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The isolation of trehalose and polyols from the conidia of Penicillium chrysogenum thom
ARCHIVES
OF
The
BIOCHEMISTRY
AND
Isolation
BIOPHYSICS
of Trehalose Penicillium
A. BALLIO, From
the International
Centre
and
Chemical
AND
from
April
the
Conidia
of
Thorn.
AND
Microbiology,
SERENA
Istituto
Superiore
RUSS1 di Sanitci,
Rome,
Italy
13, 1964
n-arabitol, meso-erythritol, and glycerol were isolated chrysogenum and were quantitatively assayed. Their and role during germination are discussed.
During the course of studies on acid-soluble nucleotides in conidia of Penicillium chrysogenum at this laboratory (I), it was observed that the supernatant remaining after precipitation of phosphate esters with mercuric acetate contained a considerable amount of n-mannitol, as well as other substances detected by alkaline silver nitrate on paper chromatograms. As a detailed knowledge of conidial components could be of some importance in the study of biochemical processes involved in the morphological differentiation of filamentous fungi, the nature of these substances was further investigated. The present paper deals with the identification and quantitative determination of glycerol, nzeso-erythritol, n-arabitol, n-mannitol, and trehalose in extracts of the ungerminated conidia of PenicilliuvL chrysogenwn. Some of these compounds have been detected in ethanolic extracts of the same mould by 34orita (2). MATERIALS
Polyols
V. DI VITTORIO, for
(1964)
chrysogenum
Received Trehalose, n-mannitol, the conidia of Penicillium ble mode of formation
177-183
107,
from possi-
Merck, Germany; D-arabitol from Fluka, Switzerland; L-arabitol from Pfanstiehl Chemical Co., U. S. A.; and trehalose dihydrate from Difco, U. S. A. n-Ribitol and n-xylitol were prepared, respectively, from n-ribose and n-xylose by reduction with sodium borohydride. Each pentose (60 mg) dissolved in water (4 ml) was treated at room temperature with solid sodium borohydride (76 mg). After 16 hours the solutions were deionized by shaking with Dowex 1 X 2 (100-200 mesh OHcycle) and with Dowex 50 X 1 (100-200 mesh, H+ cycle), and then filtered. After having checked that both solutions gave a negative reaction with triphenyltetrazolium chloride, they were used for paper chromatography without any further treatment. Dowex resins were purchased from Fluka, Switzerland. ANALYTICAL
METHODS
The determinations of polyols were made by periodate oxidation followed by assay of formaldehyde, according to the method of Argant (3). The assays on fractions collected from the cellulose column were made after evaporation of the solvents at reduced pressure and room temperature. Trehalose was determined polarimetrically with a Hilger polarimeter (sodium lamp) in a l-dm microtube by using an [ol]n value for the dihydrate in aqueous solution of +184”. The measurements of optical rotation in ammonium molybdate solution were made according to the method of Richtmyer and Hudson (4). Small quantities of material were estimated in the 2-dm polarimeter microtube. Melting points were determined with a Kofler hot-stage apparatus and are uncorrected. Paper chromatograms were developed by the descending method by using Whatman So. 1 filter
METHODS
CHEMICaLS All chemicals were C. Erba products of analytical grade, with the following exceptions: cellulose powder was Whatman’s ashless standard grade for chromatography; activated charcoal was a product of the British Drug Houses Ltd., England; Celite 503 was supplied by Light & Co. Ltd., England (it was washed with a large volume of 3 Ar hydrochloric acid, followed by distilled water until free of chloride ions, and finally dried at 105°C) ; meso-erythritol was purchased from 177
BALLIO,
178
DI VITTORIO,
paper. The following solvent systems were used: (a) 2-butanone-acetic acid-saturated aqueous solution of boric acid (9: l:l), (b) n-butanolacetone-water (2:7:1), (c) n-butanol-pyridinewater (6:4:3), (d) n-butanol-acetic acid-water (4: 1:5), upper layer, and (e) ethylacetate-pyridine-water (8:2: 1). Polyols and trehalose were detected either by alkaline silver nitrate (5), or by a periodate reagent followed by benzidine (6). The latter was always used when chromatograms were developed with solvent system a. Reducing substances were detected by spraying with equal volumes of triphenyltetrazolium chloride (2yi solution in water saturated n-butanol) and ethanolic 0.1 hi potassium hydroxide freshly mixed.
PRODUCTION
OF COSIDIA
Penicillium chrysogenum (49-133 Wis) was stored in sterile sand, and cultures were prepared monthly from the sand in test tubes containing an agar-sucrose-bean nutrient. After 67 days at 27” conidification in these tubes was complete, and the cultures were stored at 5” for up to 1 month. A sterile water suspension of conidia was prepared from these, and this in turn was used to inoculate Fernbach flasks (20 cm in diameter), each of which contained 150 gm of pearl barley moistened with 10 ml of the Whiffen and Savage sporulating solution (7). Prior to inoculation, the flasks were sterilized for 20 minutes at 100” and for 20 minutes at 120”. Conidification in the inoculated flasks was complete after 7 days at 25”. The yield of conidia per flask was about 3 gm (dry weight). Conidia were also produced on an agar-glycerol-beet molasses medium having the following composition (gm/liter): glycerol, 7.5; beet molasses, 7.5; yeast extract, 5; NaCl, 10; MgS04.7Hz0, 0.005; KH2P04, 0.006; (NHn)zFe(S04)2.6H20, 0.0016; CuS04.5H~0, 0.001; CaS04.2H20, 0.250; agar, 25; the pH was adjusted to 6.4. The medium was sterilized in Roux bottles for 20 minutes at 100” and for 20 minutes at 120”. Conidification was complete after 7 days at 25”.
EXTRACTION
OF CONIDIA
After conidification on pearl barley, 150-200 ml of distilled water was added to each flask, and the contents were stirred with a glass rod for a few minutes. The green aqueous suspension was decanted from the grains of barley, filtered through cotton gauze, and centrifuged. The sediment was washed 3 times with distilled water and then resuspended in a small volume. All these operations were carried out at 3”-5”. The suspension was poured into boiling aqueous ethanol to give a final alcohol content of 5Oyc. The volume of liquid corresponded to approximately 8 ml per gram dry
AND
RUSS1
weight of conidia. After boiling for 5 minutes, the suspension was cooled with tap water and centrifuged. The sediment was extracted twice more under identical conditions by using 50% ethanol. The combined extracts were acidified with nitric acid and treated with mercuric acetate according to the method of Caputto and co-workers (8). The suspension was filtered and the filtrate was treated with hydrogen sulfide to free it from mercuric ions. The clear solution, obtained after removing mercuric sulfide by filtration through Celite 503, was freed from hydrogen sulfide by treatment with a stream of air, concentrated in a rotatory evaporator to half its original volume, applied to a column of Dowex-1 X 2 (100-200 mesh, OH- cycle; the volume was 5 times the original dry weight of the conidia or j$ the volume of the conidial extract), and eluted directly with water. Fractions corresponding to s/4 of the column’s volume were collected, and each fraction was tested on paper for the presence of compounds that react with alkaline silver nitrate. Fractions giving a positive reaction were pooled and passed through a small column (j43 the volume of the first column) of Dowex 50 X 2 (100-200 mesh, H+ cycle), followed by two column volumes of water. The combined effluent and eluate were pooled and freeze-dried. The conidia produced on the agar-glycerol-beet, molasses medium were removed by wetting them with water containing a minute amount of commercial sodium lauryl sulfate and carefully scraping the surface of the medium with a plug of washed absorbent cotton-wool tied to a steel wire. After filtering through a small amount of cottonwool, to remove mycelial fragments, the conidial suspension was centrifuged; the residue was thoroughly washed with distilled water by centrifugation and then extracted with ethanol, as described above.
PARTITION CHROMATOGRAPHY ON CELLULOSE The residue (0.274 gm) obtained by freezedrying 50 ml of the deionized conidial extract was dissolved in 4 ml of water and was mixed with a small amount of cellulose. The mixture was taken to dryness over calcium chloride in ~acuo and was applied to a column of cellulose powder (440 X 22 mm) which had been packed dry, and then washed with 600 ml methanol followed by 700 ml acetoneand by2000mlof n-butanol-acetone-water (2:7: 1). The latter mixture of solvents was also used to effect elution at the rate of 1 ml per minute. After 185 fractions of 10 ml each had been collected, the column was washed with water and a further twenty lo-ml fractions were collected. Elution was followed by testing individual frac-
TREHALOSE
AND POLYOLS
tions spotted on to paper with alkaline silver nitrate; fractions giving a positive reaction were chromatographed on paper with solvent system b, along with the appropriate reference substances, and t)hen taken to dryness. The residues were weighed, taken up in a measured amount of water, and quantitatively assayed. Polyols were determined by estimating the formaldehyde derived from periodat,e oxidation and trehalose by polarimetry. IDENTIFIC~~TIOS
0~ COMPOUNDS
INDIVIDUAL
This was carried out by paper chromatography in solvent systems a and G. Both alkaline silver nitrate and periodate-benzidine gave single spots with the purified compounds; a triphenyltetrazolium chloride spray consistently showed that reducing compounds were absent. The structure of the isolated products was further confirmed by their melting points and mixed melting points with reference compounds, by their opt,ical rotation in acidified molybdate solution, and by preparing the forlowing derivatives: the tri-p-nitrobenzoate of glycerol, the tetrabenzoate of erythritol, the hexaacetate of mannitol, and the octaacetate of trehalose. RESULTS AX~~LYSIS
OF CONIDML AND A[YCELIAL EXTRACTS BY PAPER CHROMATOGRAPHT
Preliminary examination by paper chromatography of deionized extracts derived from conidia grown on barley suggestedthat glycerol, erythritol, arabitol, mannitol, and trehalose were present (Table I). The same substances were detected in extracts prepared from conidia grown on agar-glycerolbeet molasses medium ; no trace of them was found in an extract of uninoculated barley grains. Before deionization with Dowex1, conidial extracts were found to contain also free and bound glucose. Retention of reducing sugars on anion exchangers in the OH- form is well known (9, 10). The same type of analysis was also used to examine extracts prepared from washed samples of mycelium of PeniciZliu?n ch~ysogenunz collected after 24, 48, 72, and 96 hours growth on the chemically defined medium of Jarvis and Johnson (11). The ratio between dry weight of mycelium and volume of extract was always identical to that ob-
FROM
P.
li9
CHRYSOGENU~~f
tained with the conidia. On inspection of paper chromatograms developed in solvent systems a and c, it was observed that glycerol, erythritol, arabitol, and trehalose, if present, occurred in traces, whereas the amount of mannitol was comparable to that found in the conidial extracts. The occurrence of glucose and of a slow-moving spot, which liberates glucose and galactose on acid hydrolysis, was also consistently noted in the mycelial extracts. I"RAcTI~K:ATI~X
OF CONIDIAL ox
EXTRACTS
A CELLULOSE COLUMN
In order to verify the identity of the compounds detected by paper chromatography, a deionized extract was fractionated on a cellulose column. As shown in Fig. 1, four peaks were eluted with n-butanol-acetonewater (2:7: 1) and a fifth peak with water. Paper chromatography in solvent system c suggested that peaks 1, 2, 3, and 4 corresponded to glycerol, erythritol, arabitol, and mannitol, respectively and that peak 5 contained trehalose. Further evidence for the structure of the individual compounds was obtained from the following experiments. CHz4RhCTERIZATION TREHALOSE CONIDIAL
OF POLYOLS ISOLATED FROM EXTRACTS
BND
Glycerol: A portion of the liquid left after freeze-drying the eluate composing “peak 1” was reacted with p-nitrobenzoylchloride in pyridine. A derivative was obtained which melted at 198” aft,er crystallization from chloroform; the mixed melting point with a pure sample of glycerol tri-p-nitrobenzoate gave no depression. Meso-erythritol: The residue left after freeze-drying “peak 2” was crystallized from ethanol. Prisms with a melting point of 119”120” were obtained; in admixture with pure l?zeso-erythritol the melting point showed no depression.’ The henzoate, after crystallization from acetic acid, melted at 190”; the mixed melting point with a pure sample of 1 The accepted values for ?raeso-erythritol, the n-form, the n-form, and the racemic compound are 120”, 88.5”-89”, 88”, and 72”, respectively (12).
Conidial extract Trehalose D-Mannitol D-Arabitol meso-Erythritol Glycerol
Compounds
COMPARISON
a
2.14
2.51
2.15,2.48,
Solvent (mlucose)
CHROMATOGRAPHIC
0.43,1.70, 0.42 1.73
OF THE
3.05
3.00
IN
TABLE
Solvent (Rr)
b
0.41 0.52
I
0.59,0.97,1.11,1.32,1.53 0.59 0.98 1.14
Solvent c (R~~uoose)
1.35
mobilities
1.55
WITH THAT OF OF PENICILLIUM
Chromatographic
OF KNOWN COMPOUNDS EXTRACT FROM CONIDIA
0.10,0.25,0.33,0.41,0.52 0.10 0.26 0.33
BEHAVIOR A DEIONIZEU
0.08,0.17,0.22,0.30,0.41 0.08 0.18 0.22
Solvent (RJ)
CBRYSOGENUM
PERI~DATE-OXIDIZABLE
d
0.30 0.42
CONTAINED
1.3G
Solvent e Uh~uoose)
0.20,0.86,1.36, 0.20 0.87
SUBSTANCES
2.01 2.94
2.03,2.95
THEHALOSE
Ah-11
POLYOLS
FROM
l’.
181
CH/Z~‘SOGE.YL’C’.lf TABLE
COMPARISON
OF TIIE
II
CHROM.~TOORAPHIC
Chromatopraphic Compounds
l’entitol conidia D-Arabitol L-Arabitol o-Ribitol D-Xylitol
Froct~on
number
FIG. 1. Fractionation c,n a cellulose column of a deionized extract of conidia from Penicillium chrysogenum. (1) Glycerol; (2) nreso-erythritol; (3) D-arabitol; (4) D-mannitol; (5) trehalose. The arrow indicates the point at which the eluting solvent was changed from n-butanol-acetonewater (2:7:1) to water. Further det,ails are reported in MATERIALS AND METHODS.
nleso-erythritol tctrabcnzoate gave no dcpression. o-Arabitol: The residue obtained after freeze-drying “peak 3” was not sufhcicnt to permit the preparation of a crystalline derivative. The compound had the same paper chromatographic mobility in solvent systems a, b, and c as a sample of n- or L-arabitol, whereas it could be differentiated from xylitol and ribitol (Table II). The optical rotation of an amount determined by periodate oxidation was measured in 0.2 N sulfuric acid containing 4% hydrated ammonium molybdate. An [a)lD value of +130” (c = 0.167) was obtained; a pure sample of D-arabitol gave the same value. o-Mannitol: The residue obtained by freeze-drying “peak 4” was crystallized from acetic acid and melted at 166” both alone and in admixture with a pure sample of D-mannitol; its [& in 0.2 N sulfuric acid containing 4 70 hydrated ammonium molybdate was +141” (c = 0.4), which agrees well with the accepted value (4). A portion of the crystalline material was reacted with acetic anhydride and a few drops of concentrated sulfuric acid. After crystallization
frcjm
I~EHAWOR
mobilities
Solvent a (RArahinosr)
Solvent b (RArabinose)
1.75
1.05
1.34
1.75 1.74 1.80 1.83
1.07 1 06 1.17 1.02
1.35 1.35 1.48 1.17
Solvent c (R~~ucos,:)
from ethanol the product melted at 123”124”, both alone and in admixture with a pure sample of n-mannitol hexaacetatc. Trehalose: When the residue obtained by freeze-drying “peak 5” was developed by paper chromatography in solvent system c a major spot was obtained which had the same mobility as trehalose; two very small spots were also revealed, one by the periodate-benzidine spray and the other by alkaline silver nitrate. In order to purify the main component, the solid was dissolved in 60 ml of water and applied on a column of charcoal-Celite 503 (160 X 14 mm). After washing with water (40 ml), a gradient elution was carried out by using a closed mixer (initially containing 50 ml of water) and a reservoir with 100 ml of 20 % ethanol. Percolate, washings, and cluates were automatically collected in Zml fractions. The main component of the mixture emerged in six fractions (from n. 33 to n. 38); these were first concentrated in a rotatory evaporator to remove the ethanol and were then freezcdried. The residue, after crystallization from ethanol, gave white crystals with an [aID value of +180” (c = 0.15 in water), which is in good agreement with the accepted value for trehalose dihydrate (13). They melted between 130” and 140” and lost their birefringence between 100’ and 102”. A sample of commercial trehalose melted hctwecn 134” and 140”, and the birefringence was lost at 102O.f Acctylation of the product’ gave a z Reisener et al. (11) observed the same rather unusual behavior when the melting point of four different, samples of trehalose dihydrate was measured with a microscope melting block.
182
BALLIO, TABLE
DI
VITTORIO,
DISCUSSION
-
Glycerol. meso-Erythrito1. D-Arabitol. D-Mannitol Trehalose hydrate. ___~~
-
Actual weight (wx)
Weight as determined by chemical assay (a) or polarimetry (b) hd
IN
-
AXWage (4
32.8
27.8
(a)
30.30
1.5
26.9 3.4 97.6
27.2 3.3 99.1
(a) (a) (a)
27.05 3.35 98.35
1.4 0.2 5.0
86.5
80.0
(b)
83.25
4.3
247.2
237.4
di-
Total.
-
242.30
-
12.4
-
compound which melted at 104”-105” when crystallized from ethanol, with sintering at 80”-81”; the mixed melting point with a sample of trehalose octaacetate gave no depression. Furthermore, the trehalose isolated from the conidia showed the same mobility as an authentic sample when developed in solvent systems b and c. Both the isolated and the reference compounds gave a negative reaction on paper with triphenyltetrazolium chloride and liberated a single reducing sugar, identical with glucose, after acid hydrolysis with 1 N hydrochloric acid for 1 hour at 100”. QUANTITATIVE INDIVIDUAL FROM
RUSS1
III
AMOUNTS OF POLYOLS AND TREHALOSE FOUND THE CONIDIA OF PENICILLIUM CXRYSOGEYUX
Compounds
AND
DETERMINATION COMPOUNDS I~~LATIGD CONIDIAL EXTRACTS
OF
The results from a quantitative assay of the individual compounds isolated from the conidia of Penicillium chrysogenum by cellulose column chromatography are given in Table III. The values determined directly by weighing with a semimicro balance were in fair agreement with those deduced from formaldehyde assays made after periodate oxidation of the four polyols, and from the polarimetric measurements made on trehalose. Of the material applied to the cellulose column, 90% by weight was recovered in the first case and 87 % in the second.
Polyols have frequently been detected in fungi (15, 16), but the simultaneous occurrence in one organism of a trio], a tetritol, a pentitol, and a hexitol, all having the same configuration at C-2 and C-3, is not common. To our knowledge, the same compounds have only been observed in the uredospores of the wheat stem rust (Puccinia gruminis tritici) (11,17), an organism unrelated to P. chrysogenum, where the amounts were comparable to those now found in the conidia of the latter organism. It is possible that extracts from the conidia and spores of other genera, if subjected to careful chromatographic analysis, will also show a similar pattern. The mode of formation and the role during germination of these compounds is at present unknown. The polyols might arise by enzymic reduction and dephosphorylation of triose, tetrose, pentose, and hexose phosphates formed through the glycolytic pathway and the pentose phosphate cycle, both of which function in P. chrysogenum (U-20). They represent nearly 10 % of the dry weight of the conidia, and may possibly be a reserve material which is utilized as a source of carbon and energy during germination. They may also represent a potential source of reduced coenzyrnes; in this connection it is of interest that a n-mannitol-l-phosphate: NAD dehydrogenase (EC 1.1.1.17) has been recently observed in conidial extracts of P. chrysogenum in this laboratory (21). Trehalose, which is virtually ubiquitous in fungi, may also constitute a storage product that is dissimilated during the germination of conidia. It has, in fact, already been demonstrated that Neurospora tetrasperma (22) and Saccharomyces cerevisiae (23) utilize this disaccharide when they pass from a dormant or resting condition into an active metabolic phase, and it is quite possible that this is a process common to most fungi. ACKNOWLEDGMENTS The authors the preparation and F. Pieroni
wish to thank Dr. A. Carilli for of conidia, and Messrs. G. Amici for skilled technical assistance. REFERENCES
1. BALLIO, A., 1st. Super.
AND DI VITTORIO, V., Sanita 2, 232 (1962).
Sci.
Rept.
TREHALOSE 2. MORITA, [Chem. 3 ARGANT, (1949). 4.
G. 7. 8.
9. 10.
11. 12.
13.
POLYOLS
73, 909 (1952) Uiol.
31,
485
FROM
P.
K., A~VD HUDSON, C. S., ./. -4~. 73, 2249 (1951). TRNELYAN, W. E., PROCTER, 1). P., AND HARRISON, J. S., Xature 166, 444 (1950). GORDON, H. T., TIIORNRURO, W., AND WERUM, L. X., Anal. Chenr. 28, 849 (1956). WHIFFES, A. J., AND SAVAGE, G. M., J. Btccteriol. 63, 231 (1947). CAPUTTO, It., LELOIR, L. F., CARDINI, C. E., AND PALADIN, A. C., J. Biol. Chern. 184, 333 (1950). ROSEMAN, S., ABELES, R. H., ARD DORPMAN, A., Arch. Biochern. Riophys. 36, 232 (1952). AT~IS, I’. W., HARDY, F. E., BUCIIANAN, J. G., AND BADDIIXY, ,J., J. Chew Sot. 5350 (1963). JARVIS, F. G., AND JOHNSON, M. J., J. ilm. Chew Sot. 69, 3010 (1947). “Beilstein’s Handbuch der organischen Chemie,” Vol. 1, pp. 525, 527, 528. Springer Verlag, Berlin, 1918. “Specifications and Criteria for Biochemical National Academy of SciCompounds.” h‘.
Sot.
183
CHRYSOG~‘.~~~II~
cnces-National Research ington, 11. C., 1960. 14. REISESER, H. J., (:OLDSCHMID, IiAM,
RICHTMYER,
Chem.
5.
E., J. chm. Sot. Japan Abstr. 47, 4415e (1953)]. N., Ulcll. Sot. C’hinz.
AND
G.
ii.,
ASD
Council,
H. It., LEDINOA. S., f,‘un. J. 40, 1248 (1962). SIIAFV, L). R. D., Physiol. PERLIS,
Biochm. Physiol. 15. TOLSTEH, C., ASD Rev. 42, 181 (1962). 16. PI,OI~VIER, I’., U~rll. SOC. Chim. (1963). 17. PRENYICI~:, N., C[rE:NI)E1’, 1,. w.
F.,
ANI)
SMITH,
Wash-
E‘.,
Biol. S.,
46, 1079 GM)I)ES,
c/. .I ?!I. C’hWl.
S'OC.
81, (i84 (1959). 18. LEWIS, K. F., BLCMENTI~AL, H. J., WENNMR, C. E., .~ND WEINHOL-SE, S., P’ederation Proc. 13, 252 (1954). 19. SIH, C. J., H.~MILIY)S, I’. B., AND KNIOIIT, S. G., J. Bacterial. 73, 447 (1957). 10. WANG, C. II., STERIX, I., GILMOUR, C. M., KL~N(:SOYR, S., REED, 11. J., BIALY, J. .I., CHRISTENSEN, B. E., AND CHELDELIN, V. H., J. Hacteriol. 76, 207 (1958). 21. BALLIO, A., AND ~‘ABRAMO, I’., Unpublished results. 22. SUSSMAN, A. S., Quart. Rev. Biol. 36,109 (1961). 23. PANEK, A., ilxh. Biochem. Biophys. 100, 422 (19G3).
OF
The
BIOCHEMISTRY
AND
Isolation
BIOPHYSICS
of Trehalose Penicillium
A. BALLIO, From
the International
Centre
and
Chemical
AND
from
April
the
Conidia
of
Thorn.
AND
Microbiology,
SERENA
Istituto
Superiore
RUSS1 di Sanitci,
Rome,
Italy
13, 1964
n-arabitol, meso-erythritol, and glycerol were isolated chrysogenum and were quantitatively assayed. Their and role during germination are discussed.
During the course of studies on acid-soluble nucleotides in conidia of Penicillium chrysogenum at this laboratory (I), it was observed that the supernatant remaining after precipitation of phosphate esters with mercuric acetate contained a considerable amount of n-mannitol, as well as other substances detected by alkaline silver nitrate on paper chromatograms. As a detailed knowledge of conidial components could be of some importance in the study of biochemical processes involved in the morphological differentiation of filamentous fungi, the nature of these substances was further investigated. The present paper deals with the identification and quantitative determination of glycerol, nzeso-erythritol, n-arabitol, n-mannitol, and trehalose in extracts of the ungerminated conidia of PenicilliuvL chrysogenwn. Some of these compounds have been detected in ethanolic extracts of the same mould by 34orita (2). MATERIALS
Polyols
V. DI VITTORIO, for
(1964)
chrysogenum
Received Trehalose, n-mannitol, the conidia of Penicillium ble mode of formation
177-183
107,
from possi-
Merck, Germany; D-arabitol from Fluka, Switzerland; L-arabitol from Pfanstiehl Chemical Co., U. S. A.; and trehalose dihydrate from Difco, U. S. A. n-Ribitol and n-xylitol were prepared, respectively, from n-ribose and n-xylose by reduction with sodium borohydride. Each pentose (60 mg) dissolved in water (4 ml) was treated at room temperature with solid sodium borohydride (76 mg). After 16 hours the solutions were deionized by shaking with Dowex 1 X 2 (100-200 mesh OHcycle) and with Dowex 50 X 1 (100-200 mesh, H+ cycle), and then filtered. After having checked that both solutions gave a negative reaction with triphenyltetrazolium chloride, they were used for paper chromatography without any further treatment. Dowex resins were purchased from Fluka, Switzerland. ANALYTICAL
METHODS
The determinations of polyols were made by periodate oxidation followed by assay of formaldehyde, according to the method of Argant (3). The assays on fractions collected from the cellulose column were made after evaporation of the solvents at reduced pressure and room temperature. Trehalose was determined polarimetrically with a Hilger polarimeter (sodium lamp) in a l-dm microtube by using an [ol]n value for the dihydrate in aqueous solution of +184”. The measurements of optical rotation in ammonium molybdate solution were made according to the method of Richtmyer and Hudson (4). Small quantities of material were estimated in the 2-dm polarimeter microtube. Melting points were determined with a Kofler hot-stage apparatus and are uncorrected. Paper chromatograms were developed by the descending method by using Whatman So. 1 filter
METHODS
CHEMICaLS All chemicals were C. Erba products of analytical grade, with the following exceptions: cellulose powder was Whatman’s ashless standard grade for chromatography; activated charcoal was a product of the British Drug Houses Ltd., England; Celite 503 was supplied by Light & Co. Ltd., England (it was washed with a large volume of 3 Ar hydrochloric acid, followed by distilled water until free of chloride ions, and finally dried at 105°C) ; meso-erythritol was purchased from 177
BALLIO,
178
DI VITTORIO,
paper. The following solvent systems were used: (a) 2-butanone-acetic acid-saturated aqueous solution of boric acid (9: l:l), (b) n-butanolacetone-water (2:7:1), (c) n-butanol-pyridinewater (6:4:3), (d) n-butanol-acetic acid-water (4: 1:5), upper layer, and (e) ethylacetate-pyridine-water (8:2: 1). Polyols and trehalose were detected either by alkaline silver nitrate (5), or by a periodate reagent followed by benzidine (6). The latter was always used when chromatograms were developed with solvent system a. Reducing substances were detected by spraying with equal volumes of triphenyltetrazolium chloride (2yi solution in water saturated n-butanol) and ethanolic 0.1 hi potassium hydroxide freshly mixed.
PRODUCTION
OF COSIDIA
Penicillium chrysogenum (49-133 Wis) was stored in sterile sand, and cultures were prepared monthly from the sand in test tubes containing an agar-sucrose-bean nutrient. After 67 days at 27” conidification in these tubes was complete, and the cultures were stored at 5” for up to 1 month. A sterile water suspension of conidia was prepared from these, and this in turn was used to inoculate Fernbach flasks (20 cm in diameter), each of which contained 150 gm of pearl barley moistened with 10 ml of the Whiffen and Savage sporulating solution (7). Prior to inoculation, the flasks were sterilized for 20 minutes at 100” and for 20 minutes at 120”. Conidification in the inoculated flasks was complete after 7 days at 25”. The yield of conidia per flask was about 3 gm (dry weight). Conidia were also produced on an agar-glycerol-beet molasses medium having the following composition (gm/liter): glycerol, 7.5; beet molasses, 7.5; yeast extract, 5; NaCl, 10; MgS04.7Hz0, 0.005; KH2P04, 0.006; (NHn)zFe(S04)2.6H20, 0.0016; CuS04.5H~0, 0.001; CaS04.2H20, 0.250; agar, 25; the pH was adjusted to 6.4. The medium was sterilized in Roux bottles for 20 minutes at 100” and for 20 minutes at 120”. Conidification was complete after 7 days at 25”.
EXTRACTION
OF CONIDIA
After conidification on pearl barley, 150-200 ml of distilled water was added to each flask, and the contents were stirred with a glass rod for a few minutes. The green aqueous suspension was decanted from the grains of barley, filtered through cotton gauze, and centrifuged. The sediment was washed 3 times with distilled water and then resuspended in a small volume. All these operations were carried out at 3”-5”. The suspension was poured into boiling aqueous ethanol to give a final alcohol content of 5Oyc. The volume of liquid corresponded to approximately 8 ml per gram dry
AND
RUSS1
weight of conidia. After boiling for 5 minutes, the suspension was cooled with tap water and centrifuged. The sediment was extracted twice more under identical conditions by using 50% ethanol. The combined extracts were acidified with nitric acid and treated with mercuric acetate according to the method of Caputto and co-workers (8). The suspension was filtered and the filtrate was treated with hydrogen sulfide to free it from mercuric ions. The clear solution, obtained after removing mercuric sulfide by filtration through Celite 503, was freed from hydrogen sulfide by treatment with a stream of air, concentrated in a rotatory evaporator to half its original volume, applied to a column of Dowex-1 X 2 (100-200 mesh, OH- cycle; the volume was 5 times the original dry weight of the conidia or j$ the volume of the conidial extract), and eluted directly with water. Fractions corresponding to s/4 of the column’s volume were collected, and each fraction was tested on paper for the presence of compounds that react with alkaline silver nitrate. Fractions giving a positive reaction were pooled and passed through a small column (j43 the volume of the first column) of Dowex 50 X 2 (100-200 mesh, H+ cycle), followed by two column volumes of water. The combined effluent and eluate were pooled and freeze-dried. The conidia produced on the agar-glycerol-beet, molasses medium were removed by wetting them with water containing a minute amount of commercial sodium lauryl sulfate and carefully scraping the surface of the medium with a plug of washed absorbent cotton-wool tied to a steel wire. After filtering through a small amount of cottonwool, to remove mycelial fragments, the conidial suspension was centrifuged; the residue was thoroughly washed with distilled water by centrifugation and then extracted with ethanol, as described above.
PARTITION CHROMATOGRAPHY ON CELLULOSE The residue (0.274 gm) obtained by freezedrying 50 ml of the deionized conidial extract was dissolved in 4 ml of water and was mixed with a small amount of cellulose. The mixture was taken to dryness over calcium chloride in ~acuo and was applied to a column of cellulose powder (440 X 22 mm) which had been packed dry, and then washed with 600 ml methanol followed by 700 ml acetoneand by2000mlof n-butanol-acetone-water (2:7: 1). The latter mixture of solvents was also used to effect elution at the rate of 1 ml per minute. After 185 fractions of 10 ml each had been collected, the column was washed with water and a further twenty lo-ml fractions were collected. Elution was followed by testing individual frac-
TREHALOSE
AND POLYOLS
tions spotted on to paper with alkaline silver nitrate; fractions giving a positive reaction were chromatographed on paper with solvent system b, along with the appropriate reference substances, and t)hen taken to dryness. The residues were weighed, taken up in a measured amount of water, and quantitatively assayed. Polyols were determined by estimating the formaldehyde derived from periodat,e oxidation and trehalose by polarimetry. IDENTIFIC~~TIOS
0~ COMPOUNDS
INDIVIDUAL
This was carried out by paper chromatography in solvent systems a and G. Both alkaline silver nitrate and periodate-benzidine gave single spots with the purified compounds; a triphenyltetrazolium chloride spray consistently showed that reducing compounds were absent. The structure of the isolated products was further confirmed by their melting points and mixed melting points with reference compounds, by their opt,ical rotation in acidified molybdate solution, and by preparing the forlowing derivatives: the tri-p-nitrobenzoate of glycerol, the tetrabenzoate of erythritol, the hexaacetate of mannitol, and the octaacetate of trehalose. RESULTS AX~~LYSIS
OF CONIDML AND A[YCELIAL EXTRACTS BY PAPER CHROMATOGRAPHT
Preliminary examination by paper chromatography of deionized extracts derived from conidia grown on barley suggestedthat glycerol, erythritol, arabitol, mannitol, and trehalose were present (Table I). The same substances were detected in extracts prepared from conidia grown on agar-glycerolbeet molasses medium ; no trace of them was found in an extract of uninoculated barley grains. Before deionization with Dowex1, conidial extracts were found to contain also free and bound glucose. Retention of reducing sugars on anion exchangers in the OH- form is well known (9, 10). The same type of analysis was also used to examine extracts prepared from washed samples of mycelium of PeniciZliu?n ch~ysogenunz collected after 24, 48, 72, and 96 hours growth on the chemically defined medium of Jarvis and Johnson (11). The ratio between dry weight of mycelium and volume of extract was always identical to that ob-
FROM
P.
li9
CHRYSOGENU~~f
tained with the conidia. On inspection of paper chromatograms developed in solvent systems a and c, it was observed that glycerol, erythritol, arabitol, and trehalose, if present, occurred in traces, whereas the amount of mannitol was comparable to that found in the conidial extracts. The occurrence of glucose and of a slow-moving spot, which liberates glucose and galactose on acid hydrolysis, was also consistently noted in the mycelial extracts. I"RAcTI~K:ATI~X
OF CONIDIAL ox
EXTRACTS
A CELLULOSE COLUMN
In order to verify the identity of the compounds detected by paper chromatography, a deionized extract was fractionated on a cellulose column. As shown in Fig. 1, four peaks were eluted with n-butanol-acetonewater (2:7: 1) and a fifth peak with water. Paper chromatography in solvent system c suggested that peaks 1, 2, 3, and 4 corresponded to glycerol, erythritol, arabitol, and mannitol, respectively and that peak 5 contained trehalose. Further evidence for the structure of the individual compounds was obtained from the following experiments. CHz4RhCTERIZATION TREHALOSE CONIDIAL
OF POLYOLS ISOLATED FROM EXTRACTS
BND
Glycerol: A portion of the liquid left after freeze-drying the eluate composing “peak 1” was reacted with p-nitrobenzoylchloride in pyridine. A derivative was obtained which melted at 198” aft,er crystallization from chloroform; the mixed melting point with a pure sample of glycerol tri-p-nitrobenzoate gave no depression. Meso-erythritol: The residue left after freeze-drying “peak 2” was crystallized from ethanol. Prisms with a melting point of 119”120” were obtained; in admixture with pure l?zeso-erythritol the melting point showed no depression.’ The henzoate, after crystallization from acetic acid, melted at 190”; the mixed melting point with a pure sample of 1 The accepted values for ?raeso-erythritol, the n-form, the n-form, and the racemic compound are 120”, 88.5”-89”, 88”, and 72”, respectively (12).
Conidial extract Trehalose D-Mannitol D-Arabitol meso-Erythritol Glycerol
Compounds
COMPARISON
a
2.14
2.51
2.15,2.48,
Solvent (mlucose)
CHROMATOGRAPHIC
0.43,1.70, 0.42 1.73
OF THE
3.05
3.00
IN
TABLE
Solvent (Rr)
b
0.41 0.52
I
0.59,0.97,1.11,1.32,1.53 0.59 0.98 1.14
Solvent c (R~~uoose)
1.35
mobilities
1.55
WITH THAT OF OF PENICILLIUM
Chromatographic
OF KNOWN COMPOUNDS EXTRACT FROM CONIDIA
0.10,0.25,0.33,0.41,0.52 0.10 0.26 0.33
BEHAVIOR A DEIONIZEU
0.08,0.17,0.22,0.30,0.41 0.08 0.18 0.22
Solvent (RJ)
CBRYSOGENUM
PERI~DATE-OXIDIZABLE
d
0.30 0.42
CONTAINED
1.3G
Solvent e Uh~uoose)
0.20,0.86,1.36, 0.20 0.87
SUBSTANCES
2.01 2.94
2.03,2.95
THEHALOSE
Ah-11
POLYOLS
FROM
l’.
181
CH/Z~‘SOGE.YL’C’.lf TABLE
COMPARISON
OF TIIE
II
CHROM.~TOORAPHIC
Chromatopraphic Compounds
l’entitol conidia D-Arabitol L-Arabitol o-Ribitol D-Xylitol
Froct~on
number
FIG. 1. Fractionation c,n a cellulose column of a deionized extract of conidia from Penicillium chrysogenum. (1) Glycerol; (2) nreso-erythritol; (3) D-arabitol; (4) D-mannitol; (5) trehalose. The arrow indicates the point at which the eluting solvent was changed from n-butanol-acetonewater (2:7:1) to water. Further det,ails are reported in MATERIALS AND METHODS.
nleso-erythritol tctrabcnzoate gave no dcpression. o-Arabitol: The residue obtained after freeze-drying “peak 3” was not sufhcicnt to permit the preparation of a crystalline derivative. The compound had the same paper chromatographic mobility in solvent systems a, b, and c as a sample of n- or L-arabitol, whereas it could be differentiated from xylitol and ribitol (Table II). The optical rotation of an amount determined by periodate oxidation was measured in 0.2 N sulfuric acid containing 4% hydrated ammonium molybdate. An [a)lD value of +130” (c = 0.167) was obtained; a pure sample of D-arabitol gave the same value. o-Mannitol: The residue obtained by freeze-drying “peak 4” was crystallized from acetic acid and melted at 166” both alone and in admixture with a pure sample of D-mannitol; its [& in 0.2 N sulfuric acid containing 4 70 hydrated ammonium molybdate was +141” (c = 0.4), which agrees well with the accepted value (4). A portion of the crystalline material was reacted with acetic anhydride and a few drops of concentrated sulfuric acid. After crystallization
frcjm
I~EHAWOR
mobilities
Solvent a (RArahinosr)
Solvent b (RArabinose)
1.75
1.05
1.34
1.75 1.74 1.80 1.83
1.07 1 06 1.17 1.02
1.35 1.35 1.48 1.17
Solvent c (R~~ucos,:)
from ethanol the product melted at 123”124”, both alone and in admixture with a pure sample of n-mannitol hexaacetatc. Trehalose: When the residue obtained by freeze-drying “peak 5” was developed by paper chromatography in solvent system c a major spot was obtained which had the same mobility as trehalose; two very small spots were also revealed, one by the periodate-benzidine spray and the other by alkaline silver nitrate. In order to purify the main component, the solid was dissolved in 60 ml of water and applied on a column of charcoal-Celite 503 (160 X 14 mm). After washing with water (40 ml), a gradient elution was carried out by using a closed mixer (initially containing 50 ml of water) and a reservoir with 100 ml of 20 % ethanol. Percolate, washings, and cluates were automatically collected in Zml fractions. The main component of the mixture emerged in six fractions (from n. 33 to n. 38); these were first concentrated in a rotatory evaporator to remove the ethanol and were then freezcdried. The residue, after crystallization from ethanol, gave white crystals with an [aID value of +180” (c = 0.15 in water), which is in good agreement with the accepted value for trehalose dihydrate (13). They melted between 130” and 140” and lost their birefringence between 100’ and 102”. A sample of commercial trehalose melted hctwecn 134” and 140”, and the birefringence was lost at 102O.f Acctylation of the product’ gave a z Reisener et al. (11) observed the same rather unusual behavior when the melting point of four different, samples of trehalose dihydrate was measured with a microscope melting block.
182
BALLIO, TABLE
DI
VITTORIO,
DISCUSSION
-
Glycerol. meso-Erythrito1. D-Arabitol. D-Mannitol Trehalose hydrate. ___~~
-
Actual weight (wx)
Weight as determined by chemical assay (a) or polarimetry (b) hd
IN
-
AXWage (4
32.8
27.8
(a)
30.30
1.5
26.9 3.4 97.6
27.2 3.3 99.1
(a) (a) (a)
27.05 3.35 98.35
1.4 0.2 5.0
86.5
80.0
(b)
83.25
4.3
247.2
237.4
di-
Total.
-
242.30
-
12.4
-
compound which melted at 104”-105” when crystallized from ethanol, with sintering at 80”-81”; the mixed melting point with a sample of trehalose octaacetate gave no depression. Furthermore, the trehalose isolated from the conidia showed the same mobility as an authentic sample when developed in solvent systems b and c. Both the isolated and the reference compounds gave a negative reaction on paper with triphenyltetrazolium chloride and liberated a single reducing sugar, identical with glucose, after acid hydrolysis with 1 N hydrochloric acid for 1 hour at 100”. QUANTITATIVE INDIVIDUAL FROM
RUSS1
III
AMOUNTS OF POLYOLS AND TREHALOSE FOUND THE CONIDIA OF PENICILLIUM CXRYSOGEYUX
Compounds
AND
DETERMINATION COMPOUNDS I~~LATIGD CONIDIAL EXTRACTS
OF
The results from a quantitative assay of the individual compounds isolated from the conidia of Penicillium chrysogenum by cellulose column chromatography are given in Table III. The values determined directly by weighing with a semimicro balance were in fair agreement with those deduced from formaldehyde assays made after periodate oxidation of the four polyols, and from the polarimetric measurements made on trehalose. Of the material applied to the cellulose column, 90% by weight was recovered in the first case and 87 % in the second.
Polyols have frequently been detected in fungi (15, 16), but the simultaneous occurrence in one organism of a trio], a tetritol, a pentitol, and a hexitol, all having the same configuration at C-2 and C-3, is not common. To our knowledge, the same compounds have only been observed in the uredospores of the wheat stem rust (Puccinia gruminis tritici) (11,17), an organism unrelated to P. chrysogenum, where the amounts were comparable to those now found in the conidia of the latter organism. It is possible that extracts from the conidia and spores of other genera, if subjected to careful chromatographic analysis, will also show a similar pattern. The mode of formation and the role during germination of these compounds is at present unknown. The polyols might arise by enzymic reduction and dephosphorylation of triose, tetrose, pentose, and hexose phosphates formed through the glycolytic pathway and the pentose phosphate cycle, both of which function in P. chrysogenum (U-20). They represent nearly 10 % of the dry weight of the conidia, and may possibly be a reserve material which is utilized as a source of carbon and energy during germination. They may also represent a potential source of reduced coenzyrnes; in this connection it is of interest that a n-mannitol-l-phosphate: NAD dehydrogenase (EC 1.1.1.17) has been recently observed in conidial extracts of P. chrysogenum in this laboratory (21). Trehalose, which is virtually ubiquitous in fungi, may also constitute a storage product that is dissimilated during the germination of conidia. It has, in fact, already been demonstrated that Neurospora tetrasperma (22) and Saccharomyces cerevisiae (23) utilize this disaccharide when they pass from a dormant or resting condition into an active metabolic phase, and it is quite possible that this is a process common to most fungi. ACKNOWLEDGMENTS The authors the preparation and F. Pieroni
wish to thank Dr. A. Carilli for of conidia, and Messrs. G. Amici for skilled technical assistance. REFERENCES
1. BALLIO, A., 1st. Super.
AND DI VITTORIO, V., Sanita 2, 232 (1962).
Sci.
Rept.
TREHALOSE 2. MORITA, [Chem. 3 ARGANT, (1949). 4.
G. 7. 8.
9. 10.
11. 12.
13.
POLYOLS
73, 909 (1952) Uiol.
31,
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FROM
P.
K., A~VD HUDSON, C. S., ./. -4~. 73, 2249 (1951). TRNELYAN, W. E., PROCTER, 1). P., AND HARRISON, J. S., Xature 166, 444 (1950). GORDON, H. T., TIIORNRURO, W., AND WERUM, L. X., Anal. Chenr. 28, 849 (1956). WHIFFES, A. J., AND SAVAGE, G. M., J. Btccteriol. 63, 231 (1947). CAPUTTO, It., LELOIR, L. F., CARDINI, C. E., AND PALADIN, A. C., J. Biol. Chern. 184, 333 (1950). ROSEMAN, S., ABELES, R. H., ARD DORPMAN, A., Arch. Biochern. Riophys. 36, 232 (1952). AT~IS, I’. W., HARDY, F. E., BUCIIANAN, J. G., AND BADDIIXY, ,J., J. Chew Sot. 5350 (1963). JARVIS, F. G., AND JOHNSON, M. J., J. ilm. Chew Sot. 69, 3010 (1947). “Beilstein’s Handbuch der organischen Chemie,” Vol. 1, pp. 525, 527, 528. Springer Verlag, Berlin, 1918. “Specifications and Criteria for Biochemical National Academy of SciCompounds.” h‘.
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CHRYSOG~‘.~~~II~
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RICHTMYER,
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5.
E., J. chm. Sot. Japan Abstr. 47, 4415e (1953)]. N., Ulcll. Sot. C’hinz.
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H. It., LEDINOA. S., f,‘un. J. 40, 1248 (1962). SIIAFV, L). R. D., Physiol. PERLIS,
Biochm. Physiol. 15. TOLSTEH, C., ASD Rev. 42, 181 (1962). 16. PI,OI~VIER, I’., U~rll. SOC. Chim. (1963). 17. PRENYICI~:, N., C[rE:NI)E1’, 1,. w.
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ANI)
SMITH,
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46, 1079 GM)I)ES,
c/. .I ?!I. C’hWl.
S'OC.
81, (i84 (1959). 18. LEWIS, K. F., BLCMENTI~AL, H. J., WENNMR, C. E., .~ND WEINHOL-SE, S., P’ederation Proc. 13, 252 (1954). 19. SIH, C. J., H.~MILIY)S, I’. B., AND KNIOIIT, S. G., J. Bacterial. 73, 447 (1957). 10. WANG, C. II., STERIX, I., GILMOUR, C. M., KL~N(:SOYR, S., REED, 11. J., BIALY, J. .I., CHRISTENSEN, B. E., AND CHELDELIN, V. H., J. Hacteriol. 76, 207 (1958). 21. BALLIO, A., AND ~‘ABRAMO, I’., Unpublished results. 22. SUSSMAN, A. S., Quart. Rev. Biol. 36,109 (1961). 23. PANEK, A., ilxh. Biochem. Biophys. 100, 422 (19G3).