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Identification of indigo produced in submerged culture of Agaricus campestris, mutant culture
ARCHIVES
OF BIOCHEMISTRY
AND
Identification
96, 430-433
RIOI'HYSIC'S
of Indigo Agaricus
Produced
From
Mutant AND
August
Escola Paula,
EXPERIMENTAL AND
Superior Brazil
de
21, 1961 produced a blue was identified.
pig-
by shaking. Two milliliters of the suspension so obtained was aseptically inoculated in 500-ml. Erlenmeyer flasks, each containing 200 ml. of the following culture medium (3) : 40 g. glucose monohydrate; 1 g. ammonium tartrate; 0.5 g. monopotassium phosphate; 0.5 g. magnesium sulfate heptahydrate; 0.003 g. ferrous sulfate heptahydrate; 0.003 g. zinc sulfate heptshydrate; 0.002 g. mnnganous sulfate monohydrate; 0.05 g. calcium chloride; 1 mg. pyridoxine; I mg. p-aminobenzoic acid; 0.5 mg. thiamine; water up to 1000 ml., pH 5.0. The Erlenmeyer flasks, after 2 days resting, were transferred to a rotatory shaker developing 240 rpm. with an eccentricity of 1 in. where the cultures ‘rvere grown at 22” in the dark. After 1 week the mycelium was separated by filtration and washed with distilled water. Since coloring matter was still retained by the mycelium, it was blended in a Waring blendor with some distilled water. The resultant, suspension was centrifuged at 2,000 r.p.m. The slightly turbid, blue snpcrnatant liquor sepnrated from the pigment-free mpcelium was combined with the filtrate and extracted several times with chloroform. The chloroform extracts dried o\-er anhydrous N&O1 were evaporstcd almost to tlryncss, and pct,roleurn ether (30-50”) was added to precipitate the blue pigment. which was collected and dried, The petroleum et.her-chloroform residual solution was a rctl-brown color, and after elimination of 11~1; colwnls a gummy residue was obtained.
During the study of dgaricus campestris in submerged culture (1) it was observed that in some experiments the mycelium and medium became green colored and finally turned to blue. The same blue color was observed in some transferred cultures from the stock culture. Although several workers have reported isolation and identification of pigments from fungi (2) and other microorganisms, no report has been made of blue pigments in Agnricus campestris in submerged culture. The coloring matter isolated from Agaricus cnmpestris could be separated into three main fractions. The first one was identified as indigo, the second one tentatively ident,ified as indirubin, and a third brown-colored fraction not, identified.
~IATERIALS
of
P. A. BOBBIO
culture of a mutant culture of Agaricus campestris could be resolved into three fractions in which indigo
INTRODUCTION
Culture
Culture
the Chemistry Department, Instituto Zimotknico, Agricultura “L&z de QueZroz,” University of Silo Received
Submerged ment which
in Submerged
campestris,
H. FALAKGHE
(1962)
METHODS
Tile organism uwtl in this work was the Agrrric?(s caozpc.stris (I,) Fr. CBS, mutant culture originatcd during our esperimc,nts, and named by us A. cc~mpcslris (I,) Fr. blue mutant. The inowlunl was produced by the transference of mycelium bits, from mycelium tlereloped in P.D.A. slants at 15°C. in the dark, to 250~ml. Erlenmcyer flasks containing 40 ml. of a 5% malt-extract. broth. rlftcr incubation for 10 days at 22°C. in the dark, the inoculum was washed with sterile distilled water and aseptically transferred to 250-ml. glass-stoppered Erlenmeyer flasks containing glass beads and 40 ml. of sterile distilled water; and the mycelium \vas broken up
P~RIE'ICATION
AND THE
BLUE
CHARACTERIZ.~TIOS
OF
PIGMEKT
The pigment was practically insoluble in water, dilute mineral acids and alkali, in methanol (MeOH), ethanol (EtOH), ace430
INDIGO
1% MUTANT
tone, petroleum ether; slightly soluble in benzene, soluble in chloroform and cont. sulfuric acid with intense blue color. It gave a positive test for K (4), negative for sulfur and halogens. Reduction with zinc in NaOH produced discoloration of the pigment which returned to blue by passing a stream of oxygen through the discolored solution. The pigment was purified by sublimat,ion at 250°C. under pressure of 0.01 mm. Hg. h blue coppery lustrous microcrystalline product was obtained and identified as indigo by comparing its infrared absorption spectrum wit’11 the infrared spect,rum of pure indigo (Fig. 1,l , and by comparing the ult,raviolet absorption spectrum of the pigment and pure indigo in chloroform (5)) using a Beckman DG model (Fig. 2). Elementary analysis of the blue pigment agrees with the calculated values for C, H, N of indigo. Anal. Calculated: C, 73.27; H, 3.84; K, 10.68. Found: C, 72.86; H, 4.01; N, 10.41.
431
CULTURE
vcloping
with
benzene-ethyl
acetate
(10: 3
V/V).
The material from the red zone was rechromatographed on alumina (6j, and the column was developed with petroleum ether (50-WC.j-ethyl acetate (2: 1 v/v). Two zones were developed: a top narrow brown zone and a lower red one which was recovered from the column. Its identification was made by means of its absorption spectrum in benzene using a Beckman DU model (Fig. 3 J . DISCUSSIOK
Since the pigmentation only appeared in some of the transferred cultures and in submerged cultures originated from them, we considered the pigment producing Agariclbs campestris a mutant culture. The initial 2 days resting of the inoculated flasks prior to shaking is important for the pigment production. When the flasks were transferred to the rotatory shaker immediately after inoculat,ion, pigmentation PURIFI~ATIOS .ua IDENTIFICATION 0~ was slight and only in a few flasks; while THE RED PIGMEST when resting was obscrT’cd, pigmentation was intensive and in all flasks. A benzene solut#ion of the gummy reddish The experimental data show conclusively brown product separated from indigo was chromatographcd on alumina (6). The colthxt the blue pigment produced by dgclricus umn was dcvelopcd with benzcnc until a cnrnpestris is indigo not completely pure lo~-cr green band was completely eluted. =1 (Fig. 1). Possibly the impurities were artilarge brown zone at t,he top of t,he column facts due to the sublimation of the pigwas separated from a lower red zone by de- ment 16).
FIG.
Lower
1. Infrared spectrum spectrum : indigo.
in ?;uiol.
Upper
spectrum:
blue
pigment
from
A. campestiis.
432
F.~I,AIYGIIE
.\KD
BOBBIO
.-
FIG. 2. Visible and ultraviolet campeslris. Solid line : indigo.
spectra in chloroform.
Dotted line: blue pigment from A.
0.4
0.35
0.30
g
0.25
s x
0.2
ii iz 8
0.15
0.1
0.05
700
FIG. 3. Ultraviolet
650
600
550
500
spectrum of red pigment from A.
450
campestris.
43omJ.J
1I;DIGO
IN
MUTANT
The second pigment purified by chromatography on alumina could be tentatively identified as indirubin, since its absorption maximum (Fig. 3) agrees with the maximum of the absorption curve obtained by Rimington (6). There is a similarity between natural coloring matter obtained from various spcties of Indigofera and the coloring matter isolated from the higher fungus ilgarims mmpestris. In both cases at least three fractions can be separated: indigo, indirubin, and a brown pigment. There is a correlation between pigmentation and mycelium weight’ expressed by a decrease in mycclium weight when pigmentation appears. The production of pigment was accompanied by an increase in the nitrogen content of the mycclium dctcrmincd by the micro-Kjeldahl mct’hod.
CULTURE
433
ACKNOWLEDGMENTS Grants from the Rockefeller Foundation and Conselho Kacional de Pesquieas are gratefully acknowledged. The zu1t~11or.s arc indebted to Dr. 0. R. Gottlieb from the Instituto de Quimica .1gricola (Rio de Janeiro) for the infrared spectra of indigo.
1. HUMFELD, H., S&we 107, 373 (1948). 2. MII,ES, G. P., LUSD, H., AID RAPER, J. R., Arch. Riochem. Riophys. 62, l-5 (1958) ; RAISTRICX, H., 8~. Xc{). Riochcnl. 9, 571 (1940). 3. RECXSER, F., SPENCER, J. F. T., AKD SALL~XS, H. R,. Appl. Mico&oZ. 6, l-4 (1958). 4. FEIGL, F., “Spot Tests,” l-01 II. p 73. Elsevier Publ. Co., 1954. 5. BRODE, R. JV., PEARSOK, E. G., .~SD JVYMAX, G. hl.. 1. Am. Chem. Sot. 76, 1034-6 (1954). 6. ~~IMISGTOS, c., &ochem J. 40, 669-74 (1946)
OF BIOCHEMISTRY
AND
Identification
96, 430-433
RIOI'HYSIC'S
of Indigo Agaricus
Produced
From
Mutant AND
August
Escola Paula,
EXPERIMENTAL AND
Superior Brazil
de
21, 1961 produced a blue was identified.
pig-
by shaking. Two milliliters of the suspension so obtained was aseptically inoculated in 500-ml. Erlenmeyer flasks, each containing 200 ml. of the following culture medium (3) : 40 g. glucose monohydrate; 1 g. ammonium tartrate; 0.5 g. monopotassium phosphate; 0.5 g. magnesium sulfate heptahydrate; 0.003 g. ferrous sulfate heptahydrate; 0.003 g. zinc sulfate heptshydrate; 0.002 g. mnnganous sulfate monohydrate; 0.05 g. calcium chloride; 1 mg. pyridoxine; I mg. p-aminobenzoic acid; 0.5 mg. thiamine; water up to 1000 ml., pH 5.0. The Erlenmeyer flasks, after 2 days resting, were transferred to a rotatory shaker developing 240 rpm. with an eccentricity of 1 in. where the cultures ‘rvere grown at 22” in the dark. After 1 week the mycelium was separated by filtration and washed with distilled water. Since coloring matter was still retained by the mycelium, it was blended in a Waring blendor with some distilled water. The resultant, suspension was centrifuged at 2,000 r.p.m. The slightly turbid, blue snpcrnatant liquor sepnrated from the pigment-free mpcelium was combined with the filtrate and extracted several times with chloroform. The chloroform extracts dried o\-er anhydrous N&O1 were evaporstcd almost to tlryncss, and pct,roleurn ether (30-50”) was added to precipitate the blue pigment. which was collected and dried, The petroleum et.her-chloroform residual solution was a rctl-brown color, and after elimination of 11~1; colwnls a gummy residue was obtained.
During the study of dgaricus campestris in submerged culture (1) it was observed that in some experiments the mycelium and medium became green colored and finally turned to blue. The same blue color was observed in some transferred cultures from the stock culture. Although several workers have reported isolation and identification of pigments from fungi (2) and other microorganisms, no report has been made of blue pigments in Agnricus campestris in submerged culture. The coloring matter isolated from Agaricus cnmpestris could be separated into three main fractions. The first one was identified as indigo, the second one tentatively ident,ified as indirubin, and a third brown-colored fraction not, identified.
~IATERIALS
of
P. A. BOBBIO
culture of a mutant culture of Agaricus campestris could be resolved into three fractions in which indigo
INTRODUCTION
Culture
Culture
the Chemistry Department, Instituto Zimotknico, Agricultura “L&z de QueZroz,” University of Silo Received
Submerged ment which
in Submerged
campestris,
H. FALAKGHE
(1962)
METHODS
Tile organism uwtl in this work was the Agrrric?(s caozpc.stris (I,) Fr. CBS, mutant culture originatcd during our esperimc,nts, and named by us A. cc~mpcslris (I,) Fr. blue mutant. The inowlunl was produced by the transference of mycelium bits, from mycelium tlereloped in P.D.A. slants at 15°C. in the dark, to 250~ml. Erlenmcyer flasks containing 40 ml. of a 5% malt-extract. broth. rlftcr incubation for 10 days at 22°C. in the dark, the inoculum was washed with sterile distilled water and aseptically transferred to 250-ml. glass-stoppered Erlenmeyer flasks containing glass beads and 40 ml. of sterile distilled water; and the mycelium \vas broken up
P~RIE'ICATION
AND THE
BLUE
CHARACTERIZ.~TIOS
OF
PIGMEKT
The pigment was practically insoluble in water, dilute mineral acids and alkali, in methanol (MeOH), ethanol (EtOH), ace430
INDIGO
1% MUTANT
tone, petroleum ether; slightly soluble in benzene, soluble in chloroform and cont. sulfuric acid with intense blue color. It gave a positive test for K (4), negative for sulfur and halogens. Reduction with zinc in NaOH produced discoloration of the pigment which returned to blue by passing a stream of oxygen through the discolored solution. The pigment was purified by sublimat,ion at 250°C. under pressure of 0.01 mm. Hg. h blue coppery lustrous microcrystalline product was obtained and identified as indigo by comparing its infrared absorption spectrum wit’11 the infrared spect,rum of pure indigo (Fig. 1,l , and by comparing the ult,raviolet absorption spectrum of the pigment and pure indigo in chloroform (5)) using a Beckman DG model (Fig. 2). Elementary analysis of the blue pigment agrees with the calculated values for C, H, N of indigo. Anal. Calculated: C, 73.27; H, 3.84; K, 10.68. Found: C, 72.86; H, 4.01; N, 10.41.
431
CULTURE
vcloping
with
benzene-ethyl
acetate
(10: 3
V/V).
The material from the red zone was rechromatographed on alumina (6j, and the column was developed with petroleum ether (50-WC.j-ethyl acetate (2: 1 v/v). Two zones were developed: a top narrow brown zone and a lower red one which was recovered from the column. Its identification was made by means of its absorption spectrum in benzene using a Beckman DU model (Fig. 3 J . DISCUSSIOK
Since the pigmentation only appeared in some of the transferred cultures and in submerged cultures originated from them, we considered the pigment producing Agariclbs campestris a mutant culture. The initial 2 days resting of the inoculated flasks prior to shaking is important for the pigment production. When the flasks were transferred to the rotatory shaker immediately after inoculat,ion, pigmentation PURIFI~ATIOS .ua IDENTIFICATION 0~ was slight and only in a few flasks; while THE RED PIGMEST when resting was obscrT’cd, pigmentation was intensive and in all flasks. A benzene solut#ion of the gummy reddish The experimental data show conclusively brown product separated from indigo was chromatographcd on alumina (6). The colthxt the blue pigment produced by dgclricus umn was dcvelopcd with benzcnc until a cnrnpestris is indigo not completely pure lo~-cr green band was completely eluted. =1 (Fig. 1). Possibly the impurities were artilarge brown zone at t,he top of t,he column facts due to the sublimation of the pigwas separated from a lower red zone by de- ment 16).
FIG.
Lower
1. Infrared spectrum spectrum : indigo.
in ?;uiol.
Upper
spectrum:
blue
pigment
from
A. campestiis.
432
F.~I,AIYGIIE
.\KD
BOBBIO
.-
FIG. 2. Visible and ultraviolet campeslris. Solid line : indigo.
spectra in chloroform.
Dotted line: blue pigment from A.
0.4
0.35
0.30
g
0.25
s x
0.2
ii iz 8
0.15
0.1
0.05
700
FIG. 3. Ultraviolet
650
600
550
500
spectrum of red pigment from A.
450
campestris.
43omJ.J
1I;DIGO
IN
MUTANT
The second pigment purified by chromatography on alumina could be tentatively identified as indirubin, since its absorption maximum (Fig. 3) agrees with the maximum of the absorption curve obtained by Rimington (6). There is a similarity between natural coloring matter obtained from various spcties of Indigofera and the coloring matter isolated from the higher fungus ilgarims mmpestris. In both cases at least three fractions can be separated: indigo, indirubin, and a brown pigment. There is a correlation between pigmentation and mycelium weight’ expressed by a decrease in mycclium weight when pigmentation appears. The production of pigment was accompanied by an increase in the nitrogen content of the mycclium dctcrmincd by the micro-Kjeldahl mct’hod.
CULTURE
433
ACKNOWLEDGMENTS Grants from the Rockefeller Foundation and Conselho Kacional de Pesquieas are gratefully acknowledged. The zu1t~11or.s arc indebted to Dr. 0. R. Gottlieb from the Instituto de Quimica .1gricola (Rio de Janeiro) for the infrared spectra of indigo.
1. HUMFELD, H., S&we 107, 373 (1948). 2. MII,ES, G. P., LUSD, H., AID RAPER, J. R., Arch. Riochem. Riophys. 62, l-5 (1958) ; RAISTRICX, H., 8~. Xc{). Riochcnl. 9, 571 (1940). 3. RECXSER, F., SPENCER, J. F. T., AKD SALL~XS, H. R,. Appl. Mico&oZ. 6, l-4 (1958). 4. FEIGL, F., “Spot Tests,” l-01 II. p 73. Elsevier Publ. Co., 1954. 5. BRODE, R. JV., PEARSOK, E. G., .~SD JVYMAX, G. hl.. 1. Am. Chem. Sot. 76, 1034-6 (1954). 6. ~~IMISGTOS, c., &ochem J. 40, 669-74 (1946)