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Extracellular cellulase activity from Trichoderma viride grown in the presence of glucose
Notes and brief articles
52 2
CARPENTER, J. M. (1973). Virus-like particles in the take-all fungus , Gaeumannomyces graminis. Annals of Applied Biology 74, 197-209. RAWLINSON, C . J. & MUTHYALU, G. ( 1975). Similar viruses in Gaeumannomyces spp. and Phialophora spp. Rothamsted Experimental Station , Report for 1974, Part 1,256. SOMMER, S. S. & WICKNER, R. B. ( 1982). Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes {HOK}, {NEX} and {E X L } are on one of these dsRNAs. Cell 31, 429-441.
STREET, J. E ., CROXSON, M . C ., CHADDERTON, W . F. & BELLAMY, A. R. (1982) . Sequence diversity of human rotavirus strains investigated by northern blot hybridisation analysis. Journal of Virology 43 , 369-378. WALKER, J. ( 1981). Taxonomy oftake-all fungi and related genera and species. In Biology and Control of Take-all (ed . M . J. C. Asher & P. J. Shipton), pp. 15-74. London : Academic Press.
EXTRACELLULAR CELLULASE ACTIVITY FROM TRICHODERMA VIRIDE GROWN IN THE PRESENCE OF GLUCOSE BY T. RUBIDGE*
Biodeterioration Laboratory, Materials Quality Assurance Directorate, Royal Arsenal East, Woolwich, London SE18 6TD, U .K. Trichoderma viride produces both an extracellular cellulase activity in the presence of insoluble cellulose and a similar activity during growth in the presence of glucose. The latter activity was detected only after precipitation from the culture filtrate. Its function is discussed with particular reference to the mechanism of enzyme induction. It is well-established that growth of Trichoderma viride Pers. upon cellulose induces cellulase activity. However, the insoluble nature of this material has prompted an examination of the role of soluble carbohydrates in the induction process. Only cellobiose, lactose, sophorose and a trisaccharide isolated from the medium of a culture where glucose was the sole source of carbon have been shown to induce cellulase activity (Mandels & Reese, 1960). Mandels & Reese (1963) suggested that a basal extracellular cellulase activity must exist in T. viride in order that cellobiose might be released to function as an inducer. Mandels et al. (1962) reported the appearance of an extracellular cellulase activity in T. viride cultures after the cessation of growth at the expense of glucose. This paper is the first report of such an activity during growth in cultures containing glucose. T. viride was grown in the liquid mineral medium of Mandels et al. (1962) supplemented (1 % w/w) with cellulose, sodium carboxymethylcellulose (CM-cellulose) or glucose. Cellulose (microgranular, Sigma) was twice extracted with distilled water to remove soluble materials. The aqueous extract was concentrated by lyophilization. Incubation temperature was 25 °C and the stirrer speed and aeration rate adjusted so that the growth form was of dispersed mycelial fragments. Where the
* Present address : Defence Microbiology Division, Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 oJQ, U.K. Trans. Br. mycol, Soc. 83 (3), (1984)
medium was supplemented with the concentrated extract from the cellulose preparation the volume added was that equivalent to 1 % (w Iv) cellulose. Extracellular carboxymethylcellulase (C M -cellu lase, E .C. 3.2.1.4) was determined as the release of reducing sugars from CM-cellulose these being determined with dinitrosalicylic acid reagent after incubation for an hour at 50° (Pettersson & Porath, 1966). Where enzyme activity released less than 0'4 mM glucose equivalents of reducing sugars into the reaction mixture the volume of enzyme preparation was increased in order to use the linear portion of the calibration graph. When T. viride was grown on glucose no CM-cellulase activity was detected in the culture filtrates . The secretion of activity after depletion of the glucose (M an dels et al., 1962; Brown et al., 1975) was not shown. When mycelia harvested by centrifugation after 3 days growth at the expense of glucose (equivalent to the mid-exponential phase of growth) were resuspended in the mineral medium supplemented with CM-cellulose there was a secretion of CM-cellulase activity which reached a maximum (16 '7 mg glucose released pg-l protein h" ) after 3 days. When the resuspension medium was that supplemented with cellulose extracted with water, activity was slower to appear but reached a higher maximum (20'0 mg glucose released pg-l protein h -1 ) after 4 days. Resuspension in the mineral medium alone or in the medium supplemented with the concentrated aqueous extract elicited no secretion of activity. These
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Notes and brief articles Table 1. Precipitation of CM-cellulase activity from cultures of T. viride containing glucose Volume ratio (culture filtrate: acetone) 0·5 1·0 1·5 2·0 2·5 3·5
CM-cellulase activity (pg glucose released p,g-l protein h")
5.0
3 6"6
7·3 43·8 23"8 21·6 34·3 15·6
Table 2. Recovery of CM-cellulase activity from cultures of T. viride containing glucose Preparation 1. Culture filtrate 2. Acetone precipitate 3" Optically dense fraction from gel filtration 4· Acetone precipitate from preparation 3.
Volume (ml) 1860 30 133
Protein (pg) 163700 11700 4670
10
688
CM-cellulase activity* None detected 624 20 442 142
Specific activity'[
53"3 4377"3 206"4
* mg glucose released h- 1 • t p,gglucose released p,g-l protein h ".
findings are broadly in agreement with studies discussed by Sternberg (1976) and Halliwell & Lovelady (1981). Since the induction of extracellular cellulase activity is a positive reaction to the exogenous presence ofan insoluble material the possibility that a basal activity exists in cultures containing non-cellulosic substrates was investigated. Since such an activity would be inhibited by the glucose in the medium attempts were made to precipitate an extracellular protein fraction from cultures grown in the presence of glucose using ammonium sulphate. These were wholly unsuccessful. Precipitation with cold acetone ( - 10°) gave good yields of CM-cellulase activity (Table 1). Culture filtrates adjusted to pH 4.0 were mixed with different volume ratios of acetone and held at 4° until visible precipitation had ceased. The precipitate was removed by centrifugation and resuspended in citrate-phosphate buffer (pH 5.0, 0·1 M). Greatest recovery of activity was in the order of acetone to filtrate volume ratios 2·5 > 5·0> 1·0. Greatest specific activities were recovered in the order 1·0> S·O > 2·5. Both CM-cellulase and cellobiase (E.C. 3.2.1 .21) activities were precipitated from cultures grown in the presence of glucose and, although no detailed investigation of the enzyme kinetics was carried out, distinct differences were detected. Whereas cellobiase activity (determined in a similar fashion to CM-cellulase activity but Trans. Br. mycol. Soc. 83 (3), (1984)
with a cellobiose substrate) was hyperbolic with respect to substrate concentration CM-cellulase activity was sigmoid. Both activities precipitated from cultures grown in the presence of cellulose showed hyperbolic kinetics. In order to assess the role of glucose in the sigmoid kinetics two experiments were undertaken. First, activity precipitated from culture in the presence of cellulose was resuspended in glucose solution (0·01-0·1 % w Iv). Although a strong inhibitory effect was shown there was no shift from the hyperbolic relationship between enzyme activity and substrate concentration. Second, precipitated activity from a culture grown in the presence of glucose was passed through a column of Sephadex G-150 gel so that free glucose would be separated from the enzyme preparation. Whereas the bulk of the reducing sugars eluted with the terminal marker, 90 % of the CM-cellulase activity was associated with a discrete, optically dense fraction (Table 2). The CM-cellulase fraction was concentrated by a second acetone precipitation step when 99·3 % of the activity was lost. The relative instability of CM-cellulase activity in dilute solution when exposed to acetone was regularly observed. The sigmoid relationship between enzyme activity and substrate concentration was unaffected by the removal of reducing sugars. The detection of CM-cellulase activity in the medium of cultures of T. viride containing glucose has not previously been reported. This activity and
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Notes and brief articles the observation that soluble carbohydrates are unnecessary for the induction of CM-cellulase activity in the presence of cellulose suggest that cellobiose, or another inducer molecule formed from it, is released through the action of a constitutive enzyme. The role of cellobiose might be to allow glucose indirectly to inhibit extensive cellulolysis when cellulose and glucose are both present. In this context the functions of cellobiose would be centrally important in metabolic control providing a link between enzyme induction and enzyme inhibition. No conclusions can be drawn as to whether the CM-cellulase activities from cultures grown in the presence of glucose and of cellulose are brought about by the same enzyme protein or represent the product of different genes. I wish to thank Professor David Hughes for his advice and Maria Palmier for her technical assistance. REFERENCES
viride QM 9123 . In Symposium on Enzymatic Hydrolysis of Cellulose (ed . M . Bailey, T. Enari & M . Linko), pp . 137-153 . Aulanko, Finland : Technical Research Centre. HALLIWELL, G . & LOVELADY, J. ( 19 81). Utilization of carboxymethylcellulose and enzyme synthesis by Trichoderma honingii. Journal of General Microbiology 126,211-217.
MANDELS, M . & REESE, E. T . (1960 ). The induction of cellulase by cellobiose. Journal of Bacteriology 79, 816-826.
MANDELS, M . & REESE, E. T. (1963) . Inhibition of cellulases and fJ-glucosidases. In Advances in Enzymic Hydrolysis of Cellulose and Related Materials (ed . E . T. Reese), pp. 115-117. London : Pergamon Press. MANDELS, M ., PARRISH, F . W: & REESE, E. T . (1962). Sophorose as an inducer of cellulase in Trichoderma oiride . Journal of Bacteriology 83, 400-408. PETTERSSON, G . & PORATH, J. (1966) . A cellulolytic enzyme from Penicillium notatum. In Methods in Enzymology 8 (ed . S. P. Colowick & N. O . Kaplan), pp. 603-608. New York: Academic Press. STERNBERG, D . (1976). fJ-glucosidase of Trichoderma . Applied and Environmental Microbiology 31, 648-654.
BROWN, D . E ., HALSTEAD, D. J. & HOWARD, P. (1975). Studies on the biosynthesis of cellulase by Trichoderma
ORIGIN OF THE LIQUID IN BULLER'S DROP BY J. WEBSTER AND R. A. DAVEY Department of Biological Sciences, University of Exeter AND C. T. INGOLD 11,
Buckner's Close, Benson, Oxford OX9 6LR
The origin of the liquid in Buller's drop is discussed and evidence presented that Tilletiopsis washingtonensis and I. perplexans may extrude a hygroscopic substance from the punctum lacrymans, resulting in rapid absorption of liquid from a humid atmosphere. Webster, Davey, Duller & Ingold (1984) have shown that the drop which develops shortly before spore discharge at the hilar appendix in the ballistospores of ltersonilia perplexans Derx is liquid which is projected with the spore. In this fungus the drop may attain a diam of 10 /lm or more and takes about 40 s to reach maximum size. The volume of liquid represents about 60 % of the initial volume of the spore but photomicrographs and video-recordings of drops as they increase in size show no corresponding reduction in the volume of the spores to which they are attached. Several spores detached on a micromanipulator needle were shown to be capable of developing drops, indicating that the source of liquid within the drop cannot be by conduction through the sterigma. Because liquid is virtually in com p r essib le the origin of the fluid within the drop poses a problem. Webster et al, Trans. Br . mycol. Soc. 83 (3), (19 84)
( 1984) have suggested three possible explanations for the phenomenon : (a) The spore wall is hygroscopic and can acquire liquid from a saturated atmosphere to compensate for that which is extruded at the hilar appendix. (b) Local corrugation of the spore wall might be associated with a reduction in the volume of its contents. (c) Replacement of liquid from within the spore by air or by gas generated metabolically. The first of these three possibilities seemed the most likely explanation. In this paper we provide evidence of the same phenomenon in Tilletiopsis washingtonensis Nyland ( 19 50 ) .
A culture of T . washingtonensis was isolated by one of us (C. T .I.) as a contaminant during isolation
Printed in Great Britain
52 2
CARPENTER, J. M. (1973). Virus-like particles in the take-all fungus , Gaeumannomyces graminis. Annals of Applied Biology 74, 197-209. RAWLINSON, C . J. & MUTHYALU, G. ( 1975). Similar viruses in Gaeumannomyces spp. and Phialophora spp. Rothamsted Experimental Station , Report for 1974, Part 1,256. SOMMER, S. S. & WICKNER, R. B. ( 1982). Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes {HOK}, {NEX} and {E X L } are on one of these dsRNAs. Cell 31, 429-441.
STREET, J. E ., CROXSON, M . C ., CHADDERTON, W . F. & BELLAMY, A. R. (1982) . Sequence diversity of human rotavirus strains investigated by northern blot hybridisation analysis. Journal of Virology 43 , 369-378. WALKER, J. ( 1981). Taxonomy oftake-all fungi and related genera and species. In Biology and Control of Take-all (ed . M . J. C. Asher & P. J. Shipton), pp. 15-74. London : Academic Press.
EXTRACELLULAR CELLULASE ACTIVITY FROM TRICHODERMA VIRIDE GROWN IN THE PRESENCE OF GLUCOSE BY T. RUBIDGE*
Biodeterioration Laboratory, Materials Quality Assurance Directorate, Royal Arsenal East, Woolwich, London SE18 6TD, U .K. Trichoderma viride produces both an extracellular cellulase activity in the presence of insoluble cellulose and a similar activity during growth in the presence of glucose. The latter activity was detected only after precipitation from the culture filtrate. Its function is discussed with particular reference to the mechanism of enzyme induction. It is well-established that growth of Trichoderma viride Pers. upon cellulose induces cellulase activity. However, the insoluble nature of this material has prompted an examination of the role of soluble carbohydrates in the induction process. Only cellobiose, lactose, sophorose and a trisaccharide isolated from the medium of a culture where glucose was the sole source of carbon have been shown to induce cellulase activity (Mandels & Reese, 1960). Mandels & Reese (1963) suggested that a basal extracellular cellulase activity must exist in T. viride in order that cellobiose might be released to function as an inducer. Mandels et al. (1962) reported the appearance of an extracellular cellulase activity in T. viride cultures after the cessation of growth at the expense of glucose. This paper is the first report of such an activity during growth in cultures containing glucose. T. viride was grown in the liquid mineral medium of Mandels et al. (1962) supplemented (1 % w/w) with cellulose, sodium carboxymethylcellulose (CM-cellulose) or glucose. Cellulose (microgranular, Sigma) was twice extracted with distilled water to remove soluble materials. The aqueous extract was concentrated by lyophilization. Incubation temperature was 25 °C and the stirrer speed and aeration rate adjusted so that the growth form was of dispersed mycelial fragments. Where the
* Present address : Defence Microbiology Division, Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 oJQ, U.K. Trans. Br. mycol, Soc. 83 (3), (1984)
medium was supplemented with the concentrated extract from the cellulose preparation the volume added was that equivalent to 1 % (w Iv) cellulose. Extracellular carboxymethylcellulase (C M -cellu lase, E .C. 3.2.1.4) was determined as the release of reducing sugars from CM-cellulose these being determined with dinitrosalicylic acid reagent after incubation for an hour at 50° (Pettersson & Porath, 1966). Where enzyme activity released less than 0'4 mM glucose equivalents of reducing sugars into the reaction mixture the volume of enzyme preparation was increased in order to use the linear portion of the calibration graph. When T. viride was grown on glucose no CM-cellulase activity was detected in the culture filtrates . The secretion of activity after depletion of the glucose (M an dels et al., 1962; Brown et al., 1975) was not shown. When mycelia harvested by centrifugation after 3 days growth at the expense of glucose (equivalent to the mid-exponential phase of growth) were resuspended in the mineral medium supplemented with CM-cellulose there was a secretion of CM-cellulase activity which reached a maximum (16 '7 mg glucose released pg-l protein h" ) after 3 days. When the resuspension medium was that supplemented with cellulose extracted with water, activity was slower to appear but reached a higher maximum (20'0 mg glucose released pg-l protein h -1 ) after 4 days. Resuspension in the mineral medium alone or in the medium supplemented with the concentrated aqueous extract elicited no secretion of activity. These
Primed in Great Britain
Notes and brief articles Table 1. Precipitation of CM-cellulase activity from cultures of T. viride containing glucose Volume ratio (culture filtrate: acetone) 0·5 1·0 1·5 2·0 2·5 3·5
CM-cellulase activity (pg glucose released p,g-l protein h")
5.0
3 6"6
7·3 43·8 23"8 21·6 34·3 15·6
Table 2. Recovery of CM-cellulase activity from cultures of T. viride containing glucose Preparation 1. Culture filtrate 2. Acetone precipitate 3" Optically dense fraction from gel filtration 4· Acetone precipitate from preparation 3.
Volume (ml) 1860 30 133
Protein (pg) 163700 11700 4670
10
688
CM-cellulase activity* None detected 624 20 442 142
Specific activity'[
53"3 4377"3 206"4
* mg glucose released h- 1 • t p,gglucose released p,g-l protein h ".
findings are broadly in agreement with studies discussed by Sternberg (1976) and Halliwell & Lovelady (1981). Since the induction of extracellular cellulase activity is a positive reaction to the exogenous presence ofan insoluble material the possibility that a basal activity exists in cultures containing non-cellulosic substrates was investigated. Since such an activity would be inhibited by the glucose in the medium attempts were made to precipitate an extracellular protein fraction from cultures grown in the presence of glucose using ammonium sulphate. These were wholly unsuccessful. Precipitation with cold acetone ( - 10°) gave good yields of CM-cellulase activity (Table 1). Culture filtrates adjusted to pH 4.0 were mixed with different volume ratios of acetone and held at 4° until visible precipitation had ceased. The precipitate was removed by centrifugation and resuspended in citrate-phosphate buffer (pH 5.0, 0·1 M). Greatest recovery of activity was in the order of acetone to filtrate volume ratios 2·5 > 5·0> 1·0. Greatest specific activities were recovered in the order 1·0> S·O > 2·5. Both CM-cellulase and cellobiase (E.C. 3.2.1 .21) activities were precipitated from cultures grown in the presence of glucose and, although no detailed investigation of the enzyme kinetics was carried out, distinct differences were detected. Whereas cellobiase activity (determined in a similar fashion to CM-cellulase activity but Trans. Br. mycol. Soc. 83 (3), (1984)
with a cellobiose substrate) was hyperbolic with respect to substrate concentration CM-cellulase activity was sigmoid. Both activities precipitated from cultures grown in the presence of cellulose showed hyperbolic kinetics. In order to assess the role of glucose in the sigmoid kinetics two experiments were undertaken. First, activity precipitated from culture in the presence of cellulose was resuspended in glucose solution (0·01-0·1 % w Iv). Although a strong inhibitory effect was shown there was no shift from the hyperbolic relationship between enzyme activity and substrate concentration. Second, precipitated activity from a culture grown in the presence of glucose was passed through a column of Sephadex G-150 gel so that free glucose would be separated from the enzyme preparation. Whereas the bulk of the reducing sugars eluted with the terminal marker, 90 % of the CM-cellulase activity was associated with a discrete, optically dense fraction (Table 2). The CM-cellulase fraction was concentrated by a second acetone precipitation step when 99·3 % of the activity was lost. The relative instability of CM-cellulase activity in dilute solution when exposed to acetone was regularly observed. The sigmoid relationship between enzyme activity and substrate concentration was unaffected by the removal of reducing sugars. The detection of CM-cellulase activity in the medium of cultures of T. viride containing glucose has not previously been reported. This activity and
Printed in Great Britain
Notes and brief articles the observation that soluble carbohydrates are unnecessary for the induction of CM-cellulase activity in the presence of cellulose suggest that cellobiose, or another inducer molecule formed from it, is released through the action of a constitutive enzyme. The role of cellobiose might be to allow glucose indirectly to inhibit extensive cellulolysis when cellulose and glucose are both present. In this context the functions of cellobiose would be centrally important in metabolic control providing a link between enzyme induction and enzyme inhibition. No conclusions can be drawn as to whether the CM-cellulase activities from cultures grown in the presence of glucose and of cellulose are brought about by the same enzyme protein or represent the product of different genes. I wish to thank Professor David Hughes for his advice and Maria Palmier for her technical assistance. REFERENCES
viride QM 9123 . In Symposium on Enzymatic Hydrolysis of Cellulose (ed . M . Bailey, T. Enari & M . Linko), pp . 137-153 . Aulanko, Finland : Technical Research Centre. HALLIWELL, G . & LOVELADY, J. ( 19 81). Utilization of carboxymethylcellulose and enzyme synthesis by Trichoderma honingii. Journal of General Microbiology 126,211-217.
MANDELS, M . & REESE, E. T . (1960 ). The induction of cellulase by cellobiose. Journal of Bacteriology 79, 816-826.
MANDELS, M . & REESE, E. T. (1963) . Inhibition of cellulases and fJ-glucosidases. In Advances in Enzymic Hydrolysis of Cellulose and Related Materials (ed . E . T. Reese), pp. 115-117. London : Pergamon Press. MANDELS, M ., PARRISH, F . W: & REESE, E. T . (1962). Sophorose as an inducer of cellulase in Trichoderma oiride . Journal of Bacteriology 83, 400-408. PETTERSSON, G . & PORATH, J. (1966) . A cellulolytic enzyme from Penicillium notatum. In Methods in Enzymology 8 (ed . S. P. Colowick & N. O . Kaplan), pp. 603-608. New York: Academic Press. STERNBERG, D . (1976). fJ-glucosidase of Trichoderma . Applied and Environmental Microbiology 31, 648-654.
BROWN, D . E ., HALSTEAD, D. J. & HOWARD, P. (1975). Studies on the biosynthesis of cellulase by Trichoderma
ORIGIN OF THE LIQUID IN BULLER'S DROP BY J. WEBSTER AND R. A. DAVEY Department of Biological Sciences, University of Exeter AND C. T. INGOLD 11,
Buckner's Close, Benson, Oxford OX9 6LR
The origin of the liquid in Buller's drop is discussed and evidence presented that Tilletiopsis washingtonensis and I. perplexans may extrude a hygroscopic substance from the punctum lacrymans, resulting in rapid absorption of liquid from a humid atmosphere. Webster, Davey, Duller & Ingold (1984) have shown that the drop which develops shortly before spore discharge at the hilar appendix in the ballistospores of ltersonilia perplexans Derx is liquid which is projected with the spore. In this fungus the drop may attain a diam of 10 /lm or more and takes about 40 s to reach maximum size. The volume of liquid represents about 60 % of the initial volume of the spore but photomicrographs and video-recordings of drops as they increase in size show no corresponding reduction in the volume of the spores to which they are attached. Several spores detached on a micromanipulator needle were shown to be capable of developing drops, indicating that the source of liquid within the drop cannot be by conduction through the sterigma. Because liquid is virtually in com p r essib le the origin of the fluid within the drop poses a problem. Webster et al, Trans. Br . mycol. Soc. 83 (3), (19 84)
( 1984) have suggested three possible explanations for the phenomenon : (a) The spore wall is hygroscopic and can acquire liquid from a saturated atmosphere to compensate for that which is extruded at the hilar appendix. (b) Local corrugation of the spore wall might be associated with a reduction in the volume of its contents. (c) Replacement of liquid from within the spore by air or by gas generated metabolically. The first of these three possibilities seemed the most likely explanation. In this paper we provide evidence of the same phenomenon in Tilletiopsis washingtonensis Nyland ( 19 50 ) .
A culture of T . washingtonensis was isolated by one of us (C. T .I.) as a contaminant during isolation
Printed in Great Britain