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Isolation of a cellulolytic enzyme from the mold Poria vaillantii
502
LETTERS
TO THE
EDITORS
In our laboratory, this is accomplished by suspending the paper strip in a 1000 ml. glass-stoppered graduated cylinder containing 50-100 ml. of concentrated hydrochloric acid. The paper strip is held in place with the glass stopper, slightly above the level of the hydrochloric acid. Upon exposure to the acid fumes for approximately 1530 sec., the flavanones are revealed as purple to magenta spots. Using this procedure it has been possible to demonstrate the position of hesperidin, hesperetin, naringin, naringenin, homoeriodictyol and neohesperidin on chromatograms. -4s little as 2 pg. of flavanone can be detected. In practice, 5-10 pg. of flavanone are applied to the paper strip. In this manner the flavanone constituents of crude materials have been located among the many fluorescent substances usually observed on chromatograms of such compounds. This reaction has been applied to spot plate and test tube testing to determine the presence of flavanones in solutions and extracts prior to chromatography on paper. The test is carried out by dissolving 1-2 mg. of crude or 0.1 mg. of pure test material in a small amount of suitable solvent, such as methanol, and adding approximately 10 mg. of solid sodium borohydride to the mixture. Dilute hydrochloric acid (1%) is added dropwise until no further release of hydrogen is observed. Addition of concentrated hydrochloric acid to the mixture causes the development of the typical violet ho magenta color. The method outlined above has been used successfully in our laboratories in the screening of natural products, for the detection of flavanone compounds on paper chromatograms, and for following the course of their isolation. Further details will be reported in a subsequent communication. ACKNOWLEDQMENT To Mr. A. J. Merritt
for reviewing
this note.
REFERENCES 1. 2. 3. 4.
BATE-SMITH, E. C., Nature 161, 835 (1948). BATE-SMITH, E. C., Biochem. Sot. Symposia, Cambridge, Engl. No. S 62 (1949). GAQE, T. B., DOUGLASS, C. D., AND WENDER, S. H., Anal. Chem. 23,1582 (1951). WILLSTKTTER, R., Ber. 47, 2874 (1914).
Analytical and Control Laboratories, U. S. Vitamin Corporation Yonkers, New York Received March 18, 1967 Isolation
of a Cellulolytic
EDWARD EICIEN MILTON BLITZ EPHRAIM GUNSBERG
Enzyme from the Mold Poria uaiZZanfiil
The isolation of chemically unchanged lignin from wood or bagasse requires the removal of the cellulose with which it is associated. The utilization of wooddestroying fungi for the isolation of large amounts of enzymically freed lignin (1) and for the elucidation of the mechanism of the biogenesis of lignin building stones (2-4) prompted an attempt at the isolation of a cellulolytic enzyme. Such a preparation has now been obtained from the filtrates of six-week-old cultures of Poria vaillantii. The medium employed for the growth of the organism was the same as that used previously in this laboratory (1).
1 Communication
No. 327.
LETTERS
TABLE
f’urijkation Enzyme Preparation
503
TO THE EDITORS I
EzperiwsenP Activity
Specific Activity
34.8 348.0 16.7 II 166.8 44.0 440.0 III a The activity represents gamma of Somogyi reducing sugar (calculated as glucose) produced by 1 ml. of a 1% enzyme solution acting on a 1% solution of carboxymethylcellulose substrate for 2 hr. at 40°C. The specific activity is the activity per unit n-eight. I
Twenty liters of the culture filtrate were concentrated to 3 1. by perevaporation. The concentrate was cooled to 2°C. and 680/, acetone was added. The precipitate was dissolved in distilled water, dialyzed overnight, and freeze dried (I). The supernatant was found to bc inactive. A 5% solution of (I) in 0.1 M acetate buffer (pH 5.3) was clarified by centrifugation. The insoluble residue was found to be inactive. To the clear supernatant was added 5070 acetone by volume and the precipitate (II) collected as before. Acetone was added to the supernatant to make it 63% acetone by volume. The precipitate was again collected as before (III). The fractionation was carried out at about -10°C. (see Table I). Electrophoretic patterns of 1% solutions of the enzyme preparations in 0.1 iii acctatc buffer at two pH’s are shown in Fig. 1. The three preparations consist mainly of two components, one of which is practically immobile. The patterns indicate that after a series of fraction&ions, the faster-moving component increases in concentration relative to that of the slowermoving component, as shown by the reversal in the heights of the two peaks. The two components were separated electrophoretically and their activities determined (Table II). These results indicate that the cellulolytic activity resides mainly in the fast component.
pH 5.3 I FIG. 1. Electrophoretic
III
II patterns
of enzyme
preparations
(I),
(II),
and (III).
501
LETTERS
TO THE
TABLE Eleclrophoretic Enzyme preparation
II Separation
Number of drops of solution withdrawn after electrophoresis and assayed
Component
I
EDITORS
Fast Slow Fast Slow Fast
II III
TABLE dcliou
Activity
11 11 17 17 10 10
Slow
of (III)
45.0 137.5 22.0 80.0 32.0 370.0
II1
on Various
Substratcsa
Substrate
Swollen lint.ers (5j Precipitated cotton cellulose (5) Precipitat,ed oak cellulose (5) Solka-floe (commercial wood cellulose)
Per cent hydrolysis
2.8 6.5 4.6 4.1
a Ten milliliters of 1% suspensions of each substrate were incubat,ed with 1 ml. of 1% (III) for 17 hr. at 40°C. The percentage hydrolysis was calculatjed from the increase in reducing sugar titre converted to glucose. The action of (III) on various substrates is recorded in Table III. These results indicate t,hat the enzyme preparation attacks both cotton and wood cellulose. Thus, we are able to obtain a cellulolytic enzyme preparation which consists mainly of two electrophoreticallp separable component,s. One of these is practically immobile at varying pH’s and may thus be a complex polysaccharide rather than a prot.ein. The cellulolytic act,ivit.y is definit.ely associated with the mobile component. ACKNOWLEDGMENTS
The original culture of Poria vaillanfii was received from Dr. William J. Robbins of the New York Botanical Garden. This investigation was supported in part by grants of the U. S. Public Health Service, the National Science Foundation and the U. S. Atomic Energy Commission. REFERENCES
1. SCHUBERT, W. J., AND NORD, F. F., J. Am. Chem. Sot. 73, 977, 3835 (1950). 2. EBERHARDT, G., AND NORD, F. F., Arch. Biochem. atid Biophys. 66, 578 (1955); EBERHARDT, G., J. -4,. Chem. Sot. 78. 2832 (1956). 3. EBERHARDT, G., AND SCHUBERT, W. J., J. Am. Chem. Sot. 78, 2835 (1956). 4. NORD, F. F., SCHUBERT, W. J., AND ACERBO, S. N., Naturwiss. 44, 35 (1957); J. Am. Chem. Sot. 79, 251 (1957). 5. SEALES, P. M., 2. Bacterial. und Parasitenkunde 44,661 (1916). DepartmerLt of Organic Ch.emistry Fordharn I.‘niuersity New York, New I’orlk Received April 16, 1957
and Enzymology,
BIENVENIDU C. SISON, JR. WALTER J. SCHUBERT F. F. NORD
LETTERS
TO THE
EDITORS
In our laboratory, this is accomplished by suspending the paper strip in a 1000 ml. glass-stoppered graduated cylinder containing 50-100 ml. of concentrated hydrochloric acid. The paper strip is held in place with the glass stopper, slightly above the level of the hydrochloric acid. Upon exposure to the acid fumes for approximately 1530 sec., the flavanones are revealed as purple to magenta spots. Using this procedure it has been possible to demonstrate the position of hesperidin, hesperetin, naringin, naringenin, homoeriodictyol and neohesperidin on chromatograms. -4s little as 2 pg. of flavanone can be detected. In practice, 5-10 pg. of flavanone are applied to the paper strip. In this manner the flavanone constituents of crude materials have been located among the many fluorescent substances usually observed on chromatograms of such compounds. This reaction has been applied to spot plate and test tube testing to determine the presence of flavanones in solutions and extracts prior to chromatography on paper. The test is carried out by dissolving 1-2 mg. of crude or 0.1 mg. of pure test material in a small amount of suitable solvent, such as methanol, and adding approximately 10 mg. of solid sodium borohydride to the mixture. Dilute hydrochloric acid (1%) is added dropwise until no further release of hydrogen is observed. Addition of concentrated hydrochloric acid to the mixture causes the development of the typical violet ho magenta color. The method outlined above has been used successfully in our laboratories in the screening of natural products, for the detection of flavanone compounds on paper chromatograms, and for following the course of their isolation. Further details will be reported in a subsequent communication. ACKNOWLEDQMENT To Mr. A. J. Merritt
for reviewing
this note.
REFERENCES 1. 2. 3. 4.
BATE-SMITH, E. C., Nature 161, 835 (1948). BATE-SMITH, E. C., Biochem. Sot. Symposia, Cambridge, Engl. No. S 62 (1949). GAQE, T. B., DOUGLASS, C. D., AND WENDER, S. H., Anal. Chem. 23,1582 (1951). WILLSTKTTER, R., Ber. 47, 2874 (1914).
Analytical and Control Laboratories, U. S. Vitamin Corporation Yonkers, New York Received March 18, 1967 Isolation
of a Cellulolytic
EDWARD EICIEN MILTON BLITZ EPHRAIM GUNSBERG
Enzyme from the Mold Poria uaiZZanfiil
The isolation of chemically unchanged lignin from wood or bagasse requires the removal of the cellulose with which it is associated. The utilization of wooddestroying fungi for the isolation of large amounts of enzymically freed lignin (1) and for the elucidation of the mechanism of the biogenesis of lignin building stones (2-4) prompted an attempt at the isolation of a cellulolytic enzyme. Such a preparation has now been obtained from the filtrates of six-week-old cultures of Poria vaillantii. The medium employed for the growth of the organism was the same as that used previously in this laboratory (1).
1 Communication
No. 327.
LETTERS
TABLE
f’urijkation Enzyme Preparation
503
TO THE EDITORS I
EzperiwsenP Activity
Specific Activity
34.8 348.0 16.7 II 166.8 44.0 440.0 III a The activity represents gamma of Somogyi reducing sugar (calculated as glucose) produced by 1 ml. of a 1% enzyme solution acting on a 1% solution of carboxymethylcellulose substrate for 2 hr. at 40°C. The specific activity is the activity per unit n-eight. I
Twenty liters of the culture filtrate were concentrated to 3 1. by perevaporation. The concentrate was cooled to 2°C. and 680/, acetone was added. The precipitate was dissolved in distilled water, dialyzed overnight, and freeze dried (I). The supernatant was found to bc inactive. A 5% solution of (I) in 0.1 M acetate buffer (pH 5.3) was clarified by centrifugation. The insoluble residue was found to be inactive. To the clear supernatant was added 5070 acetone by volume and the precipitate (II) collected as before. Acetone was added to the supernatant to make it 63% acetone by volume. The precipitate was again collected as before (III). The fractionation was carried out at about -10°C. (see Table I). Electrophoretic patterns of 1% solutions of the enzyme preparations in 0.1 iii acctatc buffer at two pH’s are shown in Fig. 1. The three preparations consist mainly of two components, one of which is practically immobile. The patterns indicate that after a series of fraction&ions, the faster-moving component increases in concentration relative to that of the slowermoving component, as shown by the reversal in the heights of the two peaks. The two components were separated electrophoretically and their activities determined (Table II). These results indicate that the cellulolytic activity resides mainly in the fast component.
pH 5.3 I FIG. 1. Electrophoretic
III
II patterns
of enzyme
preparations
(I),
(II),
and (III).
501
LETTERS
TO THE
TABLE Eleclrophoretic Enzyme preparation
II Separation
Number of drops of solution withdrawn after electrophoresis and assayed
Component
I
EDITORS
Fast Slow Fast Slow Fast
II III
TABLE dcliou
Activity
11 11 17 17 10 10
Slow
of (III)
45.0 137.5 22.0 80.0 32.0 370.0
II1
on Various
Substratcsa
Substrate
Swollen lint.ers (5j Precipitated cotton cellulose (5) Precipitat,ed oak cellulose (5) Solka-floe (commercial wood cellulose)
Per cent hydrolysis
2.8 6.5 4.6 4.1
a Ten milliliters of 1% suspensions of each substrate were incubat,ed with 1 ml. of 1% (III) for 17 hr. at 40°C. The percentage hydrolysis was calculatjed from the increase in reducing sugar titre converted to glucose. The action of (III) on various substrates is recorded in Table III. These results indicate t,hat the enzyme preparation attacks both cotton and wood cellulose. Thus, we are able to obtain a cellulolytic enzyme preparation which consists mainly of two electrophoreticallp separable component,s. One of these is practically immobile at varying pH’s and may thus be a complex polysaccharide rather than a prot.ein. The cellulolytic act,ivit.y is definit.ely associated with the mobile component. ACKNOWLEDGMENTS
The original culture of Poria vaillanfii was received from Dr. William J. Robbins of the New York Botanical Garden. This investigation was supported in part by grants of the U. S. Public Health Service, the National Science Foundation and the U. S. Atomic Energy Commission. REFERENCES
1. SCHUBERT, W. J., AND NORD, F. F., J. Am. Chem. Sot. 73, 977, 3835 (1950). 2. EBERHARDT, G., AND NORD, F. F., Arch. Biochem. atid Biophys. 66, 578 (1955); EBERHARDT, G., J. -4,. Chem. Sot. 78. 2832 (1956). 3. EBERHARDT, G., AND SCHUBERT, W. J., J. Am. Chem. Sot. 78, 2835 (1956). 4. NORD, F. F., SCHUBERT, W. J., AND ACERBO, S. N., Naturwiss. 44, 35 (1957); J. Am. Chem. Sot. 79, 251 (1957). 5. SEALES, P. M., 2. Bacterial. und Parasitenkunde 44,661 (1916). DepartmerLt of Organic Ch.emistry Fordharn I.‘niuersity New York, New I’orlk Received April 16, 1957
and Enzymology,
BIENVENIDU C. SISON, JR. WALTER J. SCHUBERT F. F. NORD