Constitutive Cellulolytic Enzymes of Diplodia zeae

11 Constitutive Cellulolytic Enzymes of Diplodia zeae

Downloaded by PENNSYLVANIA STATE UNIV on June 8, 2012 | http://pubs.acs.org Publication Date: June 1, 1969 | doi: 10.1021/ba-1969-0095.ch011

J. N . B E M I L L E R , D I A N E O. T E G T M E I E R , and A . J. PAPPELIS Departments of Chemistry and Botany, Southern Illinois University, Carbondale, Ill. 62901

Diplodia zeae produces cellulolytic activity even when grown in media containing D-glucose as the only carbo hydrate. The cellulolytic enzymes are believed to be at tached to the surface of the fungal hyphae and released to the culture medium after the growth period, when most of the available carbohydrate is used up. It is suggested that not the synthesis of the C enzyme(s) but its release is con trolled by the presence of D-glucose. x

T t has been reported that fungal cellulases are induced enzymes and that cellulose preparations induce cellulolytic activity while easily assimil able carbon sources give the best fungal growth but less production of enzyme activity (9, 12, 14). F o r example, Horton and Keen (10) found that 7.5 X 10" M D-glucose repressed the synthesis of cellulase to a basal level i n Pyrenochaeta terrestris and suggested that cellulase synthesis was regulated by an induction-repression mechanism.

A

3

In this laboratory, we have studied stalk rot of corn (Zea mays L . ) , a disease caused i n part b y Diplodia zeae (Schw.) L e v . The organism produces cellulolytic activity, although it is of a very low level ( 3 ) , which destroys cell wall structures as the pathogen spreads through parenchyma tissue, hollowing out the corn stalk. The parenchyma tissue i n which the fungus grows, however, contains relatively large amounts of sugars. [Approximate ranges are reducing sugars 0.1-0.5M, sucrose 0.1-0.35M, total sugars 0.15-0.5M ( 4 ) ] . Hence, we became interested i n the effects of cellulose and D-glucose on the induction and repression, respectively, of the cellulolytic enzymes of D . zeae. 188

In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.

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Enzymes of Diplodia zeae

189

Experimental D . zeae Culture. Diplodia zeae (Schw.) Lev. was grown i n liquid shake culture as described b y BeMiller et al (3) with variations i n the source and amount of carbohydrate. Assay Methods. D-Glucose i n the culture media was determined with Glucostat reagent (Worthington Biochemical Corp., Freehold, N . J . ) . The assay medium contained 0.67% carboxymethylcellulose ( C M C 7 L F , Hercules Inc., Wilmington, Del.) i n p H 4.5 acetate buffer ( 0 . 2 M ) . Total cellulolytic ( C ) activity was determined b y measurement of the total reducing power i n the assay medium with sodium 3,5-dinitrosalicylate ( D N S A ) reagent. 0-Glucosidase activity was determined b y measure ment, with Glucostat reagent, of the D-glucose produced i n the assay medium. Details of the D N S A and Glucostat methods have been de scribed previously ( 3 ) .


x

Cellulolytic Enzymes in Culture Media as a Function of Growth Time. D . zeae was grown i n two kinds of media, one containing 2.0 grams/liter of D-glucose (0.01M) and 7.5 grams/liter of cellulose and the other containing 10.0 grams/liter of D-glucose (0.05M) and 7.5 grams/liter of cellulose as the carbohydrate source. Other components were 1 gram of casein hydrolysate, 0.25 gram of yeast extract, 2.5 grams of N H N 0 , 1.25 grams of K H P 0 , 0.625 grams of M g S 0 · 7 H 0 , and 0.005 grams F e C l per liter as previously reported ( 3 ) . Aliquots were taken at 12-hour intervals and determinations of D-glucose concentration, C activity, and β-glucosidase activity were made. Results of three deter minations of each were averaged. 4

3

2

4

4

2

3

x

Release of Cellulolytic Enzymes. Culture medium was filtered from 4-day-old cultures of D . zeae. The mycelial mats were rinsed with dis tilled water, then stripped from the filter paper and fragmented i n a minimum of distilled water in a semi-micro Waring Blendor cup. Portions of the mycelial suspension were pipeted into solutions of the compounds being tested, and the flasks were shaken i n the dark for 20 minutes. The suspensions were then filtered, and the filtrates were assayed for enzyme activity. To determine the amount of enzyme activity remaining on the my celium after treatment with lignosulfonate solutions, cultures were filtered and mycelial mats recovered. One-fourth of the mats were incubated for 20 minutes with a 2.0% lignosulfonate solution p H (4.5); the mixture was then filtered, the mycelial mats recovered, and the filtrates assayed. A l l of the treated mats and one-half of the untreated mats were placed i n an acetone-dry ice bath for 15 minutes, a treatment which killed the organism. Enzyme activity remaining on these mycelial mats was deter mined by placing them directly into the assay medium. Also, to determine the amount of the reducing power due to sugars being leached from the mycelium rather than enzyme activity, one-fourth of the mycelium was placed i n buffer alone, the buffer was assayed for reducing power and D-glucose, and these values were subtracted from the values found i n the enzyme assays. The assay mixtures were filtered and aliquots of the filtrates were assayed for enzyme activity to confirm that active enzyme was present and was released during assay of the mycelium. Since C x


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enzymes are not inhibited by Merthiolate, the same procedure was re peated using 1 % Merthiolate to kill the organism. Sonification. Mycelium was fragmented i n buffer using a W a r i n g Blendor. This suspension was subjected to sonification with a 125-watt Branson Sonifer i n a 4°C. bath for 20 minutes. N o cell structures could be observed under the microscope.


Results and

Discussion

The results of experiments set up to determine if the concentration of D-glucose i n culture media containing both D-glucose and cellulose reaches a steady state level owing to an induction-repression mechanism are presented i n Figures 1 and 2. Some observations can be made from these data. N o steady state concentration of D-glucose was found. Solid cellulose particles disappear from the media before more than traces of enzyme activity can be measured in the media. Since each enzyme mole cule must come into direct physical contact with its specific substrate molecule before catalysis can occur and since only traces of enzyme activity can be found in the media before and during the disappearance of cellulose, it is suggested that the cellulolytic enzymes of D . zeae occur on the surface of the hyphae.

Figure I. Concentration of Ό-glucose and activity of C and β-glucosidase enzymes in D . zeae culture media as a function of time. OHginal Ό-glucose concentration. 2.0 grams/liter; cellulose concentration, 7.5 grams/liter x



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Figure 2. Concentration of Ό-glucose and activity of C and β-glucosidase enzymes in D . zeae culture media as a function of time. Original Ό-glucose concentration, 10.0 grams/liter; cellulose concentration, 7.5 grams/liter w

Cowling and Kelman (7) reported that, i n cultures of dikaryotic decayed tissue isolates of Fomes annosus with relatively high cellulase activity, up to 34% of total activity was bound to the mycelium ( 14% ) and cellulose particles ( 2 1 % ); and Tashpulatov (15) reported that part of the cellulolytic enzymes remained i n the mycelium during growth of Aspergillus fumigatus. It can also be observed from Figures 1 and 2 that little C activity and no β-glucosidase activity could be detected i n the culture filtrates until the carbohydrate (D-glucose) had almost completely disappeared; enzyme activity appeared sooner i n cultures containing the smaller amount of D-glucose. It is suggested that easily assimilable carbohydrate such as D-glucose regulates, not the synthesis of superficial cellulolytic enzymes, but their release into the culture medium. This release is a function of the age or condition of the culture and occurs, for the most part, after the growth period. Deshpande and Deshpande (8) report that cellulase is liberated 48 hours after innoculation for Aspergillus fonsecaeus grown on 0.05M D-glucose, and Moreau and Trique (13) found a continued liberation of cellulase into the culture medium i n the absence of substrate and after the growth period of Pénicillium cyclopium x



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and Aspergillus versicolor. Isaac ( I I ) found cellulase activity around certain short, branched hyphae in older parts of mycelium of Rhizoctonia sofai, but none in either young hyphal tips or older trunk hyphae bearing active side branches. It is obvious that conclusions about the amount of enzyme synthesis could not be based on the amount of cellulolytic activity found i n the culture medium since the amount of activity found depended on the time at which measurements were made. O n the basis of the hypothesis that the cellulolytic enzymes are bound to the surface of the hyphae and are released to the culture medium only slowly after the growth period, we undertook to investigate the release of the enzymes; for, i n order to determine if an induction-repression mechanism operates, it is necessary to measure total enzyme activity. Coles and Gross (5, 6) have found that some of the penicillinase of Staphylococcus aureus is bound ionically to the surface of the cell wall and can be liberated by low concentrations of anions. Polyanions that do not penetrate the cell wall and tricarboxylic acids were particularly effective as release agents. Weimberg and Orton (16) had found earlier that acid phosphatase could be released from the cell surface of Saccharo myces mellis by chloride ions i n combination with 2-mercaptoethanol and concluded that this enzyme is held on the cell surface by a combination of electrostatic forces and disulfide bonds. Likewise, polygalacturonase is released from the surface of spores of Geotrichum candidum with poly anions and 2-mercaptoethanol (2). To investigate the release of cellulolytic enzymes from D. zeae, harvested mycelium was treated with various reagents. N o appreciable activity was released by p H 4.5 acetate buffer (0.1-0.8M ) or the following anionic reagents: 0.5M KC1, 2 M KC1, 0.15M N a H P 0 ( p H 6.0, 7.5), 0.05M succinic acid ( p H 4.5), 0.04M malic acid ( p H 4.5), 0.03M citric acid ( p H 4.5), 0.4M oxalic acid ( p H 4.5), 0.5% water-soluble poly acrylamide, 1.0% Reten A - l (Hercules Inc., Wilmington, D e l . ) . 2

4

Activity was not released by 1.0% Reten 205M (a cationic polymer) (Hercules Inc., Wilmington, D e l . ) , 0.13-0.38M 2-mercapto ethanol, liquid phenol, incubation with 0.1% lysozyme, osmotic shock ( I ) , or the following surfactants: 0.4% Triton X-100 (Rohm and Haas Co., Philadelphia, Pa.), 0.4% sodium lauryl sulfate, 0.5% digitonin i n 0.05M tris buffer ( trishydroxymethyl-aminomethane ). More C activity was found in the wash solution when the harvested mycelium was treated with 2.2-4.0% polygalacturonic acid ( p H 4.5); this activity was about 15 times that found in washes of p H 4.5 acetate buffer. Lignosulfonic acid (2.0%, p H 4.5) also either released or acti vated C activity but inhibited the glucostat reagent so that the β-gluco sidase activity could not be determined; the C activity was about five x

x

x


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times that found in washes of p H 4.5 acetate buffer. A solution of 1.0% lignosulfonic acid and 2.0% polygalacturonic acid ( p H 4.5) released no more activity than solutions of either polyanion alone. About the same amount of enzyme activity was released from mycelium of D . zeae grown in a medium containing only D-glucose as carbohydrate (no cellulose or C M C ) by solutions of either polygalacturonate or lignosulfonate. Since lignosulfonate was effective as a release agent, it was added to the growth medium; no significant increase in C activity of the medium was found. Some C and 0-glucosidase activity (two to three times that of the con trols) was released from harvested mycelium by solutions of highly substituted carboxymethylcellulose C M C 12M31P (Hercules Inc., W i l mington, D e l . ) . x


x

Table I.

Enzyme Activity Remaining on Mycelium After Treatment with Lignosulfonate Rehtive Enzyme Activity (Reducing Tower as u-Glucose per mg. Fresh wt.)

Treatment

C

β-Glucosidase

280

100

160

80

195

40

580

130

310

60

320

30

x

Acetone-dry ice Non-frozen mats in assay medium Acetone-dry ice treated mats in assay medium Acetone-dry ice treated mats previ ously washed with lignosulfonate in assay medium Merthiolate Untreated mats in assay medium Merthiolate treated mats in assay me dium Merthiolate treated mats previously washed with lignosulfonate in assay medium

A n investigation was then carried out to determine the amount of enzyme activity washed from the mycelium with lignosulfonate. Cultures were grown and mycelium was collected by filtration. Part of the my celium was washed with lignosulfonate; then the washed mycelium and part of the unwashed mycelium was killed with either acetone-dry ice or Merthiolate. The mycelium was then placed directly into the assay medium and the reducing power and D-glucose were determined. Results of this investigation, given in Table I, indicate that some of the enzyme activity is lost during the killing process but that little of the C activity is removed and/or the C enzyme(s) is activated by the lignosulfonate wash. The latter hypothesis was proved to be true by the addition of x

x


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lignosulfonic acid to active culture filtrates. The β-glucosidase activity is either removed or enough lignosulfonate remains to partially inhibit the glucostat reagent. To get a better idea of the portion of the C activity released by treatment with the lignosulfonate polyanion, mycelium was disrupted by sonification after a lignosulfonate washing. The results i n Table II again show that there was some greater removal and/or activation of the C enzyme(s) by the lignosulfonate wash than by the acetate buffer wash. However, no more than half of the total activity was solubilized even by very extensive sonification. Treatment of remaining pellets with the polyanions, surfactants, and enzymes used before removed no additional enzyme. x


x

Table II. Cx Enzyme Distribution After Sonification (20 min.) and Centrifugation Percentage of Total C

x

Cultrate filtrate Wash Supernatant after sonification and centrifugation Pellet after sonification and centrifugation

Activity

Mycelium Unwashed Before Sonification

Mycelium Washed with 0.2M Acetate Buffer (pH 4.5) Before Sonification

Mycelium Washed with 0.4% Lignosulfonate Before Sonification

35%

29% 15%

28% 22%

32%

28%

24%

33%

27%

25%

This is a preliminary report of our work. Our results to date indicate that cellulolytic enzymes may be found extracellularly, both i n the culture medium ^nd attached to the surface of hyphae, and, perhaps, intracellularly. In addition, they may be found in active and inactive forms i n all places. The binding of the enzymes to the mycelium and the natural release mechanism is unknown. In order to investigate induction and repression mechanisms and the influence of chemical and environmental factors on the synthesis of cellulolytic enzymes, total enzyme activity must be measured. Further work along these lines is i n progress. Literature Cited (1) Anraku, Y., Heppel, L. Α., J. Biol. Chem. 242, 2561 (1967). (2) Barash, I., Klein, L . , Phytopathology 59, 319 (1969). (3) BeMiller, J. N . , Tegtmeier, D . O., Pappelis, A. J., Phytopathology 58, 1336 (1968).



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(4) Betterton, H . O., M.S. Thesis, Southern Illinois University, Carbondale, Illinois (1963). (5) Coles, N . W . , Gross, R., Biochem. J. 102, 742 (1967). (6) Ibid., 102, 748 (1967). (7) Cowling, Ε. B., Kelman, Α., Phytopathology 53, 873 (1963). (8) Deshpande, Κ. B., Deshpande, D . S., Enzymologia 30, 206 (1966). (9) Horton, J. C., Keen, N. T., Phytopathology 56, 908 (1966). (10) Horton, J. C., Keen, N. T., Can. J. Microbiol. 12, 209 (1966). (11) Isaac, P. K., Can. J. Microbiol. 10, 621 (1964). (12) Mandels, M., Reese, E . T., Ann. Rev. Phytopathology 3, 85 (1965). (13) Moreau, M., Trique, Β., Compt. Rend. 263, 239 (1966). (14) Noviello, C., Ann. Fac. Sci. Agrar. Univ. Studii Napoli Portici 30, 461 (1965); Chem. Abstr. 65, 2679 (1966). (15) Tashpulatov, Zh. Mikrobiologiya 35, 27 (1966); Chem. Abstr. 64, 16322 (1966). (16) Weimberg, R., Orton, W . L . , J. Bacteriol. 90, 82 (1965). October 14, 1968. A contribution of Interdisciplinary Research in Senescence, a Cooperative Research Project of Southern Illinois University.

Discussion J. K. Alexander (Philadelphia): "Where you have increase of en zyme activity, I wondered if you have looked at the possibility of a cellulase inhibitor being released, therefore, freeing your activity?" J. N . BeMiller: 'Tes. W e are relatively confident that we can rule out inhibition as a factor. W e have considered this and can find no evidence of inhibitors being present and then being destroyed, by the various criteria that we have used. W e have also ruled out, at least i n our own minds, the idea of a precursor being present which becomes activated to form the true enzyme. However, we are trying to keep an open mind on these subjects and w i l l continue to consider them." Ε. T . Reese: " W e have found that nearly all cellulolytic organisms, where cellulase is induced, do produce small amounts of constitutive enzyme, very very low levels. Your measurements here indicate similar very low levels of enzymatic activity. Under the conditions that you use I notice that the glucose d i d not disappear very rapidly. In our work on cellulase induction, we actually try to reduce the rate at which the sub strate is consumed. I am wondering whether your conditions, where it is obvious that the organism wasn't very happy with its environment, were actually limiting growth and so stimulating induction?"


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J . N . BeMiller: "It may be. I do believe that stress is a necessary prerequisite to induction and that unfavorable growth conditions usually produce higher amounts of enzyme. However, our growth conditions are actually quite good for this organism, which is considered to be a very poor pathogen by the plant pathologists. It doesn't grow very well even in the corn stalk; it grows well enough to do considerable damage, but it is not what would be considered virulent.


Constitutive Cellulolytic Enzymes of Diplodia zeae

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