Multiple enzyme forms in the cellulase system of Trichoderma reesei during its growth on cellulose

Biochimica et BiophysicaActa, 744(1983) 135-140

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Elsevier Biomedical Press

BBA31575

MULTIPLE ENZYME FORMS IN THE CELLULASE SYSTEM OF T R I C H O D E R M A R E E S E I DURING ITS GROWTH ON CELLULOSE IVICA LABUDOVA and VLADIMIR FARKAS *

Institute of Chemistry, Slovak Academy of Sciences, D(~bravsk~ cesta, 842 38 Bratislava (Czechoslovakia) (Received September 15th, 1982)

Key words: Endo- l, 4.fl- D-glucanase; ~-Glucosidase," Heterogeneity; Extracellular enzyme," Cellulose," Cellulase," (Trichoderma reesei)

The pattern of multiple molecular forms of endo-l,4-fl-D-glucanase (EC 3.2.1.4) and of fl-glucosidase (EC 3.2.1.21) components has been followed by means of analytical isoelectric focusing during submerged growth of the fungus Trichoderma reesei QM 9414 on microcrystalline cellulose, both in buffered and in non-buffered medium. It has been found that multiple molecular forms of the cellulase enzymes appear very early in the course of cultivation. It is concluded that the presence of multiple enzyme forms is an inherent property of the cellulase system of Trichoderma and cannot be considered solely as the result of proteolytic postsecretional modification of secreted extracellular enzymes.

1. Introduction The cellulolytic enzyme system of the filamentous fungus Trichoderma reesei consists of three basic types of enzyme: endo-l,4-flD-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91) and cellobiase (fl-glucosidase, EC 3.2.1.21). In cellulase preparations these enzymes occur in multiple forms which differ in their physicochemical properties, and some of them also in their kinetic behaviour and mode of action on cellooligosaccharides [ 1-5]. The origin of multiple enzyme forms has not yet been satisfactorily explained. Some authors believe that multiple enzymes of the same type arise from the partial proteolysis of a common enzyme precursor by proteases present in the culture medium [6-8]. Another possibility is that the different enzyme forms represent distinct protein species, i.e., they are true isoenzymes. This work represents an attempt to solve the

question on the origin of multiple forms of the enzymes of the cellulase system from T. reesei by following the changes in the enzyme composition during growth of the organism on cellulose. For this purpose analytical isoelectric focusing in polyacrylamide gels has been adopted. It has been observed that multiple forms of the individual enzymes appear in the culture medium already at the early stages of growth but that their number increases with the time of cultivation, especially in the non-buffered growth media.

* To whom correspondence should be addressed. 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

2. Materials and Methods The organism used was Trichoderma reesei QM 9414 obtained from Professor E.G. Simmons, University of Massachussets, Amherst, U.S.A. The fungus was cultivated in shake flasks at 30°C on rotary shaker in medium containing 1% microcrystalline cellulose (Lachema, Czechoslovakia) as described by Mandels and Andreotti [9]. The initial pH of the medium was 5.0 in all cases. In the buffered media the pH was maintained by the presence of citrate-phosphate buffer, pH 5.0, at

136 the final concentration 0.05 M. During growth, at 2-day intervals portions of culture broths were aseptically taken off, centrifuged (5000 rpm) and the supernatants were filtered through a sinteredglass filter (G-4). The culture filtrates were immediately analyzed for pH, cellulolytic activities and protein contents, desalted by dialysis and lyophilized. The dry enzyme preparations were stored at - 15°C until further analysis. Fractionation and analysis of desalted crude cellulase preparations were carried out by isoelectric focusing (IEF) in polyacrylamide gels in 160 × 4 mm tubes as described elsewhere [10]. Each gel was sliced into 2-ram sections which were eluted in 1 ml of distilled water and, after measurement of the pH, endoglucanase and fl-glucosidase activities were determined under conditions described previously [6]. Sodium CM-cellulose (Serva, Heidelberg) and 4-nitrophenyl-fl-D-ghicopyranoside (Lachema, Czechoslovakia) were used as the respective substrates. The filter-paper degrading activity in the crude culture filtrates was measured with Whatman No. l filter paper according to Mandels et al. [11].

Results Fig. 1 depicts the changes of pH, filter-paper activity and activities of endoglucanase and fl-glucosidase as measured during the growth of Tric h o d e r m a reesei QM 9414 in the buffered and non-buffered medium. In the latter case the pH rapidly dropped to 1.8 on the 3rd day of the cultivation and maintained a low value until the 9th day. In comparison, the pH of the buffered medium only slightly increased from 5.0 to 5.7 during the 9 days of cultivation. In the nonbuffered medium the maximum of fl-glucosidase activity was recorded on the 4rd day of cultivation and thereafter it decreased, probably as a result of enzyme inactivation at the low pH of the medium. On the other hand, the fl-glucosidase did not lose its activity in the buffered medium where a certain lag in the release of cellulolytic enzymes could be noticed. The activity of endo-l,4-fl-o-glucanase in the buffered medium reached twice as high a level as that in the non-buffered one. There are some evident differences in the kinetics of release of cellulases in the buffered and non-buffered media which are reflected in the specific activity of endo1,4-fl-o-glucanase, fl-glucosidase and total celhilase in the lyophilized enzyme preparations (Ta/3'G IU/ml x10-2

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ble I). It can be seen that during cultivation in the buffered medium the specific activity of cellulolytic enzymes in the extracellularly released proteins increased. On the other hand, the specific activity of cellulase in enzyme preparations from

non-buffered medium constantly decreased during 9 days of cultivation. During growth we have regularly checked the protease activity in the culture media. From the 5th day on there was low activity against Azocoll

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detected after prolonged incubation (16 h) at pH 7.5 in all samples. There were, however, no appreciable differences found between the samples from the buffered and non-buffered media (results not shown). Resolution of the protein samples from the culture media by means of analytical IEF showed

the changes in the spectra of individual cellulase components taking place in the course of cultivation. After the first day of cultivation there were only very low single peaks of endoglucanase (pI 5.2) and #-glucosidase (pI 4.8) detected in the sample from the buffered medium while in the sample from the non-buffered medium there was a

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139 TABLE I SPECIFIC ACTIVITIES OF TOTAL CELLULASE ACTIVITY, ENDOGLUCANASE AND fl-GLUCOS1DASE IN CELLULOLYTIC ENZYME PREPARATIONS ISOLATED FROM TRICHODERMA REESE1 DURING ITS GROWTH IN NON-BUFFERED AND BUFFERED MEDIA Enzyme units are expressed as #mol product formed per min. In cellulase and exoglucanaseassays the product was measured as reducing glucose equivalents; in fl-glucosidase assays the liberated 4-nitrophenol was determined. Day of cultivation

Total cellulase (units/mg protein)

A. Non-buffered medium 3 0.14 5 0.16 7 0.13 9 0.13 B. Buffered medium 3 0.04 5 0.06 7 0.23 9 0.26

Endoglucanasefl-Glucosidase (units/mg (units/mg protein) protein)

0.3 0.32 0.22 0.24

0.009 0.006 0.005 0.009

0.13 0.29 0.32 0.31

0.007 0.014 0.25 0.23

low peak of endoglucanase at p I around 7.0 and a small peak of fl-glucosidase at p I 7.5 (results not shown). On the 3rd day of cultivation the cellulolytic activities in the growth media were readily detectable. As shown in Fig. 2, the spectra of enzyme activities in the sample from the non-buffered medium exhibited four peaks of endo-l,4-flglucanase ( p l values 4.2; 4.6; 5.0 and 6.9) and four peaks of fl-glucosidase (pl values 4.6; 5,0; 6.4; and above 7.5). From the 5th day on, the presence of two additional endoglucanase enzymes with p I values of 3.9 and 5.4 was detected. The minor endo-l,4-fl-o-glucanase with p I 6.9 disappeared from the enzyme mixture on the 9th day of cultivation. The spectrum of fl-glucosidase components also changed in the course of cultivation in the nonbuffered medium. Towards the end of cultivation (7th day) a marked decay of the fl-glucosidase with p I > 7.5 was observed. On the other hand, two fl-glucosidases with respective p I values of 5.4 and 6.7, the latter one being predominant, appeared.

The samples taken from the buffered medium differed to a certain extent from those from the non-buffered. Already on the third day of cultivation the enzyme complex contained six forms of endoglucanase ( p I values 4.2; 4.5; 4.8; 5.2; 6.3) and three fl-glucosidases; the predominant one with p I 4.8 and two minor ones with p I 6.3 and 6.5. In the course of further cultivation the number of fl-glucosidase components increased to five ( p I values 4.8; 6.3; 6.5; 6.9 and 7.5). In contrast to the non-buffered medium, the inactivation of fl-glucosidase components having their isoelectric points in the alkaline region of the p H gradient did not occur when the p H of the medium was maintained at a constant level during cultivation. The numbers of endo-l,4-fl-D-glucanase components did not change in the course of further cultivation; however, there were differences in the relative abundance of the individual enzymes (Fig. 3). Discussion The existence of multiple enzymes of the same type in the cellulase complex of Trichoderma has been the subject of numerous discussions. Gong et al. [8] expressed the widespread feeling that " . . . it was wasteful for a microorganism, which possesses limited genetic pools, to transcribe and translate so many enzymes for the same function". As an explanation for the observed enzyme multiplicity it has been suggested that multiple enzyme forms of given type may arise from the common enzyme precursor by its postsecretional modification by proteases present in the culture medium [6-8]. The results described in this paper indicate that Trichoderma reesei secretes the multiple enzyme forms practically from the very beginning of cultivation. It may be argued that after the first day of cultivation there were only single peaks of endoglucanase and fl-glucosidase detected; however, the detected peaks were too low to be considered as precursors of other enzymes. These findings are in apparent contrast with the results of G o n g et al. [8], who, using DEAE-cellulose chromatography to resolve the cellulase complex of T. reesei, found only one endoglucanase in the 6-dayold culture, while in the 14-day culture they detected four different endoglucanase peaks. We

140

ascribe this discrepancy to the better resolving power of isoelectric focusing over the ion-exchange. The same argument may be applied to the results of Gritzali and Brown [12], who, using polyacrylamide disc-gel electrophoresis, found in culture medium of T. reesei incubated with sophorose or grown for 28 h on cellulose only two cellobiohydrolases, one endoglucanase and a flglucosidase. The conclusion from our experiments is that the 'cellulase' from T. reesei is inherently a complex system containing multiple enzyme forms of the same type, which are, at least in part, genetically determined. A theoretical possibility exists that the enzyme heterogenity might be caused by immediate posttranslational modification of the precursor enzyme by its glycosylation. However, since glycosylated cellulase enzymes from Trichoderma contain mostly neutral sugars in their carbohydrate moieties [13], it can hardly be expected that glycosylation would appreciably change the electric charge on the protein and thus its isoelectric point. The alterations of the protein pattern during growth of the microorganism may be caused by several factors. (i) The changes of the pH observed in the non-buffered medium could cause inactivation of some enzymes and stimulate the secretion of others. (ii) Some enzyme forms may arise as a result of limited proteolytic degradation of preexisting enzymes, as has already been suggested by other authors [6-8]. (iii) Finally, the appearance of

additional enzyme forms at the end of the cultivation period may be the result of their liberation from cellulose. During growth of the microorganism these enzymes may have been adsorbed on the insoluble substrate and released only after the cellulose had been exhausted. References 1 Gum, E.K. and Brown, R.D. (1977) Biochim. Biophys. Acta 492, 225-23 I 2 Shoemaker, S.P. and Brown, R.D. (1978) Biochim. Biophys. Acta 523, 133-146 3 0 k a d a , G., Nisizawa, K. and Suzuki, H. (1978) J. Biochem. 63, 591-607 4 Berghem, L.E.R., Pettersson, L.G. and Axi6-Fredriksson. U.B. (1976) Eur. J. Biochem. 61,621-630 5 Weber, M., Foglietti, M.J. and Percheron. F. (1980) J. Chromatogr. 188, 377-382 6 Nakayama, M. (1975) Mem. Osaka Kyoiku Univ. 24, 55-66 7 Nakayama, M. (1975) Mem. Osaka Kyoiku Univ. 24, 127-143 8 Gong, C.S., Ladisch, M.R. and Tsao, G.T. (1979) Adv. Chem. Ser. 181,261-287 9 Mandels, M. and Andreotti, R.E. (1978) Process Biochem. 13, 6-5 10 FarkaL V., Jalanko, A. and Kolarova, N. (1982) Biochim. Biophys. Acta 705, 105-I 10 11 Mandels, M., Andreotti, R.E. and Roche, C. (1975) Biotechnol. Bioeng. Symp. No. 6, 21-33 12 Gritzali, M. and Brown, R.D, (1979) Adv. Chem. Ser. 181, 237-260 13 Shoemaker, S.P. and Brown, R.D., Jr. (1978) Biochim. Biophys. Acta 523, 147-161