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Production of substituted catechols from substituted benzenes by a Pseudomonas sp.
Production of substituted catechols from substituted benzenes by a P s e u d o m o n a s sp. James B. Johnston* and V. Renganathan Smith Kline and French Laboratories, Swedeland, PA 19479, USA (Received 8 April 1987; revised 30 June 1987)
In the presence of a halobenzene or benzonitrile, Pseudomonas T-12 can produce substituted catecholsJrom the corresponding substituted benzenes. A variety of monosubstituted benzenes with substituents containin9 up to four carbons, and some meta- and para- disubstituted benzenes, can serve as catechol precursors.
Keywords:Catechol-2,3-oxygenase;halocatechol; fluorocatechol; benzonitrile; fluorobenzene; Pseudomonas Introduction The catabolic enzymes of m a n y hydrocarbon-degrading bacteria perform adventitious reactions that can transform unnatural substrates related to their natural substrates. Usually the overall type of transformation, e.g., dehydrogenation, hydroxylation, etc. and reaction mechanism are essentially unchanged, and the reaction is considered adventitious only because an unnatural substrate is transformed. An example of such a reaction is the oxygenation of indole by the naphthalene oxygenase of Pseudomonas putida. 1 In principle, enzymes performing adventitious reactions can be recruited for the production of chemicals useful as intermediates in synthetic chemical processes. The vast technological potential of adventitious enzyme reactions in living cells has not been widely exploited, however. Factors restricting exploitation frequently include the need for a stimulus to induce enzyme synthesis, penetration of the adventitious substrate to the subcellular space where the enzyme resides, supply of essential reaction cofactors and cosubstrates, blocking the further metabolism of the reaction's product and export of the product to the culture medium. Toluene catabolism via 3-methylcatechol by species of Pseudomonas is a system where all of these restrictions can be overcome. The work presented here will show that this system can be recruited for the production of a wide variety of substituted catechols by intracellular enzymes functioning in situ. The pathway of toluene catabolism via 3-methylcatechol has been described previously 2 and is illustrated in Figure 1. In addition to natural substrates, i.e., those supporting the growth of strains harboring this pathway, all of the halobenzenes induce this pathway and are converted to the corresponding 3-halocatechols. 2-4
*Present address: Enzymatics Inc., Horsham, PA 19044, USA
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Enzyme Microb. Technol., 1 987, vol. 9, December
When the ring-opening catechol oxygenase acts on halocatechols, the product is an acylhalide that can alkylate this oxygenase and inactivate it. 4 The metabolic block introduced by inactivation of this oxygenase causes accumulation of halocatechol in the intracellular space and eventually leads to excretion of the halocatechol to the culture medium. We reasoned that if a second substituted benzene substrate were simultaneously supplied with a halobenzene, any catechol formed from the second substrate should also accumulate and be excreted to the medium. In the course of this work, benzonitrile was found to act like the halobenzenes, inducing the catechol-forming enzymes and fostering excretion of the catechols of alternate substituted benzenes.
Materials and methods Strains and culture conditions Strain T-12 is a fluorescent Pseudomonas isolated from soil by enrichment culture on toluene. It was routinely cultured on L-broth, 5 or ATCC medium 508, 6 a defined, minimal medium, supplemented with 0.2% D-glucose or L-glutamate. Production a n d analysis o f catechols All chemicals were obtained from Aldrich and were used without further purification. Catechol production from alternate substrates was tested in tube cultures. Strain T-12, grown on 508 agar over toluene vapor as the sole carbon and energy source, was suspended in 508 medium and grown for 4-8 h on glucose plus glutamate. The culture was diluted 10-fold and partitioned into 5 ml aliquots in 65 ml screw capped tubes. Two/zl offluorobenzene or benzonitrile plus 5 #1 of the alternate substituted benzene substrate were spotted on a cotton plug that was lodged in the top of the tube and the cap screwed shut. After overnight incubation at 30°C on a rotary shaker, the culture was saturated with solid NaCI, extracted with 2 ml
0141-0229/87/120706-03 $03.00 © 1987 Butterworth Publishers
Production of substituted catechols: J. B. Johnston and V. Renganathan
~ OH
OH
~
,
~"~//~OH
H Substituted Benzene
Dihydrodiol
Catechol
Ring Fission Product
Figure 1 P r o p o s e d p a t h w a y f o r m e t a b o l i s m o f s u b s t i t u t e d b e n z e n e s b y Pseuffomonas T - 1 2 G r o w t h substrates for T - 1 2 i n c l u d e R = H, C H 3, C 2 H 5. T h e p a t h w a y is a n a l o g o u s t o p a t h w a y s o f s u b s t i t u t e d b e n z e n e m e t a b o l i s m in o t h e r strains, 2 a n d is s u p p o r t e d b y t h e i s o l a t i o n o f s u b s t i t u t e d c a t e c h o l s a n d s u i c i d e i n h i b i t i o n b y R - h a l o g e n or C N ( t h i s r e p o r t )
ethyl acetate and aliquots of the organic phase were spotted on 250-micron thick silica gel thin layer plates containing fluorescent indicator. The chromatograms were developed in toluene:ethyl acetate:acetic acid (60:40: 1), dried and visualized under u.v. and sprayed with Gibb's reagent (10 mg m l - 1) in m e t h a n o l f '8 Yields were quantified by the Arnow reaction 9 after separation of substituted catechols by semipreparative thin layer chromatography (t.l.c.). Total catechols were determined in the ethyl acetate extract; the solvent was evaporated under nitrogen, and the residue redissolved to a final concentration of 20 mM. A thin band containing 0.8/~mole of total catechol was chromatographed, and the bands containing the resolved catechols were scraped from the plate. After resuspension in 0.5 N HCI and removal of silica particles by centrifugation, the isolated substituted catechols were quantified by the Arnow reaction. Catechol concentrations were determined from a standard curve constructed with unsubstituted catechol. In our hands, standard curves constructed with catechol, 4-methylcatechol and 3-cyanocatechol were identical within experimental error.
Results and discussion Table 1 displays the results of catechol-formation trials with mono-substituted benzenes, and Table 2 shows the comparable results for a number of disubstituted benzenes. For each example shown, there was abundant formation of the catechol of the inducing substrate,
fluorobenzene or benzonitrile. A positive result was scored when the catechol of the substituted benzene gave a spot of comparable intensity to that from the inducing substrate. Weak results were scored (wk) when the substituted benzene gave a catechol spot clearly less intense than that of the inducing substrate. A negative was scored when the only catechol spot detected was due to the inducing substrate. Negatives were always tested in pairs of tubes using fluorobenzene and benzonitrile as the inducer substrate. Because the catechols of these inducer substrates have different Rfs, a negative result with both inducers rules out the possibility that a new catechol was formed from the test benzene but masked on the t.l.e, plate by a cateehol from the inducer having the same Rf. The range of monosubstitutions on the benzene ring leading to new catechols in the T-12 system includes alkyl side-chains with one to four carbons, electron-donating substituents like methoxyl and ethoxyl, and electronwithdrawing substituents such as - C H O , - C N and - C F 3. This suggests that electronic factors do not strongly influence catechol formation. Benzenes with electron-donating substituents such as methoxy or methyl are as good substrates as those with electron-withdrawing substituents such as nitrile or halogen. Nitrobenzene is a possible exception to this generalization, as it does not form a catechol in this test. Moreover, it is a potent inhibitor of catechol formation from fluorobenzene and benzonitrile. Whether this inhibition is Table
2
D i s u b s t i t u t e d b e n z e n e s as c a t e c h o l p r e c u r s o r s Catechol formed
Table
1
Monosubstituted
b e n z e n e s as c a t e c h o l p r e c u r s o r s
Catechol substituent
Formed
-Halogen -CH 3 -C2H 5 C3H 7 -CH(CH3)CH 3 -CH CH2=CH 2 -CH=CH CH 3 --CH(CH3) =CH 2 --cyclopropyl
+ + + 4+ + 4+ +
- n - , iso-, sec-, tert-butyl C6H 5 -C---CH -CH =CH 2 wk - weak, +, -.
wk + See t e x t
Catechol substituent
Formed
-CN -CF 3 -OCH 3 -OC2H 5 -CHO -CH2OH -CH2CH2OH CHOHCH 3 -CHOHCH2OH -CH2CH2CH2OH CH2CHOHCH 3 -CHOHC2H 5 - C(CH3)~2OH
+ + + + + 4+ 4-
Substrate
ortho-
meta-
para-
Xylene Ethyl t o l u e n e Isopropyl toluene Fluorotoluene Chlorotoluene Difluorobenzene Dichlorobenzene Dibromobenzene Diiodobenzene Fluorobenzonitrile Chlorobenzonitrile Dicyanobenzene Fluoroanisole Fluorobenzotrifluoride Fluorobenzaldehyde Fluorobenzyl alcohol
-
NT + NT NT + -
+ + + + + wk wk + -
NT -
NT NT
-
N T = n o t tested, w k = w e a k , + , - .
See t e x t
Enzyme Microb. Technol., 1987, vol. 9, December
707
Papers
mediated by a direct action on the catechol-forming enzymes, or by inhibition of enzyme induction, is not known. Several of the halogenated benzenes led to excretion of a substituted catechol when incubated with strain T-12 in the absence of either fluorobenzene or benzonitrile. These substrates included all of the monohalogenated benzenes, para-fluorotoluene, para-chlorotoluene, paradifluorobenzene, and para-dichlorobenzene. Each of these substrates apparently induced the 3-alkylcatechol pathway, para-Xylene, trifluoromethyl benzene and fluorobenzonitriles required the presence of an inducer/ inhibitor to form catechols. Cyclopropylbenzene accumulated a yellow metabolite in the absence of an inducer/inhibitor, but yields a catechol in their presence. Among the mono-substituted alkylbenzenes that gave rise to catechols, Table 1, there was a substantial decrease in amount of catechol formed when the substituent went from three to four carbons. The propyl-, allyl-, and cyclopropyl-benzenes and methyl styrenes were all good substrates while none of the butyl benzenes was. Within the butyl benzene series there was a clear trend in visual spot intensity on t.l.c, plates going from the most intense with tert-butyl benzene to n-butyl benzene, which made a barely detectable trace of catechol. Among the phenylalkylalcohols, benzyl alcohol, 1-phenyl ethanol and 2phenyl ethanol gave good yields of catechol (Table 3), while none of the phenylpropanols gave rise to detectable catechol. These observations imply that steric factors such as the size of the substituent play a determining role in substrate acceptability. Several para-disubstituted benzenes were good precursors to catechols, as were a few meta-disubstituted benzenes (Table 2). Within a substitution pattern, substituent size appears to play a predominant role in determining what substitutions were accepted, para-Xylene was a good substrate, but para-ethyl toluene and paracymene were not. Methyl, halo- and cyano-substitutions were most often present on disubstituted benzenes that were converted to cateehols. None of the orthodisubstituted benzenes tested served as catechol precursors (Table 2). The ability of a substrate to be converted to a catechol depends on the substrate specificity of two enzymes, a toluene dioxygenase to produce a dihydrodiol, and a dehydrogenase to yield the catechol (Figure 1 ). Studies in other laboratories using toluene dioxygenase isolated from P. putida have shown that biphenyl, ortho-, rectaand para-dichlorobenzene can be transformed into the corresponding dihydrodiols. 2'1°'11 Of these, only paradichlorobenzene is a catechol precursor in strain T-12. para-Difluorobenzene, a catechol precursor for strain T12, was not tested with the P. putida system. Fused ring compounds tested with strain T-12 included naphthalene, tetralin and acenaphthene. None of these formed hydroxylated products detectable with Gibb's reagent. Indole incubated with strain T-12 and fluorobenzene gave rise to a series of colored products, presumably due to oxidation of indole ~ Two trisubstituted compounds, 3-chloro-ortho-xylene and SK&F 86466 (6-ehloro-3-methyl-2,3,4,5-tetrahydro-lH3-benzazepine), were not attacked. Catechols with substitution in the 3-position are the presumed products from monosubstituted benzenes. This has been confirmed for the products from fluorobenzene
708
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 7 , v o l . 9, D e c e m b e r
Table 3
Total and isolated yields of selected catechols
Catechol precursor
para-Xylene Cumene Benzaldehyde Benzyl alcohol 2-Phenyl ethanol 1-Phenyl ethanol Anisole Phenetole para- Fluorobenzonitrile meta- Fluorobenzonitrile
Total catechol a (raM)
Fractional yield b from: Inducer c
Precursor
0.86 2.0 1.3 2.0 1.4 0.89 1.9 1.6
0.41 0.18 0.54 0.15 0.29 0.12 0.37 c 0.78 c
0.02 0.40 0.35 0.58 0.43 0.62 0.37 0.02
0.98
0.61
0.10
0.77
0.13
0.52
aTotal catechol concentration in the culture broth b#moles of catechol isolated from t.l.c, plate scrapings divided by /~moles of total catechol applied to the t.l.c, plate CFluorobenzene was the inducer for all compounds except anisole and phenetole; benzonitrile was the inducer/inhibitor for anisole and phenetole
and benzonitrile. For the product from metadisubstituted benzenes, it is not known whether one or both of the possible isomeric products is produced. Analyses of the structure of these products will be communicated in the future. Total and isolated yields of a number of substituted catechols are described in Table 3. Quantification employed the Arnow reaction, which is specific for catechols. 9 Spiking culture broths with unsubstituted catechol demonstrated that the Arnow reaction was a reliable, quantitative measure of total catechol in the broth. Using the protocol for Table 3, 80% of the catechol in the broth was obtained in the ethyl acetate extract of cultures receiving only fluorobenzene or benzonitrile. Recovery of these catechols following t.l.c, averaged 75%, for an overall isolated yield of 60%. These results were obtained with a simple, unoptimized protocol that potentially involves competition between inducer and test substrate in the organism, variable recovery by a single solvent extraction and variable loss during t.l.c, separation and recovery of the catechol products. Nevertheless, it is clear that this biochemical process could be used as a preparative method for these catechols. Moreover, optimization of the fermentation, extraction and separation steps will increase the overall yields.
References 1 Ensley, B. D. et al. Science 1983, 222, 167-169 2 Gibson, D. T. and Subramanian, V. in Microbial Degradation of Organic Compounds (Gibson, D. T., ed.) Marcel Dekker, New York, 1984, pp. 181 252. 3 Gibson, D. T. et al. Biochemistry 1968, 7, 3795-3802 4 Bartels, I., Knackmuss, H-J, and Reineke, W. Appl. Environ. Microbiol. 1984, 47, 500-505 5 Lennox, E. S. Virology 1955, 1, 190-206 6 American Type Culture Collection in Catalogue of Strains 1, 15th Edition, ATCC, Rockville, Md., 1982, p. 623 7 Gibbs, H. D. J. Biol. Chem. 1927, 72, 649 664 8 Josephy, P.D. andVanDamme, A. Anal. Chem. 1984,.~,813 814. 9 Arnow, L. E. J. Biol. Chem. 1937, 118, 531 537 10 Ziffer, H. et al. Tetrahedron 1977, 33, 2491 2496 11 Safe, S. H. in Microbial Degradation of Organic Compounds (Gibson, D. T., ed.) Marcel Dekker, New York, 1984, pp. 361 369
In the presence of a halobenzene or benzonitrile, Pseudomonas T-12 can produce substituted catecholsJrom the corresponding substituted benzenes. A variety of monosubstituted benzenes with substituents containin9 up to four carbons, and some meta- and para- disubstituted benzenes, can serve as catechol precursors.
Keywords:Catechol-2,3-oxygenase;halocatechol; fluorocatechol; benzonitrile; fluorobenzene; Pseudomonas Introduction The catabolic enzymes of m a n y hydrocarbon-degrading bacteria perform adventitious reactions that can transform unnatural substrates related to their natural substrates. Usually the overall type of transformation, e.g., dehydrogenation, hydroxylation, etc. and reaction mechanism are essentially unchanged, and the reaction is considered adventitious only because an unnatural substrate is transformed. An example of such a reaction is the oxygenation of indole by the naphthalene oxygenase of Pseudomonas putida. 1 In principle, enzymes performing adventitious reactions can be recruited for the production of chemicals useful as intermediates in synthetic chemical processes. The vast technological potential of adventitious enzyme reactions in living cells has not been widely exploited, however. Factors restricting exploitation frequently include the need for a stimulus to induce enzyme synthesis, penetration of the adventitious substrate to the subcellular space where the enzyme resides, supply of essential reaction cofactors and cosubstrates, blocking the further metabolism of the reaction's product and export of the product to the culture medium. Toluene catabolism via 3-methylcatechol by species of Pseudomonas is a system where all of these restrictions can be overcome. The work presented here will show that this system can be recruited for the production of a wide variety of substituted catechols by intracellular enzymes functioning in situ. The pathway of toluene catabolism via 3-methylcatechol has been described previously 2 and is illustrated in Figure 1. In addition to natural substrates, i.e., those supporting the growth of strains harboring this pathway, all of the halobenzenes induce this pathway and are converted to the corresponding 3-halocatechols. 2-4
*Present address: Enzymatics Inc., Horsham, PA 19044, USA
706
Enzyme Microb. Technol., 1 987, vol. 9, December
When the ring-opening catechol oxygenase acts on halocatechols, the product is an acylhalide that can alkylate this oxygenase and inactivate it. 4 The metabolic block introduced by inactivation of this oxygenase causes accumulation of halocatechol in the intracellular space and eventually leads to excretion of the halocatechol to the culture medium. We reasoned that if a second substituted benzene substrate were simultaneously supplied with a halobenzene, any catechol formed from the second substrate should also accumulate and be excreted to the medium. In the course of this work, benzonitrile was found to act like the halobenzenes, inducing the catechol-forming enzymes and fostering excretion of the catechols of alternate substituted benzenes.
Materials and methods Strains and culture conditions Strain T-12 is a fluorescent Pseudomonas isolated from soil by enrichment culture on toluene. It was routinely cultured on L-broth, 5 or ATCC medium 508, 6 a defined, minimal medium, supplemented with 0.2% D-glucose or L-glutamate. Production a n d analysis o f catechols All chemicals were obtained from Aldrich and were used without further purification. Catechol production from alternate substrates was tested in tube cultures. Strain T-12, grown on 508 agar over toluene vapor as the sole carbon and energy source, was suspended in 508 medium and grown for 4-8 h on glucose plus glutamate. The culture was diluted 10-fold and partitioned into 5 ml aliquots in 65 ml screw capped tubes. Two/zl offluorobenzene or benzonitrile plus 5 #1 of the alternate substituted benzene substrate were spotted on a cotton plug that was lodged in the top of the tube and the cap screwed shut. After overnight incubation at 30°C on a rotary shaker, the culture was saturated with solid NaCI, extracted with 2 ml
0141-0229/87/120706-03 $03.00 © 1987 Butterworth Publishers
Production of substituted catechols: J. B. Johnston and V. Renganathan
~ OH
OH
~
,
~"~//~OH
H Substituted Benzene
Dihydrodiol
Catechol
Ring Fission Product
Figure 1 P r o p o s e d p a t h w a y f o r m e t a b o l i s m o f s u b s t i t u t e d b e n z e n e s b y Pseuffomonas T - 1 2 G r o w t h substrates for T - 1 2 i n c l u d e R = H, C H 3, C 2 H 5. T h e p a t h w a y is a n a l o g o u s t o p a t h w a y s o f s u b s t i t u t e d b e n z e n e m e t a b o l i s m in o t h e r strains, 2 a n d is s u p p o r t e d b y t h e i s o l a t i o n o f s u b s t i t u t e d c a t e c h o l s a n d s u i c i d e i n h i b i t i o n b y R - h a l o g e n or C N ( t h i s r e p o r t )
ethyl acetate and aliquots of the organic phase were spotted on 250-micron thick silica gel thin layer plates containing fluorescent indicator. The chromatograms were developed in toluene:ethyl acetate:acetic acid (60:40: 1), dried and visualized under u.v. and sprayed with Gibb's reagent (10 mg m l - 1) in m e t h a n o l f '8 Yields were quantified by the Arnow reaction 9 after separation of substituted catechols by semipreparative thin layer chromatography (t.l.c.). Total catechols were determined in the ethyl acetate extract; the solvent was evaporated under nitrogen, and the residue redissolved to a final concentration of 20 mM. A thin band containing 0.8/~mole of total catechol was chromatographed, and the bands containing the resolved catechols were scraped from the plate. After resuspension in 0.5 N HCI and removal of silica particles by centrifugation, the isolated substituted catechols were quantified by the Arnow reaction. Catechol concentrations were determined from a standard curve constructed with unsubstituted catechol. In our hands, standard curves constructed with catechol, 4-methylcatechol and 3-cyanocatechol were identical within experimental error.
Results and discussion Table 1 displays the results of catechol-formation trials with mono-substituted benzenes, and Table 2 shows the comparable results for a number of disubstituted benzenes. For each example shown, there was abundant formation of the catechol of the inducing substrate,
fluorobenzene or benzonitrile. A positive result was scored when the catechol of the substituted benzene gave a spot of comparable intensity to that from the inducing substrate. Weak results were scored (wk) when the substituted benzene gave a catechol spot clearly less intense than that of the inducing substrate. A negative was scored when the only catechol spot detected was due to the inducing substrate. Negatives were always tested in pairs of tubes using fluorobenzene and benzonitrile as the inducer substrate. Because the catechols of these inducer substrates have different Rfs, a negative result with both inducers rules out the possibility that a new catechol was formed from the test benzene but masked on the t.l.e, plate by a cateehol from the inducer having the same Rf. The range of monosubstitutions on the benzene ring leading to new catechols in the T-12 system includes alkyl side-chains with one to four carbons, electron-donating substituents like methoxyl and ethoxyl, and electronwithdrawing substituents such as - C H O , - C N and - C F 3. This suggests that electronic factors do not strongly influence catechol formation. Benzenes with electron-donating substituents such as methoxy or methyl are as good substrates as those with electron-withdrawing substituents such as nitrile or halogen. Nitrobenzene is a possible exception to this generalization, as it does not form a catechol in this test. Moreover, it is a potent inhibitor of catechol formation from fluorobenzene and benzonitrile. Whether this inhibition is Table
2
D i s u b s t i t u t e d b e n z e n e s as c a t e c h o l p r e c u r s o r s Catechol formed
Table
1
Monosubstituted
b e n z e n e s as c a t e c h o l p r e c u r s o r s
Catechol substituent
Formed
-Halogen -CH 3 -C2H 5 C3H 7 -CH(CH3)CH 3 -CH CH2=CH 2 -CH=CH CH 3 --CH(CH3) =CH 2 --cyclopropyl
+ + + 4+ + 4+ +
- n - , iso-, sec-, tert-butyl C6H 5 -C---CH -CH =CH 2 wk - weak, +, -.
wk + See t e x t
Catechol substituent
Formed
-CN -CF 3 -OCH 3 -OC2H 5 -CHO -CH2OH -CH2CH2OH CHOHCH 3 -CHOHCH2OH -CH2CH2CH2OH CH2CHOHCH 3 -CHOHC2H 5 - C(CH3)~2OH
+ + + + + 4+ 4-
Substrate
ortho-
meta-
para-
Xylene Ethyl t o l u e n e Isopropyl toluene Fluorotoluene Chlorotoluene Difluorobenzene Dichlorobenzene Dibromobenzene Diiodobenzene Fluorobenzonitrile Chlorobenzonitrile Dicyanobenzene Fluoroanisole Fluorobenzotrifluoride Fluorobenzaldehyde Fluorobenzyl alcohol
-
NT + NT NT + -
+ + + + + wk wk + -
NT -
NT NT
-
N T = n o t tested, w k = w e a k , + , - .
See t e x t
Enzyme Microb. Technol., 1987, vol. 9, December
707
Papers
mediated by a direct action on the catechol-forming enzymes, or by inhibition of enzyme induction, is not known. Several of the halogenated benzenes led to excretion of a substituted catechol when incubated with strain T-12 in the absence of either fluorobenzene or benzonitrile. These substrates included all of the monohalogenated benzenes, para-fluorotoluene, para-chlorotoluene, paradifluorobenzene, and para-dichlorobenzene. Each of these substrates apparently induced the 3-alkylcatechol pathway, para-Xylene, trifluoromethyl benzene and fluorobenzonitriles required the presence of an inducer/ inhibitor to form catechols. Cyclopropylbenzene accumulated a yellow metabolite in the absence of an inducer/inhibitor, but yields a catechol in their presence. Among the mono-substituted alkylbenzenes that gave rise to catechols, Table 1, there was a substantial decrease in amount of catechol formed when the substituent went from three to four carbons. The propyl-, allyl-, and cyclopropyl-benzenes and methyl styrenes were all good substrates while none of the butyl benzenes was. Within the butyl benzene series there was a clear trend in visual spot intensity on t.l.c, plates going from the most intense with tert-butyl benzene to n-butyl benzene, which made a barely detectable trace of catechol. Among the phenylalkylalcohols, benzyl alcohol, 1-phenyl ethanol and 2phenyl ethanol gave good yields of catechol (Table 3), while none of the phenylpropanols gave rise to detectable catechol. These observations imply that steric factors such as the size of the substituent play a determining role in substrate acceptability. Several para-disubstituted benzenes were good precursors to catechols, as were a few meta-disubstituted benzenes (Table 2). Within a substitution pattern, substituent size appears to play a predominant role in determining what substitutions were accepted, para-Xylene was a good substrate, but para-ethyl toluene and paracymene were not. Methyl, halo- and cyano-substitutions were most often present on disubstituted benzenes that were converted to cateehols. None of the orthodisubstituted benzenes tested served as catechol precursors (Table 2). The ability of a substrate to be converted to a catechol depends on the substrate specificity of two enzymes, a toluene dioxygenase to produce a dihydrodiol, and a dehydrogenase to yield the catechol (Figure 1 ). Studies in other laboratories using toluene dioxygenase isolated from P. putida have shown that biphenyl, ortho-, rectaand para-dichlorobenzene can be transformed into the corresponding dihydrodiols. 2'1°'11 Of these, only paradichlorobenzene is a catechol precursor in strain T-12. para-Difluorobenzene, a catechol precursor for strain T12, was not tested with the P. putida system. Fused ring compounds tested with strain T-12 included naphthalene, tetralin and acenaphthene. None of these formed hydroxylated products detectable with Gibb's reagent. Indole incubated with strain T-12 and fluorobenzene gave rise to a series of colored products, presumably due to oxidation of indole ~ Two trisubstituted compounds, 3-chloro-ortho-xylene and SK&F 86466 (6-ehloro-3-methyl-2,3,4,5-tetrahydro-lH3-benzazepine), were not attacked. Catechols with substitution in the 3-position are the presumed products from monosubstituted benzenes. This has been confirmed for the products from fluorobenzene
708
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 7 , v o l . 9, D e c e m b e r
Table 3
Total and isolated yields of selected catechols
Catechol precursor
para-Xylene Cumene Benzaldehyde Benzyl alcohol 2-Phenyl ethanol 1-Phenyl ethanol Anisole Phenetole para- Fluorobenzonitrile meta- Fluorobenzonitrile
Total catechol a (raM)
Fractional yield b from: Inducer c
Precursor
0.86 2.0 1.3 2.0 1.4 0.89 1.9 1.6
0.41 0.18 0.54 0.15 0.29 0.12 0.37 c 0.78 c
0.02 0.40 0.35 0.58 0.43 0.62 0.37 0.02
0.98
0.61
0.10
0.77
0.13
0.52
aTotal catechol concentration in the culture broth b#moles of catechol isolated from t.l.c, plate scrapings divided by /~moles of total catechol applied to the t.l.c, plate CFluorobenzene was the inducer for all compounds except anisole and phenetole; benzonitrile was the inducer/inhibitor for anisole and phenetole
and benzonitrile. For the product from metadisubstituted benzenes, it is not known whether one or both of the possible isomeric products is produced. Analyses of the structure of these products will be communicated in the future. Total and isolated yields of a number of substituted catechols are described in Table 3. Quantification employed the Arnow reaction, which is specific for catechols. 9 Spiking culture broths with unsubstituted catechol demonstrated that the Arnow reaction was a reliable, quantitative measure of total catechol in the broth. Using the protocol for Table 3, 80% of the catechol in the broth was obtained in the ethyl acetate extract of cultures receiving only fluorobenzene or benzonitrile. Recovery of these catechols following t.l.c, averaged 75%, for an overall isolated yield of 60%. These results were obtained with a simple, unoptimized protocol that potentially involves competition between inducer and test substrate in the organism, variable recovery by a single solvent extraction and variable loss during t.l.c, separation and recovery of the catechol products. Nevertheless, it is clear that this biochemical process could be used as a preparative method for these catechols. Moreover, optimization of the fermentation, extraction and separation steps will increase the overall yields.
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