FUNGAL CELLULASES. By M. A. JERMYN\:' [Manuscript received October 6, 1952] Summary

Transcription

1 FUNGAL CELLULASES. ST ACHYBOTRYS ATRA: GROWTH AND ENZYME PRODUCTON ON NON-CELLULOSC SUBSTRATES By M. A. JERMYN\:' [Manuscrpt receved October 6, 1952] Summary The growth of the mould Stachybotrys atra OCl modfed vvaksman-carey medum has been studed. ron, znc, copper, and manganese have been shown to be norganc mcronutrents for the 'mould but ther essental nature has not been conclusvely demonstrated. Certan other supplements appear to stmulate growth. When ammona s the ntrogen source, the mould makes good growth only wth sugars and related compounds as carbon sources; the only other carbon source readly utlzed s acetate. Replacement of ammona by ntrate asssts the utlzaton of a range of organc acds. A wde range of ntrogen sources can be utlzed for growth; protens are good sources of both carbon and ntrogen, but ndvdual amno acds are nadequate. The course of growtn on sucrose, starch, and both buffered and unbuffered glucose meda has been followed. Enzymes wth f1-glucosdase actvtes were produced on all meda and the presence of substrates contanng f1-glucosdc lnkages was unnecessary for the producton of actvty smulatng the "Cx enzyme" (enzyme splttng polymerc f1-glucosdc lnkages) of Reese, Su, and Levnson (1949). Hgh actvty for the hydrolyss of salcn and sodum carboxymethyl cellulose appears n the medum at and just after the germnaton of the mould. n unbuffered meda a second maxmum s assocated wth the attanment of maxmum mycelal weght. Enzymc actvty aganst p-ntrophenyl-f1-glucosde does not appear to be closely correlated wth the other two actvtes. The meanng of these results s dscussed n terms of the hypothess of multple f1-glucosdase actvtes and the non-exstence of a specfc "Cx" enzyme (Jermyn 1952b).. NTRODUCTON Stachybotrys atra Corda s typcally north temperate n ts dstrbuton accordng to Whte, Yeager, and Shotts (1949) but s also a very frequent solate n the Melbourne area (Cox et al. 1945). n tropcal areas S. atra s replaced by the very closely related M emnonella echnata (Rv.) Gallow. The complete elmnaton of nvald speces n Stachybotrys has not yet been accomplshed and Bsby (1945) beleves that many of the speces descrbed wll eventually be reduced to synonymy n the hghly varable S. atra. The uncertanty about. dentfcatons n ths genus must be borne n mnd n comparng the varous physologcal studes n the lterature. Su (1951) classfes S. atra among the «strong" cellulolytc fung wth abundant solatons from decomposng cellulosc \:' Bochemstry Unt, Wool Textle Research Laboratory, C.S..R.O., Melbourne.

2 FUNGAL CELLULASES. 49 materals n the feld; the "very strong" M yrothecum verrucara on whch the majorty of studes on fungal cellulase have been made n the past few years (Saunders, Su, and Genest 1948; Whtaker 1951) seems unable to mantan tself on cellulosc substrates n the feld. For ths reason S. atra has been selected for fundamental studes n an Australan laboratory on the producton of cellulolytc enzymes by mcroorgansms. Reese, Su, and Levnson (1949) have postulated that two types of enzymes are requred for the breakdown of cellulose. The frst ("C. enzyme"), whch s present only n cellulolytc fung, degrades natve cellulose to a form that can be further hydrolysed by the second ("Cx enzyme") whch s present n all cellulolytc and most non-cellulolytc fung. t has been shown (Jermyn 1952a, 1952b) that the non-cellulolytc fungus Aspergllus oryzae releases Cx actvty nto the culture medum and that ths actvty s due to the presence of some eght,b-glucosdases, whch have dfferent degrees of specfcty for dfferent substrates. Producton of these enzymes took place n the absence from the culture medum of substrates havng,b-glucosdc lnkages. The work presented n ths paper s desgned to test whether the "Cx" actvty of S. atra s produced n the culture medum n the absence of,b-glucosdc carbon sources and to dscover condtons for producng the enzyme or enzymes nvolved n ths actvty n suffcent concentratons to warrant purfcaton. t also extends the results of Perlman (1948) to gve a wde enough prelmnary survey of the growth of S. atra n shake culture to serve as a bass. for a programme of physologcal research on the fungus.. CULTURAL CONDTONS To smplfy ths prelmnary study only certan of the possble varables n culture condtons have been nvestgated. Except where specfc menton of the fact s made, the condtons specfed n the descrpton below were used throughout. ncubaton was carred out n a warm room mantaned at 28 ±: 0.5 C., whch s approxmately the optmum temperature for growth of S. atra (Basu 1948). The shaker used was adapted from the desgn of Jennson et az. (1950); t had a recprocatng acton wth a total excurson of 2 n. and a speed of 96 cycles per mnute. Where a contnuous record of the ph of the culture medum was to be taken, a Leeds and Northrup Mcromax recordng ph meter wth shelded leads was used. t was found that the drectly determned ph of the culture medum and that recorded on the chart were n agreement for runs of up to 3 weeks. A culture of S. atra was obtaned from Mr. F. M. Crook of the Defence Research Laboratores, Melbourne. t agreed n all partculars wth the lterature descrptons of the speces. The fungus was mantaned by seral transfer on potato-dextrose sugar slopes. After 10 days growth at 30 C, tubes were kept n a refrgerator at 5 C. tll requred. A spore suspenson from the tubes was used to noculate a layer of potato-dextrose agar medum n 250 m!. Roux bottles. After days ncubaton at 28 C., the contents of the Roux bottle were used to prepare spore suspensons for noculatng the culture flasks.

3 50 M. A. JERMYN The basc mneral medum used throughout was that of Waksman and Carey (1926) as modfed by Fahraeus (1947). n accordance wth the fndngs of Marsh and Bollenbacher (1946), Perlman (1948), and Buston and Basu ( 1948), 12 p.g. of botn was routnely added per ltre of medum. After experments on mneral nutrton had ndcated the stmulatng effect of znc ons, the Waksman-Carey medum was further modfed by the addton of :2 mg.!l. of ZnS04.7H20. The fnal composton of the medum was as follows: (NH4)2HP04 MgS0 4 7H:P KC CaCl 2 FeS04 7HO ZnS0 4 7H 2 0 MuS04.4H 2 0 D-botn g.! X X X X X 10-6 Usng ths medum experments were performed to test: () Ablty of S. atra to Use Varous Carbon and Ntrogen Sources.-The medum was dstrbuted nto 250-m!. concal flasks, 50 m!. beng added per flask and, after autoclavng, 0.2 m1. of spore suspenson contanng 10 9 spores/ml. was added to each. () Enzyme Producton by S. atra on Varous Meda.-A three-necked, 2-1. Woulffe bottle contanng 500 m1. of medum was autoclaved, one of the necks beng ftted wth a potassum chlorde brdge extenson from a calomel electrode dppng nto the lqud. On coolng, an extended glass electrode, sterlzed n dstlled water, was ntroduced nto a second neck. The contents of the bottle were then noculated and the electrodes connected to the recordng ph meter. Samples were wthdrawn as requred through the thrd neck of the bottle. A contnuous record of mycelal weght could not be obtaned from ths apparatus; a set of 250-ml. flasks was therefore noculated n parallel groups of four flasks chosen by lot removed from the shaker from tme to tme, and the bulked mycelal weght calculated to gve that from 500 m!. of medum. Growth n the flasks was n general a lttle ahead of that n the bottle and Fgures 3-8 must be nterpreted wth ths fact n mnd.. DETERMNATON OF ENZYMES AND OTHER COMPONENTS OF THE CULTURE MEDUM (a) Ca: Enzyme and Other f3-glucosdases The procedure for the Cx enzyme usng sodum carboxymethyl cellulose as substrate has been outlned by Jermyn (1952a). A smlar procedure wth 1 per cent. soluton of sodum cellulose sulphate (Eastman Kodak Co.) as sub <;trate was also used. The measurement of salcnase and p-ntrophenyl-f3-glucosdase has also been outlned by Jermyn (1952a).

4 _._-- FUNGA CELLULASES. 51 ( b) Amylase and Sucrase The procedure of Crewther and Lennox (1952) was used. ( c) Proteases The actvty of enzyme solutons n reducng the vscosty of gelatn was measured accordng to the method of Lennox and Ells (1945). Ther acton on gelatn was also measured by the gravmetrc technque of Crewther (1952). These are the "vscometrc" and "gravmetrc" enzymes of Crewther and Lennox (1953 ). TABLE 1 GROWTH OF S. ATRA ON ACETATE MEDA, CONTANNG THE SAME MNERAL ELEMENTS AS THE WAKSMAN-CAREY MEDUM AND FURTHER SUPPLEMENTED BY THE ADDTON OF POSSBLE MCRONUTRENTS ntal ph 7.0; cultured 5 days at 28 C. Medum Composton of Medum (g./.) Yeld (mg.jml.) (mean of 6 flasks) C, 8 0; N, 0 6; K, 14 8; P, 0,2; Na, 200 X 10-3 ; Cl, X 10-3 ; S, 66 X 10-3 ; Mg, 50 X 10-3 ; Ca, 7 X 10-3 ; Fe, 2xlO-"; Zn, 450 X 10-6 ; Mn, 250x0-6 ; botn, lox As 1 plus Co, 20 X 10-6 ; Cu, 12 X 10-6 ; Mo, 25 X 10-6 As 2 plus, 30 X 10-6 ; B, 3 X 10-6 As 3 plus ncotnamde, 500 X 10-6 ; thamn hydrochlorde, 200 X 10-6 ; p-amnobenzoc acd, 100 X 10-6 ; rboflavn, cholne chlorde, sodum pantothenate, pyrdne hydrochlorde; all 50 X 10-6 As 4 plus peptone, 5 X As 5 plus F, 45xlO-6 ; Br, 35xlO-6 ; Cd, 20xlO-6 ; N, 12 X 10-6 ; A, 4 X As 6 plus Hg, 40 X 10-6 ; Ba, 25 X 10-6 ; Cr, 10 X ( d) Esterase Ths enzyme was measured by ts ablty to decompose the dacetyl dervatve of chlorphenol red (3 X 1O-5M) at ph 7.0 and 3TC. ( e),reducng Sugar The reducng sugar n the sutably dluted culture fltrate was estmated by the Nelson-Somogy colormetrc method (Somogy 1945). All substances present capable of reducng alkalne copper reagents would be estmated as "reducng sugar" by ths technque. Snce the apparent reducng sugar n the

5 52 M. A. JERMYN medum usually fell to zero, error from ths source could not have been very great. (f) Sucrose Sucrose was determned as n (e) after nverson wth dlute hydrochlorc acd. TABLE 2 GROWTH OF S. ATRA N A BASAL MEDUM CONTANNG ACETC.ACD A.R. 15 ML., KNO. A.R. 1.0 G., MgSO. 7H 20 A.R. 0.5 G., KH"pO. A.R. 3.0 G., KOH A.R G., AND D-BOTN 10 f-tg/l. Medt:m adjusted to ph 7.0 (KOH) and trace elements removed accordng to the procedure of Stenberg (1950). Addtons Yeld of Mycelum (mg./ml.) Mter 5 Fe Zn Mn Cu Mo Other Days at 28 C. (f-tg./l.) (f-tg./l.) (f-tg./l.) (f-tg./l.) (f-tg./.) Addtons ' ' 300 f-tg./., B' f-tg./l., B 25 f-tg./1. Plant ash (from wood of Eucalyptus 0 42 sp.) mg./ Mxed B vtamns 1 96 at f-tg./ Peptone 10 mg./ All the above add tons (g) Total Carbohydrates The orcnol method of Pre (1936) was sutable for the estmaton of starch and ts degradaton products n S. atra culture fltrates. t h) Mycelal Weght The culture medum was fltered through a par of matched ashless flter papers (Whatman No. 541), the mycelum washed once wth dstlled water, the whole dred 48 hr. at 55 C., and the mycelal weght obtaned by dfference.

6 FUNGAL CELL ULASES. 53 V. MNERAL NUTRTON OF S. ATRA The modfed Waksman-Carey soluton of Fahraeus (1947) s defcent n certan elements known to be mportant n the nutrton of fung, ncludng znc, copper, and molybdenum (Stenberg: 1950). When S. atra was grown n ths medum wth glucose or sucrose as a carbon source, addton of other elements dd not affect the fnal yeld of mycelum. The addton of ZnS04.7H:!O (2 mg.!!.) was found to elmnate the lag perods of many days between the germnaton of the spores and the onset of rapd growth, whch occurred n an unpredctable way n the absence of znc. Apart from ths regulatory effect, ordnary "purfed" sugars and A.R. chemcals apparently contaned enough of the mcronutrent elements for growth of the fung. TABLE 3 COMPARSON OF MNERAL NUTRTON OF S. ATRA WTH THAT OF M. ECHNATA (PERUvAN 19,48) S. atra, Present Work, Acetate M. echnata, Perlman (1948), Glucose Meda Meda Metal Optmum Rato of Mycelal Optmum Concentra- Rate of Mycelal Concen- Yeld at Optmum to Concen- ton n Basc Yeld at Optmum to traton Mycelal Yeld on traton Medum Mycelal Yeld on (flg fl.) Basc Medum (flg fl. ) (flg fl.) Basc Medum ron c Znc >800 At least 6 5 c Manganese >400 At least 4 c Copper c.2 2 c When S. atra was grown on acetate meda ts dependence on varous norganc mcronutrents became apparent. t was possble to show that the presence of all the mneral components of the Waksman-Carey medum except calcum was necessary for optmum growth when usng unpurfed reagents. Table 1 shows that certan further addtons to the medum brought about stmulaton of growth under the condtons used by a further 40 per cent. These observatons were amplfed by testng certan mcronutrents n the more rgorous experments set out n Table 2. Specally cleaned, slcone-treated glassware was used, the basal medum was purfed by the calcum carbonate method of Stenberg (1950) and washed-spore suspensons were used for noculaton. Sgnfcant but not absolute dependence on ron, manganese, copper, and znc was demonstrated, together wth an apparently nhbtory effect of molybdenum n excess of 40 flg.!l. Far growth of the mould n "purfed" basal medum apparently means that Stenberg's procedure s not completely effectve n removng trace metals from acetate meda. n Table 3 the results for the growth of S. atra are compared wth those of Perlman (1948) for the growth of M. echnata n glucose meda.

7 54 M. A. JERMYN TABLE 4 UTLZATON OF VAROUS CARBON SOURCES BY S. ATRA GROWNG ON A WAKSMAN-CAREY MNERAL MEDUM Concentraton of carbon source, 1 per cent.; ntal ph 7.0; grown 5 days at 28 C. Carbon Source (a) Sugars D-Fructose Maltose D-Galactose D-Glucose.. D-Mannose D-Xylose L-Rhamnose Lactose Sucrose Cellobose Trehalose.. Raffnose.. D-Lyxose " D-Arabnose D-Rbose L-Arabnose D-Fucose L-Xylose.. L-Sorbose D-Glucoseamne (b) Sugar alcohols Glycerol Manntol Arabtol Dulctol Adontol nostol Sorbtol Erythrtol.. (c) Glucosdes,8-Methyl-glucosde a-methyl-glucosde Salcn (d) Polysacchardes nuln Sodum carboxymethyl cellulose Starch Dextrn Pectn Arabnc acd Xylan Chtn Cellulose: cotton wool "After 13 days ncubaton. t After 10 days ncubaton. t After 7 days ncubaton. Yeld (mg.jml) ' '9 1 8 O g '8* o g 0 8t 0 3t g t 1 3t [ Carbon Source Cellulose: flter paper sawdust Sodum algnate.. Cellulose aceta te.. (e) Mscellaneous sugar dervatves Gluconc acd Galactonc acd Gulonc acd Rbonc acd Saccharc acd Mucc acd Fructose dphosphate Cellobose octa-acetate (f) Alcohols Methanol.. Ethanol so-propanol so-butanol n-butanol tert-pentanol n-pentanol n-hexanol Ethylene glycol Propylene glycol (g) Organc acds Acetate Proponate Butyrate.. so-butyrate Valerate Oxalate Malonate Succnate Adpate Malate Lactate Ctrate Benzoate Salcylate Formate Glycollate Tartrate Glycerophosphate Pyruvate.. Resdual nsoluble substrate prevented mycelal weght determnaton. 1f "" ndcates growth n non-weghable amounts. Yeld (mg.jml.) 3 0t

8 FUNGAL CELLULASES. 55 TABLE 4 (Contnued) Carbon Source Yeld \mg./mj.) (h) Organc acds; ntrate as N source Acetate 1 0 Malonate 0 5 Malate 0 4 Tartrate 0 1 Succnate 0 1 so-butyrate Butyrate Lactate Ctrate Oxalate Formate Valerate Mxed B vtamns and proten ntrogen exert an apparent stmulatory effect on the growth of S. atra; n vew of the results of Marsh and Bollenbacher ( 1946) and Perlman (1948) ths s unlkely to reflect the exstence of absolute requrements. V. UTLZATON OF CARBON AND NTROGEN SOURCES (a) Carbon Sources The substance to be tested was added to the basal medum at a fnal concentraton of 1 per cent., and where necessary the ph of the medum was readjusted to 7.0. The results of tests usng a number of substrates are set out n Table 4. (b) Ntrogen Sources The carbon source used was 1 per cent. glucose and a few experments wth acetate meda are also reported. The medum was vared by addng K2HP04 (3.3 g./l.) n place of (NH4 hhp04 and a ntrogen source equvalent to ph 7.0 The results are set out n Table 5. ( c) Sngle Substrates as both Carbon and Ntrogen Sources A certan number of the amno acds and protens were tested as sources of both carbon and ntrogen. The (NH4hHP04 n the medum was agan replaced by K2HP04 and the nutrent was added n an amount suffcent to gve 0.57 g. NL These results are set out n Table 6. ( d) Adaptaton to Varous Meda The results quoted n Tables 4, 5, and 6 are for drect transfer from growth on the potato-dextrose agar sold medum to growth n lqud culture. All meda on whch far growth was not apparent by the ffth day were retaned

9 56 M. A. JERMYN TABLE 5 UTLZATON OF VAROUS NTROGEN SOURCES BY S. ATRA GROWNG ON A W AKSMAN-CAREY MNERAL MEDUM Carbon source: 1 per cent. glucose; ntal ph 7.0; ncubated 5 days at 28 C. Ntrogen Source Yeld (mg.jml.) Ntrogen Source Yeld (mg.jml.) (a) norganc Potassum ntrate Potassum ntrte.. Ammonum phosphate Ammonum ntrate Ammonum sulphate Ammonum chlorde Ammonum carbonate Potassum thocyanate Potassum cyande Hydroxylamne hydrochlorde.. Hydrazne sulphate (b) Amnes Methylamne Dmethylamne Trmethylamne Tetramethylammonum bromde Ethylamne n-propylamne so-butylamne n-amylamne Ethanolamne Ethylenedamne.. Putrescne Cadaverne Pyrdne Pperdne a-amno pyrmdne ndole (e) Urea and related substances Methyl urea Ethyl urea Guandne hydrochlorde Alloxan Allanton.. Urea Methylsothourea sulphate Ntroguandne Phenylurea Creatne Creatnne Thourea.. (d) Amdes Acetamde ' O g * O g o g Potassum phthalmde Urethane Hppurc acd (e) Purnes Xanthne Urc acd Caffene Theobromne (f) Growth factors Cholne chlorde Ncotnamde Pyrdoxne hydror.hlorde p-amnobenzoc acd Thamne chlorde (g) Amno acds Prolne Argnn Glycne Valne Asparagne Phenylalanne Leucne Lysne soleucne Hstdne.. Cystne Hydroxyprolne Aspartc acd Glutamc acd (h) Pro tens Gelatn Peptone () Varous N sources wth acetate meda Ammonum ntrate Potassum ntrate Ammonum phosphate Urea Argnne.. Guandne Prolne Methylurea Urc acd Glycnepotassum ntrate Glycne Potassum ntrte g t >/> "" ndcates growth n non-weghable amounts. t Resdual soluble substrate prevented mycelal weght determnaton.

10 FUNGAL CELLULASES. 57 on the shakng machne for 3 weeks; f no growth was apparent after ths perod the cultures were dscarded. n a fw cases, noted n the tables, scanty growth took place n ths perod. TABLE 6 UTLZATON OF VAROUS SUBSTRATES AS CARBON AND NTROGEN SOURCES BY S. ATRA GROW-"G ON WAKSMAN-CAREY MEDUM ntal ph 7.0, ncubated 5 days at 28 C. Subtrate Yeld (mg.jml.) (a) (b) Amno acds Alanne 0 3 Valne 0' 15 Prolne 0 04 Argnne 0 03 Hstdne 0 03 Glycne * Lysne soleucne Asparagne Phenylalanne Cystne Leucne Hydroxyprolne Protens Haemoglobn 3 0 Gelatn 2 8 Tryptone 2 1 Zen 2 0 Egg albumen.. ].g Peptone ].g "Mould.. enzyme" ] 2 Casen 0 5 Ground wool.. t.. "" ndcates growth n non-weghable amounts. t Resdual nsoluble matter prevented mycelal weght determnaton. Mycelal transfers were made from sparse cultures to a fresh batch of medum and ths process was contnued so long as there were sgns of adaptaton to the new substrate. Seral transfers were also made from cultures on varous meda that had supported measurable amounts of growth n the orgnal experments. The results of these subcultures may be summarzed as follows: () Meda supportng "good" growth (better than about 0.5 mg./ml. n 5 days) cortnued to support growth at least as well on contnued subculture. () Some meda that gave scanty growth (" ") supported luxurant growth on contnued subculture; n other cases the amount of vsble growth decreased to zero after two or three subcultures. Thus adaptaton was made to growth

11 58 M. A. JERMYN on meda wth proponate and malate as carbon sources and on cyande as a ntrogen source, but subculture proved mpossble on meda wth succnate and lactate as carbon sources. t was eventually possble to transfer to the correspondng sold medum S. atra that had become adapted to growth n lqud meda; the fungus has, for nstance, been mantaned for over a year on proponate - Waksman-Carey agar. The range of ntrogen sources that could be utlzed by these adapted strans was much dmnshed, ntrate beng strongly preferred. () The adaptaton of S. atra to growth on meda contanng salcylate and benzoate was accompaned by vsble "mutaton" and after about 20 days of culture on the salcylate medum, S. atra began to show sparse growth. Subculture on salcylate medum led to profuse growth wth the producton of large quanttes of nsoluble yellow pgment. Transfer of the culture to agar slopes, whether of soldfed salcylate medum or potato dextrose agar, gave olve colones that showed no sgn of reverson to the wld type. Mcroscopcally the cultures were characterzed by globose spores borne on exceptonally long condophores. Whte, Yeager, and Shotts (1949) have shown that aggregates of Stachybotrys spores always contan a few globose spores of the 'Memnonella type; ther observatons have been confrmed n ths laboratory. When spores from successve subcultures on lqud salcylate meda were examned, t was found that replacement of the ellpsod Stachybotrys type by the globose Memnonella type occurred over three transfers. ntermedate cultures showed both spore forms. t would seem that S. atra s heterokaryotc, wth salcylate actng as a selectng agent. (> V. GROWTH AND ENZYME PRODUCTON ON V AROUS MEDA (a) General Observatons The composton of the modfed Waksman-Carey medum s such that growth of a mcoorgansm upon t wth the consequent depleton of the ammonum ntrogen must lead to a sharp fall n ph. Wth S. atra the onset of growth s sudden and the consequent fall n ph extremely sharp; wth no expermental technque so far devsed has t been possble to synchronze ths change n a large number of flasks. The occurrence of the second smaller drop n ph durng growth. on sugar meda s partcularly erratc and no account of the factors controllng ts appearance can yet be gven. Dscrepances between the "chronologcal" and "physologcal" ages of cultures vtated all experments n whch flasks were removed from the shaker at ntervals and the culture fltrates examned. The W oulffebottle method for followng a sngle culture was therefore devsed to overcome ths dffculty. t mght appear that these troubles could be elmnated by bufferng the medum; n Table 7 are set out the results of a seres of experments n whch '" Morphologcal detals of the:: new stran and a full account of the above and further experments wll be publshed elsewhere.

12 FUNGAL CELLULASES. 59 meda were prepared contanng ncreasng proportons of 1.0M K"Ha-xPo'., ph 7.0. The table shows that reproducble growth s not obtaned even when the ph of the medum s held relatvely steady. "'" s "... '" 0 6'0 5'0 4'0. -:!.:.:-::::.:::::-- 0-._._.-...,..., ", ' " ', \ \ \ \ \ \ \ \ \ \ \ \ \, \ 40., '. \!, \!,!, \! \ \ \ \, : ' \\ \ ' \, \."'. \ t. \ \ \... NCUBATON TME (HR,) 80 \! \ Fg..-Changes n ph durng the frst 100 hr. of growth of S. atra on Waksman-Carey medum contanng 2 per cent. of glucose. Four separate Woulffe-bottle cultures at 28 C.; ph followed on the recordng ph meter. The causes of the nherent varablty n growth cannot at present be defned; n the experments on the nutrton of the mould small amounts of vtamns or protens added to the medum gave cultures wth much reduced varablty (d. Table 1). t s possble that random varaton n the carry-over of such stmulatory substances n the noculum was responsble for the observed results. Examnaton of culture fltrates by solvent extracton after the addton of excess mneral acd, followed by chromatographc nvestgaton of the extract, showed the absence at all stages of organc acds n other than trace amounts. Ths s n agreement wth the observatons of Perlman (1948) on M. echnata. The producton of organc acds by S. atra thus plays no part n the observed ph changes of the medum. (b) Changes n ph Durng Growth The varablty of the tme-ph curve for the frst 100 hr. of growth on a medum contanng 2 per cent. glucose s llustrated n Fgure 1 for a number

13 60 M. A. JERMYN TABLE 7 GROWTH OF S. ATRA ON WAKSMAN-CAREY MEDUM CONTANNG VAROUS CARBON SOURCES AND DLUTED WTH VARYNG AMOUNTS OF loom PHOSPHATE BUFFER OF ph 7.0 ndvdual 250-ml. flasks n shake culture at 28 C., contanng 50 ml. of medum. "Enzyme actvty" s the hydrolytc actvty aganst sodum carboxymethyl cellulose Carbon Source Volume per cent. of Cotton Wod (60 Phosphate Glucose (2%) Sucrose (2%) mesh: 2%) Days Buffer n ncu- Fnal bated Medum ph of Enzyme Mycel- ph of Enzyme Mycel- ph of Enzyme Medum Actvty al Med"ffl Actv<y,u Med" ll Actv 'y Weght Weght l (mg.) (mg.) '

14 FUNGAL CELLULASES. 61 TABLE 7 (Contnued) Carbon Source Volume per cent. of Cotton Wool (60 Phosphate Glucose (2%) Sucrose (2%) mesh: 2%) Days Buffer n ncu- Fnal bated Medum ph of Enzyme Mycel- ph of Enzyme Myce1- ph of Enzyme a1 Medum Actvty a1 MCd=1 Act""y l MCd;uml Act,,;,y Weght Weght (mg.) (mg.) of experments. Fgure 2 llustrates the complete course of the tme-ph curves durng typcal growth on meda contanng 1, 3, a:ld 5 per cent. glucose. ( c) Enzyme Producton on Varous M eela n Fgures 3 and 4 are set out the essental detals of growth on unmodfed Waksman-Carey meda wth 2 per cent. glucose and sucrose, respectvely, as the carbon sources. There s an overall smlarty n the relaton between Cx actvty, mycelal weght, ph of medum, and utlzaton of sugar for cultures on the two meda. Fgure 4 shows the tendency, whch has been general n these experments, for salcnase to follow the same course as Cx and the p-ntrophenyl-j3-glucosdase to pursue an ndependent course. The data on whch Fgure 3 s based were secured before the mportance of checkng the actvty of a number of enzymes was realzed and no further contnuous runs on ths smple unmodfed medum have been made. Later ndependent experments have shown that the proteases (gravmetrc and vscometrc) are not demonstrable at any stage of growth; sporadc and extremely small actvtes can be demonstrated for esterase, amylase, and sucrase between the ffth and tenth days of growth.

15 62 M. A. JERMYN 7.orl , S " "... 0 " '0 4'0... '. c"\ \! :-.-:::::: ".... o NCUBATON TME (DAYS) 12 Fg. 2.-Changes n ph durng the growth of S. atra on Waksman Carey medum plus glucose. Woulffe-bottle cultures at 28 C.; ph followed on the recordng ph meter. A, 1 per cent. glucose; B, 3 per cent. glucose; C, 5 per cent. glucose. u "1-0 u '" " u :E S " :E ". '" " u 12 2'0 g ::E ::> S :E z z D 1'0 u 8 '" "... C.. C '" > ::> u g '"!!; :c! '" CD.. > '" ::> u NCUBATON TME (DAYS) Fg. 3.-Growth of S. atra on 2 per cent. glucose medum. A, ph of medum; B, mycelal weght; C, "C x " actvty; D, sugar content of medum.

16 FUNGAL CELLULASES. 63 The growth of S. atra was also followed on glucose meda modfed n varous ways; the results are set out n Fgures 5, 6, and 7. t s apparent that each modfcaton nduces ts own partcular varaton n the pattern -of growth and enzyme producton. n no case could the formaton of sgnfcant amounts of amylase, sucrase, esterase, or protease be detected.!; :!:s;....,., ::5 "'>...,. :a 0< 0.5 ::: "'> >", "''' au >.... s: >;: u., j: LoJ 1 0 «g 1:;... u> "'" u '" \ \ \ \ \, ? 'la. lt V -$( '.-, E \ 'e e. \,., \ -- \ 4,' \), \. f' \ \.Jt '/ '...\. " 1/ " '\, \ '... " '" ' V \ 4.0 = " "' a 2 0 ff Q.2 z c- o., la.. u " 2 0 S<..., -g... ag ".,... Z:c 03 U...J 1.0 "'u u> ",..,.... u <... "'> >", '" =>u " u ---0' p\, \, \, \ \, : /'" ' Q-" '0... A 7'0 " " s o.. > u '".--',, NCUBATON TME (DAYS) Fg. 4.-Growth of S. atta on 2 per cent. sucrose medum. A, sugar content of medum; B, ph of medum; C, mycelal weght; D, Cx actvty; E, salcnase actvty; F, p-ntrophenyl-p-glucosdase actvty; G, sucrase actvty; H, esterase actvty;, amylase actvty. When glucose s replaced by starch (Fg. 8), esterase, amylase, and sucrase are produced n some quantty and there s a general tendency for perods of maxmum and mnmum actvty of these enzymes n the culture fltrate to concde wth those of the enzymes splttng,b-glucosdc lnkages. Of these latter enzymes ex and salcnase do not exceed the maxmum actvtes found n sugar meda, but there s great ncrease n the p-ntrophenyl-,b-glucosdase.

17 .., 64 M. A. JERMYN (d) Comparson of Certan f3-glucosdase Actvtes n the Culture Meda The comparatve hydrolytc actvty aganst salcn, p-ntrophenyl-f3-glucosde, sodum carboxymethyl cellulose, and sodum cellulose sulphate was determned n bulked culture fltrates wth the extra accuracy allowed by large sample sze. The results are set out n Table '5 '> ;::!:: u 0> «;:: :; 1.0 x u u.. :J C ;:.. ;w :;) l.lj 0'5 u u,..e /,;/,e" / ; ;:: w., '" C n 0 3 " 0'2, 0'1 c,0----_'... Q..._... " '" / 'b-, ";'-.. J: 0 := Co 2 0,. 0 '" C 0 os... Z J: S2 Z W o ;;: u w., 2 a 1"0 u u '",. ".. <* u w W 0> > '" '" '" a u 0' P /, NCUBATON TME (DAYS) 12 W 0'0 > gj u 7'0 8,. 6'0 0 '. Fg. 5.--Growth of S. atm on 2 per cent. glucose medum plus 115 vol. of l.om phosphate buffer of ph 7.0. A, sugar content of medum; B, ph of medum; C, mycelal weght; D, ex actvty; E, salcnase actvty; F, p-n tropheny l-tl-glucosdase actvty. '" > u '" V. DSCUSSON There s no evdence of any unusual physologcal characterstcs n S. atra. The lsts of avalable ntrogen and carbon sources supplement those of Perlman ( 1948) wthout any essental dsagreement. The mould s not restrcted to any type of ntrogen source, but as carbon sources shows a marked preference for sugars, polysacchardes, glucosdes, and sugar alcohols, smpler molecules beng utlzed only n the presence of ntrate ntrogen.

18 FUNGAL CELLULASES. (5 TABLE 8 COMPARSON OF VAROUS,a-GLUCOSDASE ACTVTES N BULKED CULTURE FLTRATES Hydrolytc Actvty Aganst Carbon Source Days of Growth p-ntrophenyl-,8-glucosde Salcn Sodum Cellulose Sulphate Sodum Carboxymethyl Cellulose Arbtrary Unts Arbtrary Unts Arbtrary Unts SCMCase=1 SCMCase=1 SCMCase=1 Arbtrary Unts Glucose Sucrose Growth on unbuffered sugar meda shows a defnte sequence of phases: (a) Germnaton, wth consderable lberaton of,b-glucosdases before there s much apparent mycelal growth. g 1'0 r:=:7tt-,-'- )00... _ <J-,'' / F (J-:-_-'-'" - cr g " ---,- " Q < - - " --e-:- rr: ';'! W 0'5 < _-(J e '-. _ 8 / '" u 0'0 ()/". _ 2'0 4'0,. J,. 0 ::> l f :E :!E :J: " l- ;; Z o 3: U 1-0..J :::; '" " 0... u u >- 3. :( u '" > > a: a: :::> u u " 0'0 0'0-0-"'0 "" :...-,C o.o, NCUBATON TME (DAYS) =-.:-o, -'>- >- -- 0'2?! :J: - o. U 0< 0'1 0'0 7'0 z<, c 0: V u 3 0: " ::> ' u"'- f "... o 6'0 "-.. Fg. 6.-Growth of S. atra on 2 per cent. glucose medum plus 1 per cent. (w/v) of CaC0 3 A, sugar content of medum; B, ph of medum; C, mycelal weght; V, C x actvty; E, salcnase actvty; F, p-ntrophenyl-,b-glucosdase actvty > a: ::> u

19 66 M. A. JERMYN ( b) Rapd growth, wth fall n ph, consumpton of sugars, and loss of ex and salcnase actvty. ( c) A steady perod at ph 3-4, wth a new rse n ex and salcnase actvty and lttle or no utlzaton of sugar. (d) Renewed utlzaton of sugar, wth a further slght decrease n ph and loss of ex and salcnase actvty. >- 1 5 t: >- s; ;::.. '-0 M uu.. ::J c < v. 1& =>.. u:; -.e, \,0 _ <1... F..."\ '" ,,.... e a,(1-- ;; 2) / 10'0 : 5" 0 "g z.. - ",>- 0'" u", 0 -,-' <5 =>-' c.. ;Cu... U U 0'0',0",! /,/",, " Q...--o... C '0' '!! NCUBATON TME (DAYS), "'-..\ o 0 1 a o ' u Fg. 7.-Growth of S. atra on 2 per cent. glucose medum modfed by replacng (NH4)2HP04 wth K 2 HP0 4 KN0 3 A, sugar content of medum; B, ph; C, mycelal weght; D, ex actvty; E, salcnase actvty; F, p-ntrophenyl-,b-glucosdase actvty. t s noteworthy that the frst of these phases s not much altered by the addton of bufferng agents or the replacement of ammonum by ntrate; secreton of relatvely large amounts of,b-glucosdases at, or just after, germnaton appears to be a constant feature of the metabolsm of S. atra. Snce the "ex" actvty s part of "cellulase" actvty ths early secreton of enzyme wll be of the utmost mportance for the mould n ganng a foothold on cellulosc substrates, and may be decsve n securng for S. atra the domnant poston n the cellulolytc flora whch t so often occupes n the feld. The secreton of "ex" and salcnase appears to occur n bursts, wth slow nactvaton of the secreted enzyme thereafter; ths s shown most clearly by the data for the two buffered glucose meda, where the hghest peak of actvty occurs early n the hstory of the culture, and s followed by a nearly constant declne. On the lmted evdence of the starch medum, ths concluson appears to be true for other enzymes also. The factor lmtng growth n the meda used was ntrogen; calculatons show that approxmately enough s present to synthesze about 1.6 g. of proten per 500 ml. of medum. nspecton of the

20 FUNGAL CELLULASES. 67 fgures shows that except for the medum contanng CaC03 growth ceased at a fgure of about 2 g. of mycelum per 500 m., followed by a drop n mycelal weght, and the lberaton of enzymes. There were marked changes n ph, and the sugar contnued to be utlzed (presumably by respraton to CO2 ), Ths partal autolyss could not be pushed further, however, and ncubaton for ndefnte perods showed an almost constant mycelal weght. The resdual mycelum was autolysed n all experments (18 hr. wth ethyl ether at 1 DC.) but sgnfcant amounts of saccharases were not recovered. 0'3 : B 0-2 < "' < t..?.:- > '> ;::: U > c( 1'0 gg u 0 5 :E:E g 1'5,1 :Ez " L: 1.0 ;: z u:; 0" ww u 0 5 Q.» "'''' :.-- --" r'".." )(-.. \ ( /, v..\ ".,v,. \ //x > '> ;::: 2-0 w / / \ / 1 0 /.. H w /.""..' :.': "", '!5 ':.--':.,"; -v.._.. <;> U ';-',, lt / f \,/ " f / -! \,/ : \,.. " '.... ::'._._._._._._.-e--...,..,d \()_/ ' e...,...; V >t:..-e "'z 10.0 a h,...o..\,.0-.0' t, -- CJf /,t:l... (5 a 0'0' a NCUBATON TME (OAYS) ff 1 5 <' n 8 too :3 " t "-0>- 1..a:t: 7 0 o ::; '.'" 4 0 u. 1:; :> U Fg. 8.-Growth of S. atra on 1 per cent. starch medum. A, carbohydrate content of medum; B, ph of medum; C, mycelal weght; D, C x actvty; E, salcnase actvty; F, p-ntrophenyl-{:l-glucosdase actvty; G, sucrase actvty; H, esterase actvty;, amylase actvty. Shu and Blackwood (1951) have studed the producton of certan carbohydrases by A. nger growng n submerged culture. Alpha-amylase and maltase actvty were strongly dependent on the carbon source used, beng hgher on starch, dextrn, or maltose than on carbon sources not contanng a-glucosdc lnkages; "lmt-dextrnase" actvty on the other hand was almost ndependent of the carbon source. Ths stuaton s very smlar to that found n ths study for S. atra, amylase and sucrase producton beng dependent on the carbon source, and that of the Cx enzyme and other,b-glucosdases beng relatvely

21 68 M. A. JERMYN ndependent. Jermyn (1952a) has already shown that n general somewhat hgher levels of the Cx enzyme are produced on meda contanng carbon sources wth,8-glucosdc lnkages, so that ths enzyme mght be classfed as "adaptve" f a few results only were consdered. Contrary to the conclusons of Reese, Su, and Levnson (1949) for cellulolytc moulds n general, however, ts formaton n large quanttes by S. atra s possble n the absence of cellulose dervatves as carbon sources. The results obtaned are apparently dependent on the balance of a number of factors, and ths type of stuaton s farly general; Gershenfeld and Wasserman (1950) found that, by choosng certan ntal condtons, t was possble to demonstrate an apparently hgher producton of sucrase by Neurospora crassa growng on xylose, raffnose, and fructose than on sucrose, although a more complete study showed ths enzyme to be partally adaptve. The salcnase and Cxenzyme actvtes of S. atra follow courses that are suffcently close to make t credble that the same enzymes are nvolved, although nspecton of Fgures 3-8 and of Table 8 shows that ths relatonshp s not absolute; p-ntrophenyl-,8-glucosdase appears to bear lttle or no relatonshp to the other two actvtes. The explanaton advanced by Jermyn (1952b) for the propertes of the A. oryzae,8-glucosdases may therefore be applcable to those of S. atra and we may here have another example of a group of,8-glucosdases each wth ts own rato of actvtes towards dfferent substrates, the sum of these actvtes beng measured as the apparent actvtes of such "enzymes" as "salcnase" and "Cx'" Ths hypothess receves support from the tendency of salcnase and the C x enzyme to have parallel maxma and mnma of actvty that are a day or two apart, as f a certan set of condtons was operatng to produce or nactvate the,8-glucosdases, but ndvdual enzymes responded slghtly dfferently. The present study therefore gves no support for the exstence of "Cx" as separate enzyme, hydrolysng polymerc,8-glucosdc lnkages only, and produced by the mould solely n response to the presence of these lnkages. V. ACKNOWLEDGMENTS The author s deeply ndebted to Mss B. Lyons and Mrs. M. C. Wlknson for ther able techncal assstance. X. REFERENCES BASU, S. N. (1948).-J. Text. nst. 39: T237. BSBY, G. R. (1945).-Trans. Brt. Mycol. Soc. 28: 11. BUSTON, H. W., and BASU, S. N. (1948).-J. Gen. Mcrobol. 2: 162. Cox, A. B., HNDSON, W. R., MORTENSEN, K. N., and WADE, G. C. (1945).-Dep. Supply, Melb., Muntons Supply Lab. Rep. No CREWTHER, W. G. (952).-Aust. J. Sc. Res. B 5: 290. CREWTHER, W. G., and LENNOX, F. G. (1953).-Aust. J. Bol. Sc. 6 (3) (n press). FAHRAEUS, G. (1947).-Symb. Bot. Upsalens.9 (2). GERSHENFELD, L., and WASSERMAN, A. E. (1950).-Amer. J. Pharm. 122: 146.

22 FUNGAL CELLULASES. 69 JENNSON, M. W., HENDERSON, R., DERRCK, M., O'NELL, R., and Rupp, F. (1950). Physology of the wood-rottng fung. Summary Tech. Rep. U.S. Off. Naval Res., Jan.-Dec JERMYN, M. A. (1952a).-Aust. J. Sc. Res. B 5: 409. JERMYN, M. A. (1952b).-Amt. J. Sc. Res. B 5: 433. LENNOX, F. G., and ELLS, W. J. (1945).-Bochem. J. 39: 465. MARSH, P. B., and BOLLENBACHER, K. (1946).-Amer. J. Bot. 33: 245. PERLMAN, D. (1948).-Amer. J. Bot. 35: 36. PRE, N. W. (1936).-Brt. J. Exp. Path. 17: 269. REESE, E. T., SU, R. G. H., and LEVNSON, H. S. (1949).-J. Bact. 59: 485. SAUNDERS, R. R., SU, R. G. H., and GENEST, R. N. (1948).-J. Bol. Chern. 174: 697. SHU, P., and BLACKWOOD, H. C. (1951).-Canad. J. Bot. 29: 113. SU, R. G. H. (1951).-"Mcrobal Decomposton of Cellulose." (Renhold: New York.) SU, R. G. H., and SNDEN, J. W. (1951) -Arner. J. Bot. 38: 284. SOMOGY, M. (1945).-J. Bol. Chern. 161: 89. STENBERG, R. A. (1950).-Arch. Bochem. 28: ll. WAKSMAN, S. A., and CAREY, C. (1926).-J. Bact. 12: 87. WHTAKER, D. L. (1951 ).-Nature 168: WHTE, W. L., YEAGER, C. C., and SHOTTS, H. (1949).-Farlowa 3: 399.