FUNGAL

IX. GROWTH OF STACHYBOTRYS ATRA ON AND PRODUCTION OF A ,8-GLUCOSIDASE HYDROLYSING

By G. YOUATT*

[Manuscript received October 1, 1957]

Summary The growth of Stachybotrys atra on an improved Waksman-Carey medium containing cellulose as the sole carbon source is described. Under these conditions the organism produces an extracellular and an intracellular ,8-glucosidase which is capable of hydrolysing cellobiose. Some properties of this ,8-glucosidase are discussed.

I. INTRODUCTION Previous papers in this series have been concerned with the growth and production of by Strachybotrys atra. Thomas (1956) has described the pro­ duction of an extracellular cellulase which is produced by the organism when grown on cellulose and which hydrolyses poly-,8-1,4- chains to a mixture of glucose and cellobiose but which is incapable of hydrolysing cellobiose itself. Jermyn (1955) has described an extracellular ,8-glucosidase which is produced when the organism is grown on glucose or starch, but which does not hydrolyse cellobiose. The fate of the cellobiose produced by the cellulase when the organism was grown on cellulose was therefore unknown. The present paper is concerned with some improvements in the culture of S. atra on media containing cellulose as the sole source of carbon and also with some properties of an intracellular ,8-g1ucosidase which is capable of hydrolysing cellobiose and cellulose and which differs from the two enzymes already described.

II. METHODS (a) Determination of Fungal Growth Since the presence of undigested cellulose prevented the determination of mycelial dry weights, the extent of fungal growth was estimated by the amount of insoluble nitrogen formed by the culture. The culture (50 ml) was filtered, and after washing the mycelium with c. 200 ml water, its nitrogen content was determined by the method of McKenzie and Wallace (1954). Growth of the organism is expressed as mg nitrogen formed per culture. If it is assumed that the mycelium contains 4·6 per cent. nitrogen, as found by Thomas (1956) for mycelium grown on glucose, then 1 mg of nitrogen is equivalent to 21·7 mg dry weight of mycelium.

(b) Estimation of Cellulase Activity The viscometric method of Thomas (1956), using sodium carboxymethylcellulose as substrate, was employed. 'It was found that over the restricted range of cellulase

*Biochemistry Unit, Wool Textile Research Laboratories, C.S.I.R.O., Parkville, Vic. 210 G. YOUATT activities encountered in untreated culture filtrates the equation used by Thomas to relate concentration to rate of decrease of viscosity could be simplified and the following equation was used for calculating cellulase activities:

E = (1Jt~o)1.25d(lr1))/dt, where E = enzyme concentration in units/g solution, and 1J = specific viscosity.

(c) Estimation of Oellobiase Activity The determination of cellobiase activity by measurement of the increase in titre is insensitive and a manometric method based on that of Kellin and Hartree (1948) was used. In this technique, the glucose formed by the of cellobiose was oxidized in the presence of glucose oxidase and the oxygen con­ sumption measured by standard Warburg techniques. The Warburg flasks contained 1 ml McIlvaine sodium phosphate-citric acid buffer, pH 5·4, 1 ml 1 per cent. w/v glucose oxidase (Sigma Chemical Co., U.S.A.), 0·5 ml 1 per cent. w/v catalase (L. Light & Co. Ltd., England), 0·1 ml ethanol, and 1 ml of the enzyme preparation; the side-arm of the flask contained 1 ml4 per cent. w/v cellobiose solution. Measure­ ments of the oxygen consumption, which under these conditions is directly pro­ portional to time, were made at 5-min intervals. The rate of oxygen consumption was calculated by the method of Aldridge, Berry, and Davies (1949) and converted to mg glucose formed per minute. One unit of enzyme activity is that quantity of enzyme which liberates 1 mg of glucose per minute under these conditions.

(d) Preparation of Enzyme Solutions Cellobiase activity is found in the mycelium of S. atra; occasionally activity has been found in the medium but such occurrences have been sporadic and generally associated with old cultures-the enzyme is characteristically intracellular. Enzyme solutions were prepared by filtering off the mycelium, washing with water, and then grinding it with 10-20 ml of water in a Potter-Elvehjem homogenizer. The extract was filtered to remove debris and the clear solution used. Culture filtrates were used as a source of cellulase without further treatment.

III. GROWTH EXPERIMENTS (a) Effect of Oultural Oonditions The was maintained by serial transfer on cellulose-agar slopes (McQuade, unpublished datlJ,). Spore suspensions prepared from these slopes were used to inoculate 50-ml amounts of medium contained in 250-ml conical flasks. Incubation was at 28°C and the reciprocating shaker used had a total excursion of 2·5 in. and a rate of 98 c/min unless otherwise stated. Work in this Laboratory has shown that greatly improved growth of S. atra can be obtained by modifying the original Waksman-Carey medium (McQuade, unpublished data). Though this work has been done using glucose as the carbon source it appeared reasonable to assume that similar improvements could also be FUNGAL OELLULASES. IX 211

made with cellulose media. Accordingly an investigation was made of the effect of changes in the levels of cellulose, nitrogen, phosphate, and shaking rate on the growth and enzyme production of the fungus. The minor constituents of the medium, which appeared to be at adequate concentrations, were not investigated.

TABLE 1 EFFECT OF CELLULOSE CONCENTRATION AND SHAKING RATE ON THE GROWTH OF S. ATRA AFTER 7 DAYS

Mean Mycelial Mean Mycelial Cellulose Shaking Rate Nitrogen Nitrogen (gil) (c/min) (mg) (mg)

5 3·87 98 7·70 10 5·31 120 4·36 20 8·64

In order to examine the effect of changes in the concentration of media constit­ uents, a number of flasks representing the various treatment combinations were set up in factorial arrangements (Cochran and Cox 1950), and determinations made ofthe extent of growth and level of enzyme production. Table I shows the increasing amount of growth with increasing cellulose concentration and the deleterious effect of a high rate of shaking.

TABLE 2 EFFECT OF CELLULOSE CONCENTRATION AND AGE OF CULTURE ON THE GROWTH AND ENZYME PRODUCTION OF s. ATRA

Mean Mycelial Nitrogen Mean Cellulase Mean Cellobiase (mg) Activity Activity

Cellulose (gil) 10 6·77 18·40 0·26 20 1l·72 24·18 0·39 ------Age (days) 6 8·04 10·37 0·41 12 9·36 19·13 0·24 18" 10·33 31·84

Effects due to ammonium chloride and dipotassium hydrogen phosphate were o bserved in those experiments in which the levels ofthese salts were in the range 5-10 gil and 3-6 gil respectively. However, when the concentration of ammonium chloride was decreased (2·5-5 gil) and that of potassium phosphate increased (6-12 gil) no effects attributable to these salts were observed. In order to determine whether the effects of changes in the composition of the medium were influenced by the age of the culture, an experiment was carried out in which the cultures were harvested after 6, 12, and 18 days. The levels of ammonium 212 G. YOUATT chloride and potassium phosphate were in the ranges prev:iously found to be optimal, and under these conditions it was found that the extent of growth was dependent only on the concentration of cellulose and the length of time for which the culture was allowed to grow (Table 2). The effects of these various treatments on the yield of enzymes were very similar to those produced in growth. Production of cellulase appeared to run parallel with growth and, at the concentration of ammonium chloride and potassium phosphate finally chosen, the factors influencing the yield were cellulose concentration and age of the culture. Similar results were found with cellobiase except that, in contrast with cellulase, the yield of enzyme decreased with age of the culture.

40 (a) O·B (b)

30 0·6 UI !:z 20 ::I ~ I /I III UI . ! « ~ 20 :l . '0, III io.~ I/~ 0 ;:v~o. '0, lO~ / ./ ./ /

6 12 o 6 12 DAYS DAYS

Fig. I.-Production of (a) cellulase and (b) cellobiase by S. afJra in media containing high levels of cellulose. Enzyme activities refer to the total activity of the culture and the cellulose concentrations (gil) are given on the figures.

Thus the following standardized medium was chosen as being the most suitable for the general growth of S.atra on cellulose and for the production of the cellulolytic enzymes: gil mgll Cellulose 20 CaC12 20 NH4Cl 5 ZnS04·7H2O 2 K 2HP04 7·5 MnS04·4H2O 1 MgS04·7H2O 1 Iron-alum lO Biotin 0·02

(b) Enzyme Production in the Presence of High Ooncentrations of Oellulose Thomas (1956) reported a diminished yield of cellulase when the amount of cellulose in the medium was greater than 2 gil. Since no evidence ofthis was obtained FUNGAL CELLULASES. IX 213 during the experiments just described, an experiment was carried out to see if this effect could be observed at higher levels of cellulose. The results are shown in Figures l(a) and l(b). The concentration of cellulose in the medium may be raised to over 20 gil before there is any decrease in the amount of cellulase detectable. Cellobiase production appears to be relatively unaffected by this high level of cellulose.

(c) Enzyme Production during the First 12 Days of Growth Growth of the organism on this medium is fairly rapid and the cultures mature at about 7 days. Figure 2 illustrates the growth, production of enzymes, and pH of the medium during a 12-day growth period.

32 ,.9

'"r12 24 /-k" l' § ~ 10 20 ..III ~ z :J Z 8 '- « "~ ~/ - .J g :J :t 0'6 tn "I12 J .. 6 !:: w ~ rz U °11: :J " 4 0'4 ~ B ~ iJ m i70~: 0 21- 21- /" I 10'2 ::j-l4 W U

oL It ~ I I I I I 10 Jo 2 4 6 B 10 12 ° DAYS Fig. 2.-Growth and enzyme production of S. atm on cellu­ lose. A, growth (mg N); B, cellulase activity of the medium; G, cellobiase activity of mycelium; D, pH of the medium.

IV. PROPERTIES OF CELLOBIASE (a) Adaptive Nature of Oellobiase Since cellulase activity is not found when S. atra is grown on glucose and the ,8-glucosidase of Jermyn is not found in cultures growing on cellulose it was of interest to examine the production of cellobiase under these two sets of conditions. It was found that it is only when cellobiose, either added as such or produced by the break­ down of cellulose, is present that the enzyme is produced in any great quantity (Table 3). (b) Extractability of the Enzyme Though grinding of the mycelium with water in a Potter-Elvehjem homogenizer was used as the standard technique for extracting the enzyme, other methods were tried. Of these, grinding the mycelium with silica flour and water in a mortar gave equally active extracts but dispersing the mycelium in a Waring Blendor, with or without the addition of abrasives, gave lower yields. It was also possible to freeze-dry the mycelium and extract the resulting powder. 214 G. YOUATT

An attempt was made to effect a partial purification of the enzyme by selective extraction of the mycelium with cold dilute ethanol. Freeze-dried mycelium (200 mg) was ground at 5~C with lO-ml aliquots containing 1 ml of O·lM acetate buffer, pH 5, and ethanol to give concentrations of 0, 10, 20, and 40 percent. v/v.

TABLE 3 PRODUCTION OF CELLOBIASE BY S. ATRA ON VARIOUS CARBON SOURCES

Cellobiase Activity Mycelial Weight Carbon Source I of Culture (mg) ------Cellulose 0·59 270* Glucose 0·04 273 Maltose 0·02 265 Starch 0·01 239 Cellobiose 0·66 320

*Calculated value assuming 4·6 per cent. nitrogen in mycelium.

The mycelial debris was removed by centrifugation and the supernatants assayed for cellobiase activity. Table 4 lists the activities of the extracts and also the amounts of protein extracted, using an arbitrary scale deduced from the optical densities of the solutions at 275 mfl-. From these results it appears that by extraction of the mycelium with 20 per cent. ethanol, discarding the extract, and re-extracting the mycelium with 10 per cent. ethanol it should be possible to obtain c. 85 per cent. of the enzyme activity with only c. 25 per cent. of the total extractable protein, a purification factor of about 4. It was found, however, that when the mycelium from 21. of culture fluid was fractionated between 10 and 20 per cent. ethanol in this way, it gave rise to only 5 mg of crude protein. In view of this very small amount of material further fractionation experiments were not attempted.

TABLE 4 FRACTIONAL EXTRACTION OF CELLOBIASE FROM THE MYCELIUM OF S. ATRA

Percentage ethanol 0 10 20 40 Cello biase activity of extract 0·64 0·58 0·05 0·02 Protein in extract (arbitrary units) 1·00 0·68 0·41 0·62

_.------_._---

(c) Hydrolysis of Various Glycosides Mycelial extracts prepared in this way hydrolysed cellobiose and p-nitrophenyl ,B-glucoside readily but had only slight activity towards methyl ,B-glucoside. There was only a trace of a-glucosidase activity as shown by action against p-nitrophenyl a-glucoside but in spite of this there was considerable hydrolysis of p-nitrophenyl a-cellobioside. However, the hydrolysis of the cellobioside does not go to completion and if a correction is made for the slight a-glucosidase activity it appears that only FUNGAL CELLULASES. IX 215 one mole of glucose is being liberated per mole of cellobioside. The ~-linkage between the glucose residues is readily split but the a-linkage of the aglucone is almost com­ pletely resistant. Lactose, which differs from cellobiose solely in the configuration of the terminal C4 hydroxyl, is essentially unattacked.

(d) Effect of Time and Enzyme Concentration Under the conditions given above for the.assay of the enzyme the rate of hydro­ lysis of cellobiose is linear with time and proportional to enzyme concentration (Figs. 3(a), 3(b)).

1'51- lal

0'12 0'06 Ib) _ 0·10 _0'05 Z Z ~0.08 ~0'04 :E :E ~O'06 ~O'03 III IIIo o UO·04 gO'02 (//0 ~ .J .J (!) (!)0'02 0·01 "(\~

~ OV f! , 0 1 ! I I ! o 5 10 15 20 25 ENZYME CONCN. 4 567 TIME IMINI (ARBITRARY UNITS) ~ Fig. 3.-(a) Hydrolysis of cellobiose by various concentrations of cellobiase; (b) effect of enzyme concentration on the rate of hydrolysis of cellobiose by cellobiase; (e) pH-activity curve for cellobiase.

(e) Effect of pH In Figure 3(c) is set out the pH-activity curve for the hydrolysis of cellobiose. McIlvaine buffers were used throughout this experiment as they covered the required pH range. The limits of this pH curve may be affected by the pH-activity relation­ ships of the glucose oxidase used in the assay system. Since, however, the rate of hydrolysis at· the optimum pH observed is within the range where the cellobiase concentration is the limiting factor, this optimum will not be influenced by the other enzymes in the system.

(f) Effect of Substrate Concentration The relation between the substrate concentration and the rate of hydrolysis for the two substrates cellobiose and p-nitrophenyl ~-glucoside were investigated. The results lead to rectilinear plots according to the method of Lineweaver and Burk and the Michaelis constants were found to be 3·9 X 1O-4M and 2·0 X 1O-4M respectively. The extracellular ~-glucosidase has a Michaelis constant of 3 X lO-fiM when acting on p-nitrophenyl ~-glucoside under comparable conditions (Jermyn 1955).

(g) Hydrolysis of Cellulose Oligosaccharides It was found that the oligosaccharides from cellobiose to cellopentaose inclusive and also a of mean chain length 11, which was prepared by sulphuric acid 216 G. YOUATT degradation of cellulose, were readily hydrolysed and at comparable rates. Some of the results are given in Figure 4. Attempts to demonstrate the hydrolysis of cellulose itself, either in the form of alkali-swollen cellulose or as a soluble derivative such as carboxymethylcellulose were inconclusive.

_____ oC

100 _____ os

______0 A

90

80

70

I/) iii ~ 60 0 1l: 0 > X 50

C) '"« >-z U'" 40 1l: '"II. 30

20

10

o 10 20 30 40 50 60 70 80 90 MINUTES

Fig. 4.-Hydrolysis of cellobiose (A), cellotetraose (B), and a cello~ dextrin of chain length 11 units (0) by S. atra cellobiase.

V. DISOUSSION Compared with the original Waksman-Carey medium used there has been about a sixfold increase in yield of mycelium. Correlation of the results of this paper with previous ones in the series leads to the recognition of at least three well-defined enzymes in S. atra which are capable of hydrolysing the fJ-1, 4-glucosidic linkage, but which differ in their specificity towards the rest of the substrate molecule. They are (1) fJ-glucosidase (Jermyn 1955) which attacks aryl fJ-glucosides but not cellobiose or cellobiosides; (2) cellulase (Thomas 1956) which attacks chains of fJ-l, 4-linked glucosaccharides having a chain length of at least three units, and soluble cellulose derivatives; (3) cellobiase which hydrolyses cellobiose, fJ-l,4-linked glucosaccharides up to a chain length of at least 11 units, and some aryl fJ-glucosides. FUNGAL CELLULASES. IX 217

The production of cellulase is very closely linked with the production of mycelium, and the finding that proportionately more cellulase is produced as the age of the culture increases may not be due so much to increased production of the enzyme as to the liberation of absorbed enzyme from the diminishing amount of cellulose in the medium. Since the cellobiase is capable of hydrolysing a cellodextrin and on this basis could be classified as a cellulase, it is difficult to account for the failure to demonstrate any hydrolysis of either swollen cellulose or carboxymethylcellulose and it is not possible to set any upper limit to the number of units of a 11-1, 4-linked glucosaccharide chain beyond which it is not attacked by the enzyme. However, under the normal physiological conditions of growth when the enzyme is within the mycelium only those oligosaccharides sufficiently small to pass through the would be presented to it and it seems unlikely that a molecule of any great chain length would be involved.

VI. ACKNOWLEDGMENTS I am extremely grateful to Mr. W. B. Hall for the statistical work required in this paper, to Mr. A. B. McQuade for allowing me to draw upon his extensive know­ ledge of the growth habits of S. atra, and to Dr. M. A. Jermyn for the cello-oligosac­ charides.

VII. REFERENCES ALDRIDGE, W. N., BERRY, W. K., and DAVIES, D. R. (1949).-Nature 164: 925. COOHRAN, W. G., and Cox, G. M. (1950).-"Experimental Design." (John Wiley & Sons, Inc.: New York.) JERMYN, M. A. (1955).-Auat. J. Biol. Sci. 8: 541. KElLIN, D., and HARTREE, E. F. (1948).-Biochem. J. 42: 230. MOKENZIE, H. A., and WALLAOE, H. S. (1954).-Auat. J. Ohem. 7: 55. THOMAS, R. (1956).-Aust. J. Biol. Sci. 9: 159. -