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Pectic enzymes of Erwinia carotovora
Pectic Enzymes of Erwinia carotovora Albert J. KraghV From
the Department
of Bacteriology,
and Mortimer University
Received
March
P. Starr
of California,
Davis,
California
6, 1952
In recent years the study of bacterial pectic enzymes has lagged behind its counterpart in plant and mold enzymes. From work on enzymes derived principally from citrus, alfalfa, and tomatoes, plants have been shown to be rich in pectinesterase (PE) but poor in polygalacturonase (PG). Similarly, studies on pectic enzymes derived from mold mycelium and mold culture filtrates have provided good evidence that some molds produce abundant quantities of both enzymes (I). Although the statement that the soft-rot bacterial group produce both PE and PG is encountered frequently, the published evidence supporting this opinion is, unfortunately, very fragmentary. Davison and Willaman (a), who were pioneer workers in this field, found that Erwinia carotovora, grown on carrot decoction, produced a moderately strong PG but they could not demonstrate the presence of PE by the calcium pect,ate method. Since then, Kertesz (3) has shown that this method is unreliable in the presence of PG because of interference by t,he latter enzyme. Norman (4) studied the rate at which pectin disappeared from a pectin medium inoculated with soft-rot bacteria by noting t,he time interval between inoculation and failure to obtain a calcium pectate precipitate in the culture medium. His experiment may be regarded as an indirect demonstration of PG production. Later, Oxford (5) reported active PG preparations obtained by alcoholic precipitation of the enzyme from cultures of plant pathogenic pseudomonads growing in a pectin medium. Mills (6) reported PE production by a strain of Pseudomonas prunicola only when the carbon source in the growth medium was pectin, pectic acid, or galacturonic acid. No PE activity was detected when the carbon substrate was either glucose or galactose. 1 Present
address:
Wallace
and
Tiernan 271
Company,
Monrovia,
Calif.
272
A. J. KRAGHT
AND M. P. STARR
Recently, Wood (7) reported that the pectic enzyme or enzymes produced by Erwinia (Bacterium) aroideae, grown on synthetic medium, contained little PE, but did contain an enzyme, activated by calcium ions, which rapidly reduced the viscosity of high-methoxyl pectin solutions, yet was unable to increase substantially the reducing power of the solution. The latter property suggests that this enzyme is different from bacterial PG reported previously (2, 5). The present study was undertaken as part of an investigation (8,9) on the biochemical behavior of the soft-rot bacteria. Evidence is presented that the strain of Erwinia carotovora used in this study produced both PE and PG when grown in a pectin medium. The amount of PG detected in the culture filtrate was materially greater when the organism was grown in pectin rather than glucose media. EXPERIMENTAL
METHODS
Culture Used The strain of Erwinia carotovora used in this study was isolated from soft-rot spoilage in a commercial shipment of green peppers grown in Los Mochis, Mexico, and shipped to the Los Angeles market in 1949. This otherwise typical strain produced little or no visible gas in the usual sugar fermentation tests. A report on the end products of utilization, by the culture, of galacturonic acid and glucose has been prepared (9).
Pectin Growth Medium The organism was grown in a semisynthetic pectin medium containing 10 g. pectin National Formulary (N.F.), 4 g. (NH&S04, 13 g. KHzPOI, 9 g. Na2HPOI, 0.8 g. MgSOc.HzO, 1 mg. HaB03, 2.mg. CuSOa.5H20, 20 mg. FeCh*6H20, 2 mg. MnS04*HsO, 2 mg. Na&IoO(.2HzO, 26 mg. CaCh, and 10 mg. ZnCln per liter. The pectin and mineral components of the medium were sterilized separately by steaming and added aseptically when needed. The completed medium was adjusted to approximately pH 6.9 with sterile.0.25 N NaOH. Incubation temperature was 30”, unless otherwise specified. To inoculate 106 ml. of the medium, 0.05 ml. was transferred from a 24-hr. culture of the organism growing on a medium of the same composition.
Assay of PE Activity Production of PE by Erwinia carotovora, while growing on the pectin medium, was followed by determining the amount of methyl alcohol in the pectin medium at various time intervals after inoculation. Methyl alcohol was estimated after oxidation to formaldehyde with potassium permanganate in the presence of phosphoric acid by a slight modification of the chromotropic acid (1,3-dihydroxynaphthalene-3,6-disulfonic acid) method of Boos (10). This method was found
PECTIC ENZYMES OF ERWINIA
CAROTOVORA4
273
satisfactory, although its weakness lies in the oxidation procedure. For this reason, known amounts of methanol were included in each determination. Preliminary separation of the methanol from the medium was accomplished by steam dist#illation; five volumes of distillate were collected.
Assay of PC Activity Polygalacturonase was demonstrated, as is commonly done with plant and fungal extracts, by showing an increase in reducing groups when the enzyme is allowed to act on pectic acid. The assay was carried out in the presence of fluoride in the following manner: eqch 250-ml. reaction flask contained 10 ml. of a 2% solution of sodium pectate, 20 ml. of the supernatant resulting from centrifugation of a 72-hr. culture grown at 30” (usually in the pectin medium, unless stated otherwise), 0.08 ml. of 1 M sodium fluoride, sufficient 1 A; HCl or NaOH to adjust to the desired pH (usually about 5.8, unless otherwise stated), and distilled water to make a total volume of 40ml. The mixture was held at 25” and its reducing power determined periodically by the hypoiodite method described by Jansen and MacDonnell (11). All values were correct.ed for the small amount of reducing power found in the heat-inactivated crude enzyme preparation.
Deiermination of Relative Viscosity The relative viscosity of the pectin medium at various times after inoculation was determined in an Ostwald viscometer held in a constant-temperature water bath at 30”. .411 determinations were made in duplicate.
Ethyl Alcohol Precipitate The ethyl alcohol precipitation test was performed by adding 10 ml. of 95% ethyl alcohol to 5 ml. of culture liquor and observing the nature of the precipitate formed. EXPERIMENTAL
RESULTS
Pectinesterase In the experimental data given in Table I, the accumulation of methyl alcohol in the medium, as pectin becomes more and more degraded, may be taken as conclusive evidence of PE activity. Changes in the relative viscosity of the medium, coupled with decreasing ability to obtain a gelatinous alcohol precipitate of pectin, are evidence of degradation of the pectin in the inoculated medium. The pH of the growth medium dropped from 6.9 to 6.2 in the 72-hr. experimental period. Only an insignificant amount of de-esterification occurred during the first 24 hr. despite a 700-fold increase in bacterial numbers and a substantial decrease in viscosity of the pectin medium. Within 48 hr., approximately two thirds of the potential methanol had been liberated,
274
A.
J.
KRAGHT
AND
M.
and by the end of the experiment hydrolysis but, progressively increasing, amount of from the uninoculated medium incubated instability of pectin in combination with medium. Additional experiments showed that thd was not unique in producing PE; the carotovora were tested: a) E. carotovora, Division of Plant Pathology, Ijniversity
inoculation
-___~-
0 24 48 72
Relative
STARR
was 94% complete. A small, methyl alcohol was obtained at 30”, which may be due to the other ingredients of the Mexican green pepper isolate following strains of Erwinia obtained in 1948 from the of California, Davis, Cali-
TABLE I of Pectin by a Growing Culture of Erwinia the 1% Pectin N.F.mMedium
Progressive Degradation
:rtz
P.
viscosity
Alcohol
precipitate*
Number
carotovora
in
Methyl alcohol liberatedi mg.fg. pectznc
of bacteria/ml.
-I-
A“
B=
C=
A ____-
12.4 11.8 12.1 4.8 4.6 9.1 1.4 1.3 7.7 1.3 1.2 7.0
t++ +++ ? -
-
B
+++ +++ ? -
c
A
+++29 x +++19 x +++ 29 X +++26 X
B
10429 10718 10711 lo825
x x X X
C -__
104 107 lo8 lo*
a A and B inoculated; C uninoculated. * Plus (+) indicates a gelatinous precipitate, while minus flocculent precipitate. c Theoretical amount is 92.1 mg. CH,OH/g. pectin N.F.
A
B
0 1.1 1.0 0 2.1 1.7 0 63.7 60.0 0 87.4 86.0 (-)
indicates
C
1.0 1.2 6.5 9.0 a
fornia; b) E. carotovoru (ATCC 495) ; c) E. curotovora (EC104), originally isolated by Dr. P. A. Ark in 1946 from soft-rot of potatoes; and d) E. carotovora var. delphinii (EC108), originally isolated by Dr. P. A. Ark from larkspur in 1935. Each strain was grown in a medium whose composition was 0.5% pectin N. F., 0.1% peptone, and 0.1% yeast extract. Forty-eight hours after incubation at 30”, portions of the culture liquor were steam-disl tilled, and methyl alcohol in the distillate was determined by the chromotropic acid method. Methyl alcohol, in amounts considerably in excess of that found in the control medium, was detected in all of the culture fluids. Therefore, it seemsthat PE was produced by all the tested strains of Ewinia carotovora.
PECTIC
ENZYMES
OF
ERWINIA
CAROTOVORA
275
Polygalacturonase Polygalacturonase (PG) could not be demonstrated directly in the growing culture because galacturonic acid is utilized rapidly by Ihe organism (9) and does not accumulate in the medium. Removal of the bacterial cells remaining after centrifugation by passage of the supernatant culture liquor through a fine-porosity Mandler filter candle yielded an active PG preparation as shown by an increase in reducing groups when the filtrate was added to a solution of sodium pectate. The addition
FIG.
in the
1. Effect of carbon source (loJo glucose or 0.5(% glucose growth medium on the production of polygslacturonase
plus 0.5% pectin) by Erwinia caro-
tovora.
of 0.02 M sodium fluoride or sodium azide to the supernatant of the centrifuged culture liquor without first removing the remaining bacterial cells similarly permitted demonstration of PG activity. The effect of pH from 3.3 to 7.9 on PG activity was determined. At intervals of 5, 10, 15, 20, 30, 60 and 90 min., 5-ml. aliquots of the reaction mixture were removed for the determination of reducing power. PG was found to be active over a wide pH range ext,ending from 3.9 to i.9; it has a broad optimum around pH 5.8. The effect of pectic substances in the growth medium on the formation of PG was demonstrated by growing E. carotovora in media in which glu-
276
A.
J.
KRAGHT
AND
M.
P. STARR
case had been substituted for all or for half of the pectin in the growth medium. A 24-hr. inoculum for both media was prepared by transferring the culture from beef-yeast extract agar storage. slants to the glucose medium. Growth in each medium during the 72-hr. incubation period was comparable as evidenced by identical turbidities, expressed as percentage transmission, of 15% on the Evelyn calorimeter with filter 420. From the data given in Fig. 1, it can be seen that only a small amount of PG was produced by the organism when grown on the glucose medium. PG activity was increased greatly when the organism was grown on the combination medium containing both pectin and glucose. Since there had been no prior exposure of the inoculum to pectin, the increased PG activity, when pectin was added to the growth medium, may be considered as stimulation. Yet it was necessary to exclude the possibility that greater PG activity in the glucose culture had occurred at some earlier stage of bacterial growth and was disappearing from the medium when the sample was taken. This point was checked by growing the organism in the synthetic glucose medium and determining PG activity as a function of the age of the culture. It was found that at no prior time were pectic enzymes produced in any larger quantity than was found at the end of the 72-hr. incubation period. Therefore, it seems valid to hold that PG activity was greatly increased by the incorporation of pectin in the medium. DISCUSSION
The demonstration of methyl alcohol liberation by several strains of E. carotovora growing in a high-methoxyl pectin medium may be considered as conclusive evidence of PE activity. Although in this work PE was demonstrated in the presence of growing cells only, it seems reasonable to expect that cell-free preparations of the culture fluid would contain an active PE capable of hydrolyzing the methoxyl groups in pectin. The success achieved by Mills (6) with partially purified PE from cultures of Pseudomonas prunicola can be taken as a basis for the above assumption. Polygalacturonase production by E. carotovora cultures has been demonstrated by previous workers (2). However, its optimum pH of 5.8 is now shown to be lower than the optimum of pH 7-8 reported by Oxford (5) for PseudomonasPG, and higher than the optimum pH of about 3.54.0 reported for yeast (12) and fungal (13) PG.
PECTIC
ENZYMES
OF
ERWINIA
CAROTOVORA
277
The effect of pectic substances in the medium on the formation of PG is consistent with previous reports (13, 14). Certainly any report that an organism does or does not produce PG should be considered in light of the growth medium used. The difficulty in demonstrating PO in the present study, when the organism was grown in a glucose medium, amply illustrates the validity of the above statement. The present study clearly places E. carotovora in the same category as some molds in that both PE and PG are formed. That Erwinia soft-rot bacteria may produce other pectic enzymes is suggested by the aforementioned work of Wood (7). STJMMARY
A culture of Erwinia carotovora isolated from rotted Mexican bell pepper liberated methanol practically quantitatively from pectin when growing in a pectin medium. This phenomenon is considered to be positive evidence of pectinesterase activity. Polygalacturonase could not be demonstrated directly in the culture medium containing actively growing cells because the subsequent rapid utilization of galacturonic acid made positive identification of the enzyme uncertain. Because polygalacturonase is insensitive to both fluoride and azide, and the galacturonic acid degrading system is inhibited by these substances, it was possible to demonstrate and study the polygalacturonase in the presence of these metabolic inhibitors. E. carotovora polygalacturonase is active over a wide pH range, the optimum being around 5.8. Its production or secretion into the medium is greatly increased by the addition of pectic substances to the growth medium. REFERENCES 1. 2. 8. -I. 5. 6. 7. 8. 0. 10. 11. 12. 13.
14.
LINEWEAVER, H., AND JANSEN, E. F., Advances in Enzymol. 11, 267 DAVIEON, F. R., AND WILLAMAN, J. J., &tan. Gaz. 83,329 (1927). KERTESZ, Z. I., J. BioZ. Chem. 121, 589 (1937). NORMAN, A. G., Ann. Botany 43, 233 (1929). OXFORD, A. E., Nature 164, 271 (1944). MIWS, B. G., Biochem. J. 44,302 (1949). WOOD, R. li. S., Nature 167, 771 (1951). STARR, M. P., AND MANDEL, M., J. Bact. 60, 669 (1950). KRAGHT, A. J., AND STARR, M. P., J. Bacf. 64, 259 (1952). Roes, R. N., .4naZ. Chem. 20, 964 (1948). JANSEN, E. I?., AND MACDONNELL, L. R., Arch. Biochem. 8,97 (1945). LUH, B. S., AND PHAFF, H. J., Brch. Biochem. and Biophys. 33, 212 PHAFF, H. J., AND JOSLYN, M. A., Walterstein Labs. Communs. 10, 133 PHAFF, H. J., Arch. Biochem. 13, 67 (1947).
(1951).
(1951). (1947).
the Department
of Bacteriology,
and Mortimer University
Received
March
P. Starr
of California,
Davis,
California
6, 1952
In recent years the study of bacterial pectic enzymes has lagged behind its counterpart in plant and mold enzymes. From work on enzymes derived principally from citrus, alfalfa, and tomatoes, plants have been shown to be rich in pectinesterase (PE) but poor in polygalacturonase (PG). Similarly, studies on pectic enzymes derived from mold mycelium and mold culture filtrates have provided good evidence that some molds produce abundant quantities of both enzymes (I). Although the statement that the soft-rot bacterial group produce both PE and PG is encountered frequently, the published evidence supporting this opinion is, unfortunately, very fragmentary. Davison and Willaman (a), who were pioneer workers in this field, found that Erwinia carotovora, grown on carrot decoction, produced a moderately strong PG but they could not demonstrate the presence of PE by the calcium pect,ate method. Since then, Kertesz (3) has shown that this method is unreliable in the presence of PG because of interference by t,he latter enzyme. Norman (4) studied the rate at which pectin disappeared from a pectin medium inoculated with soft-rot bacteria by noting t,he time interval between inoculation and failure to obtain a calcium pectate precipitate in the culture medium. His experiment may be regarded as an indirect demonstration of PG production. Later, Oxford (5) reported active PG preparations obtained by alcoholic precipitation of the enzyme from cultures of plant pathogenic pseudomonads growing in a pectin medium. Mills (6) reported PE production by a strain of Pseudomonas prunicola only when the carbon source in the growth medium was pectin, pectic acid, or galacturonic acid. No PE activity was detected when the carbon substrate was either glucose or galactose. 1 Present
address:
Wallace
and
Tiernan 271
Company,
Monrovia,
Calif.
272
A. J. KRAGHT
AND M. P. STARR
Recently, Wood (7) reported that the pectic enzyme or enzymes produced by Erwinia (Bacterium) aroideae, grown on synthetic medium, contained little PE, but did contain an enzyme, activated by calcium ions, which rapidly reduced the viscosity of high-methoxyl pectin solutions, yet was unable to increase substantially the reducing power of the solution. The latter property suggests that this enzyme is different from bacterial PG reported previously (2, 5). The present study was undertaken as part of an investigation (8,9) on the biochemical behavior of the soft-rot bacteria. Evidence is presented that the strain of Erwinia carotovora used in this study produced both PE and PG when grown in a pectin medium. The amount of PG detected in the culture filtrate was materially greater when the organism was grown in pectin rather than glucose media. EXPERIMENTAL
METHODS
Culture Used The strain of Erwinia carotovora used in this study was isolated from soft-rot spoilage in a commercial shipment of green peppers grown in Los Mochis, Mexico, and shipped to the Los Angeles market in 1949. This otherwise typical strain produced little or no visible gas in the usual sugar fermentation tests. A report on the end products of utilization, by the culture, of galacturonic acid and glucose has been prepared (9).
Pectin Growth Medium The organism was grown in a semisynthetic pectin medium containing 10 g. pectin National Formulary (N.F.), 4 g. (NH&S04, 13 g. KHzPOI, 9 g. Na2HPOI, 0.8 g. MgSOc.HzO, 1 mg. HaB03, 2.mg. CuSOa.5H20, 20 mg. FeCh*6H20, 2 mg. MnS04*HsO, 2 mg. Na&IoO(.2HzO, 26 mg. CaCh, and 10 mg. ZnCln per liter. The pectin and mineral components of the medium were sterilized separately by steaming and added aseptically when needed. The completed medium was adjusted to approximately pH 6.9 with sterile.0.25 N NaOH. Incubation temperature was 30”, unless otherwise specified. To inoculate 106 ml. of the medium, 0.05 ml. was transferred from a 24-hr. culture of the organism growing on a medium of the same composition.
Assay of PE Activity Production of PE by Erwinia carotovora, while growing on the pectin medium, was followed by determining the amount of methyl alcohol in the pectin medium at various time intervals after inoculation. Methyl alcohol was estimated after oxidation to formaldehyde with potassium permanganate in the presence of phosphoric acid by a slight modification of the chromotropic acid (1,3-dihydroxynaphthalene-3,6-disulfonic acid) method of Boos (10). This method was found
PECTIC ENZYMES OF ERWINIA
CAROTOVORA4
273
satisfactory, although its weakness lies in the oxidation procedure. For this reason, known amounts of methanol were included in each determination. Preliminary separation of the methanol from the medium was accomplished by steam dist#illation; five volumes of distillate were collected.
Assay of PC Activity Polygalacturonase was demonstrated, as is commonly done with plant and fungal extracts, by showing an increase in reducing groups when the enzyme is allowed to act on pectic acid. The assay was carried out in the presence of fluoride in the following manner: eqch 250-ml. reaction flask contained 10 ml. of a 2% solution of sodium pectate, 20 ml. of the supernatant resulting from centrifugation of a 72-hr. culture grown at 30” (usually in the pectin medium, unless stated otherwise), 0.08 ml. of 1 M sodium fluoride, sufficient 1 A; HCl or NaOH to adjust to the desired pH (usually about 5.8, unless otherwise stated), and distilled water to make a total volume of 40ml. The mixture was held at 25” and its reducing power determined periodically by the hypoiodite method described by Jansen and MacDonnell (11). All values were correct.ed for the small amount of reducing power found in the heat-inactivated crude enzyme preparation.
Deiermination of Relative Viscosity The relative viscosity of the pectin medium at various times after inoculation was determined in an Ostwald viscometer held in a constant-temperature water bath at 30”. .411 determinations were made in duplicate.
Ethyl Alcohol Precipitate The ethyl alcohol precipitation test was performed by adding 10 ml. of 95% ethyl alcohol to 5 ml. of culture liquor and observing the nature of the precipitate formed. EXPERIMENTAL
RESULTS
Pectinesterase In the experimental data given in Table I, the accumulation of methyl alcohol in the medium, as pectin becomes more and more degraded, may be taken as conclusive evidence of PE activity. Changes in the relative viscosity of the medium, coupled with decreasing ability to obtain a gelatinous alcohol precipitate of pectin, are evidence of degradation of the pectin in the inoculated medium. The pH of the growth medium dropped from 6.9 to 6.2 in the 72-hr. experimental period. Only an insignificant amount of de-esterification occurred during the first 24 hr. despite a 700-fold increase in bacterial numbers and a substantial decrease in viscosity of the pectin medium. Within 48 hr., approximately two thirds of the potential methanol had been liberated,
274
A.
J.
KRAGHT
AND
M.
and by the end of the experiment hydrolysis but, progressively increasing, amount of from the uninoculated medium incubated instability of pectin in combination with medium. Additional experiments showed that thd was not unique in producing PE; the carotovora were tested: a) E. carotovora, Division of Plant Pathology, Ijniversity
inoculation
-___~-
0 24 48 72
Relative
STARR
was 94% complete. A small, methyl alcohol was obtained at 30”, which may be due to the other ingredients of the Mexican green pepper isolate following strains of Erwinia obtained in 1948 from the of California, Davis, Cali-
TABLE I of Pectin by a Growing Culture of Erwinia the 1% Pectin N.F.mMedium
Progressive Degradation
:rtz
P.
viscosity
Alcohol
precipitate*
Number
carotovora
in
Methyl alcohol liberatedi mg.fg. pectznc
of bacteria/ml.
-I-
A“
B=
C=
A ____-
12.4 11.8 12.1 4.8 4.6 9.1 1.4 1.3 7.7 1.3 1.2 7.0
t++ +++ ? -
-
B
+++ +++ ? -
c
A
+++29 x +++19 x +++ 29 X +++26 X
B
10429 10718 10711 lo825
x x X X
C -__
104 107 lo8 lo*
a A and B inoculated; C uninoculated. * Plus (+) indicates a gelatinous precipitate, while minus flocculent precipitate. c Theoretical amount is 92.1 mg. CH,OH/g. pectin N.F.
A
B
0 1.1 1.0 0 2.1 1.7 0 63.7 60.0 0 87.4 86.0 (-)
indicates
C
1.0 1.2 6.5 9.0 a
fornia; b) E. carotovoru (ATCC 495) ; c) E. curotovora (EC104), originally isolated by Dr. P. A. Ark in 1946 from soft-rot of potatoes; and d) E. carotovora var. delphinii (EC108), originally isolated by Dr. P. A. Ark from larkspur in 1935. Each strain was grown in a medium whose composition was 0.5% pectin N. F., 0.1% peptone, and 0.1% yeast extract. Forty-eight hours after incubation at 30”, portions of the culture liquor were steam-disl tilled, and methyl alcohol in the distillate was determined by the chromotropic acid method. Methyl alcohol, in amounts considerably in excess of that found in the control medium, was detected in all of the culture fluids. Therefore, it seemsthat PE was produced by all the tested strains of Ewinia carotovora.
PECTIC
ENZYMES
OF
ERWINIA
CAROTOVORA
275
Polygalacturonase Polygalacturonase (PG) could not be demonstrated directly in the growing culture because galacturonic acid is utilized rapidly by Ihe organism (9) and does not accumulate in the medium. Removal of the bacterial cells remaining after centrifugation by passage of the supernatant culture liquor through a fine-porosity Mandler filter candle yielded an active PG preparation as shown by an increase in reducing groups when the filtrate was added to a solution of sodium pectate. The addition
FIG.
in the
1. Effect of carbon source (loJo glucose or 0.5(% glucose growth medium on the production of polygslacturonase
plus 0.5% pectin) by Erwinia caro-
tovora.
of 0.02 M sodium fluoride or sodium azide to the supernatant of the centrifuged culture liquor without first removing the remaining bacterial cells similarly permitted demonstration of PG activity. The effect of pH from 3.3 to 7.9 on PG activity was determined. At intervals of 5, 10, 15, 20, 30, 60 and 90 min., 5-ml. aliquots of the reaction mixture were removed for the determination of reducing power. PG was found to be active over a wide pH range ext,ending from 3.9 to i.9; it has a broad optimum around pH 5.8. The effect of pectic substances in the growth medium on the formation of PG was demonstrated by growing E. carotovora in media in which glu-
276
A.
J.
KRAGHT
AND
M.
P. STARR
case had been substituted for all or for half of the pectin in the growth medium. A 24-hr. inoculum for both media was prepared by transferring the culture from beef-yeast extract agar storage. slants to the glucose medium. Growth in each medium during the 72-hr. incubation period was comparable as evidenced by identical turbidities, expressed as percentage transmission, of 15% on the Evelyn calorimeter with filter 420. From the data given in Fig. 1, it can be seen that only a small amount of PG was produced by the organism when grown on the glucose medium. PG activity was increased greatly when the organism was grown on the combination medium containing both pectin and glucose. Since there had been no prior exposure of the inoculum to pectin, the increased PG activity, when pectin was added to the growth medium, may be considered as stimulation. Yet it was necessary to exclude the possibility that greater PG activity in the glucose culture had occurred at some earlier stage of bacterial growth and was disappearing from the medium when the sample was taken. This point was checked by growing the organism in the synthetic glucose medium and determining PG activity as a function of the age of the culture. It was found that at no prior time were pectic enzymes produced in any larger quantity than was found at the end of the 72-hr. incubation period. Therefore, it seems valid to hold that PG activity was greatly increased by the incorporation of pectin in the medium. DISCUSSION
The demonstration of methyl alcohol liberation by several strains of E. carotovora growing in a high-methoxyl pectin medium may be considered as conclusive evidence of PE activity. Although in this work PE was demonstrated in the presence of growing cells only, it seems reasonable to expect that cell-free preparations of the culture fluid would contain an active PE capable of hydrolyzing the methoxyl groups in pectin. The success achieved by Mills (6) with partially purified PE from cultures of Pseudomonas prunicola can be taken as a basis for the above assumption. Polygalacturonase production by E. carotovora cultures has been demonstrated by previous workers (2). However, its optimum pH of 5.8 is now shown to be lower than the optimum of pH 7-8 reported by Oxford (5) for PseudomonasPG, and higher than the optimum pH of about 3.54.0 reported for yeast (12) and fungal (13) PG.
PECTIC
ENZYMES
OF
ERWINIA
CAROTOVORA
277
The effect of pectic substances in the medium on the formation of PG is consistent with previous reports (13, 14). Certainly any report that an organism does or does not produce PG should be considered in light of the growth medium used. The difficulty in demonstrating PO in the present study, when the organism was grown in a glucose medium, amply illustrates the validity of the above statement. The present study clearly places E. carotovora in the same category as some molds in that both PE and PG are formed. That Erwinia soft-rot bacteria may produce other pectic enzymes is suggested by the aforementioned work of Wood (7). STJMMARY
A culture of Erwinia carotovora isolated from rotted Mexican bell pepper liberated methanol practically quantitatively from pectin when growing in a pectin medium. This phenomenon is considered to be positive evidence of pectinesterase activity. Polygalacturonase could not be demonstrated directly in the culture medium containing actively growing cells because the subsequent rapid utilization of galacturonic acid made positive identification of the enzyme uncertain. Because polygalacturonase is insensitive to both fluoride and azide, and the galacturonic acid degrading system is inhibited by these substances, it was possible to demonstrate and study the polygalacturonase in the presence of these metabolic inhibitors. E. carotovora polygalacturonase is active over a wide pH range, the optimum being around 5.8. Its production or secretion into the medium is greatly increased by the addition of pectic substances to the growth medium. REFERENCES 1. 2. 8. -I. 5. 6. 7. 8. 0. 10. 11. 12. 13.
14.
LINEWEAVER, H., AND JANSEN, E. F., Advances in Enzymol. 11, 267 DAVIEON, F. R., AND WILLAMAN, J. J., &tan. Gaz. 83,329 (1927). KERTESZ, Z. I., J. BioZ. Chem. 121, 589 (1937). NORMAN, A. G., Ann. Botany 43, 233 (1929). OXFORD, A. E., Nature 164, 271 (1944). MIWS, B. G., Biochem. J. 44,302 (1949). WOOD, R. li. S., Nature 167, 771 (1951). STARR, M. P., AND MANDEL, M., J. Bact. 60, 669 (1950). KRAGHT, A. J., AND STARR, M. P., J. Bacf. 64, 259 (1952). Roes, R. N., .4naZ. Chem. 20, 964 (1948). JANSEN, E. I?., AND MACDONNELL, L. R., Arch. Biochem. 8,97 (1945). LUH, B. S., AND PHAFF, H. J., Brch. Biochem. and Biophys. 33, 212 PHAFF, H. J., AND JOSLYN, M. A., Walterstein Labs. Communs. 10, 133 PHAFF, H. J., Arch. Biochem. 13, 67 (1947).
(1951).
(1951). (1947).