Biochemical Society Transactions ( 1 992) 20 Cellulase production by thermophilic fungi LUCIA R. DURRANT, VALERIA REGINATTO
ALESSANDRA 8 .
HELLO and
Department of Food Science, Food Engineering Faculty, UNICAMP 13081 Campinas-SP Brazil Cellulolytic enzymes have been reported to be produced by a variety of microorganisms. Among the fungi, Trichoderm filamentous mesophilic fungus, and mutants derived from it, are the best studied organisms which are able to produce cellulases [l-21. There have been many reports of thermophilic microorganisms which produce cellulases (3-61. Thermophilic microorganisms are thought to be promising new sources of cellulases because they can elaborate enzymes which are, in general, more active at high temperatures and more thermostable than those produced by mesophilic microorganisms and also because they may offer faster growth and higher rates of cellulase production than the mesophiles [41. We have been searching for thermophilic microorganisms which are able to grow and produce enzymes at the temperature of 45 C. A screening of samples from naturally biodegraded cellulosic materials, was carried out on a cellulose medium. Twentynine fungal strains were pre-selected after being able to degrade a strip (10x1 cm) of filter paper, Whatman N 1, in a test-tube containing mineral solution, in a period of less than to two weeks. These strains were then inoculated in ( 2 . 0 9 ) of wheatbran : vater (l:l), for five days. Those strains which produced better cellulose hydrolysing activities were chosen for enzyme production in liquid media. Extracellular cellulase production was carried out under shaking conditions in media containg (5.0 g/l) carbon source, (1.0 ml/l) Tween 80, and minerals. The carbon sources used were cellulose microcrystalline (Merck) and Solka- Flok BW 40 FCC (James River Corporation). Filter paper degrading activity (FPA) was estimated according to the procedures of Mandels et al. (7). Carboxymethyl cellulose hydrolising activity (CMCase) was determined by the increase in reducing sugar after 6 0 minutes of reaction of a mixture of (0.5 ml) extracellular extract and (1.0 m1) of (1%) carboxymethylcellulose at pH 5.0 in acetate buffer, incubated at 50°C. Microcrystalline cellulose degrading activity (MCDa) was assayed in a similar way as for CMCase, using (1%) microcrystalline cellulose. One unit of enzyme activity was defined as the number of micromoles of reducing sugars released by one millilitre of enzyme per minute. Reducing sugar expressed as glucose vas determined by the DNS (3,sdinitrosalicylic acid) method 1 8 1 . After solid state fermentation in wheatbran: water (l:l), six fungi strains were selected, since they exhibited reasonable CMCase and microcrystalline cellu ose degrading activity (results not shown) and were cultured in liquid media as descr bed above.
227s
Table 1 shows the results for cellulase activities in the liquid medium vhere the carbon source was microcrystalline cellulose whereas, in Table 2, the results shown were obtained after growth in Solka Flok BW 40. Table 1 Cellulase activities in medium containing microcrystalline cellulose
............................................
Strains
Cellulase Activities (umoles glucose/ml/min)
............................................ MCDA CMCase FPA ............................................ F 7.14
0.51 0.50 0.49 0.43 0.50 0.51
F 5.22 F 5.18 F 9.11 F 11.12 F 5.12
0.65 0.61 0.75 0.62 0.61 0.64
.....................................
0.16 0.11 0.18 0 0 0
Table 2 Cellulase activities in containing Solka Flok BW 40 as the source
..................................... Strain
Cellulase Activities (umoles glucose/ml/min
In general, the the strains Isolated showed better microcrystalline cellulose degrading activity (MCDA) when grown on Solka Flok as the carbon source. This was particularly true of the strain F 5.22, which showed twice as much activity than when it was grown on cellulose microcrystalline. The use of other carbon sources are worthy of investigation and production o f cellulases may be increased depending on the source. We acknowledge financial support from FAPESP (A.B.M.) and CAPES (V.R.). 1. Reese, E.T. 6 Mandels, M. (1984) in Annual Reports on Fermentation Processes 1, 1-20 2. Hontenecourt, B. S. (1983) Trends in Biotechnology &( 51,156-160 3. Shekhar Sharma, H. (1989) Appl. Microbial. Biotechnol. 1-10 4. Margaritis, A., and Merchant, R. F. Critical Reviews in Biotechnol. (1986) 4 (3I , 327-367 5. Kavamori, M. Takayama, K. 6 Takasawa, S. (1987) Agric. Biol. Chem. 5a(3), 647-654 6. Feldman, K. A., Lovett, J. S. 6 Tsao, G. T. (1988) Enzyme Microb. Technol. u ( 5 ) , 262-272 7. Mandels, M., Andreotti, R. 6 Roche, C. (1976) in Enzymatic Conversion of Cellulosic Materials: Technology and Applications (Gaden, E.L., Mandels, M., Reese, E.L. 6 Spano, L. A., eds.), pp. 21-33 John Wiley 6 Sons, New York 8. Miller, G. L. (1959) Anal. Chem. 426428
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ALESSANDRA 8 .
HELLO and
Department of Food Science, Food Engineering Faculty, UNICAMP 13081 Campinas-SP Brazil Cellulolytic enzymes have been reported to be produced by a variety of microorganisms. Among the fungi, Trichoderm filamentous mesophilic fungus, and mutants derived from it, are the best studied organisms which are able to produce cellulases [l-21. There have been many reports of thermophilic microorganisms which produce cellulases (3-61. Thermophilic microorganisms are thought to be promising new sources of cellulases because they can elaborate enzymes which are, in general, more active at high temperatures and more thermostable than those produced by mesophilic microorganisms and also because they may offer faster growth and higher rates of cellulase production than the mesophiles [41. We have been searching for thermophilic microorganisms which are able to grow and produce enzymes at the temperature of 45 C. A screening of samples from naturally biodegraded cellulosic materials, was carried out on a cellulose medium. Twentynine fungal strains were pre-selected after being able to degrade a strip (10x1 cm) of filter paper, Whatman N 1, in a test-tube containing mineral solution, in a period of less than to two weeks. These strains were then inoculated in ( 2 . 0 9 ) of wheatbran : vater (l:l), for five days. Those strains which produced better cellulose hydrolysing activities were chosen for enzyme production in liquid media. Extracellular cellulase production was carried out under shaking conditions in media containg (5.0 g/l) carbon source, (1.0 ml/l) Tween 80, and minerals. The carbon sources used were cellulose microcrystalline (Merck) and Solka- Flok BW 40 FCC (James River Corporation). Filter paper degrading activity (FPA) was estimated according to the procedures of Mandels et al. (7). Carboxymethyl cellulose hydrolising activity (CMCase) was determined by the increase in reducing sugar after 6 0 minutes of reaction of a mixture of (0.5 ml) extracellular extract and (1.0 m1) of (1%) carboxymethylcellulose at pH 5.0 in acetate buffer, incubated at 50°C. Microcrystalline cellulose degrading activity (MCDa) was assayed in a similar way as for CMCase, using (1%) microcrystalline cellulose. One unit of enzyme activity was defined as the number of micromoles of reducing sugars released by one millilitre of enzyme per minute. Reducing sugar expressed as glucose vas determined by the DNS (3,sdinitrosalicylic acid) method 1 8 1 . After solid state fermentation in wheatbran: water (l:l), six fungi strains were selected, since they exhibited reasonable CMCase and microcrystalline cellu ose degrading activity (results not shown) and were cultured in liquid media as descr bed above.
227s
Table 1 shows the results for cellulase activities in the liquid medium vhere the carbon source was microcrystalline cellulose whereas, in Table 2, the results shown were obtained after growth in Solka Flok BW 40. Table 1 Cellulase activities in medium containing microcrystalline cellulose
............................................
Strains
Cellulase Activities (umoles glucose/ml/min)
............................................ MCDA CMCase FPA ............................................ F 7.14
0.51 0.50 0.49 0.43 0.50 0.51
F 5.22 F 5.18 F 9.11 F 11.12 F 5.12
0.65 0.61 0.75 0.62 0.61 0.64
.....................................
0.16 0.11 0.18 0 0 0
Table 2 Cellulase activities in containing Solka Flok BW 40 as the source
..................................... Strain
Cellulase Activities (umoles glucose/ml/min
In general, the the strains Isolated showed better microcrystalline cellulose degrading activity (MCDA) when grown on Solka Flok as the carbon source. This was particularly true of the strain F 5.22, which showed twice as much activity than when it was grown on cellulose microcrystalline. The use of other carbon sources are worthy of investigation and production o f cellulases may be increased depending on the source. We acknowledge financial support from FAPESP (A.B.M.) and CAPES (V.R.). 1. Reese, E.T. 6 Mandels, M. (1984) in Annual Reports on Fermentation Processes 1, 1-20 2. Hontenecourt, B. S. (1983) Trends in Biotechnology &( 51,156-160 3. Shekhar Sharma, H. (1989) Appl. Microbial. Biotechnol. 1-10 4. Margaritis, A., and Merchant, R. F. Critical Reviews in Biotechnol. (1986) 4 (3I , 327-367 5. Kavamori, M. Takayama, K. 6 Takasawa, S. (1987) Agric. Biol. Chem. 5a(3), 647-654 6. Feldman, K. A., Lovett, J. S. 6 Tsao, G. T. (1988) Enzyme Microb. Technol. u ( 5 ) , 262-272 7. Mandels, M., Andreotti, R. 6 Roche, C. (1976) in Enzymatic Conversion of Cellulosic Materials: Technology and Applications (Gaden, E.L., Mandels, M., Reese, E.L. 6 Spano, L. A., eds.), pp. 21-33 John Wiley 6 Sons, New York 8. Miller, G. L. (1959) Anal. Chem. 426428
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~
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