound to microcrystalline cellulose (Avicel) was inversely proportional to the Avicel concentration; (2) pretreatment of Avicel with CBH Ilcp increased the rate of its hydrolysis by crude cellulase; and (3) dispersion of crystalline cotton linters by CBH IIcp was visually and microscopically apparent.

Keywords:Cellobiohydrolase II; core protein; cellulose binding; dispersion; hydrolysis; Trichoderma reesei

Introduction It has been over 40 years since Elwyn Reese proposed his C1-Cx hypothesis to explain why some fungal cellulase enzymes are able to hydrolyse crystalline cellulosic materials to glucose, whereas others only degrade amorphous or soluble cellulose derivatives (e.g. carboxymethylcellulose, CMC). ~ It states that cellulases capable of hydrolysing native crystalline cellulose require a factor, termed C~, that physically renders cellulose susceptible to their catalytic activity, termed Cx. Cellulases unable to hydrolyse crystalline cellulose lack C~ and, consequently, the ability to physically disrupt cellulose structure. The identity of C~ is not yet known, nor, indeed, is it known whether it is a single

This manuscript has been authored by a contractor of the U.S. Government under contract DE-AC05-84OR21400.Accordingly,the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes Research sponsored by the Chemical Sciences Division, Office of Basic Energy Sciences, U.S. Department of Energy Managed by Martin Marietta Energy Systems, Inc., for the U.S. Department of Energy under contract DE-AC05-84OR21400 Address reprint requests to Dr. Woodward at the ChemicalTechnology Division, Oak Ridge National Laboratory, Oak Ridge, TN 378316194, USA Received 19 November 1991;revised 12 February 1992;accepted 20 February 1992

© 1992 Butterworth-Heinemann

entity. Also, the mechanism of the disruption o f cellulose structure is not understood. Research over the past four decades, aimed at improving our understanding o f fungal cellulase action, has established that three types o f catalytic activity are required to hydrolyse crystalline cellulose to glucose, referred to as cellobiohydrolase (CBH), endoglucanase (EG), and /3-glucosidase (fiG) (for an overview, see reference 2). The identity of C~ has been associated with CBH, since the latter is absent in cellulases exhibiting little activity toward crystalline c e l l u l o s e ) It has also been suggested that the presence o f C1 is an inherent property of both C B H and EG, evidenced by their ability to be adsorbed tightly to cellulose. 4 In other words, only those enzymes with high affinity for crystalline cellulose render it susceptible to h y d r o l y s i s ) Although the mechanism w h e r e b y C B H and/or E G can physically disrupt the structure o f cellulose is not known, recent studies on the structure and function of Trichoderma reesei C B H and E G isoenzymes now suggest how this may be achieved. The main C B H and E G isoenzymes (CBH I and II, E G I, and II) possess a c o m m o n structural organization: a short cellulose-binding domain (CBD) consisting of 36 amino acid residues linked through a glycosylated region (hinge) to the larger catalytic domain or core protein. 6 Small-angle x-ray scattering studies have shown C B H I and II to be tadpoleshaped (18-21 nm in length), with the head comprising the catalytic domain and the tip of the tail comprising the CBD. 7'8

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Papers The CBD structure of CBH I (a synthetic 36-residue), as determined by nuclear magnetic resonance spectroscopy, is that of a compact wedge (three main hydrogen-bonded antiparallel/3-sheets) containing two disulfide bridges: cysteines 8 and 25, 19 and 35. The latter are conserved in all T. reesei CBDs, which also exhibit 70% overall homology. 9 Such a wedgelike structure may have the ability to pry apart individual cellulose macrofibrils from cellulose fibers and, as such, be a likely candidate for the role of C1. During our studies to determine whether CBD alone has the ability to disperse or disrupt cellulose structure, we found that a protein preparation isolated from a papain digest of T. reesei cellulase, tentatively identified as CBH Ilcp, apparently possessed the ability to disperse cellulose macrofibrils. The results of this investigation and their significance are presented here.

Materials and methods The crude cellulase preparation (celluclast) used in these studies was a gift from NOVO Enzymes, Danbury, Connecticut. Microcrystalline cellulose (Avicel PH-105) was generously provided by FMC Corporation, Philadelphia, Pennsylvania. Crystalline cotton linters were kindly donated by Professor D. R. Dimmel, Institute of Paper Science and Technology, Atlanta, Georgia.

Papain digestion o f crude celluclast The crude enzyme solution was diluted five times with 50 mM sodium acetate buffer, pH 5.0, and subjected to gel filtration using a PD-10 column containing Sephadex G-25 M gel (Pharmacia) equilibrated with the same buffer. The filtered enzyme solution ( - 6 mg ml-1 protein) was then digested with thiol-activated papain (Sigma) at 37°C for 30 min, at a weight ratio ofcellulase to papain of 30: 1, according to the method of van Tilbeurgh et al., which has been shown to remove Cterminal and N-terminal glycopeptides from CBH I and II, respectively (i.e. their CBDs).l°'ll Digested cellulase (1.0 ml) was subjected to gel filtration on a fast protein liquid chromatograph (FPLC) equipped with a Superdex 75 HR 10/30 column (Pharmacia) equilibrated with buffer, pH 5.0.

Transmission electron microscopy ( TEM) Cotton linters or Avicel (2.5 mg) were placed in separate test tubes with I ml of 50 mM sodium acetate buffer (control) and 1 ml o f C B H Ilcp plus buffer. Each sample was gently shaken, and a drop that was taken from the cloudy liquid portion was placed on a Formvar-coated copper grid, which had a thin layer of evaporated carbon. The samples were negative-stained by putting a drop of 1% phosphotungstic acid (PTA, pH 7.5) on the grid and allowing it to interact for 2 min. Excess acid was then carefully wicked off. TEM observations were made with a Hitachi H-600 electron microscope operated at 100 kV. Magnifications of 8,000 to 60,000 were used to survey the action of CBH IIcp on the cotton linters.

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Analytical techniques The ability of protein fractions obtained after gel filtration to hydrolyse 1% (w/v) Avicel to reducing sugar and glucose, to reduce the viscosity of 0.5% (w/v) barley/3glucan, and to hydrolyse I0 mM cellobiose at pH 5.0 was determined (for further details, see Results section and figure legends). Protein concentration was ascertained using the Coomassie Blue protein dye reagent (Bio-Rad). The adsorption of protein to Avicel was determined by measuring the absorbance at 280 nm of the free protein solution after incubation with Avicel (see legend to Figure 4). Reducing sugar and glucose concentrations were measured using the dinitrosalicylic (DNS) and hexokinase reagents, respectively. 12.~3 Gel electrophoresis of various enzyme fractions was carried out by standard procedures using the Phastsystem'" (Pharmacia).

Results and discussion

Separation of CBH Ilcp from cellulase A comparison of a typical chromatogram for native and papain-digested cellulase (Figure 1A and B) indicated the separation of a lower-molecular-weight species in fractions 12-14 for the digested enzyme. These fractions were rechromatographed as described above (Figure 1C). This species was believed, initially, to be CBD from CBH I and II because it was eluted by the same volume of buffer as was bovine heart cytochrome C, possessing a molecular weight of 12,327. However, analytical isoelectric focusing revealed it to be an acidic protein with a pI o f - 4 . 6 (Figure 2, lane 8). Tentatively, this suggested that it was CBH IIcp for the following reasons: (1) papain digestion of CBH II (pI 5.9) has been shown previously to reduce its pI to 4.411; (2) it is not CBH I core protein (CBH Icp), because the pI of the latter is much less than pI 4.4 (Figure 2, lane 2)14; CBH Icp elutes before native CBH I upon gel filtration, which is in complete contrast to CBH IIcplS; (3) EG I and II are not susceptible to papain digestion (P. Tomme, personal communication). The molecular weight of CBH IIcp by SDS-PAGE was estimated to be -36,000, and the discrepancy between this value and that obtained by gel filtration was also observed by Tomme et al. lt for CBH Ilcp. Approximately 15% of the cellulase protein was recovered as CBH Ilcp. This is consistent with the expected percentage of CBH II protein in a T. reesei cellulase system. 2 Isoelectric focusing of the large fraction obtained after papain digestion of cellulase (Figure 1B) revealed the major band of protein to be CBH Icp (Figure 2, lane 4) and to be clearly devoid of CBH II, possessing a pI of approximately 5.9 (see Figure 2, lane 6), T M which is the published pI value for CBH II.

Enzymatic activity of CBH llcp CBH IIcp possessed no cellobiase activity. When Avicel was incubated with CBH IIcp for 7 h at 40°C, generation of soluble reducing sugar was undetectable using the DNS reagent, and glucose formation was negligible.

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Tomme et al. found CBH Ilcp to possess 40% of the Avicelase activity of the intact enzyme, H in disagreement with our finding. The reason for this discrepancy is unknown but could be due to differences in the cellulase/CBH II-to-papain ratios used. In our case we used a cellulase/papain ratio of 30 : 1, whereas they used a CBH II/papain ratio of 300 : 1. As a result, the ability of our preparation to hydrolyse Avicel may be greatly impaired. The same result was obtained when crystalline cotton linters were incubated for 72 h at 23°C with CBH Ilcp. These data indicated that CBH Ilcp did not cause the release of reducing sugar. It was also determined that significant numbers of reducing end

groups on the substrate itself were not generated when CBH IIcp was incubated with crystalline cotton linters. CBH IIcp possessed the ability to decrease the viscosity of a/3-glucan solution, but at a much slower rate than an endoglucanase preparation (Figure 3). This capability distinguishes CBH enzymes and their core proteins from endoglucanases.17 The specific activity toward this substrate was 3.6 and 34.3 units mg-1 protein for CBH IIcp and endoglucanase, respectively, using the viscosity measurement after 5 min as the basis for activity calculation. CBH IIcp lacked the ability to reduce the viscosity of a solution of I% (w/v) carboxymethylcellulose (CMC), adding further support that it lacked any significant endoglucanase activity. It is noteworthy that the rate at which the viscosity of B-glucan decreased when incubated with 0.2/~g of endoglucanase is similar to that caused by 20 /~g of CBH IIcp (20/~g). Wood et al. 17explain the ability of CBH II to decrease the viscosity of/3-glucan as being due to a tendency of CBH II to form a complex with endoglucanase. While this may be true, it is also noteworthy that recombinant T. reesei CBH II expressed in yeast has also been shown to degrade fl-glucan. The recombinant form of CBH II should be free of any contaminating endoglucanase. Upon incubation of CBH IIcp (20/~g) with cellotetraose (1 mM), no glucose formation was observed; however, the concentration of reducing sugar measured after 10 min of incubation was determined to be 2 mM. At longer incubation times, no glucose was formed either. These data are consistent with the generally accepted action of a cellobiohydrolase. E n z y m e M i c r o b . T e c h n o l . , 1992, v o l . 14, A u g u s t

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Kinetics of the adsorption of CBH Ilcp to Auicel The kinetics of the adsorption of CBH IIcp to Avicel indicated that adsorption was rapid with maximal binding after 3 h (Figure 4A); furthermore, the amount of CBH IIcp bound was inversely proportional to the Avicel concentration (Figure 4B). This was surprising, because it was expected that saturation of low concentrations of Avicel by CBH IIcp would have been achieved. In this regard, native CBH I, CBH II, and CBD from CBH I have been reported to saturate Avicel (10-50 mg m1-1) at a level of 50-70/zg mg-'. 18J9 Our data, however, show that saturation of Avicel (0.5 mg ml-') by CBH IIcp did not occur, even at a level of 239 /zg mg- 1. We explain these data as follows: The binding of CBH IIcp results in an increase in the surface area of Avicel; this is more prevalent at a high CBH IIcp (added)-to-Avicel ratio, and under these conditions more CBH IIcp will be bound. It follows, therefore, that the initial rate of CBH IIcp-pretreated Avicel hydrolysis by cellulase should be higher than for untreated Avicel because of the higher number of cellulase-binding sites available. This was found to be the case (Figure 4C). The percentage increase of glucose formation was higher in the initial stages of the reaction, which would not have been expected if there was a synergistic reaction between CBH IIcp and the crude cellulase. In the latter case, the degree of synergism is constant over the course of hydrolysis. 2°'2]

Dispersion of cellulose macrofibrils Visible dispersion of Avicel upon treatment with CBH Ilcp was not easily apparent because of its small partic-

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Papers ulate nature. However, upon treatment with CBH Ilcp, dispersion of crystalline cotton linters (macrofibrils) was observed visually (Figure 5) and by TEM (Figure 6). Interpretation of the TEM data is made as follows: the cellulose macrofibrils (2-20/zm) are composed of microfibrils ( - 2 5 - 5 0 nm), which are clearly visible in Figure 6A. Upon treatment with CBH Ilcp, the macrofibrils become dispersed (Figure 6B). Sprey and Bo* chem 22 showed that an endoglucanase (pI 7.5) from T. reesei was able to split microfibrils into subfibrils.

Concluding remarks The data presented here suggest that a protein, tentatively identified as the catalytic core domain of CBH II, can be easily obtained by papain digestion of the crude cellulase followed by gel filtration. It possesses the ability to be adsorbed on cellulose, which is in agreement with the work of others, TMbut the difference here is that the amount adsorbed is inversely proportional to the Avicel concentration. A key question that arises from this work is whether or not CBH Ilcp actually disperses cellulose macrofibrils, or if the dispersion (and even binding) is caused by a small amount of endoglucanase with which it may be complexed. The latter would seem unlikely, however, in view of the extensive dispersion of the cotton linters. Based on the/3-glucanase assay, the level of endoglucanase in CBH Ilcp would only be about 1%. Also, CMC is not degraded by CBH Ilcp. Future studies will determine the precise identity of this interesting protein. Din et al. 23 recently reported that the CBD of a bacterial endoglucanase disrupted the surface of Ramie cotton fibers and that the catalytic core domain had a smoothing or polishing effect on the surface. We have also seen a similar smoothing effect on Ramie cotton fibers by native CBH I (T. reesei) and CBH Ilcp (not shown). Based on their observations, Din and co-workers suggest CBD can be identified as Reese's C1 factor. From this study, however, it appears that T. reesei CBH Ilcp can also be labeled as C1, at least with regard to its dispersive action on crystalline cotton linters (macrofibrils) and Avicel. Neither study, however, addresses the question of the effect of CBD or core protein on the crystallinity of a cellulosic substrate (i.e. effects at the atomic level or disruption of H-bonding between individual cellulose chains).

manuscript was reviewed by Milica Petek and Carol Tevault. The secretarial assistance of Debbie Weaver and Sylvia Hoglund is acknowledged. This work was supported by the Chemical Sciences Division, Office of Basic Energy Sciences, U.S. Department of Energy. Oak Ridge National Laboratory is managed by Martin Marietta Energy Systems, Inc., under contract DE-AC05-84OR21400 with the U.S. Department of Energy.

References 1 2 3

4 5 6 7 8 9 10 11 12 13 14 15

16 17 18 19

Acknowledgements

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We are grateful to Richard W. Williams of the University of Tennessee, Knoxville, under whose guidance and expertise the transmission electron micrograph of the cellulose samples were performed. The fl-glucanase assays were performed by Katrina Gardner, and the

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Reese, E. T., Siu, R. G. H. and Levinson, H. S. J. Bacteriol. 1950, 59, 485-497 Goyal, A., Ghosh, B. and Eveleigh, D. Bioresource Technol. 1991, 36, 37-50 Wood, T. M. and McCrae, S. I. in Advances in Chemistry Series, Vol. 181 (Brown, R. D., Jr., and Jurasek, L., eds.) American Chemical Society, Washington, D.C., 1978, pp. 181- 209 Klyosov, A. A., Mitkevich, O. V. and Sinitsyn, A. P. Biochemistry 1986, 25, 540-542 Klyosov, A. A. Biochemistry 1990, 29, 10577-10585 Penttila, M., Lehtovaara, P. and Knowles, J. in Yeast Genetic Engineering (Barr, P. J., Brake, A. J. and Valenuela, P., eds.) Butterworths, Boston, 1989, pp. 247-267 Abuja, P. M., Pilz, I., Claeyssens, M. and Tomme, P. Biochem. Biophys. Res. Comm. 1988, 156, 180-185 Abuja, P. M., Schmuck, M., Pilz, I., Tomme, P., Claeyssens, M. and Esterbauer, H. Eur. Biophys. J. 1988, 58, 339-342 Kraulis, P. J., Clore, G. M., Nilges, M., Jones, T. A., Pettersson, G., Knowles, J. and Gronenborn, A. M. Biochemistry 1989, 28, 7241-7257 Van Tilbeurgh, H., Tomme, P., Claeyssens, M., Bhikhabhai, R. and Pettersson, G. FEBS Lett. 1986, 204, 223-227 Tomme, P., Van Tilbeurgh, H., Pettersson, G., Van Damme, J., Vandekerckhove, J., Knowles, J., Teeri, T. and Claeyssens, M. Eur. J. Biochem. 1988, 170, 575-581 Mandels, M., Andreotti, R. and Roche, C. Biotechnol. Bioeng. 1976, 6, 21-33 Woodward, J. and Arnold, S. L. Biotechnol. Bioeng. 1981,23, 1553-1562 Offord, D. A., Lee, N. E. and Woodward, J. Appl. Biochem. Biotechnol. 1991, 28/29, 377-386 Baker, J. O., Mitchell, D. J., Grohmann, K. and Himmel, M. E. in Enzymes in Biomass Conversion (Lealtham, G. F. and Himmel, M. E., eds.) American Chemical Society, Washington, D.C., 1991, pp. 313-330 Shoemaker, S., Watt, K., Tsitovsky, G. and Cox, R. Bio/ Technology 1983, 1, 687-690 Wood, T. M., McCrae, S. I. and Bhat, K. M. Biochem. J. 1989, 260, 37-43 Tomme, P., Heriban, V. and Claeyssens, M. Biotechnol. Lett. 1990, 12, 525-530 Stahlberg, J., Johansson, G. and Pettersson, G. Bio/Technology 1991, 9, 286-290 Woodward, J., Hayes, M. K. and Lee, N. E. Bio/Technology 1988, 6, 301-304 Woodward, J., Lima, M. and Lee, N. E. Biochem. J. 1988, 255, 895-899 Sprey, B. and Bochem, H. P. F E M S Microbiol. Lett. 1991,78, 183-188 Din, N., Gilkes, N. R., Tekant, B., Miller, R. C., Jr., Warren, R. A. J. and Kilburn, D. G. Bio/Technology 1991,9, 1096-1099