World

Journal

of Microbiology

and Biotechnology,

9, 251-254

Saccharification of native and degraded cotton cellulose and commercial microcrystalline cellulose by Trichoderrna viride cellobiohydrolase I M. Finetti, M. Daz, G. Ellenrieder*

and M. Marx-Figini

The degree of polymerization of samples of acid degraded cotton cellulose has no appreciable influence on the saccharification by cellobiohydrolase I from Trichoderma viride. The increase in the number of cellulose molecule ends, achieved by a so-fold decrease in molecular weight, does not produce the effect which could be expected for a pure end-wise mode of action of this exoglucanase. Microcrystalline celluloses saccharified by the same enzyme yield considerably more reducing sugars than cotton cellulose, either with a similar degree of polymerization or one of about 7000. It appears, therefore, that the difference in the susceptibility of the commercial substrates is not a consequence of their low degree of polymerization. Key words: Cellulose,

cotton, polymerization,

saccharification,

The enzymatic hydrolysis of cellulose has been intensively studied (Klyosov 1990). However, no clear idea exists about the influence of the degree of polymerization (DP) of the substrate on the reaction mechanism. It is difficult to compare the hydrolysis of high molecular weight cotton cellulose with that of a commercial microcrystalline cellulose of low molecular weight, such as Avicel, because hydrolysis of the latter could be affected by other modifications produced by the industrial processing. More appropriate substrates for studying the influence of DP in the saccharification of cellulose can be obtained by degrading native cotton cellulose with acids under controlled conditions (Marx-Figini & Coun-Matus 1981; Marx-Figini 1986). Such methods have been employed in the present study. Of the three types of cellulase activity that have been traditionally assigned to the fungal cellulase systems, only that of exoglucanase, which acts on molecular chain ends, could be affected by the DP of the substrate. An exoglucanase was therefore selected for this study. The M. Finetti, M. Daz and G. Ellenrieder are with the lnstituto de Investigaciones para la lndustria Quimica (INIQUI), Universidad Naciowl de Salta, Buenos Aires 177, 4400 Salta. Argentina; fax: 54 087 251006. M. Marx-Figini is with the lnstituto de lnvestigaciones Fisicoquimicas Te6ricas y Aplicadas. Universidad National de La Plats, Diag. 113 63-64, 1900 La Plata. Argentina. ‘Corresponding author. @ 1993 Rapid

Communications

of Oxford

Trichodema. exoglucanase of Trichoderma acts on the non-reducing ends of the cellulose molecule, liberating cellobiose units; (Wood & McCrae 1972; Berghem & Pettersson 1973; Enari & Niko Paavola 1987). It is a P-1,4-D glucan cellobiohydrolase I (EC 3.2.1.91) (CBH I) though its exoglucanase character has been recently questioned by some authors. Okada & Tanaka (1988) indicated that, in high concentration, the enzyme produces other sugars besides cellobiose, principally glucose and Schmid & Wandrey (1990) concluded from kinetic studies that the enzyme’s action pattern on soluble cellodextrins did not correspond to that of an exoglucanase. One aim of the present study was to study how the cellobiohydrolase I responded to different cellulosic substrates with varying numbers of chain ends and whether this action corresponded to that of a genuine exoglucanase. Another objective was to compare the enzymatic action of the cellobiohydrolase I on commercial microcrystalline cellulose, especially ‘Avicel’ with that on degraded cotton cellulose of comparable degree of polymerization.

Materials

and Methods

The various cotton cellulose samples, of varying degrees of polymerization (Table 1). were prepared by acid-catalysed

Ltd

World ]oumal of Microbiology and Biokchnology. Vol 9, 1993

251

M. Fineffi et al. Table 1. Degree samples used.

of polymerization

Sample

DP,(CuEn)’

Acid degraded cotton cellulose 1 2 3 4 5 Commercial microcrystalline cellulose Avicel Sigmacel Mallinckrodt

7525 3375 1158 680 290

246 2651273 2681275

and

DP,(SEC)t

polydispersity

of the

Polydisperslfy$

-

1.67 1.93 2.00 2.00 2.00

2581244 2601255

2.56 2.74

* Average degree of polymerization determined viscometrically in copper ethylenediamine. t Viscosimetric degree of polymerization determined by size exclusion chromatography. $ DPJDP,, where DP, is the average degree of polymerization deteimined by size exclusion chromatography.

DEAE Sephadex A-50 column (that had been previously equilibrated with the dialysis buffer and washed with I 1 of the same buffer at pH 5.0) and eluted with a bu#er of pH 3.6 which contained 0.5 M NaCl. The fractions containing the enzyme, were pooled, dialysed and freeze dried. The enzyme activity on carboxymethylcellulose (CMC) (Fluka, substitution degree 0.7) was determined in accordance with IUPAC (1987) recommendations that include measuring the reducing sugars with dinitrosalicylic acid (Miller 1959). The enzymatic activity on crystalline cellulose was determined using acid degraded cotton, by treating a substrate sample (DP 3500) of 25 mg in 1 ml of 0.05 M citric acid/sodium citrate buffer, pH 4.8, with 0.25 ml of the enzyme for 1 h at 50°C. The amount of enzyme taken for the test was that necessary to liberate 0.25 mg of reducing sugars. The reducing sugars were determined as before and expressed as glucose equivalents. Assays

The saccharification assays were carried out in agitated conical flasks which contained 250 mg cellulosic substrate and 50 ml of enzyme solution in 0.05 M citric acid/sodium citrate buffer, pH 4.8, at a temperature of 40°C. Supematant samples were withdrawn at selected times, and the reducing sugars determined by the Somogyi-Nelson method (Somogyi 1952).

Results degradation of isolated cotton cellulose of nearly native degree of polymerization. Either 0.5 M KHSO, at 60°C or 1 M HCl at 80°C were used as degrading agents, according to the required degree of polymerization (Marx-Figini & Coun-Matus 1981; Marx-Figini 1986). To avoid ‘homification’ of the cellulose surfaces which can occur during drying in vacuum above room temperature, and which would make the cellulose less accessible, the degraded celluloses were freeze-dried. Before freeze drying, each degraded sample was immersed first in water and then a series of solvents of decreasing polarity (water; water/methanol I: I v/v; methanol/

benzene I:I v/v; benzene). Eramination of Celluloses The degrees of polymerization were determined viscosimetrically in CuEn (copper ethylene diamine complex) and the measured intrinsic viscosities converted into DP according to Marx-Figini (1978). Size exclusion chromatography was carried out on the nitrates of the respective samples, using p-Styragel columns (IO-~, IO-~, 10m6, 10e7 m in pore size), tetrahydrofuran as eluent and a flow velocity of 0.4 ml/min. Elution volume was calibrated against DP with cotton samples of different DP (Soubelet et al. 1990). The following commercial microcrystalline celluloses were used: Avicel (Merck); Sigmacell Type 20, particle size approx. 20 pm (Sigma); and microcrystalline cellulose, particle size < 100 pm (Mallinckrodt). Enzyme Purification The cellobiohydrolase

I was purified from a commercial preparation cellulase (Miles-Kyowa, Japan) using a modification of the procedure described by Alurralde & Ellenrieder (1984). The purification was essentially by affinity chromatography in an Avicel column and chromatography in a DEAE Sephadex A-50 column. The final gel filtration of the original method was replaced by dialysis in an AMICON cell with a PM 10 membrane (cut off 10000 Da) against 50 mM succinic acid/sodium succinate buffer, pH 5.33. The dialysate was applied to a second 3.6 x 8 cm

of

Trichodema

uiride

and Discussion

Although the enzyme purification was essentially that of Alurralde & Ellenrieder (1984) additional dialysis and chromatography produced an electrophoretically homogensteps did not affect the eous enzyme. The additional enzyme’s specific activity on cotton cellulose (0.024 U/mg) but did decrease that measured on CMC from 0.46 to 0.30 IU/mg. (the values of activity on CMC obtained by the IUPAC recommended method are not comparable with those in previous reports). The poly-dispersity (DP/DPn) of the degraded cellulose samples was shown to coincide with those expected for acid degraded celluloses in the corresponding ranges of DP (Marx-Figini & Coun-Matus 1981; Marx-Figini 1986) by size-exclusion chromatography. From Figure I saccharification of acid-degraded cotton cellulose with the enzyme preparation showed no appreciable variation when the DP of the substrates ranged from 290 to 7525. Further, the amounts of reducing sugars released with different enzyme concentrations showed no significant variation with different DP values of the substrate (Figure 2). The degree of polymerization, and consequently the number of chain ends of the cellulose, therefore, has no appreciable influence on the saccharifying action of the cellobiohydrolase I. The enzyme, therefore, may not be acting as an exoglucanase. This would agree with other recent reports on this enzyme. The authors of these reports, on product formation (Okada & Tanaka 1988) and kinetics of cellodextrin hydrolysis (Schmid & Wandrey 1990), concluded that the enzyme was not a cellobiohydrolase, as

Saccharificafion

Time

Time

(mid

of

degraded cotton

( min 1

1. Time course of the saccharification, by Trichoderma cellobiohydrolase I, of degraded cotton cellulose of varying degrees of polymerization (A-7525; O-3375; n-1158; V-290) and Avicel cellulose (Q) of 246 degrees of polymerization. The reaction mixture was of 5.0 mg substrate/ml and 0.09 mg enzyme/ml in citric acid/sodium citrate buffer, pH 4.8, at 48°C.

Figure 3. Time course of saccharification, by Trichoderma hide cellobiohydrolase I, of three commercial microcrystalline celluloses [0-Avicel; ‘-6igmacell; +-micro-crystalline cellulose (Mallinckrodt)] and degraded cotton cellulose of 290 degrees of polymerization (V). See Figure 1 for reaction conditions.

defined by the International Union of Biochemistry, such a cellobiohydrolase should only release cellobiose from cellulose or cellotetraose. Although the Trichoderma enzyme is probably not a genuine cellobiohydrolase, there exists a pronounced difference in its mode of action and that of the endoglucanases. Experiments with exoglucanases do not show any systematic degradation: amorphous (Berghem et al. 1975) and native cotton cellulose (M. Figini, M. Finetti & G. Ellenrieder, unpublished work) undergo a fast and strong decrease in DP with an endoglucanase and only an insignificant quantity of solubilized material can be detected. The difference can be explained by repetitive attack on a single chain, also called ‘Multiple Scission Attack’ (Azhari

81 Lotan 1991), by the CBH I. This can lead to appreciable production of reducing sugars with a small decrease in the DP of the substrate, even for an initially random scission. Commercial microcrystalline celluloses were more easily saccharified, by the cellobiohydrolase I, than the degraded cotton celluloses of similar DP (Figures I, 2 and 3). Comparison of the production of reducing sugar from commercial microcrystalline celluloses and from degraded cotton celluloses (Figures I and 2), indicates that low DP values do not cause low resistance to hydrolysis. Moreover, as the crystallinities of the two substrates are comparable (E. Macchi, unpublished work) the different susceptibilities to enzymatic attack are probably caused by modifications to the commercial microcrystalline celluloses which occur during production, such as changes in pore structure and surface area. Saccharifications of different commercial crystalline celluloses are scarcely comparable (Figure 3). Selection of a standard crystalline substrate therefore seems impossible. Use of substrates obtained under controlled conditions, such as the degraded cotton cellulose, would be more appropriate for use as a standard cellulosic substrate.

Figure

viride

0.280

References

Enzyme

concentration

(mglml)

Figure 2. Effect of enzyme concentration on the saccharification of degraded cotton cellulose of varying degrees of polymerization (LL A-7525; V, V-290) and Avicel cellulose (0, +) of 246 degrees of polymerization after 300 min (A, V. 0) and 24 h

(A v. l I.

Alurralde, J.L. & Ellenrieder, G. 1984 Effect of attached carbohydrates on the activity of Trichodema viride cellulases. Enzyme and Microbial Technology 6, 467-470. Azhari, R. & Lotan, N. 1991 Enzvmatic depolvmerization processes. Reaction pathways as a basis for a neh classification and nomenclature. ]ownal of Materials Science Letters IO, 243-245. Berghem, L.E.R. & Pettersson, L.G. 1973 enzymatic cellulose degradation. Purification

The mechanism of a cellulolytic

of

M.

Finetfi

et

al.

enzyme active on highly ordered cellulose. European Jotrrnul of Biochemistry 37, 21-30. Berghem, L.E.R., Pettersson, L.G. & Axio-Fredriksson, U.B. 1975 The mechanism of enzymatic cellulose degradation. Characterization and enzymatic properties of a /%1,4-D glucan cellobiohydrolase from Trichoderma viride. European Journal of Biochemisfry 53, 55-62. Enari, F.M. & Niku-Paavola, M.L. 1987 Enzymatic hydrolysis of cellulose. Is the current theory of the mechanism of hydrolysis valid? CRC Crificul Reviews in Biotechnology 5, 67-87. IUPAC 1987 (Ghose T.K. ed) Measurement of cellulase activities. Pure and Applied Chemistry 59, 268-275. Klyosov, A.A. 1990 Trends in biochemistry and enzymology of cellulose degradation. Biochemistry 29, 10577-10585. Marx-Figini, M. 1978 Significance of the intrinsic viscosity ratio of unsubstituted and nitrated cellulose in different solvents. Angewundfe Mukromolekulure Chemie 72, 161. Marx-Figini, M. 1986 The acid catalyzed degradation of cellulose in the range of medium and low degrees of polymerization. Mukromolekulare Chemie 187, 679-687. Marx-Figini, M. & CounMatus, M. 1981 On the kinetic of the hydrolytic degradation of native cellulose. Mukromolekulure Chemie 182.3603-3616.

254

World Journal

of

Microbiology and Biotechnology, Vol 9. 1993

Miller, G.L. determination

1959 of

use of reducing

dinitrosalicylic acid reagent sugar. Analytical Chemistry

for 31,

426-428. Okada, G. & Tanaka, Y. 1988 Non-existence of Exocellobiohydrolase (CBH) in the cellulase system of Trichodermu viride. Agricultural and Biological Chemistry 52, 2981-2984. Schmid, G. & Wandrey, C. 1990 Evidence for the lack of exocellobiohydrolase activity in the cellulase system of Trichodermu reesei QM 9414. Journal of Biotechnology 14,

393-410. Somogyi, M. 1952 Notes on sugar determination. Journal of Biological Chemistry 195, 19-23. Soubelet, O., Presta, M.A. 81 Marx-Figini M. 1990 SEC on cellulose nitrate: DP-Va Relationship and evaluation of different methods to determine the calibration parameters. Angewundte Mukromolekulure Chemie 175, 117-128. Wood, T.M. & McCrae, S.I. 1972 The purification and properties of Cl component of Trichoderma koningii cellulase. Biochemical Journal 128, 1183-1192.

(Received 1992)

in revised

form

23 October

7 992;

accepted

26 October

Saccharification of native and degraded cotton cellulose and commercial microcrystalline cellulose by Trichoderma viride cellobiohydrolase I.

The degree of polymerization of samples of acid degraded cotton cellulose has no appreciable influence on the saccharification by cellobiohydrolase I ...
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