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Phosphatase and pyrophosphatase activities of Aspergilli
ARCHIVES
Phosphatase
OF
BIOCHEMISTRY
AND
BIOPHYSICS
and Pyrophosphatase
69, 366-372 (1966)
Activities
of Aspergilli
M. A. Pathak and A. Sreenivasan From the Department
of Chemical Technology,
University
of Bombay,
India
Received April 11, 1955 INTRODUCTION
The presence of phosphatases in mold mycelia has been known (l-4) Their exact metabolic significance is not however well understood. Or the basis of observations that phosphatase activity varies widely during the life of the organism and as a result of cultural modifications, it ha2 been assumed that phosphatases, along with other enzymes, play a definite role in mold metabolism and are reponsible for or associated with the altered biochemical performance of the organism with changes in environmental conditions (2, 5). The present report relates to studies with two amylase-elaborating strains of aspergilli and it is shown that, under conditions of maximum amylase synthesis, there is also an increase in oxidative metabolism and in phosphatase activities. Preliminary observations on the properties of the phosphatase are also included. MATERIALS
AND METHODS
The organisms employed were a strain of Aspergillus oryzae (National Cal. lection of Type Cultures, Bangalore, India, No. 653) grown as a surface culture (6) and another of Aspergillus niger (NRRL, No. 337) cultivated under submerged conditions (7). The organisms were grown on either of the following media: (a) glucose-Czapek-Dox medium (8); (b) a corn steep liquor medium composed of (percentages) corn steep liquor 2, glucose 1, and calcium carbonate 0.26; and (c) a sprouted mung bean (Phaseolus radiatus) extract medium consisting of (percentages) 4%hr. mung bean seedling extract 2, and glucose 1. In other work (unpublished), the superiority of corn steep liquor for amylase elaboration by both organisms was established; it was also observed that mung bean seedling extract could substitute for corn steep liquor, best results being obtained with a-day-old seedlings. Pregerminated spore suspensions of the organisms, maintained on Czapek-Dox agar slants, were used to inoculate 5Oml. lots of sterilized (at 15 lb. steam pressure for 15-20 min.) media contained in 250-ml. conical flasks which were then incubated at 30°, those with A. niger on a reciprocating shaker (50 cycles/min. with a stroke length of 3 in.).
ACTIVITIES
OF ASPERGILLI
367
The mycelia were harvested after growth for 72 hr., washed free of themedium, pressed between folds of filter paper, and ground to a fine paste in a glass mortar. Acetone powders were obtained by treatment of the ground mycelia with successive small amounts of chilled acetone and removal of excess acetone under reduced pressure inside a vacuum desiccator. Phosphatase and pyrophosphatase activities were determined in terms of inorganic phosphate liberated from sodium fi-glycerophosphate (5 ml. of a 5% solution) and sodium pyrophosphate (5 ml. of 0.1% solution), respectively, by 10 mg. of the acetone-dried powder suspended in 5 ml. of 0.2 M acetate buffer, pH 4.8, unless otherwise stated, when incubated under toluene for 3 hr. at 30”. Reaction was stopped by adding 1 ml. of 1 N NaOH. Inorganic phosphorus was determined according to Fiske and SubbaRow (9). Necessary control sets were kept for enzyme and substrate blanks. Saccharogenic activity was determined in toluenated water extracts of the ground mycelia by the procedure outlined earlier (6) and expressed as total activity in terms of Lintner units (10) per gram of dry mycelial growth in 50 ml. of growth medium. Oxygen-uptake measurements were carried out in single side-arm Warburg flasks using homogenized suspensions of the ground mycelia as enzyme source. The main chamber of each flask contained 1 ml. of 0.05 M phosphate buffer, pH 6.8, and 1 ml. of mycelial cell suspension equivalent to about 10 mg. dry wt.; the side arm had 1 ml. of a solution (0.02 M) of the appropriate substrate: glucose, sodium malate, sodium succinate, sodium pyruvate, or sodium citrate; and, to the center well was added 0.2 ml. of 10% KOH solution. The total flask volume was adjusted to 3.2 ml. in all cases. The flasks were shaken at 120 oscillations/min. and the temperature was kept at 37”. After 10 min. equilibration, the substrate from the side arm was tipped in and the reaction continued for 1 hr. during which period oxygen-uptake data were recorded at IO-min. intervals. Necessary controls were run alongside. All final data were corrected for endogenous respiration and expressed as &oz or microliters oxygen/g. mycelia/hr. In the Warburg experiments, the values for Qop with freshly harvested cells were low due to high endogenous oxygen uptake. Attempts were made to reduce endogenous metabolism. Acetone-dried mycelial preparations were not very active, and dialysis in the cold or washing with normal physiological saline was likewise unsatisfactory. Aging to remove endogenous substrates was found to result in considerably reduced endogenous uptake values. The harvested cells were washed free of culture filtrate by twice centrifuging with chilled distilled water and suspended in 0.9% saline overnight at 5”; after 4-6 hr. of further aging at room temperature, they were washed twice with water, homogenized to a fine suspension, and made to volume. RESULTS
pH Optimum for Phosphatase Action Using acetate buffer (0.2 M) for the pH range 4.0-5.8 and Verona1 buffer (0.2 M) for pH 6.8-10.0, it was observed (Table I) that, with
368
M.
A.
PATHAK
Optimum pH
AND
A.
SREENIVASAN
TABLE I for Phosphatuse Action
pH of incubation mixture
4.0 4.8 5.8 6.8 7.0 7.5 8.0 8.4 9.0 10.0
8 10 8 7 2 2 2 Nil Nil Nil
-I
33 44 22 18 18 10 6 6 Nil Nil
30 41 21 22 18 8 10 6 Nil Nil
L
25 30 22 16 12 10 8 5 Nil Nil
both A. niger and A. oryzue, the pH optimum for phosphatase action on sodium fl-glycerophosphate was near about 4.8 irrespective of the composition of the medium. Activity with Dijerent
Substrates
The enzyme was active against the phosphate esters tried (Table II). The results are given in comparison with the values obtained for glycerophosphate, all at pH 4.8 and in the concentrations indicated against each substrate. Amylase Activity
and Oxidative Metabolism oj Mold Mycelia
A comparison of the changes in phosphatase activities in the different media (Tables I and II) with the corresponding activities for amylase (Table III) revealed a parallelism between the two enzymes; thus, under cultural conditions most favorable for amylase synthesis, there was also more activity for phosphatase. Similar enhanced rates of oxidation of substrates by A. niger and A. oryzue were observable under conditions of culture which result in increased phosphatase activities (Table IV). The effect of corn steep liquor or sprouted mung bean extract supple-
ACTIVITIES
369
OF ASPERGILLI
TABLE
II
Phosphatase Action 072Different Substrates A. ni;m grown on
A.orysacgrownon
I
Substrate
Phosphatase activity: micrograms inorg. P liberated by 10 mg. enzyme preparation in 3 hr.
-
Sodium @-glycerophosphate (50 mg./ml.) Adenylic a&d, yeast (1 mg./W Hexose diphosphate (1 mg./ml:) Ribosenucleic acid depolymerized (Schwarz Lab.) (1 mg./mU Deoxyribonucleic acid depolymerized (Schwarz Lab.) (1 mg./ml.)
-T-
10
51
41
20
40
40
20
36
32
22
30
26
24
56
40
18
44
42
20
32
32
20
28
26
26
51
51
26
40
30
TABLE
III
Effect of Composition of Medium WJ Amylase Activity
Amylase activity
A. niger A. oryzae
195 196
in Lintner units at 40’ per half hour in 50 ml. medium (per gram dry cells)
552 465
521 400
is apparently due to an over-all stimulation of cell activities; protein concentrations in the mycelia were not determined under the different cultural conditions, this stimulation is evident from the increased growth of the organisms on these media as shown by their ,nycelial dry weight (Table IV). Activities in general were of a higher order with A. niger under submerged conditions than in the surfacegrowing A. oryzue cultures. mentation
although
370
M.
A.
PATHAK
AND
,4. SREENIVASAN
TABLE IV Oxidation
of Substrates
by Enzymes
of
A. niger and A. oryzae
Substrate
Qor microliters
Glucose (0.02M) Sodium malate (0.02 1M) Sodium succinate (0.02 M) Sodium pyruvate (0.02 M) Sodium citrate (0.02 M) Dry wt. of cells, g., in 50 ml. of medium
2600 2570 2180 1030 1360 0.113
3940 3600 3470 1330 1570 0.259
oxygen/g. mycelia/hr
3760 3320 3440 1460 1560 0.238
1920 1660 1820 920 1100 0.106
2600 2200 2630 1460 1360 -~ 0.201
2350 2ooo 1860 1100 1200 0.170
Nature oj the Phosphatase It was observed that treatment with cyanide at a concentration of 0.002 M for 30 min. inactivated the phosphatase preparation from A. niger. This suggested the possible association of the enzyme with a metal ion. Regeneration of activity on addition of various cations after cyanide inactivation was therefore attempted. A weighed quantity ( 2 g.) of acetone-dried mycelial preparation of A. niger was extracted with toluenated water (250 ml.) for 24 hr. in the cold (5”). The enzyme was precipitated from the extract by addition of an equal volume of acetone. The precipitate was centrifuged, resuspended in distilled water (25 ml.), and dialyzed in the cold for 24 hr. against several changes of distilled water. A l-ml. aliquot of the dialyzed enzyme solution was treated with cyanide (0.002 M final concentration), and several portions of similarly inactivated preparation were supplemented with different salt solutions. Activities were determined using sodium fi-glycerophosphate as substrate in a total volume of 10 ml. made up with acetate buffer. Some of the results are given in Table V. It was observed that addition of Zn+ could regenerate activky of cyanide-inactivated enzyme to a considerable extent. Other cations tried, viz., Co++, Mgtt , Ca++, Fe++, and Mn++ had no effect. Mere dilution of the cyanide-treated enzyme solution did not appreciably alter the extent of inactivation. The reversal of cyanide inactivation of phos-
ACTIVITIES
371
OF ASPERGILLI
TABLE V Regeneration of Cyanide-Inactivated
Enzyme Phosphatase activity: inorg. P liberated enzyme solution
Treatment
1. Enzyme solution (dialyzed) 2. As in 1, heated 3. As in 1, inactivated with cyanide (0.002M) 4. As in J, plus Z&l01 soln. (0.008M) 5. As in 8, plus ZnSOd soln. (0.004M) 6. As in 8, plus ZnSOdsoln. (0.002M)
micrograms by 1 ml. of in 3 hr.
99 9 23 56 84 82
TABLE VI of A. niger and A. oryzae micrograms inorganic P liberated by 10 mg. of
Pyrophosphatase Activity
Pyrophosphataze activity: enzyme preparation in 3 hr.
Sprouted extract
Organism
A. niqer A. oryzae
128 64
’ 205 164
mung bean medium
164 144
phatase activity by Zn++ therefore points to the possible involvement of the latter in the enzyme make-up. Pyrophosphatuse of A. niger and A. oryzae The existence of pyrophosphatases of different pH optima is known (4). It was observed in preliminary experiments that the pyrophosphatase of both A. niger and A. oryzae had a pH optimum between 4 and 5. The activities of the mycelia of the two organisms grown in t,he three media were compared (Table VI), and it was observed that as with amylase and phosphatase activities, pyrophosphatase activities of mycelia harvested from the corn steep liquor and the sprouted mung beanextract media were much higher than that from the glucose-CzapekDox medium. Pyrophosphatase was also inactivated by cyanide and reactivated by Zn++. DISCUSSION
The phosphatase in the aspergilli is similar to the phosphatase of Penicillum chrysogenum (2) in its property of *inactivation by cyanide and reactivation by Zn*. However, unlike the latter, it is an acid phosphatase. It has limited substrate specificity. Whether the same enzyme exhibits both phosphatase and pyrophosphatase activities has not been studied.
372
M.
A.
PATHAK
AND
A.
SREENIVASAN
Studies on the changes in the elaboration of amylases by the two organisms and in their oxidative metabolism show a parallelism with phosphatase activity changes and it seems likely therefore that, phosphatases in molds are more than mere structural const,ituents and have physiological significance. ACKNOWLEDGMENT This work was assisted by a research grant from the Bombay State Industrial Research Committee of the Department of Industries, Government of Bombay. SUMMARY
Phosphatase and pyrophosphatase of amylase-elaborating Aspergillus niger (submerged growth) and Aspergillus oryzae (surface culture) have a pH optimum of 4.8, no activity being observable at alkaline pH range. With increase in amylase production in a medium like corn steep liquor or sprouted mung bean extract, there is a corresponding increase in phosphatase and, more particularly, pyrophosphatase activities with both organisms as well as in the oxidation of various substrates. Phosphatase activity is inactivated by cyanide and could be regenerated by addition of Zn* but not by Co++, Mg-++, Ca++, Mn*, or Fe++. REFERENCES 1. MANN
T.,
2. SADASIVAN, 3. SADASIVAN, 4. KRISHNAN, 5. KRISENAN, 6. RAO, R. 8.
Biochem.
J. 38, 339 (1944).
V., Arch. Biochem. 28, 100 (1950). V., Arch. Biochem. and Biophys. 87, 172 (1952). P. S., Arch. Biochem. and Biophys. 89,230 (1951). P. S., AND BAJAJ, V., Arch. Biochem. and Biophys. 47.39 (1953). AND SREENIVASAN, A. Trans. Am. Assoc. Cereal Chemists 8, 46
J.,
(1950). 7. ADAMS,
S. L.,
BALANKURA,
B.,
ANDREASF.N,
Eng. Chem. 29.1615 (1947). 8. SMITE, G., “An Introduction to Industrial
A. A.,
AND STARK,
Mycology,”
W. H., Ind.
2nd ed., p. 172. Ed-
ward Arnold and Co., Ltd., London, 1942. 9. FISKE, C., AND SUBBAROW, Y., J. Biol. Chem. 81,629 (1929). AND STEELE, H. K., Ind. Eng. Chem., Anal. Ed. 7, 324 (1935).
10. GORE, H. C.,
Phosphatase
OF
BIOCHEMISTRY
AND
BIOPHYSICS
and Pyrophosphatase
69, 366-372 (1966)
Activities
of Aspergilli
M. A. Pathak and A. Sreenivasan From the Department
of Chemical Technology,
University
of Bombay,
India
Received April 11, 1955 INTRODUCTION
The presence of phosphatases in mold mycelia has been known (l-4) Their exact metabolic significance is not however well understood. Or the basis of observations that phosphatase activity varies widely during the life of the organism and as a result of cultural modifications, it ha2 been assumed that phosphatases, along with other enzymes, play a definite role in mold metabolism and are reponsible for or associated with the altered biochemical performance of the organism with changes in environmental conditions (2, 5). The present report relates to studies with two amylase-elaborating strains of aspergilli and it is shown that, under conditions of maximum amylase synthesis, there is also an increase in oxidative metabolism and in phosphatase activities. Preliminary observations on the properties of the phosphatase are also included. MATERIALS
AND METHODS
The organisms employed were a strain of Aspergillus oryzae (National Cal. lection of Type Cultures, Bangalore, India, No. 653) grown as a surface culture (6) and another of Aspergillus niger (NRRL, No. 337) cultivated under submerged conditions (7). The organisms were grown on either of the following media: (a) glucose-Czapek-Dox medium (8); (b) a corn steep liquor medium composed of (percentages) corn steep liquor 2, glucose 1, and calcium carbonate 0.26; and (c) a sprouted mung bean (Phaseolus radiatus) extract medium consisting of (percentages) 4%hr. mung bean seedling extract 2, and glucose 1. In other work (unpublished), the superiority of corn steep liquor for amylase elaboration by both organisms was established; it was also observed that mung bean seedling extract could substitute for corn steep liquor, best results being obtained with a-day-old seedlings. Pregerminated spore suspensions of the organisms, maintained on Czapek-Dox agar slants, were used to inoculate 5Oml. lots of sterilized (at 15 lb. steam pressure for 15-20 min.) media contained in 250-ml. conical flasks which were then incubated at 30°, those with A. niger on a reciprocating shaker (50 cycles/min. with a stroke length of 3 in.).
ACTIVITIES
OF ASPERGILLI
367
The mycelia were harvested after growth for 72 hr., washed free of themedium, pressed between folds of filter paper, and ground to a fine paste in a glass mortar. Acetone powders were obtained by treatment of the ground mycelia with successive small amounts of chilled acetone and removal of excess acetone under reduced pressure inside a vacuum desiccator. Phosphatase and pyrophosphatase activities were determined in terms of inorganic phosphate liberated from sodium fi-glycerophosphate (5 ml. of a 5% solution) and sodium pyrophosphate (5 ml. of 0.1% solution), respectively, by 10 mg. of the acetone-dried powder suspended in 5 ml. of 0.2 M acetate buffer, pH 4.8, unless otherwise stated, when incubated under toluene for 3 hr. at 30”. Reaction was stopped by adding 1 ml. of 1 N NaOH. Inorganic phosphorus was determined according to Fiske and SubbaRow (9). Necessary control sets were kept for enzyme and substrate blanks. Saccharogenic activity was determined in toluenated water extracts of the ground mycelia by the procedure outlined earlier (6) and expressed as total activity in terms of Lintner units (10) per gram of dry mycelial growth in 50 ml. of growth medium. Oxygen-uptake measurements were carried out in single side-arm Warburg flasks using homogenized suspensions of the ground mycelia as enzyme source. The main chamber of each flask contained 1 ml. of 0.05 M phosphate buffer, pH 6.8, and 1 ml. of mycelial cell suspension equivalent to about 10 mg. dry wt.; the side arm had 1 ml. of a solution (0.02 M) of the appropriate substrate: glucose, sodium malate, sodium succinate, sodium pyruvate, or sodium citrate; and, to the center well was added 0.2 ml. of 10% KOH solution. The total flask volume was adjusted to 3.2 ml. in all cases. The flasks were shaken at 120 oscillations/min. and the temperature was kept at 37”. After 10 min. equilibration, the substrate from the side arm was tipped in and the reaction continued for 1 hr. during which period oxygen-uptake data were recorded at IO-min. intervals. Necessary controls were run alongside. All final data were corrected for endogenous respiration and expressed as &oz or microliters oxygen/g. mycelia/hr. In the Warburg experiments, the values for Qop with freshly harvested cells were low due to high endogenous oxygen uptake. Attempts were made to reduce endogenous metabolism. Acetone-dried mycelial preparations were not very active, and dialysis in the cold or washing with normal physiological saline was likewise unsatisfactory. Aging to remove endogenous substrates was found to result in considerably reduced endogenous uptake values. The harvested cells were washed free of culture filtrate by twice centrifuging with chilled distilled water and suspended in 0.9% saline overnight at 5”; after 4-6 hr. of further aging at room temperature, they were washed twice with water, homogenized to a fine suspension, and made to volume. RESULTS
pH Optimum for Phosphatase Action Using acetate buffer (0.2 M) for the pH range 4.0-5.8 and Verona1 buffer (0.2 M) for pH 6.8-10.0, it was observed (Table I) that, with
368
M.
A.
PATHAK
Optimum pH
AND
A.
SREENIVASAN
TABLE I for Phosphatuse Action
pH of incubation mixture
4.0 4.8 5.8 6.8 7.0 7.5 8.0 8.4 9.0 10.0
8 10 8 7 2 2 2 Nil Nil Nil
-I
33 44 22 18 18 10 6 6 Nil Nil
30 41 21 22 18 8 10 6 Nil Nil
L
25 30 22 16 12 10 8 5 Nil Nil
both A. niger and A. oryzue, the pH optimum for phosphatase action on sodium fl-glycerophosphate was near about 4.8 irrespective of the composition of the medium. Activity with Dijerent
Substrates
The enzyme was active against the phosphate esters tried (Table II). The results are given in comparison with the values obtained for glycerophosphate, all at pH 4.8 and in the concentrations indicated against each substrate. Amylase Activity
and Oxidative Metabolism oj Mold Mycelia
A comparison of the changes in phosphatase activities in the different media (Tables I and II) with the corresponding activities for amylase (Table III) revealed a parallelism between the two enzymes; thus, under cultural conditions most favorable for amylase synthesis, there was also more activity for phosphatase. Similar enhanced rates of oxidation of substrates by A. niger and A. oryzue were observable under conditions of culture which result in increased phosphatase activities (Table IV). The effect of corn steep liquor or sprouted mung bean extract supple-
ACTIVITIES
369
OF ASPERGILLI
TABLE
II
Phosphatase Action 072Different Substrates A. ni;m grown on
A.orysacgrownon
I
Substrate
Phosphatase activity: micrograms inorg. P liberated by 10 mg. enzyme preparation in 3 hr.
-
Sodium @-glycerophosphate (50 mg./ml.) Adenylic a&d, yeast (1 mg./W Hexose diphosphate (1 mg./ml:) Ribosenucleic acid depolymerized (Schwarz Lab.) (1 mg./mU Deoxyribonucleic acid depolymerized (Schwarz Lab.) (1 mg./ml.)
-T-
10
51
41
20
40
40
20
36
32
22
30
26
24
56
40
18
44
42
20
32
32
20
28
26
26
51
51
26
40
30
TABLE
III
Effect of Composition of Medium WJ Amylase Activity
Amylase activity
A. niger A. oryzae
195 196
in Lintner units at 40’ per half hour in 50 ml. medium (per gram dry cells)
552 465
521 400
is apparently due to an over-all stimulation of cell activities; protein concentrations in the mycelia were not determined under the different cultural conditions, this stimulation is evident from the increased growth of the organisms on these media as shown by their ,nycelial dry weight (Table IV). Activities in general were of a higher order with A. niger under submerged conditions than in the surfacegrowing A. oryzue cultures. mentation
although
370
M.
A.
PATHAK
AND
,4. SREENIVASAN
TABLE IV Oxidation
of Substrates
by Enzymes
of
A. niger and A. oryzae
Substrate
Qor microliters
Glucose (0.02M) Sodium malate (0.02 1M) Sodium succinate (0.02 M) Sodium pyruvate (0.02 M) Sodium citrate (0.02 M) Dry wt. of cells, g., in 50 ml. of medium
2600 2570 2180 1030 1360 0.113
3940 3600 3470 1330 1570 0.259
oxygen/g. mycelia/hr
3760 3320 3440 1460 1560 0.238
1920 1660 1820 920 1100 0.106
2600 2200 2630 1460 1360 -~ 0.201
2350 2ooo 1860 1100 1200 0.170
Nature oj the Phosphatase It was observed that treatment with cyanide at a concentration of 0.002 M for 30 min. inactivated the phosphatase preparation from A. niger. This suggested the possible association of the enzyme with a metal ion. Regeneration of activity on addition of various cations after cyanide inactivation was therefore attempted. A weighed quantity ( 2 g.) of acetone-dried mycelial preparation of A. niger was extracted with toluenated water (250 ml.) for 24 hr. in the cold (5”). The enzyme was precipitated from the extract by addition of an equal volume of acetone. The precipitate was centrifuged, resuspended in distilled water (25 ml.), and dialyzed in the cold for 24 hr. against several changes of distilled water. A l-ml. aliquot of the dialyzed enzyme solution was treated with cyanide (0.002 M final concentration), and several portions of similarly inactivated preparation were supplemented with different salt solutions. Activities were determined using sodium fi-glycerophosphate as substrate in a total volume of 10 ml. made up with acetate buffer. Some of the results are given in Table V. It was observed that addition of Zn+ could regenerate activky of cyanide-inactivated enzyme to a considerable extent. Other cations tried, viz., Co++, Mgtt , Ca++, Fe++, and Mn++ had no effect. Mere dilution of the cyanide-treated enzyme solution did not appreciably alter the extent of inactivation. The reversal of cyanide inactivation of phos-
ACTIVITIES
371
OF ASPERGILLI
TABLE V Regeneration of Cyanide-Inactivated
Enzyme Phosphatase activity: inorg. P liberated enzyme solution
Treatment
1. Enzyme solution (dialyzed) 2. As in 1, heated 3. As in 1, inactivated with cyanide (0.002M) 4. As in J, plus Z&l01 soln. (0.008M) 5. As in 8, plus ZnSOd soln. (0.004M) 6. As in 8, plus ZnSOdsoln. (0.002M)
micrograms by 1 ml. of in 3 hr.
99 9 23 56 84 82
TABLE VI of A. niger and A. oryzae micrograms inorganic P liberated by 10 mg. of
Pyrophosphatase Activity
Pyrophosphataze activity: enzyme preparation in 3 hr.
Sprouted extract
Organism
A. niqer A. oryzae
128 64
’ 205 164
mung bean medium
164 144
phatase activity by Zn++ therefore points to the possible involvement of the latter in the enzyme make-up. Pyrophosphatuse of A. niger and A. oryzae The existence of pyrophosphatases of different pH optima is known (4). It was observed in preliminary experiments that the pyrophosphatase of both A. niger and A. oryzae had a pH optimum between 4 and 5. The activities of the mycelia of the two organisms grown in t,he three media were compared (Table VI), and it was observed that as with amylase and phosphatase activities, pyrophosphatase activities of mycelia harvested from the corn steep liquor and the sprouted mung beanextract media were much higher than that from the glucose-CzapekDox medium. Pyrophosphatase was also inactivated by cyanide and reactivated by Zn++. DISCUSSION
The phosphatase in the aspergilli is similar to the phosphatase of Penicillum chrysogenum (2) in its property of *inactivation by cyanide and reactivation by Zn*. However, unlike the latter, it is an acid phosphatase. It has limited substrate specificity. Whether the same enzyme exhibits both phosphatase and pyrophosphatase activities has not been studied.
372
M.
A.
PATHAK
AND
A.
SREENIVASAN
Studies on the changes in the elaboration of amylases by the two organisms and in their oxidative metabolism show a parallelism with phosphatase activity changes and it seems likely therefore that, phosphatases in molds are more than mere structural const,ituents and have physiological significance. ACKNOWLEDGMENT This work was assisted by a research grant from the Bombay State Industrial Research Committee of the Department of Industries, Government of Bombay. SUMMARY
Phosphatase and pyrophosphatase of amylase-elaborating Aspergillus niger (submerged growth) and Aspergillus oryzae (surface culture) have a pH optimum of 4.8, no activity being observable at alkaline pH range. With increase in amylase production in a medium like corn steep liquor or sprouted mung bean extract, there is a corresponding increase in phosphatase and, more particularly, pyrophosphatase activities with both organisms as well as in the oxidation of various substrates. Phosphatase activity is inactivated by cyanide and could be regenerated by addition of Zn* but not by Co++, Mg-++, Ca++, Mn*, or Fe++. REFERENCES 1. MANN
T.,
2. SADASIVAN, 3. SADASIVAN, 4. KRISHNAN, 5. KRISENAN, 6. RAO, R. 8.
Biochem.
J. 38, 339 (1944).
V., Arch. Biochem. 28, 100 (1950). V., Arch. Biochem. and Biophys. 87, 172 (1952). P. S., Arch. Biochem. and Biophys. 89,230 (1951). P. S., AND BAJAJ, V., Arch. Biochem. and Biophys. 47.39 (1953). AND SREENIVASAN, A. Trans. Am. Assoc. Cereal Chemists 8, 46
J.,
(1950). 7. ADAMS,
S. L.,
BALANKURA,
B.,
ANDREASF.N,
Eng. Chem. 29.1615 (1947). 8. SMITE, G., “An Introduction to Industrial
A. A.,
AND STARK,
Mycology,”
W. H., Ind.
2nd ed., p. 172. Ed-
ward Arnold and Co., Ltd., London, 1942. 9. FISKE, C., AND SUBBAROW, Y., J. Biol. Chem. 81,629 (1929). AND STEELE, H. K., Ind. Eng. Chem., Anal. Ed. 7, 324 (1935).
10. GORE, H. C.,