Folia Microbiol (2013) 58:561–568 DOI 10.1007/s12223-013-0245-7
Purification and biochemical characterization of glucose–cellobiose-tolerant cellulases from Scytalidium thermophilum Jean Carlos Rodrigues Silva & Luis Henrique Souza Guimarães & José Carlos Santos Salgado & Rosa Prazeres Melo Furriel & Maria Lourdes T. M. Polizeli & José César Rosa & João Atilio Jorge
Received: 12 June 2012 / Accepted: 18 March 2013 / Published online: 7 April 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013
Abstract Two cellulases from Scytalidium thermophilum were purified and characterized, exhibiting tolerance to glucose and cellobiose. Characterization of purified cellulases I and II by mass spectrometry revealed primary structure similarities with an exoglucanase and an endoglucanase, respectively. Molecular masses were 51.2 and 45.6 kDa for cellulases I and II, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellulases I and II exhibited isoelectric points of 6.2 and 6.9 and saccharide contents of 11 and 93 %, respectively. Optima of temperature and pH were 60–65 °C and 4.0 for purified cellulase I and 65 °C and 6.5 for purified cellulase II. Both cellulases maintained total CMCase activity after 60 min at 60 °C. Cysteine, Mn2+, dithiotreitol and ß-mercaptoethanolstimulated cellulases I and II. The tolerance to cellulose J. C. R. Silva : J. C. S. Salgado Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil J. C. R. Silva Instituto Federal de Educação, Ciência e Tecnologia de São Paulo, Matão, São Paulo, Brazil L. H. S. Guimarães : R. P. M. Furriel : M. L. T. M. Polizeli : J. A. Jorge (*) Departamento de Biologia e Química, Faculdade de Filosofia, Ciências de Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-901, Ribeirão Preto, São Paulo, Brazil e-mail:
[email protected] J. C. Rosa Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
hydrolysis products and the high thermal stabilities of Scytalidium cellulases suggest good potential for industrial applications.
Introduction The most accepted model for the mechanism of cellulose enzymic hydrolysis at present involves the synergistic action of endoglucanases (endo-1,4-β-D-glucanases; EC 3.2.1.4), exoglucanases (cellobiohydrolases; 1,4-β-D-glucan-cellobiohydrolases; EC 3.2.1.91), and β-glucosidades (cellobiases; β-D-glucoside-glucohydrolases; EC 3.2.1.21). Endoglucanases randomly hydrolyze glycosidic bonds of cellulose chains to produce new chain ends; exoglucanases progressively cleave cellulose chains at their extremities to release soluble cellobiose; and β-glucosidases hydrolyze cellobiose and short celloligosaccharides to glucose (Bath and Bath 1997; Saha 2004). This multienzyme system is usually inhibited by the final products glucose, cellobiose, and short celloligosaccharides, which limit the efficiency of separate cellulose hydrolysis and fermentation processes (Hahn-Hagerdal et al. 2006; Berlin et al. 2007; Kumar et al. 2008). However, our group has recently reported the production of glucose, xylose-stimulated β-glucosidases by thermophilic molds (Nascimento et al. 2010). Moreover, preliminary studies with Scytalidium thermophilum crude culture filtrates showed that it produces cellulases that are not inhibited by either glucose or cellobiose. Furthermore, filamentous thermophilic fungi may be good sources of enzymes with high thermal stability, a very interesting property for industrial purposes (Haki and Rakshiti 2003; Viikari et al. 2007).
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Therefore, the aim of this work was to better investigate the tolerance of the purified S. thermophilum cellulases to glucose and cellobiose, as well as other biochemical properties that may be relevant for industrial applications.
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The S. thermophilum (CBS 619.91) strain was a gift from Dr. G. Straatsma (Mushroom Experimental Station, The Netherlands). The fungus was maintained at 40 °C on slants of solid medium containing 4 % (w/v) oatmeal baby food (Quaker®). Conidia (106 cells/mL) from 10-day-old cultures were inoculated into liquid medium containing 0.1 % KH2PO4, 0.05 % MgSO4·7H2O, Vogel’s trace-elements solution (25 μL/50 mL), 0.04 % peptone, 0.4 % yeast extract, and 1 % microcrystalline cellulose (Avicel®), with pH adjusted to 6.0. Cultures were incubated at 40 °C and 110 rpm, and after 72 h the mycelia were filtered through synthetic foam on a Büchner funnel. The filtrate was used as source of crude cellulases.
collected and monitored for absorbance at 280 nm and CMCase activity. Most cellulase activity was eluted during initial washing with buffer A, which was continued until CMCase activity was negligible. The column was then eluted with a linear NaCl gradient (0–0.5 mol/L, 100 mL) in buffer A. The CM-Fractogel chromatography resulted in four separated activity peaks, denominated I, II, III, and IV. The most active fractions of peak II were pooled, dialyzed against water, lyophilized, dissolved in a small volume of 50 mmol/L Tris–HCl buffer, pH 7.0, containing 150 mmol/L NaCl (buffer B) and applied onto a Sephacryl S-200 column (20×800 mm) equilibrated and eluted with buffer B at a flow rate of 0.3 mL/min, collecting fractions of 1 mL. The most active fractions of the single CMCase activity peak eluted from the gel filtration column were pooled and stored at 4 °C. The most active fractions of peak III from CMFractogel chromatography were pooled and applied onto a CM-cellulose (10×120 mm) column equilibrated with buffer A, and the column was eluted at a flow rate of 70 mL/h with a linear gradient of NaCl (0–1 mol/L, 100 mL) in the same buffer. Fractions of 4 mL were collected and a single peak of CMCase activity was eluted. High-activity fractions were pooled and stored at 4 °C.
Enzyme assays
Polyacrylamide gel electrophoresis
Endoglucanase (CMCase) activity was routinely assayed by incubating 0.5 mL of enzyme preparation with 0.5 mL of 2 % carboxymethylcellulose (CMC) dissolved in 100 mmol/L sodium acetate buffer, pH 4.0. Exoglucanase (Avicelase) activity was determined using 2 % microcrystalline cellulose (Avicel®) as substrate, suspended in 100 mmol/L sodium acetate buffer, pH 6.0. Filter paper activity (FPase) was determined in 100 mmol/L sodium acetate buffer (pH 5.0) using a strip (10×30 mm) of Whatman No. 1 filter paper as substrate. The reactions were carried out at 55 °C for 30 min, and enzyme preparations were diluted conveniently to assure the estimation of initial velocities. The reducing sugars released were determined by the DNS method (Miller 1959). One enzyme unit (U) was defined as the amount of enzyme that releases 1 μmol of reducing sugars/min under standard assay conditions.
The homogeneity and the apparent molecular masses of the enzymes were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), according to Laemmli (1970), using 10 % acrylamide. The gels were stained with Coomassie Brilliant Blue. Isoelectric focusing was performed according to O’Farrel et al. (1977) at 500 V for 6 h in 5 % acrylamide gels.
Materials and methods Microorganism and growth conditions
Molecular mass estimation The apparent molecular masses of the native purified cellulases were estimated by gel filtration using a Bio-Sil Sec400 (Bio-Rad, USA) HPLC column, according to the manufacturer’s instructions. Characterization of purified cellulases by mass spectrometry analysis
Purification of S. thermophilum cellulases Solid (NH4)2SO4 was added to the crude filtrate to attain 90 % saturation and the mixture stood overnight at 4 °C. The precipitated proteins were collected by centrifugation (12,000×g, 30 min, 4 °C), dissolved in 50 mmol/L sodium acetate buffer, pH 4.0 (buffer A), and dialyzed against buffer A. The dialyzed sample was applied onto a CM-Fractogel column (10×110 mm) equilibrated in buffer A, which was eluted at a flow rate of 18 mL/h. Fractions of 4 mL were
Purified cellulases were excised from SDS-PAGE and subjected to trypsin digestion for 18 h at 37 °C using 0.5 mg of modified trypsin (Promega Co.). MS analyses were carried out using an Axima Performance MALDITOF/TOF mass spectrometer (Shimadzu-Kratos, Shimadzu Corp., Kyoto, Japan). The peptides’ mass fingerprint was obtained and the sequences of tryptic peptides were deduced from series of b- and y-ion fragments produced by highenergy collision-induced dissociation.
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563
b
Cel I
0.4
0.8
0.2
0.4
0.1
0.2
The effect of glucose and cellobiose on purified enzymes activity was evaluated by two methods: medium viscosity reduction and cellulose-azure (Sigma Chem. Co., USA) hydrolysis. In both cases, the enzymic reaction was performed at 55 °C in 50 mmol/L sodium acetate buffer (pH 5.0) containing 1 % (w/v) substrate and glucose or cellobiose at desired concentrations. In the first case, the activity was expressed as viscosity reduction relative to the specific viscosity of the complete reaction medium containing the thermo-inactivated enzyme (Wood and Bhat 1988). Cellulose-azure hydrolysis was stopped by the addition of 4.0 mL of alcohol reagent solution (0.5 mL calcium chloride saturated solution in methanol plus 99.5 mL ethanol). The mixture was centrifuged at 3,000×g, and the absorbance of the limpid supernatant was measured at 575 nm.
0.02
c
Cel II
_ NaCl (0-1M)
Absorbance at 280 nm
0.3
0.04
The products of Avicel hydrolysis by the purified cellulases were analyzed by thin-layer chromatography (TLC) using silica gel plates (DC-Alufolien Kieselgel 60, Merck),
III
0.4
Effect of glucose, cellobiose, and ions on purified cellulases activity
Chromatography of cellulose hydrolysis products
0.8 II
I
_
40
80
CMCase (U/mL)
0.8
The optimum pH was determined at 55 °C using citric acid (pH 2.5–4.0), sodium acetate (pH 4.0–5.0), MES (pH 5.5– 7.0), Tris–HCl (pH 8.0–9.0), CAPS (pH 10.0–11.0), and boric acid (pH 12.0–13.0) buffers at 50 mmol/L concentration. The optimum reaction temperature was determined in 50 mmol/L sodium acetate buffer (pH 5.0) in the range 40– 80 °C. Thermal stabilities were evaluated by measuring residual CMCase activity immediately after incubation of purified enzymes diluted in MilliQ water for different time intervals at different temperatures. The half-life at 70 °C was calculated from a linear plot of log (residual activity) versus incubation time, according to Segel (1975).
IV
a
NaCl (0-0.5M)
Effect of pH and temperature on purified cellulases activity and stability
0.4
0.2
120
Fraction (Nº) Fig. 1 Chromatographic profiles of S. thermophilum cellulases in CMFractogel (a), Sephacryl S-200 (b), and CM-cellulose (c). After precipitation of crude culture filtrate with 90 % ammonium sulfate, the precipitate was dissolved in a small buffer volume, dialyzed, and applied onto a CM-Fractogel column. Activity peaks II and III from CM-Fractogel column were applied onto Sephacryl S-200 and CMcellulose columns, respectively. Filled circles, absorbance at 280 nm; empty circles, CMCase activity; Cel I cellulase I, Cel II cellulase II. Arrows indicate polled fractions
subjected to two sequential ascending chromatographic runs using butanol/ethanol/water (5:3:2) as the solvent system. The spots were developed by spraying 0.2 % orcinol solution in concentrated H2SO4 and methanol (1:9), followed by heating at 100 °C.
Table 1 Purification of two cellulases from Scytalidium thermophilum Step Crude filtrate (NH4)2SO4 (90 %) CM-fractogel
Sephacryl S-200 (peak II) CM-cellulose (peak III)
Peak
I II III IV
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification (fold)
Yield (%)
125.5 50.3 18.4 7.1 1.3 5.0 1.0 0.08
193.4 151.8 9.2 30.0 16.4 9.2 11.7 4.1
1.5 3.0 0.5 4.2 12.6 1.8 11.7 51.3
1.0 2.0 0.3 2.8 8.4 1.2 7.8 34.2
100 78.5 4.7 15.5 8.5 4.7 6.0 2.1
Data are the means of six different preparations
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M
Cel I
Cel II
66 45
29 24
Estimation of neutral saccharides and protein Total neutral saccharides were estimated according to Dubois et al. (1956) using mannose as standard. Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard.
a
125
14
Determination of kinetic parameters The kinetic parameters KM and Vmax for CMC hydrolysis by the purified enzymes were determined using the SigrafW software (Leone et al. 2005), for substrate concentrations from 2 to 20 mg/mL. Product formation was linear in response to reaction time and 4.25 and 8.50 μg of cellulase I and 0.10 and 0.20 μg of cellulase II were used, respectively, for reactions in the presence and absence of ß-mercaptoethanol.
100
Relative Activity (%)
Fig. 2 SDS-PAGE analysis of S. thermophilum-purified cellulases. Duplicate samples containing 2 μg of cellulases I (Cel I) and II (Cel II) were analyzed. M, low molecular mass protein standards (Sigma Chem. Co.)
75
50
25 40
50
60
70
80
Temperature (ºC)
b
125
Relative Activity (%)
100
75
50
25
3
6
9
12
p H Fig. 3 TLC analysis of the products of Avicel hydrolysis by S. thermophilum-purified cellulases. a cellulase I; b cellulase II; M, mixture containing glucose, cellobiose, cellotriose, and cellotetraose
Fig. 4 Optima of temperature (a) and pH (b) for CMCase activity of S. thermophilum-purified cellulases. Data are means±SD of three different experiments (n=3). Empty circles, cellulase I; filled circles, cellulase II
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Results and discussion Purification and molecular properties of S. thermophilum cellulases Two cellulases from S. thermophilum were successfully purified from crude culture filtrate by (NH4)2SO4 precipitation followed by CM-Fractogel and Sephacryl S-200 or CM-Cellulose column chromatography (Table 1).
b
125 100 Log (residual activity)
Residual Activity (%)
a
75 50
2,0 1,5 1,0 0,5 0 15 30 45 60
Time (min)
25
CM-Fractogel chromatography of (NH4)2SO4 concentrated sample resulted in four major peaks with CMCase activity, named after elution order as I, II, III, and IV. Enzymes in peaks I, II, and III weakly interacted with the resin and were eluted with a large volume of equilibrating buffer (Fig. 1a). Pooled active fractions from peaks I and IV were not further purified due to their low specific activities while those from peaks II and III were applied on Sephacryl S-200 (Fig. 1b) and CM-Cellulose (Fig. 1c) columns, respectively. In both cases, a single activity peak was eluted after the chromatography, and pooled active fractions were denominated cellulases I and II. At the end of the purification procedure, cellulases I and II were purified 7.8- and 34-fold, respectively, reaching specific activities of 11.7 and 51.3 U/mg protein (Table 1). SDS-PAGE analyses of the purified enzymes revealed single protein bands corresponding to molecular masses of about 51.2 kDa for cellulase I and 45.6 kDa for cellulase II (Fig. 2), indicating homogeneity of the preparations. Apparent molecular masses of the purified enzymes estimated by molecular sieving were 50.4 and 38 kDa for cellulases I and II, respectively, and the good agreement with the values found by SDS-PAGE suggested that the enzymes were
Table 2 Influence of ions, EDTA, cysteine, DTT, and ßmecaptoethanol on the activity of cellulases purifed from Scytalidium thermophilum
125
100
Relative activity (%) Cellulase I
Log (residual activity)
Residual Activity (%)
Addition
75
50
25
1,0 0,5 0 15 30 45 60
Time (min) 0
15
1 mM
10 mM
1 mM
10 mM
Control NH4Cl NaBr NaCl CoCl2 CuCl2 MnCl2 AlCl3 KCl
100±6 70±8 107±9 103±8 123±11 94±6 155±11 96±8 97±7
100±6 42±5 98±12 101±9 69±4 55±3 207 ±14 12±2 89±9
100±8 84±8 97±11 80±6 116±7 72±4 177±13 103±8 112±9
100±5 67±7 105±8 120 ±10 123±10 5±1 151±8 77±9 131±11
CaCl2 FeCl2 BaCl2 MgCl2 EDTA β-mercaptoethanol Cysteine DTT HgCl2
94±8 94±9 102±6 90±10 94±9 177±16 132±10 158±16 0
95±11 7±2 103±7 100±9 78±10 193±16 308±27 322±24 0
100±9 107±7 107±8 98±8 96±8 242±19 174±15 189±17 0
108±8 70±9 114±10 128±12 75±9 321±30 255±18 518±61 0
2,0 1,5
30
45
60
Time (min) Fig. 5 Thermal stabilities of purified cellulase I (a) and cellulase II (b) from S. thermophilum. Samples of the purified enzymes in water were incubated at 50 (filled squares), 60 (empty circles), and 70 °C (filled triangles) for different time intervals, and CMCase residual activity was estimated at 65 °C as described in “Materials and methods.” Insets, linear plots to estimate the half-lives at 70 °C. a y=−0.01688x+1.988; b fast phase, y=−0.05236x+1.963962; slow phase, y=−0.00939x+ 1.2519). Data are means±SD of three different experiments (n=3)
Cellulase II
Data are the means±SD of four different enzymatic assays
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Control
Vmax (U/mg protein) KM (mg/mL) Vmax/KM
Cellulase I
Cellulase II
Cellulase I
10.1±1.2 21.8±2.8 0.5±0.06
54.3±4.9 49.7±3.9 1.1±0.09
15.6±1.7 7.2±0.6 2.2±0.2
monomeric. Most fungal cellulases are monomeric, with molecular masses in the range 40–60 kDa (Maheshwari et al. 2000; Jorgensen et al. 2003; Saha 2004; Jabbar et al. 2008). Interestingly, isoelectric focusing of the purified cellulases yielded pI values of 6.2 for cellulase I and 6.9 for cellulase II, contrasting with many studies reporting acidic isoelectric points (3.5–5.0) for cellulases from different sources (Maheshwari et al. 2000; Jorgensen et al. 2003; Saha 2004; Jabbar et al. 2008). The purified enzymes were characterized by MS after hydrolysis using trypsin. For purified cellulase I, four tryptic peptides were detected in the spectrum of MALDI-TOF/TOF-MS and their respective precursor ion masses and amino acid sequences are listed as follows: m/z 1,608.8163 (FVTKHEYGTNIGSR), 1,133.5414 (HEYGTNIGSR), 1,196.5951 (LSQFFVQDGR), and 1,324.7079 (LSQFFVQDGRK). For purified cellulase II, three tryptic peptides were detected: m/z 1,029.5544 (VIGATNWLR), 1,320.537 (YAMLDPHNFGR), and 1,505.6787 (GKYAMLDPHNFGR). The peptide mass fingerprint and amino acid sequencing of tryptic peptides showed that cellulases I and II share primary structure similarities with an exoglucanase from Humicola grisea var. thermoidea and an endoglucanase 3 from Humicola insolens, respectively. In both cases, the tryptic fragments analyzed covered about 5 % of total amino acid sequence from the identified enzymes. Total saccharide contents of purified cellulases I and II were about 11 and
Table 4 Substrate specificity of purified cellulases Substrates (2 %)
CMC Avicel Filter paper PNP-C Xylan Starch Cellobiose
Specific activity (U/mg of protein) Cellulase I
Cellulase II
9.3±1.9 3.2±0.4 0.05±0.007 ND ND ND ND
49.2±4.3 8.2±0.7 ND ND ND ND ND
10 mmol/L ß-mercaptoethanol
FPase was determined using a strip (10×30 mm) of Whatman No. 1 filter paper. Data are the means±SD of four different assays PNP-C 4-nitrophenyl-β-D-cellobioside, ND not detectable by the method in the assayed conditions
Cellulase II 187.3±16.9 19.8±2.0 12.2±1.3
93 %, respectively. High glycosylation levels are usually found in enzymes from thermophilic molds. The products of Avicel hydrolysis by cellulases I and II were analyzed by TLC (Fig. 3). Confirming the exo-hydrolytic character of cellulase I, only cellobiose was detected up to two hours (Fig. 3a). In contrast, the products of cellulase II action were mainly celloligosaccharides with longer chains than cellobiose (Fig. 3b), confirming its endo-hydrolytic character.
a
125 100
Relative Activity (%)
Data are the means±SD of four different assays
Parameter
75 50 25
0,0
0,5
1,0
1,5
2,0
2,5
2,0
2,5
Effector (%)
b Relative Activity (%)
Table 3 Kinetic parameters for the hydrolysis of CMC by purified cellulase I and II from Scytalidium thermophilum
80
60
40
20
0,0
0,5
1,0
1,5
Effector (%)
Fig. 6 Effect of glucose and cellobiose on cellulase activity of purified cellulases from S. thermophilum. The effect was analyzed by the methods of cellulose-azure hydrolysis (a) and medium viscosity reduction (b), as described in “Materials and methods.” One hundred percent activity (control) was estimated in the absence of the effectors, and for the viscosity reduction method, it corresponded to the specific viscosity of the reaction media containing thermo-inactivated crude filtrate. Data are means±SD of three different experiments (n=3). Cellulase I (circles), cellulase II (squares), glucose addition (solid symbols), and cellobiose addition (open symbols)
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Effects of temperature and pH on purified S. thermophilum cellulases activity and stability Optima of temperature and pH were 60–65 °C, pH 4.0 for cellulase I and 65 °C, pH 6.5 for cellulase II (Fig. 4). These data are in accordance with the literature, since most cellulases show optimum temperature in the range 50–75 °C and optimum pH of about 3.0–6.5 (Maheshwari et al. 2000; Jorgensen et al. 2003; Saha 2004; Jabbar et al. 2008). Both purified cellulases maintained total CMCase activity after 60 min when incubated in water at 60 °C (Fig. 5). At 70 °C, however, thermal inactivation kinetics showed very different results. Cellulase I exhibited a monophasic profile, with a half-life of 17.9 min. In contrast, cellulase II exhibited a biphasic profile and half-lives of 5.76 and 32.08 min were calculated for the fast and the slow phases, respectively. It is well accepted that biphasic thermoinactivation curves may result from the coexistence of different isoenzymes or reflect the ability of an enzyme to exist in more than a stable conformational state. Effect of potential inhibitors and activators, and substrate specificities of S. thermophilum purified cellulases Cysteine and sulphydryl reducing agents greatly stimulated CMCase activity of both S. thermophilum purified cellulases (Table 2), strongly suggesting that sulfhydryl groups are important for their catalytic action. Accordingly, Sandgren et al. (2003) have shown that three cysteines are important for the thermal stability of an endoglucanase from H. grisea, although they are not involved in disulfide bonds. Moreover, Yin et al. (2010) have also described a cysteine and βmercaptoethanol-activated endoglucanase produced by a Cellulomonas strain, although the activation by 10 mmol/L DTT reached a maximum of 32 % only. Several ions differently affected the activity of cellulases I and II (Table 2). At 10 mmol/L concentration, Mn2+ stimulated cellulases I and II by 207 and 151 %, respectively. Activation by Mn 2+ was also described for the endoglucanases purified from other fungi (Siddiqui et al. 1997; Tao et al. 2010; Jabbar et al. 2008) and apparently this metal ion enhances substrate-binding affinity. In contrast, 10 mmol/L Fe2+ and Al3+ were potent inhibitors of cellulase I, but inhibited cellulase II activity by only 30 and 23 %, respectively, while Cu2+ inhibited 45 and 95 % of cellulases I and II activity, respectively. These different responses to ions of the purified Scytalidium cellulases suggested that they are different enzymes, and thus their production may be physiologically meaningful for the fungus. The kinetic parameters for the hydrolysis of CMC by the purified enzymes were determined both in the absence and the presence of 10 mmol/L β-mercaptoethanol (Table 3). Interestingly, β-mercaptoethanol increased the apparent affinity and
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Vmax for CMC hydrolysis by both Scytalidium cellulases, resulting in increased catalytic efficiencies (Vmax/KM). To the best of our knowledge, this is the first report on fungal cellulases highly activated by β-mercaptoethanol, cysteine, and DTT. The substrate specificity analysis of purified cellulases I and II (Table 4) revealed high preference for CMC as substrate and undetectable hydrolytic activity on 4-nitrophenyl-β-Dcellobioside, xylan, starch and cellobiose. Both purified S. thermophilum cellulases showed excellent tolerance to the potential inhibitors glucose and cellobiose (Fig. 6). Using cellulose-azure as substrate, the activity of purified cellulase I was unaffected by cellobiose and glucose at concentrations up to 2.5 %, while cellulase II activity was inhibited by 20 % only in the presence of 2.5 % glucose or cellobiose (Fig. 6a). Moreover, CMC hydrolysis by cellulase II was increased up to 1.5-fold in the presence of glucose in the range 0.5–2.5 % (Fig. 6b). These data confirm the highly desirable and uncommon property of tolerance, and even activation, of S. thermophilum cellulases by cellulose saccharification products. Acknowledgments This work was supported by a grant from CNPq. J.C.R.S. received a Ph.D. scholarship from FAPESP, and M.L.T.M.P., R.P.M.F., and J.A.J. are research fellows of CNPq. This work was part of the Doctoral thesis of J.C.R.S (Dept. Bioquímica-FMRP-USP).
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