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Polymerization of 1-naphthol and related phenolic compounds by an extracellular fungal enzyme
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
Polymerization Compounds ROY D. SJOBLAD, Laboratory
6, 457463
(1976)
of I-Naphthol and Related Phenolic by an Extracellular Fungal Enzyme ROBERT
of Soil Microbiology, The Pennsylvania State
D. MINARD,
AND JEAN-MARC
of Agronomy, and Department University Park, Pennsylvania
Department University,
BOLLAG of Chemistry, 16802
Received October 15, 1975; accepted January 27, 1976 An extracellular phenol oxidase was isolated from the culture filtrate of the fungus Rhizoctoniu praticola by gel filtration on Sephadex G-200. The enzyme catalyzed the polymerization of I-naphthol to dimeric, trimeric, tetrameric, and pentameric compounds as determined by mass spectrometry. One product with a molecular weight of 286 was identified as 4,4’-bi-1-naphthol. Other compounds that were dimerized or polymerized by the enzyme were phenol, o-methoxyphenol (guaiacol), p-methylphenol (p-cresol), 2,6-dimethoxyphenol, and 1,3- and 1,5-dihydroxynaphthalene. The product from 2,6-dimethoxyphenol was isolated and identified as 3,5,3’,5’tetramethoxydiphenoquinone. INTRODUCTION
Phenolic compounds can be polymerized by several chemical reagents as well as by enzymatic activity. In nature biological oxidation and coupling of phenols are key reactions that result in the formation of products such as lignins, melanins, tannins, alkaloids, and antibiotics. Although polymerization by various microorganisms is recognized as an important reaction, relatively few basic studies exist on this subject, and little is known about the enzymes involved in these reactions (1). Our limited understanding about the formation of the natural products mentioned above has to cause concern if one considers that the numerous wastes and new chemicals used in the environment may not only be difficult to biodegrade but may also be enzymatically transformed to more complex products. These products might be even more recalcitrant to biodegradation and lead to the accumulation of potentially dangerous and, as of yet, undefined environmental pollutants. 457 Copright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
In our laboratory we studied the microbial transformation of the widely used insecticide carbaryl (I-naphthyl-N-methylcarbamate), and it was established that this compound is hydrolyzed biologically as well as chemically to 1-naphthol (2). This chemical can be more toxic to organisms than the compounds from which it originates (2, 3), and its fate in the environment is not yet clearly elaborated. It was shown that bacteria can metabolize 1-naphthol to COZ, but another pathway occurred simultaneously resulting in the accumulation of various products, one of which was identified as 4-hydroxy-l-tetralone (4). Bollag and Liu (5) also found that the mycelial cell extract of Fusarium solani mineralized 1-naphthol. When 1-naphthol was added to the growth medium of the soil fungus Rhizoctonia praticola, it was possible to detect a transformation of 1-naphthol to highmolecular-weight compounds which were formed even by the culture filtrate indicating the presence of an extracellular
-+;ss
RJOBLAD,
MINAHD
enzyme (6). Further, it was shown that upon some purification of the culture filtrate, an active enzymatic fraction was obt,ained which produced ether- or benzeneclxtractable compounds that differed from the water-soluble products obtained with thtb original culture filt’rate. In this investigation it was att,emptcd to characterize the compounds which wre formed by the partially purified enzymatic fraction from R. praticola. MATERIALS
AND
METHODS
Fungal growth ill liquid medium. Rhizoctwniu praticoEa (Vaartaja No. 1347) was grown in a modified Czapek Dox medium containing 2.0 g of L-asparagine, 0.5 mg of thiamine, 25 mg of CaS04+2Hz0, and 1 ml of a tract elements solution (Na2B*07 . 10HzO, 40 mg; R2nS01.2Hz0, 80 mg; FeSOJ.7H20, 40 mg ; ZnSOA.7Hz0, 800 mg; Na&IoO*.2HzO, 80 mg; CuSOa. 5Ha0, 40 mg/lOO ml of distilled water) dissolved in 1 liter of distilled water (final pH 7.3). The cultures were grown at 25°C on a rotary shaker (150 oscillations/min). Enzyme preparation al&d assay. R. praticola was grown for 5-7 days after which t,he mycelium was separated from the growth medium by filtration, and the culture filt,rate was lyophilized. The freezcdried filtrate was redissolved in 0.1 &’ phosphate buffer, pH 6.9, to give a final concentration of 100 mg dry wt/ml, and 10 ml were applied to a column (4 X 45 cm) of Scaphadtx G-200 (Pharmacia Fine Chemicals, Inc., New Market, nT.J.) equilibratcd with the buffer. The void volume was 180 ml and the flow rate was 1 ml/min. Fractions of 8 ml were collected and their activity was tested by incubation for 3 hr at 25°C with 50 pg/ml of 1-naphthol (containing 0.005 PCi l-[l-14C]naphthol.) I-Naphthol was dissolved in ethanol and the final concent.ration in an assay was 2%. Subsequcnt.ly, each fraction was extracted with benzene and the organic phase, which clontaincd all of the radioactivity, was analyzed by thin-layer chromatography
AND
BOLL4G
(TIE)’ in an &her--hexam> t.1: 1, v/v) solwnt system. I-Naphthol \v:ts visualizcld on the plate by exposure to rlltraviolrt (uv) light at, 254 nm, and its Ri-arc&a w:w scraped from the plattl :rnd t hc> radioactivity ~3s mcbasurcad. TOP most wtivc I-napthol-transforming fractions \V(‘rY used in subwqwnt invrstigations. Five milliliters of an assay mixture wit,h 50 pg/ml of 1-naphthol as subst r:tt.e ww extracted with an equal volun~~ of brnzfw and 4 ml of the bwzcncl mtrwt wtw washed thrw times \vith an w11t;tl volume of distilled water. Aftw b(bing dric*d ovw anhydrous I\ra$O,, the solvent was waporated under a stream of h$+um, and that product) mixtuw n-as anaI.wx~l bv mass spwtromrtry. Other phPnolic* compounds wcw addt>d to t,he clnxymc’ fractions to givcx ;L final concentration of 30 kg/ml, c~swpt. phenol 2,(i-dirnclt,h(,x~phcnol (100 Hg/ml) and (1 mg/ml). Transformation products from phenol, o-mc?thoxyph(~nol, and 1 .:3- and 1,5-dihydroxynaphthalen(s w(w wt,ract’ed wit,h chloroform. 1’rodwt.s from p-met,hylphenol ww clxtractrd with &It h!.l c%htlr and the prwipitstrd product from 2,fi-dimcthoxyphenol was cwllwtc~d b>, filtration. The product, extracts \v(w w:tsh(bd with distilled watcar, dried, and :m:tlyzt~d 1~) mass spwtromc+ry. Control (~xperimt~nts c~mpioying boilrd clnzyme fractions and thch above\ suhstratw ww inactiw and, after cxtrwtitbn, th(h subst,ratcs wrc thr only cwmp~~unds d(Jt,ccted by thin-layc>r analysis. Isolatiolr of I-~~aphthol polymers. Thv actlvts canzymr frac*tions uxw incbubatcd for 5 hr at 23°C with 50 gg, ml of i-naphthol as substratf>. Thrl assay mixture was then subjected t,o a dual c~xtrwtictn with equal volumes of bcnzr~nc~ and th(k wmbined extracts were dric>d over anhydrous Na2SOd and evaporatcld in :I flash waporator at 37°C. Subsrqucntly, the produc:ts 1 Abbreviations wed: TLC: graphy, IIV : rlltraviolet , MM’: tables and figlues 1.
thin-layer rnolrc~~lar
chromatew&ght iin
POLYMERIZATION
were redissolved in ether and separated on a preparative thin-layer plate with a triethylamine solvent system. Products were visualized by uv light at 254 nm. The compound being isolated was removed from the thin-layer plate, extracted from the silica gel with acetone, and rechromatographed in triethylamine. Final purification of the products was performed by repeated TLC in an ether-hexane (4: 1, v/v) solvent system and the isolated by mass compounds were analyzed spectrometry. Analytical methods and chemicals. Radioactivity was determined with an Isocap 300 liquid scintillation counter (Nuclear Chicago, Chicago, Ill.). Samples were measured in a modified Bray solution composed of 60 g of napthalene, 100 ml of methanol, and 8 g of Omnifluor (98yo PPO [2,5-diphenyloxazole] and 2% bisMSB [p-bis- (0-methylstyryl)-benzene] ; New England Nuclear Corp., Boston, Mass.) in 1 liter of dioxane. Precoated TLC plates (Brinkman Instruments, Inc., Westbury, N.Y.) with a thickness of 0.25 and 0.50 mm of silica gel F-254 were used for the routine thinlayer analyses and for the preparative separations, respectively. Mass spectra were determined using an AEI MS-902 mass spectrometer at an ionization potential of 70 eV and at a temperature of 350°C using the direct insertion probe. 1-Naphthol and o-methoxyphenol were purchased from Eastman Organic Chemicals (Rochester, N.Y.), and l-[l-‘“C Jnaphthol with a specific activity of 3.84 mCi/mmol from Mallinckrodt (St. Louis, MO.). Crystalline 1,3-dihydroxynaphthalene was obtained from Fisher Scientific Co. (Pittsburgh, Pa.) ; 2,6-dimethoxypheno1 (99%) from Aldrich Chemical Co. (Cedar Knolls, N.J.) ; and 1,5-dihydroxynaphthalene from J. T. Baker Chemical Co. (Pittsburgh, Pa.). All chemicals that were not of sufficient purity when purchased were purified by charcoal addition and recrystallization. Ferric chloride oxida-
459
OF I-NAPRTHoL
tion of 1-naphthol was used to synthesize 4,4’-bi-1-naphthol in a procedure described by Dianin (7). RESULTS
When the culture filtrate from the fungus R. praticola grown in a modified Czapek Dox medium was concentrated by lyophilization and subsequently chromatographed on a column of Sephadex G-200, fractions were obtained that were able to transform 1-naphthol as substrate. The formation of a purple color was evident in the active fractions, and TLC analysis of the benzene extract of these fractions clearly showed a decrease of radioactive material in the Rf-area of I-naphthol (Fig. 1). The radioactivity which was lost from the R,-area of I-naphthol during incubation was recovered completely on the TLC plate in areas with an R,-value less than that of I-naphthol. Since the strongest enzyme activity was found in fractions 14 through 19, all the subsequent experiments were performed using the eluate of these six fractions. Heated active fractions were devoid of activity on the tested substrate. The benzene extract of the active fractions after incubation with I-naphthol was
FIQ. 1. Transformation after Sephadex G-Z?OO gel culture filtrate from R. radioactivity (in percent of l-naphthol after TLC in solvent system. Fraction 1 Further details see text.
oj I-naphthol (50 pg/ml) filtration of the lyophilized praticola. Data represent remaining at the Rf-area an ether-hexane (4~1,v/v) starts after the void volume.
460
SJOBLAD,
MINARD
subjected to mass spectrometric analysis to establish some of the characteristics, particularly the molecular weights, of the formed products. A mass spectrum of the reaction mixture after incubation for three hours is reproduced in Fig. 2. Major ,m/e peaks were observed at 144, 286, 425, 570, and 712 in this analysis. The substrate, I-napht,hol, has a mass of 144 and a dinwrized product would be represented by the> VI/~: pc:tk at 286. The detect’ed peak at 1% coincides with the trimer, and the w le peak at, 570 would represent a tetramc~r. The highest polymer observed with thcx mass spe&ometer corresponds to a pentamer, caxhibiting a peak at 712. A fragmmt,ation product with a loss of 1S mass units (HZO) was observed from c:wh of thr oligomers formed. Attempts were then made to separate thfa individual compounds of I-naphthol transformation to establish that they are not, fragment ions from highcr-molccularwight polymers. TIC was effective in separating two dimers, a trimer, and ~1 trtramrr. The tctramer remained at the origin of the plate with triethylamine as
ASD
BOLLAG
the solvent system, but it. \V:I.S mobile in ether-hexane (Table 1). I~[RSSspwtral data of the isolated products ~wtablish~d that the mixture of compounds wprtwntt~d in Fig. 2 is composed of individual oligomcrs of I-naphthol. Two isomcric clinlcw of 1-naphthol with molecular \vcsight,t: of 286 ww isolated (Table I). 13nwrs .A :md B posstwwd different RJ v:\lw~ in t hcl thinlayer systems used, and t,hc:y g:tvc~tlifirwnt fragment&on pattclrns \vh(w :rrl:~l~~zc~lb) mass spwtromrtry. Dimc~r IS i:: 1..+‘-hi-lnaphthol, sincr th(l mass sywt*~rum, IIV spwt~rum, melting point, (SOOY’), and thin-layer c*haractchristics of the* isolattd prodwt \v(w idcnticaal with thci :tuthontic~ cht>mic:al. ,I trimcr (molwulwr vxsight 1%) and :I tc$r:unw (molrcwl:~r \vcGghl 570) ww also dtJtectc>d by masti spwtromr+ry after separation I,]- TI,C’. but the, pc*nt:k nwrica product has not >,c,t b(w is ~I:~tcd.
m/e FIG. 2. Mass qf R. praticola
spectrum obtained from the brnzcnc with I-naphlhol as substrate.
rxtrart
I$’ an assay
with
a partidly
pltri$d
WLZ!JU~V
POLYMERIZATION
OF TABLE
TLC
Compound
and Mass
461
l-NAPHTHOL 1
Spectral Data for I-Naphthol, 4,4’-Bi-1-Naphthol, Isolated Oligomers from I-Naphthol Rf in solvent
Triethylamine
system
and
Mass
Ether-hexane (4: 1, v/v)
MW
spectral
analysis
Intensity
(%)
at m/e
144
286
428
570
1-Naphthol 4,4’-Bi-1-naphthol
0.82 0.67
0.90 0.72
144 286
100 3
100
-
-
Isolated compounds Dimer A Dimer B Trimer Tetramer
0.76 0.67 0.30 0.00
0.84 0.72 0.59 0.53
286 286 428 570
19 3 -5 9
100 100 15 16
100 5
100
with different phenolic compounds. Mass spectrometry was also used as the analytical method for determining the activity of the enzyme on the various aromatic substrates. Table 2 shorn that dimerized products were detected after incubation of the active fractions with p-methylphenol, 2,6-dimethoxyphenol, and 1,3- and 1,5dihydroxynaphthalene. o-Methoxyphenol was polymerized to a trimeric and phenol to a tetrameric product. As mentioned previously, the highest polymer detected with I-naphthol by mass spectrometry was the pentamer. Polymers of higher molecular weight could well be present, but are probably too involatile to detect under the conditions used for mass spectrometric analysis. Whereas it was assumed that some of the tested phenolic chemicals formed higher polymers which were not detected, 2,6dimethoxyphenol represents a compound whose coupling possibilities are limited. In this case it was easy to establish that after incubation of 2,6-dimethoxyphenol with the enzyme, the p-$-coupled quinonoid product, 3,5,3’,5’-tetramethoxydiphenoquinone (coerulignone) resulted as the end product. This compound was formed in the enzyme solution and precipitated as steel-blue needles in a yield of 90%. Its melting point after recrystallization from
a mixture of phenol-methanol was 289291”C, and its uv spectrum (in chloroform) and its ir spectrum (BBr) were identical with authentic material. DISCUSSION
It was established that the fungus Rhizoctoniu praticola forms an extracellular enzyme which is active in polymerizing l-naphthol, as well as a variety of other phenolic compounds. The enzyme could be isolated from the culture filtrate by gel filtration on Sephadex G-200, and it is assumed that it is a phenol oxidase, most TABLE Formation Compounds
Phenolic
2
of Polymerized Products from Phenolic ajter Incubation with an Extracellular Enzyme jrom R. praticola compound
Phenol o-Methoxyphenol p-Methylphenol 2,6-Dimethoxyphenol 1-Naphthol 1,3-Dihydroxynaphthalene 1,5-Dihydroxynaphthalene
Enzymatic products as detected by mass spectrometry Dimer, trimer, tetramer Dimer, trimer Dimer Dimer Dimer, trimer, tetramer, pentamer Dimer Dimer
4fi2
SJOBLAD,
MINAHD
probably of the Iaccase t,ype which is known to be produced by different fungi (1). Thr wide range of substrate specificity of most phenol oxidases from fungi has prrviouslg been established, and F%hraeus and Ljunggrrn (8) have shown that oxygen uptakr occurred when or&o-, mefa-, and para-substituted phenols were incubated with a lactase from Polyporus versicolor. The enzymct from R. praticola also oxidized and coupled an orth.o- (o-mcthoxyphenol). a meta- (1,3-dihydroxynaphthalt>nc), and a para-substituted phenol (p-mcth.vlphrlnol). I;fthracsus recognized that white-rot fungi w(lrc’ ahIf> to transforms 1-naphthol to dark-colored products (9) and Brown and Hocks (10) suggested that a lactase from 1,. vewidor converted I-naphthol to a purple quinonoid polymer, but in neither cast was a polymerized product isolated or identified, nor were any data presented that, substantiated a polymerization r(‘action with 1-naphthol a,s a substrate. The ma,ss spectral data of the enzymatic products from I-naphthol in our investigation shoW that this substance was polymerizchd, at least, to the pentamer, and the major m/e peaks at 286, 428, and 570 rcprcsent individual compounds and not,
MW.
FIG.
I-naphthol
3. Hypothetical hg a Jungal
scheme enzyme.
.for
570
polymerization
of
AND
BOLLAG
fragments of higher mol(4ar \vc*ight PO!?mers. The peak at. VZ/C 711, in thr mass sp&rum of the crude producet mixture indicated thr presence of thca p(~nt~arncr, but t,his compound has not bacon isolatrxd. Even t,hough, for thtb most plirt,, thfh mass sprctrnl data indicatct th(b formation of rclducrd hydroxylatcd c.c)mpountls from I-naphthol, t,hf>rcb is rc’uson to b(&(~v(~ that quinoncs arc’ also produc*ckd I)!, t h(~ acation of the (lnzym(b from f?, piaf!ccdu, ‘rho (Y)I~Ipound, 2-methyl-1-naphthol is (~(~nvert(~d by 3 lac*casr from 1’. PPr.Sic0l~N 1.0 thr> product, 3,3’-dimethyl-l , 1 ‘-binal,lrt~lyl-l,l’quinonr (1 I) in 63% y&Id :iftcbr irtc.llbation \vvith th(b subst,ratr for 4 days iit 30°C~. On thrk other hand, oxidativtb (*oupling c)t’ l-naphthol hy F(Cl~ yichlds thrccb ixornchrica binaphthols (from o--0, 11 I). and ,U /) (*OIIpling) (12) and this report cLnt:lblish(ld that at least t\vo of th(i binaphthols can Of result from c>nzymatic+ c*cmplirlg I-naphthol. Th(i intt,nscl purpl(l color a~)(-i;tf(hd with that r>nzymatic products from 1-naphthol is indicativcl of quinonc f’ormaticm ; how(lv(lr, the isolated oligomt~rs w(‘r(’ ~11 colorand their m;Lss sp(lct ra less compounds, clxhibited thfl major rTl,/e pc& of the full) reduc‘rd compounds. l&&s to d:ltcl indicatcb thni t,hc polymcrization of l-naphthol b!. th(l fungal rnzgmcx yields :L c~nmplrx mixturcb of polymerized products. While muc*h inl’ormation exists on the mechanism of tlimerization of phenols, little is kno\vn caonc*trrning polymr&ation, the proc*(xss of furt,her probably hccausc separation :rnd caharartcrization of thrse higher-mol(,c:nlar-weight pol.vmflrs rclprtxsc>nts a dificult. task. ‘1 simhypnthrticnl rnod(sl tlt>picting plificd 1-naphthol polymrrizat,ion is shonn in Fig. 3. Only t.h(L p-alp-coupled binaphtlhol is rc>prescntcd as thfa dimtAr and t)nly the fully rcducard compounds arc\ shon II. In vic,n of the rcasults \\-hic*h \\ (‘I’(’ obtaincd with 1-naphthol, whicah is :III ihstablishtbd intermcdiatc in the* degradation of the inseeticidfs carharyl, it is of irny)ortunc~
P~LYMFRIxATI~N to
focus
attention
erization
of
on
the
oxidases.
Polymerization
action
of
chemicals
these
cules
products that
are
attack,
and
should
be
possible mental
therefore of
impact
resistant
with
if one respect
inter-
4.
naturally
easily
reactions
concern
by
and with
may very
polym-
compounds
phenol occurring
possible
xenobiotic
OF
form to
mole-
of this considers to
5.
biological type their
6.
environ-
pollution.
7. ACKNOWLEDGMENT
This research was authorized for publication as Paper No. 4897 in the Journal series of the Pennsylvania Agricultural Experiment Station.
8. 9.
REFERENCES
1. B. R. Brown, Biochemical aspects of oxidative coupling of phenols, in “Oxidative Coupling of Phenols” (W. I. Taylor and A. R. Battersby, Eds.), p. 167, Marcel Dekker, New York, 1967. 2. J.-M. Bollag and S.-Y. Liu, Degradation of Sevin by soil microorganisms, Soil Biol. Biochem. 3, 337 (1971). 3. N. E. Stewart, R. E. Millernan, and W. P. Breese, Acute toxicity of the insecticide Sevin and its hydrolytic product 1-naphthol
10.
11. 12.
I-NAFHTHoL
463
to some marine organisms, Trans. Amer. Fish. Sot. 96, 25 (1.967). J.-M. Rollag, E. J. Czaplicki, and R. D. Minard, Bacterial metabolism of l-naphthol, J. Agric. Food Chem. 23, 85 (1975). J.-M. Bollag and S.-Y. Liu, Fungal degradation of I-naphthol, Canad. J. Microbial. 18, 113 (1972). J.-M. Bollag, R. D. Sjoblad, E. J. Czaplicki, and R. E. Hoeppel, Transformation of I-naphthol by the culture filtrate of Rhizoctonia praticola. Soil Biol. Biochem., 8,7 (1976). A. P. Dianin, The oxidation of naphthol by ferric chloride (in Russian), J. Russ. Phys. Chem. Sot. 6, 183 (1874). G. F%hraeus and H. Ljunggren, Substrate specificity of a purified fungal lactase, Bioehim. Biophys. Acta 46, 22 (1961). G. Fahraeus, On the oxidation of phenolie compounds by wood-rotting fungi, Ann. Roy. Agric. CoZZ. Sweden 16, 618 (1949). B. R. Brown and S. M. Backs, Some new enzymic reactions of phenols, in “Enzyme Chemistry of Phenolic Compounds” (J. B. Pridham, Ed.), p. 129, Pergamon Press, New York, 1963. B. It. Brown and A. H. Todd, A new perylene synthesis, J. Chem. Sot. 5564 (1963). I. S. Joffe and B. K. Kirchevstov, Diaryls and their derivatives. XXI. Oxidation of a-naphthol (in Russian), b. Gen. Chem. USSR 9, 1136 (1939).
BIOCHEMISTRY
AND
PHYSIOLOGY
Polymerization Compounds ROY D. SJOBLAD, Laboratory
6, 457463
(1976)
of I-Naphthol and Related Phenolic by an Extracellular Fungal Enzyme ROBERT
of Soil Microbiology, The Pennsylvania State
D. MINARD,
AND JEAN-MARC
of Agronomy, and Department University Park, Pennsylvania
Department University,
BOLLAG of Chemistry, 16802
Received October 15, 1975; accepted January 27, 1976 An extracellular phenol oxidase was isolated from the culture filtrate of the fungus Rhizoctoniu praticola by gel filtration on Sephadex G-200. The enzyme catalyzed the polymerization of I-naphthol to dimeric, trimeric, tetrameric, and pentameric compounds as determined by mass spectrometry. One product with a molecular weight of 286 was identified as 4,4’-bi-1-naphthol. Other compounds that were dimerized or polymerized by the enzyme were phenol, o-methoxyphenol (guaiacol), p-methylphenol (p-cresol), 2,6-dimethoxyphenol, and 1,3- and 1,5-dihydroxynaphthalene. The product from 2,6-dimethoxyphenol was isolated and identified as 3,5,3’,5’tetramethoxydiphenoquinone. INTRODUCTION
Phenolic compounds can be polymerized by several chemical reagents as well as by enzymatic activity. In nature biological oxidation and coupling of phenols are key reactions that result in the formation of products such as lignins, melanins, tannins, alkaloids, and antibiotics. Although polymerization by various microorganisms is recognized as an important reaction, relatively few basic studies exist on this subject, and little is known about the enzymes involved in these reactions (1). Our limited understanding about the formation of the natural products mentioned above has to cause concern if one considers that the numerous wastes and new chemicals used in the environment may not only be difficult to biodegrade but may also be enzymatically transformed to more complex products. These products might be even more recalcitrant to biodegradation and lead to the accumulation of potentially dangerous and, as of yet, undefined environmental pollutants. 457 Copright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
In our laboratory we studied the microbial transformation of the widely used insecticide carbaryl (I-naphthyl-N-methylcarbamate), and it was established that this compound is hydrolyzed biologically as well as chemically to 1-naphthol (2). This chemical can be more toxic to organisms than the compounds from which it originates (2, 3), and its fate in the environment is not yet clearly elaborated. It was shown that bacteria can metabolize 1-naphthol to COZ, but another pathway occurred simultaneously resulting in the accumulation of various products, one of which was identified as 4-hydroxy-l-tetralone (4). Bollag and Liu (5) also found that the mycelial cell extract of Fusarium solani mineralized 1-naphthol. When 1-naphthol was added to the growth medium of the soil fungus Rhizoctonia praticola, it was possible to detect a transformation of 1-naphthol to highmolecular-weight compounds which were formed even by the culture filtrate indicating the presence of an extracellular
-+;ss
RJOBLAD,
MINAHD
enzyme (6). Further, it was shown that upon some purification of the culture filtrate, an active enzymatic fraction was obt,ained which produced ether- or benzeneclxtractable compounds that differed from the water-soluble products obtained with thtb original culture filt’rate. In this investigation it was att,emptcd to characterize the compounds which wre formed by the partially purified enzymatic fraction from R. praticola. MATERIALS
AND
METHODS
Fungal growth ill liquid medium. Rhizoctwniu praticoEa (Vaartaja No. 1347) was grown in a modified Czapek Dox medium containing 2.0 g of L-asparagine, 0.5 mg of thiamine, 25 mg of CaS04+2Hz0, and 1 ml of a tract elements solution (Na2B*07 . 10HzO, 40 mg; R2nS01.2Hz0, 80 mg; FeSOJ.7H20, 40 mg ; ZnSOA.7Hz0, 800 mg; Na&IoO*.2HzO, 80 mg; CuSOa. 5Ha0, 40 mg/lOO ml of distilled water) dissolved in 1 liter of distilled water (final pH 7.3). The cultures were grown at 25°C on a rotary shaker (150 oscillations/min). Enzyme preparation al&d assay. R. praticola was grown for 5-7 days after which t,he mycelium was separated from the growth medium by filtration, and the culture filt,rate was lyophilized. The freezcdried filtrate was redissolved in 0.1 &’ phosphate buffer, pH 6.9, to give a final concentration of 100 mg dry wt/ml, and 10 ml were applied to a column (4 X 45 cm) of Scaphadtx G-200 (Pharmacia Fine Chemicals, Inc., New Market, nT.J.) equilibratcd with the buffer. The void volume was 180 ml and the flow rate was 1 ml/min. Fractions of 8 ml were collected and their activity was tested by incubation for 3 hr at 25°C with 50 pg/ml of 1-naphthol (containing 0.005 PCi l-[l-14C]naphthol.) I-Naphthol was dissolved in ethanol and the final concent.ration in an assay was 2%. Subsequcnt.ly, each fraction was extracted with benzene and the organic phase, which clontaincd all of the radioactivity, was analyzed by thin-layer chromatography
AND
BOLL4G
(TIE)’ in an &her--hexam> t.1: 1, v/v) solwnt system. I-Naphthol \v:ts visualizcld on the plate by exposure to rlltraviolrt (uv) light at, 254 nm, and its Ri-arc&a w:w scraped from the plattl :rnd t hc> radioactivity ~3s mcbasurcad. TOP most wtivc I-napthol-transforming fractions \V(‘rY used in subwqwnt invrstigations. Five milliliters of an assay mixture wit,h 50 pg/ml of 1-naphthol as subst r:tt.e ww extracted with an equal volun~~ of brnzfw and 4 ml of the bwzcncl mtrwt wtw washed thrw times \vith an w11t;tl volume of distilled water. Aftw b(bing dric*d ovw anhydrous I\ra$O,, the solvent was waporated under a stream of h$+um, and that product) mixtuw n-as anaI.wx~l bv mass spwtromrtry. Other phPnolic* compounds wcw addt>d to t,he clnxymc’ fractions to givcx ;L final concentration of 30 kg/ml, c~swpt. phenol 2,(i-dirnclt,h(,x~phcnol (100 Hg/ml) and (1 mg/ml). Transformation products from phenol, o-mc?thoxyph(~nol, and 1 .:3- and 1,5-dihydroxynaphthalen(s w(w wt,ract’ed wit,h chloroform. 1’rodwt.s from p-met,hylphenol ww clxtractrd with &It h!.l c%htlr and the prwipitstrd product from 2,fi-dimcthoxyphenol was cwllwtc~d b>, filtration. The product, extracts \v(w w:tsh(bd with distilled watcar, dried, and :m:tlyzt~d 1~) mass spwtromc+ry. Control (~xperimt~nts c~mpioying boilrd clnzyme fractions and thch above\ suhstratw ww inactiw and, after cxtrwtitbn, th(h subst,ratcs wrc thr only cwmp~~unds d(Jt,ccted by thin-layc>r analysis. Isolatiolr of I-~~aphthol polymers. Thv actlvts canzymr frac*tions uxw incbubatcd for 5 hr at 23°C with 50 gg, ml of i-naphthol as substratf>. Thrl assay mixture was then subjected t,o a dual c~xtrwtictn with equal volumes of bcnzr~nc~ and th(k wmbined extracts were dric>d over anhydrous Na2SOd and evaporatcld in :I flash waporator at 37°C. Subsrqucntly, the produc:ts 1 Abbreviations wed: TLC: graphy, IIV : rlltraviolet , MM’: tables and figlues 1.
thin-layer rnolrc~~lar
chromatew&ght iin
POLYMERIZATION
were redissolved in ether and separated on a preparative thin-layer plate with a triethylamine solvent system. Products were visualized by uv light at 254 nm. The compound being isolated was removed from the thin-layer plate, extracted from the silica gel with acetone, and rechromatographed in triethylamine. Final purification of the products was performed by repeated TLC in an ether-hexane (4: 1, v/v) solvent system and the isolated by mass compounds were analyzed spectrometry. Analytical methods and chemicals. Radioactivity was determined with an Isocap 300 liquid scintillation counter (Nuclear Chicago, Chicago, Ill.). Samples were measured in a modified Bray solution composed of 60 g of napthalene, 100 ml of methanol, and 8 g of Omnifluor (98yo PPO [2,5-diphenyloxazole] and 2% bisMSB [p-bis- (0-methylstyryl)-benzene] ; New England Nuclear Corp., Boston, Mass.) in 1 liter of dioxane. Precoated TLC plates (Brinkman Instruments, Inc., Westbury, N.Y.) with a thickness of 0.25 and 0.50 mm of silica gel F-254 were used for the routine thinlayer analyses and for the preparative separations, respectively. Mass spectra were determined using an AEI MS-902 mass spectrometer at an ionization potential of 70 eV and at a temperature of 350°C using the direct insertion probe. 1-Naphthol and o-methoxyphenol were purchased from Eastman Organic Chemicals (Rochester, N.Y.), and l-[l-‘“C Jnaphthol with a specific activity of 3.84 mCi/mmol from Mallinckrodt (St. Louis, MO.). Crystalline 1,3-dihydroxynaphthalene was obtained from Fisher Scientific Co. (Pittsburgh, Pa.) ; 2,6-dimethoxypheno1 (99%) from Aldrich Chemical Co. (Cedar Knolls, N.J.) ; and 1,5-dihydroxynaphthalene from J. T. Baker Chemical Co. (Pittsburgh, Pa.). All chemicals that were not of sufficient purity when purchased were purified by charcoal addition and recrystallization. Ferric chloride oxida-
459
OF I-NAPRTHoL
tion of 1-naphthol was used to synthesize 4,4’-bi-1-naphthol in a procedure described by Dianin (7). RESULTS
When the culture filtrate from the fungus R. praticola grown in a modified Czapek Dox medium was concentrated by lyophilization and subsequently chromatographed on a column of Sephadex G-200, fractions were obtained that were able to transform 1-naphthol as substrate. The formation of a purple color was evident in the active fractions, and TLC analysis of the benzene extract of these fractions clearly showed a decrease of radioactive material in the Rf-area of I-naphthol (Fig. 1). The radioactivity which was lost from the R,-area of I-naphthol during incubation was recovered completely on the TLC plate in areas with an R,-value less than that of I-naphthol. Since the strongest enzyme activity was found in fractions 14 through 19, all the subsequent experiments were performed using the eluate of these six fractions. Heated active fractions were devoid of activity on the tested substrate. The benzene extract of the active fractions after incubation with I-naphthol was
FIQ. 1. Transformation after Sephadex G-Z?OO gel culture filtrate from R. radioactivity (in percent of l-naphthol after TLC in solvent system. Fraction 1 Further details see text.
oj I-naphthol (50 pg/ml) filtration of the lyophilized praticola. Data represent remaining at the Rf-area an ether-hexane (4~1,v/v) starts after the void volume.
460
SJOBLAD,
MINARD
subjected to mass spectrometric analysis to establish some of the characteristics, particularly the molecular weights, of the formed products. A mass spectrum of the reaction mixture after incubation for three hours is reproduced in Fig. 2. Major ,m/e peaks were observed at 144, 286, 425, 570, and 712 in this analysis. The substrate, I-napht,hol, has a mass of 144 and a dinwrized product would be represented by the> VI/~: pc:tk at 286. The detect’ed peak at 1% coincides with the trimer, and the w le peak at, 570 would represent a tetramc~r. The highest polymer observed with thcx mass spe&ometer corresponds to a pentamer, caxhibiting a peak at 712. A fragmmt,ation product with a loss of 1S mass units (HZO) was observed from c:wh of thr oligomers formed. Attempts were then made to separate thfa individual compounds of I-naphthol transformation to establish that they are not, fragment ions from highcr-molccularwight polymers. TIC was effective in separating two dimers, a trimer, and ~1 trtramrr. The tctramer remained at the origin of the plate with triethylamine as
ASD
BOLLAG
the solvent system, but it. \V:I.S mobile in ether-hexane (Table 1). I~[RSSspwtral data of the isolated products ~wtablish~d that the mixture of compounds wprtwntt~d in Fig. 2 is composed of individual oligomcrs of I-naphthol. Two isomcric clinlcw of 1-naphthol with molecular \vcsight,t: of 286 ww isolated (Table I). 13nwrs .A :md B posstwwd different RJ v:\lw~ in t hcl thinlayer systems used, and t,hc:y g:tvc~tlifirwnt fragment&on pattclrns \vh(w :rrl:~l~~zc~lb) mass spwtromrtry. Dimc~r IS i:: 1..+‘-hi-lnaphthol, sincr th(l mass sywt*~rum, IIV spwt~rum, melting point, (SOOY’), and thin-layer c*haractchristics of the* isolattd prodwt \v(w idcnticaal with thci :tuthontic~ cht>mic:al. ,I trimcr (molwulwr vxsight 1%) and :I tc$r:unw (molrcwl:~r \vcGghl 570) ww also dtJtectc>d by masti spwtromr+ry after separation I,]- TI,C’. but the, pc*nt:k nwrica product has not >,c,t b(w is ~I:~tcd.
m/e FIG. 2. Mass qf R. praticola
spectrum obtained from the brnzcnc with I-naphlhol as substrate.
rxtrart
I$’ an assay
with
a partidly
pltri$d
WLZ!JU~V
POLYMERIZATION
OF TABLE
TLC
Compound
and Mass
461
l-NAPHTHOL 1
Spectral Data for I-Naphthol, 4,4’-Bi-1-Naphthol, Isolated Oligomers from I-Naphthol Rf in solvent
Triethylamine
system
and
Mass
Ether-hexane (4: 1, v/v)
MW
spectral
analysis
Intensity
(%)
at m/e
144
286
428
570
1-Naphthol 4,4’-Bi-1-naphthol
0.82 0.67
0.90 0.72
144 286
100 3
100
-
-
Isolated compounds Dimer A Dimer B Trimer Tetramer
0.76 0.67 0.30 0.00
0.84 0.72 0.59 0.53
286 286 428 570
19 3 -5 9
100 100 15 16
100 5
100
with different phenolic compounds. Mass spectrometry was also used as the analytical method for determining the activity of the enzyme on the various aromatic substrates. Table 2 shorn that dimerized products were detected after incubation of the active fractions with p-methylphenol, 2,6-dimethoxyphenol, and 1,3- and 1,5dihydroxynaphthalene. o-Methoxyphenol was polymerized to a trimeric and phenol to a tetrameric product. As mentioned previously, the highest polymer detected with I-naphthol by mass spectrometry was the pentamer. Polymers of higher molecular weight could well be present, but are probably too involatile to detect under the conditions used for mass spectrometric analysis. Whereas it was assumed that some of the tested phenolic chemicals formed higher polymers which were not detected, 2,6dimethoxyphenol represents a compound whose coupling possibilities are limited. In this case it was easy to establish that after incubation of 2,6-dimethoxyphenol with the enzyme, the p-$-coupled quinonoid product, 3,5,3’,5’-tetramethoxydiphenoquinone (coerulignone) resulted as the end product. This compound was formed in the enzyme solution and precipitated as steel-blue needles in a yield of 90%. Its melting point after recrystallization from
a mixture of phenol-methanol was 289291”C, and its uv spectrum (in chloroform) and its ir spectrum (BBr) were identical with authentic material. DISCUSSION
It was established that the fungus Rhizoctoniu praticola forms an extracellular enzyme which is active in polymerizing l-naphthol, as well as a variety of other phenolic compounds. The enzyme could be isolated from the culture filtrate by gel filtration on Sephadex G-200, and it is assumed that it is a phenol oxidase, most TABLE Formation Compounds
Phenolic
2
of Polymerized Products from Phenolic ajter Incubation with an Extracellular Enzyme jrom R. praticola compound
Phenol o-Methoxyphenol p-Methylphenol 2,6-Dimethoxyphenol 1-Naphthol 1,3-Dihydroxynaphthalene 1,5-Dihydroxynaphthalene
Enzymatic products as detected by mass spectrometry Dimer, trimer, tetramer Dimer, trimer Dimer Dimer Dimer, trimer, tetramer, pentamer Dimer Dimer
4fi2
SJOBLAD,
MINAHD
probably of the Iaccase t,ype which is known to be produced by different fungi (1). Thr wide range of substrate specificity of most phenol oxidases from fungi has prrviouslg been established, and F%hraeus and Ljunggrrn (8) have shown that oxygen uptakr occurred when or&o-, mefa-, and para-substituted phenols were incubated with a lactase from Polyporus versicolor. The enzymct from R. praticola also oxidized and coupled an orth.o- (o-mcthoxyphenol). a meta- (1,3-dihydroxynaphthalt>nc), and a para-substituted phenol (p-mcth.vlphrlnol). I;fthracsus recognized that white-rot fungi w(lrc’ ahIf> to transforms 1-naphthol to dark-colored products (9) and Brown and Hocks (10) suggested that a lactase from 1,. vewidor converted I-naphthol to a purple quinonoid polymer, but in neither cast was a polymerized product isolated or identified, nor were any data presented that, substantiated a polymerization r(‘action with 1-naphthol a,s a substrate. The ma,ss spectral data of the enzymatic products from I-naphthol in our investigation shoW that this substance was polymerizchd, at least, to the pentamer, and the major m/e peaks at 286, 428, and 570 rcprcsent individual compounds and not,
MW.
FIG.
I-naphthol
3. Hypothetical hg a Jungal
scheme enzyme.
.for
570
polymerization
of
AND
BOLLAG
fragments of higher mol(4ar \vc*ight PO!?mers. The peak at. VZ/C 711, in thr mass sp&rum of the crude producet mixture indicated thr presence of thca p(~nt~arncr, but t,his compound has not bacon isolatrxd. Even t,hough, for thtb most plirt,, thfh mass sprctrnl data indicatct th(b formation of rclducrd hydroxylatcd c.c)mpountls from I-naphthol, t,hf>rcb is rc’uson to b(&(~v(~ that quinoncs arc’ also produc*ckd I)!, t h(~ acation of the (lnzym(b from f?, piaf!ccdu, ‘rho (Y)I~Ipound, 2-methyl-1-naphthol is (~(~nvert(~d by 3 lac*casr from 1’. PPr.Sic0l~N 1.0 thr> product, 3,3’-dimethyl-l , 1 ‘-binal,lrt~lyl-l,l’quinonr (1 I) in 63% y&Id :iftcbr irtc.llbation \vvith th(b subst,ratr for 4 days iit 30°C~. On thrk other hand, oxidativtb (*oupling c)t’ l-naphthol hy F(Cl~ yichlds thrccb ixornchrica binaphthols (from o--0, 11 I). and ,U /) (*OIIpling) (12) and this report cLnt:lblish(ld that at least t\vo of th(i binaphthols can Of result from c>nzymatic+ c*cmplirlg I-naphthol. Th(i intt,nscl purpl(l color a~)(-i;tf(hd with that r>nzymatic products from 1-naphthol is indicativcl of quinonc f’ormaticm ; how(lv(lr, the isolated oligomt~rs w(‘r(’ ~11 colorand their m;Lss sp(lct ra less compounds, clxhibited thfl major rTl,/e pc& of the full) reduc‘rd compounds. l&&s to d:ltcl indicatcb thni t,hc polymcrization of l-naphthol b!. th(l fungal rnzgmcx yields :L c~nmplrx mixturcb of polymerized products. While muc*h inl’ormation exists on the mechanism of tlimerization of phenols, little is kno\vn caonc*trrning polymr&ation, the proc*(xss of furt,her probably hccausc separation :rnd caharartcrization of thrse higher-mol(,c:nlar-weight pol.vmflrs rclprtxsc>nts a dificult. task. ‘1 simhypnthrticnl rnod(sl tlt>picting plificd 1-naphthol polymrrizat,ion is shonn in Fig. 3. Only t.h(L p-alp-coupled binaphtlhol is rc>prescntcd as thfa dimtAr and t)nly the fully rcducard compounds arc\ shon II. In vic,n of the rcasults \\-hic*h \\ (‘I’(’ obtaincd with 1-naphthol, whicah is :III ihstablishtbd intermcdiatc in the* degradation of the inseeticidfs carharyl, it is of irny)ortunc~
P~LYMFRIxATI~N to
focus
attention
erization
of
on
the
oxidases.
Polymerization
action
of
chemicals
these
cules
products that
are
attack,
and
should
be
possible mental
therefore of
impact
resistant
with
if one respect
inter-
4.
naturally
easily
reactions
concern
by
and with
may very
polym-
compounds
phenol occurring
possible
xenobiotic
OF
form to
mole-
of this considers to
5.
biological type their
6.
environ-
pollution.
7. ACKNOWLEDGMENT
This research was authorized for publication as Paper No. 4897 in the Journal series of the Pennsylvania Agricultural Experiment Station.
8. 9.
REFERENCES
1. B. R. Brown, Biochemical aspects of oxidative coupling of phenols, in “Oxidative Coupling of Phenols” (W. I. Taylor and A. R. Battersby, Eds.), p. 167, Marcel Dekker, New York, 1967. 2. J.-M. Bollag and S.-Y. Liu, Degradation of Sevin by soil microorganisms, Soil Biol. Biochem. 3, 337 (1971). 3. N. E. Stewart, R. E. Millernan, and W. P. Breese, Acute toxicity of the insecticide Sevin and its hydrolytic product 1-naphthol
10.
11. 12.
I-NAFHTHoL
463
to some marine organisms, Trans. Amer. Fish. Sot. 96, 25 (1.967). J.-M. Rollag, E. J. Czaplicki, and R. D. Minard, Bacterial metabolism of l-naphthol, J. Agric. Food Chem. 23, 85 (1975). J.-M. Bollag and S.-Y. Liu, Fungal degradation of I-naphthol, Canad. J. Microbial. 18, 113 (1972). J.-M. Bollag, R. D. Sjoblad, E. J. Czaplicki, and R. E. Hoeppel, Transformation of I-naphthol by the culture filtrate of Rhizoctonia praticola. Soil Biol. Biochem., 8,7 (1976). A. P. Dianin, The oxidation of naphthol by ferric chloride (in Russian), J. Russ. Phys. Chem. Sot. 6, 183 (1874). G. F%hraeus and H. Ljunggren, Substrate specificity of a purified fungal lactase, Bioehim. Biophys. Acta 46, 22 (1961). G. Fahraeus, On the oxidation of phenolie compounds by wood-rotting fungi, Ann. Roy. Agric. CoZZ. Sweden 16, 618 (1949). B. R. Brown and S. M. Backs, Some new enzymic reactions of phenols, in “Enzyme Chemistry of Phenolic Compounds” (J. B. Pridham, Ed.), p. 129, Pergamon Press, New York, 1963. B. It. Brown and A. H. Todd, A new perylene synthesis, J. Chem. Sot. 5564 (1963). I. S. Joffe and B. K. Kirchevstov, Diaryls and their derivatives. XXI. Oxidation of a-naphthol (in Russian), b. Gen. Chem. USSR 9, 1136 (1939).