FUNGAL CELLULASES IX. GROWTH OF STACHYBOTRYS ATRA ON CELLULOSE AND PRODUCTION OF A ,8-GLUCOSIDASE HYDROLYSING CELLOBIOSE 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 cellulase 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 enzymes 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-glucose 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 oligosaccharides 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 enzyme 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 reducing sugar 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 hydrolysis 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 fungus 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.