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Nephelometric and turbidometric assays of cellulase activity
ANALYTICAL
BIOCHEMISTRY
Nephelometric MARTTI Technical
Research
116,
1333136
and NUMMI, Cenire
(1981)
Turbidometric
Assays
PAUL C. Fox, MARJA-LEENA AND TOR-MAGNUS ENARI of Finland,
Biotechnical
Received
March
Labomtory,
of Cellulase
Activity
NIKU-PAAVOLA, SF-02150
Espoo
IS, Finland
12, 198 I
Extensively ball-milled cellulose fibers were used as natural substrate for the determination of cellulase activity. This physical treatment breaks the large cellulose fibers to small but insoluble particles yielding a substrate accessible for complete enzymatic breakdown. The parameters studied to estimate the actilvity of cellulases were (a) the decrease in optical density of ball-milled suspension of fibers and (b) simultaneous measurement of liberated sugars during hydrolysis. A good correlation was found between the initial rate of reaction and the amount of sugar released at given times.
A number of different methods have been used for the measurement of cellulolytic activity ( 1). One principle commonly employed to evaluate the effectiveness of cellulolytic enzyme preparations is to determine the extent of hydrolysis in terms of the products found at the end point of the reaction. It is known that cellulolytic enzymes do not hydrolyze efficiently well-ordered areas of cellulose, but attack only amorphous regions. Thus, methods using extended hydrolysis times in fact measure the degree of order in the substrate used. Alternatively, the initial rate of hydrolysis of substituted cellulose substrates as measured by viscosity provides a different estimate of cellulose activity (2). In the method presented here a more refined amorphous substrate has been prepared by ball milling. This substrate is completely dissolved by cellulases and can be used to measure the initial velocity of the reaction. Initial activity is followed by means of a nephelometer and the slopesof nephelometric plots are used to calculate the cellulase activity. The initial activity can also be estimated with a photometer where the activity is measured as the decrease in absorbance of suspensionsof amorphous cellulose. 133
MATERIALS
AND METHODS
Amorphous cellulose. The substrate for hydrolysis was prepared by milling Whatman CFl 1 cellulose powder in 94% ethanol solution at 20°C for 24 to 72 h with a ball mill (Schwingmtihle Vibrator, Siebtechnik GmBH, Miilheim) using steel balls (3). The substrat.e obtained was amorphous according to the index of order value (4). To obtain a homogeneous substrate for stable suspensions, which is necessary in a nephelometer, cellulose was further filtered through frittedglassfilters (Schott Nos. 1 and 2) and a fraction with fine particles (diameter 90-l 50 pm) was collected. The iron dust released from the steel balls during the milling was removed by a magnetic stirring rod during short stirring in citrate buffer. The amorphous cellulose formed a stable suspension in citrate buffer pH 5.0. No settling or aggregation occurred within a few days as shown by constancy of nephelometer readings and reproducibility of cellulase assays. Enzyme preparation. Trichoderma reesei strain VTT-D-78085 was used to produce the cellulases (3) which were precipitated with ammonium sulfate between 20 and 0003-2697/Sl/l30133-04$02.00/O Copyright io 1981 by Academic Press. Inc. All rights of reproduction tn any form reserved.
134
NUMMI
ET AL.
FIG. 2. Dependence centrations. FIG. 1. Nephelometric plots for different enzyme concentrations. The zero line (0) represents the light reflection of cellulose suspensions alone. The arrows indicate the moment of enzyme addition. The enzyme concentrations expressed as pg protein/ml reaction mixture are marked in the respective places. The calculation used to determine slopes is shown.
40% saturation. The precipitate was dissolved and dialyzed against a 0.05 M citrate buffer pH 5.0. The protein concentration in the preparation was 130 mg/ml by the Biuret method (5). Sugar estimation. The carbohydrate contents of cellulose solutions were estimated with anthron-sulfuric acid (6). Glucose was used as standard. The reducing sugars liberated during hydrolysis were estimated as glucose by the dinitrosalicylic acid method (7). Nephelometric measurement. Nephelometric estimations were performed with Perkin-Elmer amylase-lipase analyzer Model 9 1. The temperature of the incubation chamber was 40°C. The apparatus measured the light reflected at a right angle to the axis of the exciting light. Turbidity was measured as the absorbance at 620 nm. NEPHELOMETRIC CELLULASE ACTIVITY
between
slopes and enzyme
con-
0.03% calculated from total sugar estimation and it showed a nephelometric value of 80. Figure 1 presents the plots for different enzyme dilutions. The slopesare calculated for the initial velocity from the linear part of the nephelometric trace as demonstrated in the picture. It was observed that if the substrate was completely hydrolyzed during a shorter period than 5 min the enzyme concentration was too high for measurement of the initial velocity of the reaction. When the enzyme concentrations are such that 10 to 20% of the substrate is hydrolyzed during 5 min the slopes represent the initial velocity, i.e., the slopes are linearly proportional to the enzyme concentration (Fig. 2). In prolonged hydrolysis, however, the amorphous substrate should be completely (95%) dissolved. The unit of activity is defined as the decrease in nephelometric value per minute. The dependence of the nephelometric value of cellulose concentration is given in Fig. 3.
METHOD FOR DETERMINATION
Samples of 20 ~1 of appropriately diluted enzyme solutions are incubated with 3 ml cellulose suspensionin 0.05 M citrate buffer pH 5.0. The concentration of cellulose is
FIG. 3. Relationship between (A), absorbance at 620 nm (o), of cellulose.
nephelometric values and the concentration
CELLULASE
activity reducing
sugars
released
ACTIVITY
and
in the hydrolysis.
Cellulase activity as measured by the nephelometric method and the amount of reducing sugars formed in 30 min were determined for a range of enzyme concentrations (Fig. 4). Both determinations give similar estimates of cellulase activity. The cellulase activity measured by the nephelometric method can thus be converted directly to katals using this diagram. TURBIDOMETRIC METHOD FOR ESTIMATION OF CELLULOLYTIC ACTIVITY The cellulase activity as the initial velocity of the hydrolysis can also be determined with a photometer by measuring the change in absorbance at 620 nm. Enzyme solution (200 yl) is incubated
135
ASSAYS
with 3 ml cellulose suspension (0.0470, AezO = 0.400) in 0.05 M citrate buffer pH 5.0 at 50°C for 10 min. This time was short enough to measure the initial activity for a broad range of enzyme concentrations and at the same tirne the shortest one to make the measurement of a series nf samples possible. The absorbance of the solutions is measured after exactly 10 min against a citrate buffer blank. The unit of activity is defined as the change in absorbance per minute under the reaction conditions used. The concentration of the cellulose was estimated as the amount of total sugars (Fig. 3) because the cellulose amounts concerned are beyond the range of gravimetric analyses. The amount of cellulose hydrolyzed when the absorbance is decreased by 0.1 is 100 Fg,/ml according to Fig. 3. This amount of cellulose corresponds to the change of 20 nephelometric units. To test the effect of enzyme concentration activity determinations were made with a series of enzyme dilutions with protein concentrations in the reaction mixture ranging from 8.0 to 160 pg/ml (Fig. 5). A linear relationship was obtained until the enzyme concentration containing 80 PLg protein/ml. With higher enzyme concentrations the substrate was already hydrolyzed completely during the 10 min used for activity determination. To determine higher activities the enzyme solution must be diluted. The nephelometric and turbidometric assays described here measure the initial activity in hydrolysis of cellulose. Thus, they are well suited for the determination of cellulase activities. ACKNOWLEDGMENTS Thanks are due to Mrs. Kristina Eravuo, M. SC., The Finnish Pulp and Paper Research Institute, for milling of cellulose. T.-M.E. is a research professor of the Academy of Finland.
REFERENCES FIG. enzyme
5. Relationship concentration
between cellulase activity in turbidometric assay.
and
1. Enari, T. -M.. vances in
and Markkanen, P. (1977) Biochemical Engineering
in Ad(Ghose,
136
NUMMI
T. K.. Fiechter, Vol. 5, pp. I-24, 2. Almin,
K. E., and
Biophys.
Acta
A., and Springer.
Blakebrough, Berlin.
Eriksson, K. -E. 130, 238-247.
N.,
eds).
ET
5. Bergmcqer, Michal.
M.. Niku-Paavola. Raunio, V. (1980)
M. -1..
FEBS
Enari,
Letr.
T. -M.. 113, 164-
166. 4. Visapa& 582.
Faserforsch.
Te.xtilrech.
5. 579-
Whistler.
R. 1.. and
Bernfeld. P. (Colowick. I. p. 149.
Bcrnt, in
(Bergmeyer. Academic
ods of Carbohydrate Academic Press, 7.
A. (1964)
11. U.. G. (1974)
Analysis 174.- 176,
( 1967) Biochim. 6.
3. Nummi. and
AL.
H. Press,
M. L. (1962)
Chemistry, New York. Methods Kaplan, Press,
K.. and Enzymatic
U., ed.). Vol. New York.
Wolfrom.
(1955) in S. P., and Academic
E.. Gawehn. Methods of
Vol. in N.
New
1, pp. Meth-
I, p. 390,
Enzymology 0.. eds). Vol.
York.
BIOCHEMISTRY
Nephelometric MARTTI Technical
Research
116,
1333136
and NUMMI, Cenire
(1981)
Turbidometric
Assays
PAUL C. Fox, MARJA-LEENA AND TOR-MAGNUS ENARI of Finland,
Biotechnical
Received
March
Labomtory,
of Cellulase
Activity
NIKU-PAAVOLA, SF-02150
Espoo
IS, Finland
12, 198 I
Extensively ball-milled cellulose fibers were used as natural substrate for the determination of cellulase activity. This physical treatment breaks the large cellulose fibers to small but insoluble particles yielding a substrate accessible for complete enzymatic breakdown. The parameters studied to estimate the actilvity of cellulases were (a) the decrease in optical density of ball-milled suspension of fibers and (b) simultaneous measurement of liberated sugars during hydrolysis. A good correlation was found between the initial rate of reaction and the amount of sugar released at given times.
A number of different methods have been used for the measurement of cellulolytic activity ( 1). One principle commonly employed to evaluate the effectiveness of cellulolytic enzyme preparations is to determine the extent of hydrolysis in terms of the products found at the end point of the reaction. It is known that cellulolytic enzymes do not hydrolyze efficiently well-ordered areas of cellulose, but attack only amorphous regions. Thus, methods using extended hydrolysis times in fact measure the degree of order in the substrate used. Alternatively, the initial rate of hydrolysis of substituted cellulose substrates as measured by viscosity provides a different estimate of cellulose activity (2). In the method presented here a more refined amorphous substrate has been prepared by ball milling. This substrate is completely dissolved by cellulases and can be used to measure the initial velocity of the reaction. Initial activity is followed by means of a nephelometer and the slopesof nephelometric plots are used to calculate the cellulase activity. The initial activity can also be estimated with a photometer where the activity is measured as the decrease in absorbance of suspensionsof amorphous cellulose. 133
MATERIALS
AND METHODS
Amorphous cellulose. The substrate for hydrolysis was prepared by milling Whatman CFl 1 cellulose powder in 94% ethanol solution at 20°C for 24 to 72 h with a ball mill (Schwingmtihle Vibrator, Siebtechnik GmBH, Miilheim) using steel balls (3). The substrat.e obtained was amorphous according to the index of order value (4). To obtain a homogeneous substrate for stable suspensions, which is necessary in a nephelometer, cellulose was further filtered through frittedglassfilters (Schott Nos. 1 and 2) and a fraction with fine particles (diameter 90-l 50 pm) was collected. The iron dust released from the steel balls during the milling was removed by a magnetic stirring rod during short stirring in citrate buffer. The amorphous cellulose formed a stable suspension in citrate buffer pH 5.0. No settling or aggregation occurred within a few days as shown by constancy of nephelometer readings and reproducibility of cellulase assays. Enzyme preparation. Trichoderma reesei strain VTT-D-78085 was used to produce the cellulases (3) which were precipitated with ammonium sulfate between 20 and 0003-2697/Sl/l30133-04$02.00/O Copyright io 1981 by Academic Press. Inc. All rights of reproduction tn any form reserved.
134
NUMMI
ET AL.
FIG. 2. Dependence centrations. FIG. 1. Nephelometric plots for different enzyme concentrations. The zero line (0) represents the light reflection of cellulose suspensions alone. The arrows indicate the moment of enzyme addition. The enzyme concentrations expressed as pg protein/ml reaction mixture are marked in the respective places. The calculation used to determine slopes is shown.
40% saturation. The precipitate was dissolved and dialyzed against a 0.05 M citrate buffer pH 5.0. The protein concentration in the preparation was 130 mg/ml by the Biuret method (5). Sugar estimation. The carbohydrate contents of cellulose solutions were estimated with anthron-sulfuric acid (6). Glucose was used as standard. The reducing sugars liberated during hydrolysis were estimated as glucose by the dinitrosalicylic acid method (7). Nephelometric measurement. Nephelometric estimations were performed with Perkin-Elmer amylase-lipase analyzer Model 9 1. The temperature of the incubation chamber was 40°C. The apparatus measured the light reflected at a right angle to the axis of the exciting light. Turbidity was measured as the absorbance at 620 nm. NEPHELOMETRIC CELLULASE ACTIVITY
between
slopes and enzyme
con-
0.03% calculated from total sugar estimation and it showed a nephelometric value of 80. Figure 1 presents the plots for different enzyme dilutions. The slopesare calculated for the initial velocity from the linear part of the nephelometric trace as demonstrated in the picture. It was observed that if the substrate was completely hydrolyzed during a shorter period than 5 min the enzyme concentration was too high for measurement of the initial velocity of the reaction. When the enzyme concentrations are such that 10 to 20% of the substrate is hydrolyzed during 5 min the slopes represent the initial velocity, i.e., the slopes are linearly proportional to the enzyme concentration (Fig. 2). In prolonged hydrolysis, however, the amorphous substrate should be completely (95%) dissolved. The unit of activity is defined as the decrease in nephelometric value per minute. The dependence of the nephelometric value of cellulose concentration is given in Fig. 3.
METHOD FOR DETERMINATION
Samples of 20 ~1 of appropriately diluted enzyme solutions are incubated with 3 ml cellulose suspensionin 0.05 M citrate buffer pH 5.0. The concentration of cellulose is
FIG. 3. Relationship between (A), absorbance at 620 nm (o), of cellulose.
nephelometric values and the concentration
CELLULASE
activity reducing
sugars
released
ACTIVITY
and
in the hydrolysis.
Cellulase activity as measured by the nephelometric method and the amount of reducing sugars formed in 30 min were determined for a range of enzyme concentrations (Fig. 4). Both determinations give similar estimates of cellulase activity. The cellulase activity measured by the nephelometric method can thus be converted directly to katals using this diagram. TURBIDOMETRIC METHOD FOR ESTIMATION OF CELLULOLYTIC ACTIVITY The cellulase activity as the initial velocity of the hydrolysis can also be determined with a photometer by measuring the change in absorbance at 620 nm. Enzyme solution (200 yl) is incubated
135
ASSAYS
with 3 ml cellulose suspension (0.0470, AezO = 0.400) in 0.05 M citrate buffer pH 5.0 at 50°C for 10 min. This time was short enough to measure the initial activity for a broad range of enzyme concentrations and at the same tirne the shortest one to make the measurement of a series nf samples possible. The absorbance of the solutions is measured after exactly 10 min against a citrate buffer blank. The unit of activity is defined as the change in absorbance per minute under the reaction conditions used. The concentration of the cellulose was estimated as the amount of total sugars (Fig. 3) because the cellulose amounts concerned are beyond the range of gravimetric analyses. The amount of cellulose hydrolyzed when the absorbance is decreased by 0.1 is 100 Fg,/ml according to Fig. 3. This amount of cellulose corresponds to the change of 20 nephelometric units. To test the effect of enzyme concentration activity determinations were made with a series of enzyme dilutions with protein concentrations in the reaction mixture ranging from 8.0 to 160 pg/ml (Fig. 5). A linear relationship was obtained until the enzyme concentration containing 80 PLg protein/ml. With higher enzyme concentrations the substrate was already hydrolyzed completely during the 10 min used for activity determination. To determine higher activities the enzyme solution must be diluted. The nephelometric and turbidometric assays described here measure the initial activity in hydrolysis of cellulose. Thus, they are well suited for the determination of cellulase activities. ACKNOWLEDGMENTS Thanks are due to Mrs. Kristina Eravuo, M. SC., The Finnish Pulp and Paper Research Institute, for milling of cellulose. T.-M.E. is a research professor of the Academy of Finland.
REFERENCES FIG. enzyme
5. Relationship concentration
between cellulase activity in turbidometric assay.
and
1. Enari, T. -M.. vances in
and Markkanen, P. (1977) Biochemical Engineering
in Ad(Ghose,
136
NUMMI
T. K.. Fiechter, Vol. 5, pp. I-24, 2. Almin,
K. E., and
Biophys.
Acta
A., and Springer.
Blakebrough, Berlin.
Eriksson, K. -E. 130, 238-247.
N.,
eds).
ET
5. Bergmcqer, Michal.
M.. Niku-Paavola. Raunio, V. (1980)
M. -1..
FEBS
Enari,
Letr.
T. -M.. 113, 164-
166. 4. Visapa& 582.
Faserforsch.
Te.xtilrech.
5. 579-
Whistler.
R. 1.. and
Bernfeld. P. (Colowick. I. p. 149.
Bcrnt, in
(Bergmeyer. Academic
ods of Carbohydrate Academic Press, 7.
A. (1964)
11. U.. G. (1974)
Analysis 174.- 176,
( 1967) Biochim. 6.
3. Nummi. and
AL.
H. Press,
M. L. (1962)
Chemistry, New York. Methods Kaplan, Press,
K.. and Enzymatic
U., ed.). Vol. New York.
Wolfrom.
(1955) in S. P., and Academic
E.. Gawehn. Methods of
Vol. in N.
New
1, pp. Meth-
I, p. 390,
Enzymology 0.. eds). Vol.
York.