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Widespread occurrence of galactose oxidase and glucose oxidase in fungi
COMMUNICATIONS (RF 0.9) derivatives was obtained in cyclohexanebenzene-diethylamine (7:3: 1). The azo dye of the unknown spot from several samples of epididymal fluid and seminal plasma of the ram also had, by this method, an Rp equal to that of p-cresol.Two-dimensional co-chromatography of the unknown azo dye with that of pcresol or p-ethylphenol resulted in one spot when the p-cresol derivative was added to the unknown, and in two spots when p-ethylphenol derivative was added. Further confirmation of identity of the unknown phenol and p-cresol was obtained by comparing the spectra of the azo dyes. The azo dyes of the unknown and standard phenols were dissolved in ammonia solution (sp. gr. 0.33) and the absorption maxima of the spectrum were recorded in a Hitashi-Perkin Elmer spectrophotometer. The results were similar to those of Smith and Sullivan (9). Maxima at 355 rnp and at 555 w were observed for p-cresol and the unknown spot. The concentration of the p-cresol in the samples was measured at 555 rnb, after dissolving the spots scraped from thin-layer plates in 1 ml of concentrated ammonia. The silica gel was centrifuged off and the optical density was read in l-ml cells. The amounts were calculated from a standard curve of p-cresol. The concentration of p-cresol in epididymal fluid was found to be 50-100 rg/ml and in seminal plasma 10-20 pg/ml. In samples of the latter, a spot reacting like a phenol was always present also at the origin of the chromatogram, indicating the possibility of the presence of phenol oxidation products. The significance of the occurrence of p-cresol in the semen is under further study. This research has been financed in part by a grant from the U.S. Department of Agriculture, under P.L. 430. The skillful technical assistance of Mrs. P. Holst,ein is gratefully acknowledged.
REFERENCES 1. SUEMITSU, R., FUJITA, D., AND MATSUBARA, H., Agr. Biol. Chem. 29, 908 (1965). 2. TADMOR, A., AND SCHINDLER, H. Israel J. Agric. Res. 16, 157, (1966). 3. SMITH, I., in “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.), Vol. I, Chapter 16. William Heinemann, Medical Books, Lt,d., London (1960). 4. GANSHIRT, H., in “Thin Layer Chromatography” (E. Stahl, ed.), pp. 311-313. Springer Verlag, Berlin (1965). 5. WALDI, D., in “Thin Layer Chromatography” (E. Stahl, ed.), p. 498. Springer Verlag, Berlin (1965).
6. RANDERATH, K., in “Thin Layer Chromatography,” pp. 175-184. Academic Press, New York (1964). 7. REIO, L., J. Chromatog. 1, 338, (1958). 8. FEIGL, F., “Spot Tests in Organic Analysis,” 5th edition, p. 179. Elsevier, Amsterdam (1956). 9. SMITH, G.A.L., AND SULLIVAN, P. J., Analyst 89, 312 (1964). EUGENIA ALUMOT Division
of Animal Nutrition
VoIcani Institute of Agricultural Israel Received November 16, 1966
Research
Rehovot,
Widespread
Occurrence
and Glucose
of Galactose
Oxidase
Oxidase
in Fungi
Sugar oxidases in fungi have only been described in a few species. Galactose oxidase has been found in Dactylium dendroides (l-3) and glucose oxidase in some species of Aspergillus and Penicillium (4). To our knowledge no other enzymes are known for the direct oxidation, through molecular oxygen, of common sugars. However, products of the oxidation of sugars (hexonic and pentonic acids) have been described in the culture media of several fungi (5-7). A search for possible sugar oxidases that could account for the appearance of the reported oxidation products was undertaken. Simultaneously the distribution of galactose and glucose oxidase among fungi was studied. A screening procedure was employed with approximately one hundred different species of fungi, belonging to 27 genera. The sugars, ~-glucose, n-galactose, n-mannose, n-ribose, n-xylose, and L-arabinose were used as carbon sources for growth. In all the cases where growth was observed the culture media were assayed, as described in the table, for their capacity to oxidize any of the sugars listed above. No oxidases for n-mannose, n-ribose, n-xylose, and L-arabinose were detected in the present survey. However, it is possible that a species producing one of these enzymes would appear as negative, either because the enzyme was not released into the medium or because the enzymic activity appears within a narrow range of time during the growth curve, as is actually the case for glucose and galactose oxidases (see below). In the course of this screening galactose and glucose oxidases were detected in a variety of species not previously described as producers of these enzymes (Table I). The present findings tend to support the view that these oxidases are not
COMMUNICATIONS TABLE
I
SUGAR OXIDASE ACTIVITIES Genera
Alternaria
Helminthosporium
1 1 1 1 25 2 1
Mycosphaerella Penicillium Phytium Phytophtora
-
2
14 15
Hymenomyces
IN FUNGI~
Glucoseox&se
Aspergill us Fusarium
Glomerella
589
Galactose oxidase
sp.
Alternaria -
A. niger
-
F. moniliforme
-
Helminthosporium -
Fusarium sp .
G. cingulatu
Hymenomyces sp.
-
M. pinodes
-
6b
sp.
P. album
-
P. de baryanum P. parasitica
(1The fungi were grown with rotary shaking in the medium described by Avigad et al. (2)) except that 14.5 gm KH2P0, and 2.75 gm KzHPO, per liter were used. Oxidases were assayed by adding 1 ml of the culture medium to a mixture which contained 1 ml of the appropriate sugar (0.2 M) and 1 ml of a chromogenic system according to Keston (10). [Peroxidase (10 mg/ml) 20 ~1; 0.2 M phosphate buffer, pH 7.0, 5 ml; o-dianisidine (O.lyo in ethanol) 0.5 ml; Triton X-100 lo%, 1 ml; distilled water up to a volume of 50 ml.] Increase in optical density was followed spectrophotometrically at 340 rnp at room temperature (ca. 25”). b P. chrysogenum, P. cianeum, P. citrinum, P. cant-ardille, P. martensii, P. oxalicum. unusual, but may be widely distributed among fungi. It is interesting to note that no species have been found to produce both enzymes. In connect,ion with this work the variations of galactose and glucose oxidase activities as a function of the time of growth were studied in the culture media of Fusarium moniliforme and Aspergillus niger, respectively (Fig. 1). It can be seen that after reaching a maximum the activities decrease very rapidly. Since galactose and glucose oxidase are rather stable in a 6ltered culture medium, it could appear that the enzymes are inactivated by the fungal mycelium. The characteristics of this process are not known, although a similar inactivation has been described for the galactose oxidase of Dactylium dendroides (8). Taking into account the fact that enzymic activity of the oxidases may appear in significant amounts only during a narrow range of time, negative results cannot be regarded as highly significant in a screening approach. The nature of the test used to detect oxidase activity does not allow us to decide the position of the sugar carbon chain in which the oxidation takes place. It is likely, however, that the galactose oxidase activities found in all the fungi correspond to similar enzymes, as the relative activities with galactose, lactose, and raffinose as substrates agree with the values reported for Dactylium dendroides (2). A similar study remains
to be done for the enzymes oxidizing glucose, in order to establish whether the oxidation occurs in all cases at the C-l position, as described earlier for the enzyme of Aspergillus niger (9). This work was partly supported by U.S. Public Health Service grant TW-00132-l. J. M. G. and C. G. were fellows of the Comisaria de Protection Escolar, Spain. REFERENCES 1. COOPER, J. A. D., SMITH, W., BACILA, M., AND MEDINA, H., J. Biol. Chem. 234, 445 (1959). 2. AVIGAD, G., AMARAL, D., ASENSIO, C., AND HORECKER, B. L., J. Biol. Chem. 237, 2736 (1962).
3. NOBLES, M. K., AND MADHOSINGH, C., Biothem. Biophys. Res. Commun. 12, 146 (1963). 4. SWOBODA, B. E. P., AND MASSEY, V., J. Biol. Chem. 240, 2209 (1965). 5. FOSTER, J. W., “Chemical Activities of Fungi,” pp. 464-465. Academic Press, New York (1949). 6. PIGMAN, W., “The Carbohydrates,” p. 365. Academic Press, New York (1957). of Fungi,” pp. 7. C~~HRANE, V,. “Physiology 135-136. Wiley, New York (1958). 8. MARKUS, Z., MILLER, G., AND AVIGAD, G., Appl. Microbial. 13, 636 (1965). 9. BENTLEY, R., AND NEUBERGER, A., Biochem. J. 46, 534 (1949).
3r
590
COMMUNICATIONS
Glucose
oxidase
Galactose
d
Asperqillus
niqer
Fusarium
I’ c I
i
t
oxidase
moniliforme
:
I A
: : I I : I I I I
1
I : I r’ 0
I’
\
?
: I , ,
/
24
40
/’
,
72
t
96
HOURS
FIG. 1. Glucose and galactose oxidase activities as a function of growth time. From 2-liter flasks containing 500 ml of culture medium, samples of about 5 ml were withdrawn as indicated in the figure. The samples were dialyzed for 3 hours against 0.02 M phosphate buffer, pH 7.0, at 4”. Activities were determined as indicated in the table, one unit being the quantity of enzyme which yields an optical density of 1 .O in 10 minutes. Glucose oxidase activity was also tested in mycelium extracts with similar results. 10. KESTON, A. S., Abstracts Society, 129th Meeting,
American Chemical 31 C (1956). JUANA M. GANCEDO CARLOS GANCEDO CARLOS ASENSIO
Department of Enzymology Institute G. MaraAh Madrid, Spain Received November 22, 1966
Conversion
of myo-lnositol-2J4C
I-0-Methyl-glucuronic of Maize
to Labeled
Acid in the Cell Wall Root Tips
In plant tissues undergoing cell wall deposition, myo-inositol-2.i4C or -2-3H is incorporated into cell wall polysaccharides as uranic acid and pentose residues of pectic substance and hemicellulose (1, 2). This conversion involves an oxidative
cleavage of myo-inositol between carbons 1 and 6. The product, n-glucuronic acid, is utilized as a precursor of n-galacturonic acid, n-xylose, and L-arabinose residues in cell wall polysaccharides (3). To complete this investigation of the precursor potential of myo-inositol as a source of uranic acid and pentose units, we have studied labeled myo-inositol metabolism in root tips of Zea mays, a plant rich in (4-O-methylglucurono)xylans and (glucurono)xylans (4-10). Our results indicate that both types of xylan are labeled by myoinositol-2-*4C. Carbon-14 is found in 4-O-methylglucuronic acid and glucuronic acid residues as well as in xylose. Sixty root tips (1 cm) from 3-day maize seedlings were incubated in 3 ml of 8.5 X lO+ M myoinositol-2-W (2.96 PC) at 28” for 24 hours. Over 80% of the “C! was taken up by the root tips during the first 8 hours. Very little i4C02 was respired during the first 6 hours but in the next, A hours, the 1% content of respired CO:! rose rapidly
REFERENCES 1. SUEMITSU, R., FUJITA, D., AND MATSUBARA, H., Agr. Biol. Chem. 29, 908 (1965). 2. TADMOR, A., AND SCHINDLER, H. Israel J. Agric. Res. 16, 157, (1966). 3. SMITH, I., in “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.), Vol. I, Chapter 16. William Heinemann, Medical Books, Lt,d., London (1960). 4. GANSHIRT, H., in “Thin Layer Chromatography” (E. Stahl, ed.), pp. 311-313. Springer Verlag, Berlin (1965). 5. WALDI, D., in “Thin Layer Chromatography” (E. Stahl, ed.), p. 498. Springer Verlag, Berlin (1965).
6. RANDERATH, K., in “Thin Layer Chromatography,” pp. 175-184. Academic Press, New York (1964). 7. REIO, L., J. Chromatog. 1, 338, (1958). 8. FEIGL, F., “Spot Tests in Organic Analysis,” 5th edition, p. 179. Elsevier, Amsterdam (1956). 9. SMITH, G.A.L., AND SULLIVAN, P. J., Analyst 89, 312 (1964). EUGENIA ALUMOT Division
of Animal Nutrition
VoIcani Institute of Agricultural Israel Received November 16, 1966
Research
Rehovot,
Widespread
Occurrence
and Glucose
of Galactose
Oxidase
Oxidase
in Fungi
Sugar oxidases in fungi have only been described in a few species. Galactose oxidase has been found in Dactylium dendroides (l-3) and glucose oxidase in some species of Aspergillus and Penicillium (4). To our knowledge no other enzymes are known for the direct oxidation, through molecular oxygen, of common sugars. However, products of the oxidation of sugars (hexonic and pentonic acids) have been described in the culture media of several fungi (5-7). A search for possible sugar oxidases that could account for the appearance of the reported oxidation products was undertaken. Simultaneously the distribution of galactose and glucose oxidase among fungi was studied. A screening procedure was employed with approximately one hundred different species of fungi, belonging to 27 genera. The sugars, ~-glucose, n-galactose, n-mannose, n-ribose, n-xylose, and L-arabinose were used as carbon sources for growth. In all the cases where growth was observed the culture media were assayed, as described in the table, for their capacity to oxidize any of the sugars listed above. No oxidases for n-mannose, n-ribose, n-xylose, and L-arabinose were detected in the present survey. However, it is possible that a species producing one of these enzymes would appear as negative, either because the enzyme was not released into the medium or because the enzymic activity appears within a narrow range of time during the growth curve, as is actually the case for glucose and galactose oxidases (see below). In the course of this screening galactose and glucose oxidases were detected in a variety of species not previously described as producers of these enzymes (Table I). The present findings tend to support the view that these oxidases are not
COMMUNICATIONS TABLE
I
SUGAR OXIDASE ACTIVITIES Genera
Alternaria
Helminthosporium
1 1 1 1 25 2 1
Mycosphaerella Penicillium Phytium Phytophtora
-
2
14 15
Hymenomyces
IN FUNGI~
Glucoseox&se
Aspergill us Fusarium
Glomerella
589
Galactose oxidase
sp.
Alternaria -
A. niger
-
F. moniliforme
-
Helminthosporium -
Fusarium sp .
G. cingulatu
Hymenomyces sp.
-
M. pinodes
-
6b
sp.
P. album
-
P. de baryanum P. parasitica
(1The fungi were grown with rotary shaking in the medium described by Avigad et al. (2)) except that 14.5 gm KH2P0, and 2.75 gm KzHPO, per liter were used. Oxidases were assayed by adding 1 ml of the culture medium to a mixture which contained 1 ml of the appropriate sugar (0.2 M) and 1 ml of a chromogenic system according to Keston (10). [Peroxidase (10 mg/ml) 20 ~1; 0.2 M phosphate buffer, pH 7.0, 5 ml; o-dianisidine (O.lyo in ethanol) 0.5 ml; Triton X-100 lo%, 1 ml; distilled water up to a volume of 50 ml.] Increase in optical density was followed spectrophotometrically at 340 rnp at room temperature (ca. 25”). b P. chrysogenum, P. cianeum, P. citrinum, P. cant-ardille, P. martensii, P. oxalicum. unusual, but may be widely distributed among fungi. It is interesting to note that no species have been found to produce both enzymes. In connect,ion with this work the variations of galactose and glucose oxidase activities as a function of the time of growth were studied in the culture media of Fusarium moniliforme and Aspergillus niger, respectively (Fig. 1). It can be seen that after reaching a maximum the activities decrease very rapidly. Since galactose and glucose oxidase are rather stable in a 6ltered culture medium, it could appear that the enzymes are inactivated by the fungal mycelium. The characteristics of this process are not known, although a similar inactivation has been described for the galactose oxidase of Dactylium dendroides (8). Taking into account the fact that enzymic activity of the oxidases may appear in significant amounts only during a narrow range of time, negative results cannot be regarded as highly significant in a screening approach. The nature of the test used to detect oxidase activity does not allow us to decide the position of the sugar carbon chain in which the oxidation takes place. It is likely, however, that the galactose oxidase activities found in all the fungi correspond to similar enzymes, as the relative activities with galactose, lactose, and raffinose as substrates agree with the values reported for Dactylium dendroides (2). A similar study remains
to be done for the enzymes oxidizing glucose, in order to establish whether the oxidation occurs in all cases at the C-l position, as described earlier for the enzyme of Aspergillus niger (9). This work was partly supported by U.S. Public Health Service grant TW-00132-l. J. M. G. and C. G. were fellows of the Comisaria de Protection Escolar, Spain. REFERENCES 1. COOPER, J. A. D., SMITH, W., BACILA, M., AND MEDINA, H., J. Biol. Chem. 234, 445 (1959). 2. AVIGAD, G., AMARAL, D., ASENSIO, C., AND HORECKER, B. L., J. Biol. Chem. 237, 2736 (1962).
3. NOBLES, M. K., AND MADHOSINGH, C., Biothem. Biophys. Res. Commun. 12, 146 (1963). 4. SWOBODA, B. E. P., AND MASSEY, V., J. Biol. Chem. 240, 2209 (1965). 5. FOSTER, J. W., “Chemical Activities of Fungi,” pp. 464-465. Academic Press, New York (1949). 6. PIGMAN, W., “The Carbohydrates,” p. 365. Academic Press, New York (1957). of Fungi,” pp. 7. C~~HRANE, V,. “Physiology 135-136. Wiley, New York (1958). 8. MARKUS, Z., MILLER, G., AND AVIGAD, G., Appl. Microbial. 13, 636 (1965). 9. BENTLEY, R., AND NEUBERGER, A., Biochem. J. 46, 534 (1949).
3r
590
COMMUNICATIONS
Glucose
oxidase
Galactose
d
Asperqillus
niqer
Fusarium
I’ c I
i
t
oxidase
moniliforme
:
I A
: : I I : I I I I
1
I : I r’ 0
I’
\
?
: I , ,
/
24
40
/’
,
72
t
96
HOURS
FIG. 1. Glucose and galactose oxidase activities as a function of growth time. From 2-liter flasks containing 500 ml of culture medium, samples of about 5 ml were withdrawn as indicated in the figure. The samples were dialyzed for 3 hours against 0.02 M phosphate buffer, pH 7.0, at 4”. Activities were determined as indicated in the table, one unit being the quantity of enzyme which yields an optical density of 1 .O in 10 minutes. Glucose oxidase activity was also tested in mycelium extracts with similar results. 10. KESTON, A. S., Abstracts Society, 129th Meeting,
American Chemical 31 C (1956). JUANA M. GANCEDO CARLOS GANCEDO CARLOS ASENSIO
Department of Enzymology Institute G. MaraAh Madrid, Spain Received November 22, 1966
Conversion
of myo-lnositol-2J4C
I-0-Methyl-glucuronic of Maize
to Labeled
Acid in the Cell Wall Root Tips
In plant tissues undergoing cell wall deposition, myo-inositol-2.i4C or -2-3H is incorporated into cell wall polysaccharides as uranic acid and pentose residues of pectic substance and hemicellulose (1, 2). This conversion involves an oxidative
cleavage of myo-inositol between carbons 1 and 6. The product, n-glucuronic acid, is utilized as a precursor of n-galacturonic acid, n-xylose, and L-arabinose residues in cell wall polysaccharides (3). To complete this investigation of the precursor potential of myo-inositol as a source of uranic acid and pentose units, we have studied labeled myo-inositol metabolism in root tips of Zea mays, a plant rich in (4-O-methylglucurono)xylans and (glucurono)xylans (4-10). Our results indicate that both types of xylan are labeled by myoinositol-2-*4C. Carbon-14 is found in 4-O-methylglucuronic acid and glucuronic acid residues as well as in xylose. Sixty root tips (1 cm) from 3-day maize seedlings were incubated in 3 ml of 8.5 X lO+ M myoinositol-2-W (2.96 PC) at 28” for 24 hours. Over 80% of the “C! was taken up by the root tips during the first 8 hours. Very little i4C02 was respired during the first 6 hours but in the next, A hours, the 1% content of respired CO:! rose rapidly