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Hyperproduction of thermostable β-glucosidase by Sporotrichum (Chrysosporium) thermophile
Hyperproduction of thermostable fl-glucosidase by Sporotrichum (Chrysosporium) thermophile W. Grajek Station de Gbnie Microbiologique, I N R A , 17 rue Sully-21034 Dijon, France,* and Institute o f Food Technology o f Plant Origin, Academy of Agriculture, 60-624 P O Z N A N , ul. Wojska Polskiego 31, Poland
(Received 8 April 1987; revised 29 June 1987)
The effect of the growth of temperature, pH, carbon source, nitrogen supplementation and inoculum size were examined in shake-flask scale studies to determine the optimum conditions for fl-glucosidases production by Sporotrichum (Chrysosporium) thermophile. Wheat bran and sugar-beet pulp were selected as the best carbon sources and ( N H 4 ) 2 S O 4, NH4CI and K N O 3 a s the best nitrogen supplementation. Ten liter fermentations were carried out to study the kinetics of product formation. It was found that S. thermophile is able to produce high thermostable extracellular cellobiase and aryl-fl-glucosidase. Very high aryl-fl-glucosidase (PNPG) activities in the range from 30 to 40 U ml-1 and cellobiase activities of 2,45 U ml-t in the 3-day batch fermentations were obtained. The K,, for aryl-fl-glucosidase and its thermal properties were also estimated.
Keywords:Thermophilicfungi; Sporotrichum thermophile; fl-glucosidaseproduction;thermostability Introduction The cellulase enzyme system of microorganisms consists of three main components: exo-fl-D-glucanases, endo-fl1,4-glucanases and fl-glucosidase. The action of these enzymes is synergistic and the whole multi-enzyme system is required for the complete hydrolysis of cellulosic materials. The enzyme fl-D-glucosidase can catalyse the hydrolysis of cellobiose and aryl fl-D-glucosides (e.g., pnitrophenyl fl-D-glucoside and methyl fl-D-glucoside). Although numerous microorganisms are known to be able to produce high levels of extracellular cellulases, the best cellulase producers of Trichoderma sp. are fairly deficient in fl-glucosidase. Recently, some mutants of Trichoderma reesei, particularly Rut-C30 and QM9414, have been demonstrated to have potential for practical use to cellulose hydrolysis. It was also reported that the cellulolytic system of T. reesei, consisting mainly of exoand endo-glucanases, could be successfully supplemented with fl-glucosidase from Aspergillus cultures. 1 A great effort has also been made to find other sources of microbial cellulase and many papers in this field have been published. Woodward and Wieseman 2 and Gokhale et al. 3 have showed a comparison of fl-glucosidase production by different fungi. However, all enzyme preparations obtained from mesopholic strains show a lack of thermostability, which results in low hydrolysis efficiences.
* Location to which correspondenceshould be addressed
744
An attractive alternative is to apply the thermostable enzymes produced by thermophilic microorganisms. The use of such cellulases in enzymatic hydrolysis might enhance the reaction rate, reduce the contamination hazard and prolong the periods of reaction time. Recently, interest has increased in thermophilic microorganisms, in particular in fungi, and some species are reported to be good ceUulase producers (e.g., Thielavia terrestris, Sporotrichum thermophile, Talaromyces emersonii and Aspergillus fumigatus). The enzymes produced by these organisms demonstrate the maximum of activities at elevated temperatures between 60 and 80°C, whereas the optima of cellulolytic activities from mesophilic fungi appear at temperatures from 40 to 50°C. The present work was undertaken to investigate the influence of cultue conditions on the fl-glucosidase production by Sporotrichum (Chrysosporium) thermophile. This fungus has been previously reported to be able to excrete the thermostable cellulases when it grew in liquid and solid state fermentations. 4- 8 Some references to the particular thermal properties of fl-glucosidase are also made.
Materials and methods Microorganism
A wild strain of Sporotrichum (Chrysosporium) thermophile Apinis was isolated from wooden waste materials chosen for their cellulolytic activities. The subcultures were maintained on yeast-starch agar at 45°C, stored at 4°C and transferred every month.
Enzyme M icrob. Technol., 1987, vol. 9, December
0141~0229/87/120744435 $03.00 © 1987 Butterworth Publishers
Hyperproduction of thermostable fl-glucosidase: W. Grajek
Inoculum
protein content in the water washed solid culture residues was calculated from N total-N mineral and expressed as N × 6.25. The total and mineral nitrogen were determined by the Kjeldahl procedure.
The 5-day-old cultures were transferred from agar slants into 500 ml Erlenmeyer flasks with 100 ml of liquid medium containing (g 1- z): soluble starch, 10; Ca(NO3)z, 2.0; K2HPO 4, 0.9; KH2PO 4, 1.1; MgSO4.7HzO, 0.5; NaCI, 0.1 ; yeast extract, 0.5, pH 6.5. Each flask was seeded with 107 spores. The cultures were incubated on a rotary shaker, 200 rev m i n - ~, at 43°C. After 3 days of cultivation the cultures were used to inoculate the enzyme production media. In the study of the effect of inoculum size the flasks were seeded with 10 7, 5.10 7 and 108 spores per 100ml of the medium.
Enzyme assay The cultures were harvested by centrifugation at 8000 g for 10 min. The clear supernatants were diluted to a convenient volume with the appropriate buffer and enzyme activities were estimated. Aryl-fl-glucosidase activity (fl-D-glucosidase glucohydrolase, EC 3.2.1 21) was measured at 65°C in an assay containing 1.0 ml 2 mM p-nitrophenyl-fl-Dglucoside (PNPG, Sigma) dissolved in 0.05 sodium acetate buffer, pH 5.0. Afterward 0.1 ml enzyme solution was added and after 15 min of incubation the reaction was stopped with 2.0ml 1 M Na2CO 3. The p-nitrophenol release was measured at 400 nm. In some cases cellobiase activity was also estimated using cellobiose as the reaction substrate according to the IUPAC method. 9 The Dglucose concentrations were detected by D-glucose oxidase-peroxidase method. One unit of activity was expressed as the #M of p-nitrophenol or glucose produced per min per ml of the culture filtrate, respectively. The test on the thermal stability was carried out at 65 °C, pH 6.5, by incubation of the enzyme solution without substrate by different periods of time followed by the enzyme activity assay.
Enzyme production Shake-flask experiments were applied for the evaluation of the culture conditions and medium compositions. The cultures were incubated in 500 ml Erlenmeyer flasks containing 100 ml liquid media at 43°C for 3 days on a rotary shaker, 200 rev min- ~. A standard medium had the following composition (g 1-~): dry powdered sugar-beet pulp obtained after sugar extraction, 20.0; (NH4)2SO4, 1.0; K2HPO 4, 2.2; KH2PO 4, 2.75; buffer, KC1, 0.5; MgSO4-7H20, 0.5; yeast extract, 0.1, pH 6.5. Each flask was inoculated with 10 ml starter culture. The studies with various carbon sources were performed replacing sugar-beet pulp in the standard medium by 20g 1-~: cellulose MN 30 (Machery Nagel Co, Diiren, Germany); 10 g l - ~: Solka floc cellulose SW40 (Brown Co, Berlin NH) 30 g 1-1: Wheat bran, 20 g 1-~; wheat straw, 10 g 1- ~ cellulose microcrystalline Avicel (Merck, Darmstadt, Germany); 20 g 1-1 xylan from oat spelts (Sigma Chemical Co, USA); 10 g 1-1 soluble starch (Merck); 10 g 1-1 glucose (Sigma); 10 g 1-1 cellobiose (Sigma); 10 g 1- ~ lactose (Sigma); and 10 g 1- x glycerol (Sigma). The dry sugar-beet pulp, wheat bran and wheat straw were used in the powdered form with the particle size below 0.1 mm. The effect of nitrogen sources was tested using the standard medium supplemented with N a N O 3, K N O 3, NH4NO 3, Ca(NO3)z, NH4C1, (NH4)2SO 4, (NH4)2HPO 4 and urea. The amount of additional nitrogen was 13 g per kilogram of a dry pulp. The effect of pH was investigated using the standard medium buffered by phosphate-phosphate buffer prepared by mixing the solution of KH2PO4 (5 g 1- ~) with the solution of KEHPO 4 (5 g 1- ~) to obtain the media in the range of pH from 5.5 to 7.5.
Results and discussion
Influence of cultural conditions on enzyme production S. thermophile is a thermophilic Deuteromycete growing at the temperature range between 28 and 55°C. The optimum temperature for the growth of the strain investigated was previously determined as 43°C and the optimum pH as 6.5? o It was earlier reported that the maximum of the endo- and exo-fl-glucanase production appeared at 45°C in 2 to 4 days in the presence of 1% Solka/Floc as a substrate. The results of our experiments on the influence of pH and temperature on aryl-flglucosidase production are shown in Figure 1. The
2.4
_,?.4
Fermentor-scale growth The enzyme production kinetics were studied in a 12 1 Biolafitte fermentor. The conditions of fermentation were as follows: working volume 81, pH 6.5, temperature 43°C, aeration 601 1- a h-~ and agitation 250 rev min-~. The culture medium contained 3 % powdered wheat bran and mineral solution (g 1-~): (NH4)2SO,,, 2.0; KH2PO4, 1.1; KzHPO4, 0.9 (pH 6.5); M g S O 4 . 7 H 2 0 , 0.5; KC1, 0.5; CaC12"2H20, 0.1. Sodium hydroxide 2 N solution was used to control pH. The medium was inoculated with 10% v/v 3-day-old liquid culture. The samples were taken in 12h intervals, and protein contents and enzyme activities were determined.
Crude protein analysis The growth of S. thermophile, with powdered wheat bran as carbon source, was estimated by the crude protein content evolution in the culture medium. The crude
16 12. 8
.4 I TEMPERRTURE (=E) pH Effect of pH and temperature on aryl-fl-D-glucosidase production in batch cultures of Sporotrichum (Chrysosporium) therrnophile. (Enzyme activities were determined toward pnitrophenyI-D-glucoside in filtrates from 3-day-old cultures growing in liquid medium containing 2 % powdered sugar-beet pulp)
Figure 1
Enzyme Microb. Technol., 1987, vol. 9, December
745
Papers fermentation with different pH values of media were carried out at 43°C. The optimum pH value for extracellular aryl-fl-glucosidase production was around 6.5. This value corresponds well to the pH favoring mycelial growth. It was also observed that more alkaline pH values caused a strong decrease of aryl-fl-glucosidase production. At pH 7.0, only 5.1 U ml-1 of enzyme activity was detected, whereas no decrease of mycelium production was observed. The aryl-fl-glucosidase activity declining versus acidic pH was much smaller, and at pH 5.5 about 8.7 U ml-1 were still determined. Thus, the optimal pH conditions for the aryl-fl-glucosidase production are similar to those that appear to be the best for culture growth. Data reported show that the biosynthesis of endo- and exo-fl-glucanase by S. thermophile are favored by alkaline conditions, with the pH optimum between 7.0 and
Carbon source Cellulose MN30 Solka floc Avicel Wheat bran Wheat straw Sugar-beet pulp Xylan Starch Cellobiose Glucose Lactose Glycerol
7.5 6 .
The pH of culture medium is a more critical factor in aryl-fl-glucosidase biosynthesis than the temperature. The maximum enzyme production was obtained at 40°C, but activity determined in cultures growing at 38°C and 46°C was estimated to be only 14 %, and 8 % below the maximum value. Coutts and Smith 6 also reported that the best temperature for cellulase production for this microorganism was determined at 40°C. To examine the effect of inoculum size on aryl-fl-glucosidase production, three levels of seeding were used. The production of this enzyme in the media seeded with 107, 5.107 and 108 spores per 100 ml of culture medium were 22.6, 23.1 and 23.5 U ml- 1, respectively. These results show that the influence of inoculum size on aryl-fl-glucosidase production in the cultures containing sugar-beet pulp as carbon source can be considered a s insignificant.
Effect o f carbon sources Enzyme production in presence of various cellulosic and other carbon sources is shown in Table 1. Chemically pure substrates as well as heterogeneous materials for the investigation were chosen. The quantities of cellulosic substrates added were calculated to have ~ 10 g 1-1 of cellulose in the media. It was observed that aryl-flglucosidase, was produced in the presence of all the substrates tested, but only heterogenous substrates enhanced significantly the enzyme production. Wheat bran was found to be the best carbon source for aryl-flglucosidase production. After 3 days of cultivation a very high level of enzyme activities, reaching up to 36.6 U ml-1, was noted (Table 1). A good enzyme formation in the medium with sugar-beet pulp was also observed. Both substrates mentioned above have relatively rich chemical composition, in particular the high protein contents. Wheat bran is well known to contain many stimulators of microbial biosynthesis, such as microelements and vitamins. All media containing pure forms of cellulose, such as cellulose MN30, Solka floc and Avicel, have shown a very low inductible effect on aryl-fl-glucosidase formation. The enzyme activities in these media and in the media with glucose and cellobiose appeared at very low levels, between 0.1 and 0.3 U m1-1. Starch, lactose and glycerol induced the enzyme activities better than pure source of cellulose. Effect o f nitrogen source In the fermentations the inorganic nitrogen sources and urea were also tried. The amount of supplemented
746
Table 1 Effect of carbon source on aryl-/~-glucosidase production by Sporotrichum thermophile Aryl-/Y-glucosidase activity U m1-1 0.3 0.3 0.3 36.6 4.2 22.3 0.2 3.1 0.1 0.1 0.5 0.5
Ary-]Y-glucosidase activity was determined toward p-nitrophenyl-/~D-glucoside in the supernatants from 3-day-old cultures of
S. thermophile
nitrogen was calculated to result in 40 g of total nitrogen for each kilogram of carbohydrates contained in sugarbeet pulp. Assuming that carbohydrate contents in sugarbeet pulp was about 70% dry matter (cellulose 21%, hemicellulose 23 % and pectin 30°,/0) and total nitrogen content was about 1.5 %, it was presumed that 1 kg of the pulp contained ~700g of carbohydrates and 15 g of nitrogen. Thus, to have the desired proportion between sugars and nitrogen, about 13 g nitrogen per 1 kg pulp were added. This quantity corresponds to C:N ratio a s 100:6. The fl-glucosidase activities (PNPG) produced in the media containing different nitrogen supplementation are presented in Table 2. The preliminary experiments, carried out with the citric acid-sodium dihydrophosphate (Mcllvaine) buffer used to stabilize the pH level, demonstrated a potassium deficit. For this reason the Mcllvaine buffer was replaced by the KHzPO 4 K2HPO 4 buffer. Nitrogen supplementation strongly affected the enzyme biosynthesis. Among the nitrogen sources, (NHa)2SO4, NH4C1 and KNO3 appeared to be the best. Aryl-//glucosidase activities determined in the supernatants obtained from three day-old cultures approached 22-24 U ml- 1. The ammonium ions better stimulated the aryl//-glucosidase production than the nitrate ions. Calcium nitrate, reported to be the best nitrogen source for the culture growth, is gave very poor enzyme activities. The
Table 2 Effect of nitrogen supplementation on aryl-/~-glucosidase production by Sporotrichum thermophile Nitrogen source Urea Urea + ammonium sulfate Ammonium hydrogenophosphate Ammonium sulfate Ammonium chloride Ammonium nitrate Potassium nitrate Sodium nitrate Calcium nitrate
Aryl-/Y-glucosidase activity U m1-1 12.9 20.7 22.5 23.7 23.6 17.4 22.9 10.9 5.8
Aryl-fY-glucosidase activity was determined toward p-nitrophenyl, -]Y-Dglucoside in the supernatants from 3-day-old cultures of
S. thermophile
Enzyme Microb. Technol., 1987, vol. 9, December
Hyperproduction of thermostable ~-glucosidase: W. Grajek addition of urea in the mixture with (NH4)2SO4, used to stabilize the pH of the media, allowed reasonable activities, higher than those in the media supplemented with urea, but a little below those obtained with (NH4)2SO4. The literature has reported that urea and NaNO3 are suitable for cellulase production by S. thermophile and that the variation of the concentration of NaNO3 between 0.05 and 0.4 % has little effect on cellulase activity. 6 No data were found on the effect of nitrogen sources on arylfl-glucosidase formation. In this study both the nitrogen sources mentioned above gave low yield of aryl-/Yglucosidase.
Fermenter-scale growth To study the enzyme production kinetics, cultures in 101 fermenter scale were performed. The results of these experiments are presented in the batch fermentation digram (Figure 2). Very short lag-phase period, limited to 4 h, and next the rapid exponential growth accompanied by large oxygen consumption and acidification of medium, were noticed. The beginning of an intensive extracellular flglucosidases production was observed between 18 and 24 h of culture for the both activities. A high increase of enzyme activities were detected from 24 to 72 h of culture and afterward the rate of enzyme synthesis markedly decreased. At the end of day 3 of incubation the cellobiase activity reached the highest level of 2.45 U m l - 1 and then a small decrease of the activity was observed. Aryl-flglucosidase (PNPG) activity was increasing during the whole time of fermentation, but after 3 days of culture the activity increase was very poor. The extracellular aryl-flglucosidase achieved a very high activity level determined as 36.6 U ml-1 after 72 h of incubation and 40.2 U m1-1 after 144h, respectively. Therefore, the enzyme productivity in a 3-day-old batch culture was about 500 U 1-1 h-1. It was observed that/%glucosidase release appeared in the end of the exponential and deceleration phases of the growth. At the same time the production of a green pigment adsorbed on the surface of the solid substrate was found. These observations agree with the remarks of other authors reporting the accelerated cellulase release by S.
o4
^
thermophile in the deceleration phase of culture growth. 6 In comparison with the data published, the activity of aryl-/%glucosidase obtained in this work can be estimated as a hyperproduction (Table 3). The production of aryl-/% glucosidase with Aspergillus sp., which were recognized up to now as the best fl-glucosidase producers, varied from 11U m1-1 to 13U ml 1.1,3,10 Thus, the results obtained with S. thermophile are about three times higher. However, activity toward cellobiose produced by Aspergillus sp is higher than the one obtained with S. thermophile. 1,3 Aryl-fl-glucosidase takes part in the catabolism of plant cellular aryl-fl-glucosidase arrived at a very high activity level sugar residue bounded often with phenolic derivatives. The enzymatic release of the phenolic groups is involved in the resistance system against plant diseases and the pathogens. 16 It is possible that some enzyme of microbial origin, including enzymes from S. thermophile, can also be useful to biological protection of plants. This hypothesis needs verification.
Some properties of fl-glucosidase Studies on intra- and extracellular cellulases from S.
thermophile report data related to composition and biochemical properties of individual fractions of cellulase complex.4,5,17 Meyer and Canevascini found that intracellular fl-glucosidase from S. thermophile can be fractionated into component A, with aryl-/~-glucosidase activity (ONPG), and component B, showing cellobiase activity. s Both fractions differ considerably in molecular weight, KIn, and substrate specifity. In this work the Michaelis constants of aryl-/Yglucosidase was estimated as 1.37 mM using pnitrophenyl-fl-D-glucoside as the substrate. The thermal properties of fl-glucosidase seem to be interesting. The data reported show that the thermal optimum for the flglucosidase hydrolytic action was estimated to appear at temperatures between 50 and 72°C, and the optimal pH vary in the range from 5.0 to 6.3. 7'17 These data are completed with the study on the effect of the incubation time at elevated temperature on the thermo-resistance of aryl-fl-glucosidase. The experiments were performed at 65°C using the 0.05 M McIlvaine buffer, pH 5.0 and two enzyme concentrations: the original culture filtrate and the filtrate diluted 50-fold with the buffer solution. To compare the results, the first enzyme solution was also Table 3
Comparison of/~-glucosidase production with different fungal cultures
,.,
=/=>
~
0 [¢
=
,-n.~
c
0
0
Organisms
Aspergillus sp
24
7Z 9g lZ0 TIME (hour's)
F i g u r e 2 Production of cellobiase and aryl-/Y-D-glucosidase with Sporotrichum thermophile in the batch culture. A , aryl-/~glucosidase; I , cellobiase; ©, crude protein content in the medium with 3 % wheat bran as carbon source
A. phoenicus A. niger A. ustus A. solfosfi T. viride Basidiomycetes T. terrestris S. thermophile
/~-glucosidase U ml 1
Substrate used in determination
9 11 22.6 11.6 7.4 13 23 6 13 25 36.6 2.4
PN PG Cellobiose Cellobiose Cellobiose PNPG Cellobiose PNPG Cellobiose Salici PNPG Cellobiose
Reference
3 1 10 11 12 13 14 this report
The enzyme activities are expressed as the amount of/~m of products of reactions released per min per ml of culture filtrate
Enzyme Microb. Technol., 1 987, vol. 9, December
747
Papers
diluted in the same ratio after incubation and the enzyme activities in both the enzyme solutions were assayed. It was observed that the enzyme solution used without dilution appeared to be more resistant to high temperature incubation than this previously diluted. After 2 h of incubation, 65% of initial activity was found in the nondiluted samples, whereas only 40 % of initial activity remained in the diluted samples. Conclusions
S. thermophile showed to be a good producer of aryl-flglucosidase in the submerged culture. The strain used in this study produced a very high activity of aryl-flglucosidase, reaching up to 36.6 U ml- 1 in the 3 day batch fermentation. The carbon source and the pH of culture medium seem to be the most important cultural factors determining fl-glucosidase production. Acknowledgements The author would like to thank Catherine Vergoignan for her technical assistance.
748
References 1 Stenberg, D., Vijayakumar, P. and Reese, E. T. Can. J. MicrobioL 1977, 23, 139 2 WoodwardJ.andWiseman, A.EnzymeMicrob. Technol. 1982,4,73 3 Gokhale, D. V. et al. Biotechnol. Lett. 1984, 6, 719 4 Canevascini, G. et al. J. Gen. Microbial. 1979, 110, 291 5 Canevascini, G., Frachebout, D. and Meier, H. Can. J. Microbiol. 1983, 29, 1071 6 Coutts, A. D. and Smith, R. E. Appl. Environ. Microbiol. 1976, 31, 819 7 Grajek, W. Biotechnol. Lett. 1986, 8, 587 8 Meyer, H. P. and Canevascini, G. Appl. Environ. Microbiol. 1981, 41, 924 9 Commission on Biotechnology of the IUPAC. Measurement of Cellulase Activities. New Delhi, Dec. 1984 10 Shamala, T. R. and Sreekantiah, K. R. Enzyme Microb. Technol. 1986, 8, 178 11 Sadana, J. C., Shewale, J. G. and Deshparide, M. V. Appl. Environ MicrobioL 1980, 39, 935 12 Nisizawa, T. et al. J. Biochem. 11971, 70, 375 13 Shewale, J. G. and Sadama, J. C. Can. J. MicrobioL 1978.24, 1204 14 Breuil, C. Wojtczak, G. and Saddler, J. N. Biotechnol. Lett. 1986, 8, 673 15 Grajek, W. Biotechnol. Bioeng. 1987, in press 16 Garibaldi, A. and Gibbins, L. N. Can. J. MicrobioL 1975, 21, 513 17 Margaritis, A. and Creese, E. Biotechnol. Lett. 1981, 3, 471
EnzymeMicrob. Technol., 1987, vol. 9, December
(Received 8 April 1987; revised 29 June 1987)
The effect of the growth of temperature, pH, carbon source, nitrogen supplementation and inoculum size were examined in shake-flask scale studies to determine the optimum conditions for fl-glucosidases production by Sporotrichum (Chrysosporium) thermophile. Wheat bran and sugar-beet pulp were selected as the best carbon sources and ( N H 4 ) 2 S O 4, NH4CI and K N O 3 a s the best nitrogen supplementation. Ten liter fermentations were carried out to study the kinetics of product formation. It was found that S. thermophile is able to produce high thermostable extracellular cellobiase and aryl-fl-glucosidase. Very high aryl-fl-glucosidase (PNPG) activities in the range from 30 to 40 U ml-1 and cellobiase activities of 2,45 U ml-t in the 3-day batch fermentations were obtained. The K,, for aryl-fl-glucosidase and its thermal properties were also estimated.
Keywords:Thermophilicfungi; Sporotrichum thermophile; fl-glucosidaseproduction;thermostability Introduction The cellulase enzyme system of microorganisms consists of three main components: exo-fl-D-glucanases, endo-fl1,4-glucanases and fl-glucosidase. The action of these enzymes is synergistic and the whole multi-enzyme system is required for the complete hydrolysis of cellulosic materials. The enzyme fl-D-glucosidase can catalyse the hydrolysis of cellobiose and aryl fl-D-glucosides (e.g., pnitrophenyl fl-D-glucoside and methyl fl-D-glucoside). Although numerous microorganisms are known to be able to produce high levels of extracellular cellulases, the best cellulase producers of Trichoderma sp. are fairly deficient in fl-glucosidase. Recently, some mutants of Trichoderma reesei, particularly Rut-C30 and QM9414, have been demonstrated to have potential for practical use to cellulose hydrolysis. It was also reported that the cellulolytic system of T. reesei, consisting mainly of exoand endo-glucanases, could be successfully supplemented with fl-glucosidase from Aspergillus cultures. 1 A great effort has also been made to find other sources of microbial cellulase and many papers in this field have been published. Woodward and Wieseman 2 and Gokhale et al. 3 have showed a comparison of fl-glucosidase production by different fungi. However, all enzyme preparations obtained from mesopholic strains show a lack of thermostability, which results in low hydrolysis efficiences.
* Location to which correspondenceshould be addressed
744
An attractive alternative is to apply the thermostable enzymes produced by thermophilic microorganisms. The use of such cellulases in enzymatic hydrolysis might enhance the reaction rate, reduce the contamination hazard and prolong the periods of reaction time. Recently, interest has increased in thermophilic microorganisms, in particular in fungi, and some species are reported to be good ceUulase producers (e.g., Thielavia terrestris, Sporotrichum thermophile, Talaromyces emersonii and Aspergillus fumigatus). The enzymes produced by these organisms demonstrate the maximum of activities at elevated temperatures between 60 and 80°C, whereas the optima of cellulolytic activities from mesophilic fungi appear at temperatures from 40 to 50°C. The present work was undertaken to investigate the influence of cultue conditions on the fl-glucosidase production by Sporotrichum (Chrysosporium) thermophile. This fungus has been previously reported to be able to excrete the thermostable cellulases when it grew in liquid and solid state fermentations. 4- 8 Some references to the particular thermal properties of fl-glucosidase are also made.
Materials and methods Microorganism
A wild strain of Sporotrichum (Chrysosporium) thermophile Apinis was isolated from wooden waste materials chosen for their cellulolytic activities. The subcultures were maintained on yeast-starch agar at 45°C, stored at 4°C and transferred every month.
Enzyme M icrob. Technol., 1987, vol. 9, December
0141~0229/87/120744435 $03.00 © 1987 Butterworth Publishers
Hyperproduction of thermostable fl-glucosidase: W. Grajek
Inoculum
protein content in the water washed solid culture residues was calculated from N total-N mineral and expressed as N × 6.25. The total and mineral nitrogen were determined by the Kjeldahl procedure.
The 5-day-old cultures were transferred from agar slants into 500 ml Erlenmeyer flasks with 100 ml of liquid medium containing (g 1- z): soluble starch, 10; Ca(NO3)z, 2.0; K2HPO 4, 0.9; KH2PO 4, 1.1; MgSO4.7HzO, 0.5; NaCI, 0.1 ; yeast extract, 0.5, pH 6.5. Each flask was seeded with 107 spores. The cultures were incubated on a rotary shaker, 200 rev m i n - ~, at 43°C. After 3 days of cultivation the cultures were used to inoculate the enzyme production media. In the study of the effect of inoculum size the flasks were seeded with 10 7, 5.10 7 and 108 spores per 100ml of the medium.
Enzyme assay The cultures were harvested by centrifugation at 8000 g for 10 min. The clear supernatants were diluted to a convenient volume with the appropriate buffer and enzyme activities were estimated. Aryl-fl-glucosidase activity (fl-D-glucosidase glucohydrolase, EC 3.2.1 21) was measured at 65°C in an assay containing 1.0 ml 2 mM p-nitrophenyl-fl-Dglucoside (PNPG, Sigma) dissolved in 0.05 sodium acetate buffer, pH 5.0. Afterward 0.1 ml enzyme solution was added and after 15 min of incubation the reaction was stopped with 2.0ml 1 M Na2CO 3. The p-nitrophenol release was measured at 400 nm. In some cases cellobiase activity was also estimated using cellobiose as the reaction substrate according to the IUPAC method. 9 The Dglucose concentrations were detected by D-glucose oxidase-peroxidase method. One unit of activity was expressed as the #M of p-nitrophenol or glucose produced per min per ml of the culture filtrate, respectively. The test on the thermal stability was carried out at 65 °C, pH 6.5, by incubation of the enzyme solution without substrate by different periods of time followed by the enzyme activity assay.
Enzyme production Shake-flask experiments were applied for the evaluation of the culture conditions and medium compositions. The cultures were incubated in 500 ml Erlenmeyer flasks containing 100 ml liquid media at 43°C for 3 days on a rotary shaker, 200 rev min- ~. A standard medium had the following composition (g 1-~): dry powdered sugar-beet pulp obtained after sugar extraction, 20.0; (NH4)2SO4, 1.0; K2HPO 4, 2.2; KH2PO 4, 2.75; buffer, KC1, 0.5; MgSO4-7H20, 0.5; yeast extract, 0.1, pH 6.5. Each flask was inoculated with 10 ml starter culture. The studies with various carbon sources were performed replacing sugar-beet pulp in the standard medium by 20g 1-~: cellulose MN 30 (Machery Nagel Co, Diiren, Germany); 10 g l - ~: Solka floc cellulose SW40 (Brown Co, Berlin NH) 30 g 1-1: Wheat bran, 20 g 1-~; wheat straw, 10 g 1- ~ cellulose microcrystalline Avicel (Merck, Darmstadt, Germany); 20 g 1-1 xylan from oat spelts (Sigma Chemical Co, USA); 10 g 1-1 soluble starch (Merck); 10 g 1-1 glucose (Sigma); 10 g 1-1 cellobiose (Sigma); 10 g 1- ~ lactose (Sigma); and 10 g 1- x glycerol (Sigma). The dry sugar-beet pulp, wheat bran and wheat straw were used in the powdered form with the particle size below 0.1 mm. The effect of nitrogen sources was tested using the standard medium supplemented with N a N O 3, K N O 3, NH4NO 3, Ca(NO3)z, NH4C1, (NH4)2SO 4, (NH4)2HPO 4 and urea. The amount of additional nitrogen was 13 g per kilogram of a dry pulp. The effect of pH was investigated using the standard medium buffered by phosphate-phosphate buffer prepared by mixing the solution of KH2PO4 (5 g 1- ~) with the solution of KEHPO 4 (5 g 1- ~) to obtain the media in the range of pH from 5.5 to 7.5.
Results and discussion
Influence of cultural conditions on enzyme production S. thermophile is a thermophilic Deuteromycete growing at the temperature range between 28 and 55°C. The optimum temperature for the growth of the strain investigated was previously determined as 43°C and the optimum pH as 6.5? o It was earlier reported that the maximum of the endo- and exo-fl-glucanase production appeared at 45°C in 2 to 4 days in the presence of 1% Solka/Floc as a substrate. The results of our experiments on the influence of pH and temperature on aryl-flglucosidase production are shown in Figure 1. The
2.4
_,?.4
Fermentor-scale growth The enzyme production kinetics were studied in a 12 1 Biolafitte fermentor. The conditions of fermentation were as follows: working volume 81, pH 6.5, temperature 43°C, aeration 601 1- a h-~ and agitation 250 rev min-~. The culture medium contained 3 % powdered wheat bran and mineral solution (g 1-~): (NH4)2SO,,, 2.0; KH2PO4, 1.1; KzHPO4, 0.9 (pH 6.5); M g S O 4 . 7 H 2 0 , 0.5; KC1, 0.5; CaC12"2H20, 0.1. Sodium hydroxide 2 N solution was used to control pH. The medium was inoculated with 10% v/v 3-day-old liquid culture. The samples were taken in 12h intervals, and protein contents and enzyme activities were determined.
Crude protein analysis The growth of S. thermophile, with powdered wheat bran as carbon source, was estimated by the crude protein content evolution in the culture medium. The crude
16 12. 8
.4 I TEMPERRTURE (=E) pH Effect of pH and temperature on aryl-fl-D-glucosidase production in batch cultures of Sporotrichum (Chrysosporium) therrnophile. (Enzyme activities were determined toward pnitrophenyI-D-glucoside in filtrates from 3-day-old cultures growing in liquid medium containing 2 % powdered sugar-beet pulp)
Figure 1
Enzyme Microb. Technol., 1987, vol. 9, December
745
Papers fermentation with different pH values of media were carried out at 43°C. The optimum pH value for extracellular aryl-fl-glucosidase production was around 6.5. This value corresponds well to the pH favoring mycelial growth. It was also observed that more alkaline pH values caused a strong decrease of aryl-fl-glucosidase production. At pH 7.0, only 5.1 U ml-1 of enzyme activity was detected, whereas no decrease of mycelium production was observed. The aryl-fl-glucosidase activity declining versus acidic pH was much smaller, and at pH 5.5 about 8.7 U ml-1 were still determined. Thus, the optimal pH conditions for the aryl-fl-glucosidase production are similar to those that appear to be the best for culture growth. Data reported show that the biosynthesis of endo- and exo-fl-glucanase by S. thermophile are favored by alkaline conditions, with the pH optimum between 7.0 and
Carbon source Cellulose MN30 Solka floc Avicel Wheat bran Wheat straw Sugar-beet pulp Xylan Starch Cellobiose Glucose Lactose Glycerol
7.5 6 .
The pH of culture medium is a more critical factor in aryl-fl-glucosidase biosynthesis than the temperature. The maximum enzyme production was obtained at 40°C, but activity determined in cultures growing at 38°C and 46°C was estimated to be only 14 %, and 8 % below the maximum value. Coutts and Smith 6 also reported that the best temperature for cellulase production for this microorganism was determined at 40°C. To examine the effect of inoculum size on aryl-fl-glucosidase production, three levels of seeding were used. The production of this enzyme in the media seeded with 107, 5.107 and 108 spores per 100 ml of culture medium were 22.6, 23.1 and 23.5 U ml- 1, respectively. These results show that the influence of inoculum size on aryl-fl-glucosidase production in the cultures containing sugar-beet pulp as carbon source can be considered a s insignificant.
Effect o f carbon sources Enzyme production in presence of various cellulosic and other carbon sources is shown in Table 1. Chemically pure substrates as well as heterogeneous materials for the investigation were chosen. The quantities of cellulosic substrates added were calculated to have ~ 10 g 1-1 of cellulose in the media. It was observed that aryl-flglucosidase, was produced in the presence of all the substrates tested, but only heterogenous substrates enhanced significantly the enzyme production. Wheat bran was found to be the best carbon source for aryl-flglucosidase production. After 3 days of cultivation a very high level of enzyme activities, reaching up to 36.6 U ml-1, was noted (Table 1). A good enzyme formation in the medium with sugar-beet pulp was also observed. Both substrates mentioned above have relatively rich chemical composition, in particular the high protein contents. Wheat bran is well known to contain many stimulators of microbial biosynthesis, such as microelements and vitamins. All media containing pure forms of cellulose, such as cellulose MN30, Solka floc and Avicel, have shown a very low inductible effect on aryl-fl-glucosidase formation. The enzyme activities in these media and in the media with glucose and cellobiose appeared at very low levels, between 0.1 and 0.3 U m1-1. Starch, lactose and glycerol induced the enzyme activities better than pure source of cellulose. Effect o f nitrogen source In the fermentations the inorganic nitrogen sources and urea were also tried. The amount of supplemented
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Table 1 Effect of carbon source on aryl-/~-glucosidase production by Sporotrichum thermophile Aryl-/Y-glucosidase activity U m1-1 0.3 0.3 0.3 36.6 4.2 22.3 0.2 3.1 0.1 0.1 0.5 0.5
Ary-]Y-glucosidase activity was determined toward p-nitrophenyl-/~D-glucoside in the supernatants from 3-day-old cultures of
S. thermophile
nitrogen was calculated to result in 40 g of total nitrogen for each kilogram of carbohydrates contained in sugarbeet pulp. Assuming that carbohydrate contents in sugarbeet pulp was about 70% dry matter (cellulose 21%, hemicellulose 23 % and pectin 30°,/0) and total nitrogen content was about 1.5 %, it was presumed that 1 kg of the pulp contained ~700g of carbohydrates and 15 g of nitrogen. Thus, to have the desired proportion between sugars and nitrogen, about 13 g nitrogen per 1 kg pulp were added. This quantity corresponds to C:N ratio a s 100:6. The fl-glucosidase activities (PNPG) produced in the media containing different nitrogen supplementation are presented in Table 2. The preliminary experiments, carried out with the citric acid-sodium dihydrophosphate (Mcllvaine) buffer used to stabilize the pH level, demonstrated a potassium deficit. For this reason the Mcllvaine buffer was replaced by the KHzPO 4 K2HPO 4 buffer. Nitrogen supplementation strongly affected the enzyme biosynthesis. Among the nitrogen sources, (NHa)2SO4, NH4C1 and KNO3 appeared to be the best. Aryl-//glucosidase activities determined in the supernatants obtained from three day-old cultures approached 22-24 U ml- 1. The ammonium ions better stimulated the aryl//-glucosidase production than the nitrate ions. Calcium nitrate, reported to be the best nitrogen source for the culture growth, is gave very poor enzyme activities. The
Table 2 Effect of nitrogen supplementation on aryl-/~-glucosidase production by Sporotrichum thermophile Nitrogen source Urea Urea + ammonium sulfate Ammonium hydrogenophosphate Ammonium sulfate Ammonium chloride Ammonium nitrate Potassium nitrate Sodium nitrate Calcium nitrate
Aryl-/Y-glucosidase activity U m1-1 12.9 20.7 22.5 23.7 23.6 17.4 22.9 10.9 5.8
Aryl-fY-glucosidase activity was determined toward p-nitrophenyl, -]Y-Dglucoside in the supernatants from 3-day-old cultures of
S. thermophile
Enzyme Microb. Technol., 1987, vol. 9, December
Hyperproduction of thermostable ~-glucosidase: W. Grajek addition of urea in the mixture with (NH4)2SO4, used to stabilize the pH of the media, allowed reasonable activities, higher than those in the media supplemented with urea, but a little below those obtained with (NH4)2SO4. The literature has reported that urea and NaNO3 are suitable for cellulase production by S. thermophile and that the variation of the concentration of NaNO3 between 0.05 and 0.4 % has little effect on cellulase activity. 6 No data were found on the effect of nitrogen sources on arylfl-glucosidase formation. In this study both the nitrogen sources mentioned above gave low yield of aryl-/Yglucosidase.
Fermenter-scale growth To study the enzyme production kinetics, cultures in 101 fermenter scale were performed. The results of these experiments are presented in the batch fermentation digram (Figure 2). Very short lag-phase period, limited to 4 h, and next the rapid exponential growth accompanied by large oxygen consumption and acidification of medium, were noticed. The beginning of an intensive extracellular flglucosidases production was observed between 18 and 24 h of culture for the both activities. A high increase of enzyme activities were detected from 24 to 72 h of culture and afterward the rate of enzyme synthesis markedly decreased. At the end of day 3 of incubation the cellobiase activity reached the highest level of 2.45 U m l - 1 and then a small decrease of the activity was observed. Aryl-flglucosidase (PNPG) activity was increasing during the whole time of fermentation, but after 3 days of culture the activity increase was very poor. The extracellular aryl-flglucosidase achieved a very high activity level determined as 36.6 U ml-1 after 72 h of incubation and 40.2 U m1-1 after 144h, respectively. Therefore, the enzyme productivity in a 3-day-old batch culture was about 500 U 1-1 h-1. It was observed that/%glucosidase release appeared in the end of the exponential and deceleration phases of the growth. At the same time the production of a green pigment adsorbed on the surface of the solid substrate was found. These observations agree with the remarks of other authors reporting the accelerated cellulase release by S.
o4
^
thermophile in the deceleration phase of culture growth. 6 In comparison with the data published, the activity of aryl-/%glucosidase obtained in this work can be estimated as a hyperproduction (Table 3). The production of aryl-/% glucosidase with Aspergillus sp., which were recognized up to now as the best fl-glucosidase producers, varied from 11U m1-1 to 13U ml 1.1,3,10 Thus, the results obtained with S. thermophile are about three times higher. However, activity toward cellobiose produced by Aspergillus sp is higher than the one obtained with S. thermophile. 1,3 Aryl-fl-glucosidase takes part in the catabolism of plant cellular aryl-fl-glucosidase arrived at a very high activity level sugar residue bounded often with phenolic derivatives. The enzymatic release of the phenolic groups is involved in the resistance system against plant diseases and the pathogens. 16 It is possible that some enzyme of microbial origin, including enzymes from S. thermophile, can also be useful to biological protection of plants. This hypothesis needs verification.
Some properties of fl-glucosidase Studies on intra- and extracellular cellulases from S.
thermophile report data related to composition and biochemical properties of individual fractions of cellulase complex.4,5,17 Meyer and Canevascini found that intracellular fl-glucosidase from S. thermophile can be fractionated into component A, with aryl-/~-glucosidase activity (ONPG), and component B, showing cellobiase activity. s Both fractions differ considerably in molecular weight, KIn, and substrate specifity. In this work the Michaelis constants of aryl-/Yglucosidase was estimated as 1.37 mM using pnitrophenyl-fl-D-glucoside as the substrate. The thermal properties of fl-glucosidase seem to be interesting. The data reported show that the thermal optimum for the flglucosidase hydrolytic action was estimated to appear at temperatures between 50 and 72°C, and the optimal pH vary in the range from 5.0 to 6.3. 7'17 These data are completed with the study on the effect of the incubation time at elevated temperature on the thermo-resistance of aryl-fl-glucosidase. The experiments were performed at 65°C using the 0.05 M McIlvaine buffer, pH 5.0 and two enzyme concentrations: the original culture filtrate and the filtrate diluted 50-fold with the buffer solution. To compare the results, the first enzyme solution was also Table 3
Comparison of/~-glucosidase production with different fungal cultures
,.,
=/=>
~
0 [¢
=
,-n.~
c
0
0
Organisms
Aspergillus sp
24
7Z 9g lZ0 TIME (hour's)
F i g u r e 2 Production of cellobiase and aryl-/Y-D-glucosidase with Sporotrichum thermophile in the batch culture. A , aryl-/~glucosidase; I , cellobiase; ©, crude protein content in the medium with 3 % wheat bran as carbon source
A. phoenicus A. niger A. ustus A. solfosfi T. viride Basidiomycetes T. terrestris S. thermophile
/~-glucosidase U ml 1
Substrate used in determination
9 11 22.6 11.6 7.4 13 23 6 13 25 36.6 2.4
PN PG Cellobiose Cellobiose Cellobiose PNPG Cellobiose PNPG Cellobiose Salici PNPG Cellobiose
Reference
3 1 10 11 12 13 14 this report
The enzyme activities are expressed as the amount of/~m of products of reactions released per min per ml of culture filtrate
Enzyme Microb. Technol., 1 987, vol. 9, December
747
Papers
diluted in the same ratio after incubation and the enzyme activities in both the enzyme solutions were assayed. It was observed that the enzyme solution used without dilution appeared to be more resistant to high temperature incubation than this previously diluted. After 2 h of incubation, 65% of initial activity was found in the nondiluted samples, whereas only 40 % of initial activity remained in the diluted samples. Conclusions
S. thermophile showed to be a good producer of aryl-flglucosidase in the submerged culture. The strain used in this study produced a very high activity of aryl-flglucosidase, reaching up to 36.6 U ml- 1 in the 3 day batch fermentation. The carbon source and the pH of culture medium seem to be the most important cultural factors determining fl-glucosidase production. Acknowledgements The author would like to thank Catherine Vergoignan for her technical assistance.
748
References 1 Stenberg, D., Vijayakumar, P. and Reese, E. T. Can. J. MicrobioL 1977, 23, 139 2 WoodwardJ.andWiseman, A.EnzymeMicrob. Technol. 1982,4,73 3 Gokhale, D. V. et al. Biotechnol. Lett. 1984, 6, 719 4 Canevascini, G. et al. J. Gen. Microbial. 1979, 110, 291 5 Canevascini, G., Frachebout, D. and Meier, H. Can. J. Microbiol. 1983, 29, 1071 6 Coutts, A. D. and Smith, R. E. Appl. Environ. Microbiol. 1976, 31, 819 7 Grajek, W. Biotechnol. Lett. 1986, 8, 587 8 Meyer, H. P. and Canevascini, G. Appl. Environ. Microbiol. 1981, 41, 924 9 Commission on Biotechnology of the IUPAC. Measurement of Cellulase Activities. New Delhi, Dec. 1984 10 Shamala, T. R. and Sreekantiah, K. R. Enzyme Microb. Technol. 1986, 8, 178 11 Sadana, J. C., Shewale, J. G. and Deshparide, M. V. Appl. Environ MicrobioL 1980, 39, 935 12 Nisizawa, T. et al. J. Biochem. 11971, 70, 375 13 Shewale, J. G. and Sadama, J. C. Can. J. MicrobioL 1978.24, 1204 14 Breuil, C. Wojtczak, G. and Saddler, J. N. Biotechnol. Lett. 1986, 8, 673 15 Grajek, W. Biotechnol. Bioeng. 1987, in press 16 Garibaldi, A. and Gibbins, L. N. Can. J. MicrobioL 1975, 21, 513 17 Margaritis, A. and Creese, E. Biotechnol. Lett. 1981, 3, 471
EnzymeMicrob. Technol., 1987, vol. 9, December