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Efficient reduction of cyclohexanone to cyclohexanol and cyclohexane by Halobacterium halobium MMT22
Joumal of Molecular Catal@s,
63 (1990)
L15-L19
L15
Efficient reduction of cyclohexanone to cyclohexanol and cyclohexane by Halobacterium halobium MMT,, M. M. Taqui Khan* and J. P. Bhatt Discipline of Coorokatim Chemistry and Homogemaus Catalysis, Central Salt & Marine Chemicals Research Institute, Bhavnagar 364 002 (India) (Received
February 27, 1990; accepted
July 12, 1990)
Introduction Reduction of ketones to alcohols, chemically 11, 21 or electrochemically [ 3, 41, in the presence of enzymes is of current interest as a means of synthesis of optically active alcohols. Many photochemical [5-71, electrochemical and enzymatic [S-lo] systems for the regeneration of nicotinamide adenine dinucleotide (phosphate) (reduced form NAD(P)H) were reported. Among these systems, those involving enzymes have the great advantage and importance in producing compounds with high optical purity. Light-induced reduction of a variety of electron-acceptor substrates in the presence of adequate electron donors has been accomplished [ 111. Thus, the use of primary light-induced electron transfer reactions in driving the secondary enzyme-catalyzed reduction of ketones seems feasible. This system can be visualised as an endoergic energy storage system as well as a useful synthetic apparatus for the preparation of optically active alcohols and further reduction to saturated hydrocarbons. In Halobacterium halobium, photoactivation of the bacteriorhodopsin results in the transfer of protons from the interior to the exterior of the cells. These protons, when coupled to the electrons donated by hydrogenase of Escherichia coli, result in the successful evolution of hydrogen [ 12-161. The present paper describes the photobiological reduction of cyclohexanone to cyclohexanol and finally to cyclohexane with the hydrogen evolved by a coupled system of H. halobium MMTz2 and E. coli. Based on the work done previously in our laboratory, the best hydrogen-evolving strain, H. hulobium MMTaa, was selected for the present investigations. Experimental The extremehalophileH. halobium MMTzz was isolated from the brine obtained from the Salt Farm of CSMCRI in a nutrient medium containing *Author to whom correspondence
0304-5102/90/$3.50
should be addressed.
8 Elsevier
Sequoia/Printed
in The Netherlands
L16
250 g NaC1, 20 g MgS04 - 7H20, 3 g trisodium citrate. 2H20, 2 g KCl, 10 g peptone, 1000 ml distilled water at pH 7.0. Incubation was carried out at 40 + 1 “C under illumination. E. coli NCL 2065 ATCC was obtained from National Chemical Laboratory, Poona (India) and was made salt-tolerant (20% NaCl). Enrichment of E. coli was done in a medium containing nutrient broth + 20% NaCl. Photobiological studies were carried out at 40 f 1 “C in a reaction vessel wherein the system for the reduction of ketone, cyclohexanone, by hydrogen thus contains 1 mg (wet weight) of H. halobium MMTa2 and 1 mg (wet weight) of salt-tolerant E. coli suspended in a 25 ml nutrient medium with 500 mg (0.2 M) of cyclohexanone. The reaction vessel was illuminated with a light intensity of 0.3 mW cm-‘. The hydrogen evolved by the coupled system of H. hulobium MMTzz and E. coli was utilized for the reduction of the ketone. Samples were taken out at various time intervals and were made free of bacterial cells by centrifugation. The cell-free suspension was extracted with ether and the ether fraction analysed by GC and NMR. Gas chromatographic analysis was done on Shimadzu GC-9A with Porasil B column. The other conditions of GC analysis were: carrier gas Na, flow rate 60 ml min-l, column temperature 200 “C, injection temperature 175 “C, TCD temperature 200 “C, current 50 mA and detector used was a TCD. The products were characterised by 13C NMR at 24.99 MHz and ‘H NMR at 99.55 MHz frequencies, using JEOL FT-100 Fl’ NMR. ‘H NMR in DMSO-d, and 13C NMR were carried out in a 10 mm NMR tube using CO axial D20 capillary for lock. Samples for NMR studies were prepared by extracting the reaction mixture with diethyl ether. The ether extract was dried over anhydrous sodium sulphate and the ether removed. The residue was used for taking both ‘H and 13C NMR spectra. Characterisatti of products The percentage of the products cyclohexanol, cyclohexane and unchanged
cyclohexanone was determined by the area normalisation method (GC analysis) by measuring the areas of the peaks in GC and comparing them with the areas of the peaks of the authentic samples at a particular concentration. The products were also confirmed by standard ‘H and 13CNMR spectra. Results
and discussion
Evolution
of hydrogen
Bacteriorhodopsin present within the purple membrane of H. halobium functions as a light-driven proton pump which creates an electrochemical gradient across the plasma membrane. The purple membrane is oriented in such a manner that release of protons occurs into the surrounding medium. The absorption of a photon results in the deprotonation of the Schiff base linkage between retinal and lysine, and the apoprotein undergoes a conformational change which allows the unidirectional migration of a proton from the opposite side of the membrane by the ionisation of water. The
L17
protons thus released, when coupled to the electrons donated by the hydrogenase of E. co& result in the evolution of hydrogen. Reduction
of cyclohexanone
by hydrogen
Regeneration of NADH and ketone hydrogenation by hydrogen with the combination of hydrogenase and alcohol dehydrogenase is reported [17]. Ketones such as 2-pentanone, 2-butanone, 2-cyclohexanone and cyclohexanone were easily hydrogenated by hydrogen gas; after 65 h, the conversion of 2-butanone was 87% [ 171. H. halobium MMTZ2 under photobiological conditions reduced cyclohexanone to cyclohexanol and further to cyclohexane. The rate of reduction of cyclohexanone is 3.40X 10m3 M min-‘, equal to the rate of formation of cyclohexanol which is 3.42 X 10e3 M min-‘. Formation of cyclohexane starts after the build up of cyclohexanol concentration in 15 mins at the rate of 6.90 x 10e4 M ruin-‘, which is approximately five times slower than the rate of formation of cyclohexanol. Figure 1 and Table 1 show the time dependence of cyclohexanol and cyclohexane formation. It is apparent that a steady state concentration of cyclohexanone is achieved after about 50% conversion. Its further reduction to cyclohexanol stops because of the toxicity of cyclohexane to microbial cells. Scheme 1 represents the mechanism of ketone hydrogenation.
Time (mid
1. Amount of products formed through reduction of cyclohexanone catalysed by I-I. h&&km MMTz2 and E. coli. (M) cyclohexanone, (A-A) cyclohexanol, (n--O) cyclohexane. Fig.
L18 TABLE 1 Reduction of cyclohexanone to cyclohexanol and cyclohexane by Halobacterium
halobium
m22.
Products (mol)
cyclohexanone (substrate) cyclohexanol cyclohexane
PURPLE
0
10 min
15 min
30 min
45 min
0.192
0.158
0.138
0.134
0.134
0.0 0.0
0.029 0.0
0.052 0.0
0.039 0.012
0.004 0.046
COMPLEX
/ INTERMEDIATE(S)
\ INTERMEDIATE
t 42 nm COMPLEX
k 2H;
Ze-( HYDKOGENASE)
I HZ I CYCLOHEXANONE-+CYCLOHEXANX;fiCYCLOHEXANE
W-h,CO -$&+C&COH Cyclohexanone
Cyclohexanol
+/$,H,,
+ Hz0 Cyclohexane
Scheme 1. Mechanism of ketone hydrogenation
The system is very efficient in the reduction of ketoneqwith a conversion of 50% within 15 min. The turnover rates are 3.4 mol alcohol per mg of the wet weight of bacteria per minute. References 1 2 3 4
5 6 7 8 9 10
C. H. Wong, G. M. Whitesides et al., J. Am. Chem. Sot., 103 (1981) 6227. C. H. Wong and G. M. Whitesides, J. Org. CYt.em.,47 (1982) 2816. Z. Shaked, J. J. Barber and G. M. Whitesides, J. Org. Cti. 46 (1981) 4101. R. Dicosimo, C. H. Wong, L. Daniels and G. M. Whitesides, J. Org. Chem., 46 (1981) 4622. D. Mandier and I. Wiliner, J. Am. Chem. Sot., 106 (1984) 5352. P. Cuendet and M. Gratzel, Photo&em. PhotobioZ.,39 (1984) 609. R. Weinkamp and E. Steckhan, Angew. Ch.em. Int. Ed. En&, 22 (1983) 497. H. Simon, J. Bader, H. Gunther, S. Nevmann and J. Thanos, Angew. Chem. ht. Ed. En.&, 24 (1985) 539. C. H. Wong and G. H. Wbitesides, J. Am. C&m. Sot., 103 (1981) 4890. Z. Shaked and G. M. Whltesides, J. Am. Chem. Sot., 102 (1980) 7104.
LlQ 11 12 13 14 15 16 17
K. Kalyanasundamm,Coo&. Chem. Rev., 46 (1982) M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Iti. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen K. Otsuka, S. Aono, I. Okura and F. Hasumi, J. Mol.
159. Energy, 14 (1989) 643. Energy, 15 (1990) 473. Energy, 15 (1990) 477. Energy, in press. Energy, in press. Cutal., 51 (1989) 35.
63 (1990)
L15-L19
L15
Efficient reduction of cyclohexanone to cyclohexanol and cyclohexane by Halobacterium halobium MMT,, M. M. Taqui Khan* and J. P. Bhatt Discipline of Coorokatim Chemistry and Homogemaus Catalysis, Central Salt & Marine Chemicals Research Institute, Bhavnagar 364 002 (India) (Received
February 27, 1990; accepted
July 12, 1990)
Introduction Reduction of ketones to alcohols, chemically 11, 21 or electrochemically [ 3, 41, in the presence of enzymes is of current interest as a means of synthesis of optically active alcohols. Many photochemical [5-71, electrochemical and enzymatic [S-lo] systems for the regeneration of nicotinamide adenine dinucleotide (phosphate) (reduced form NAD(P)H) were reported. Among these systems, those involving enzymes have the great advantage and importance in producing compounds with high optical purity. Light-induced reduction of a variety of electron-acceptor substrates in the presence of adequate electron donors has been accomplished [ 111. Thus, the use of primary light-induced electron transfer reactions in driving the secondary enzyme-catalyzed reduction of ketones seems feasible. This system can be visualised as an endoergic energy storage system as well as a useful synthetic apparatus for the preparation of optically active alcohols and further reduction to saturated hydrocarbons. In Halobacterium halobium, photoactivation of the bacteriorhodopsin results in the transfer of protons from the interior to the exterior of the cells. These protons, when coupled to the electrons donated by hydrogenase of Escherichia coli, result in the successful evolution of hydrogen [ 12-161. The present paper describes the photobiological reduction of cyclohexanone to cyclohexanol and finally to cyclohexane with the hydrogen evolved by a coupled system of H. halobium MMTz2 and E. coli. Based on the work done previously in our laboratory, the best hydrogen-evolving strain, H. hulobium MMTaa, was selected for the present investigations. Experimental The extremehalophileH. halobium MMTzz was isolated from the brine obtained from the Salt Farm of CSMCRI in a nutrient medium containing *Author to whom correspondence
0304-5102/90/$3.50
should be addressed.
8 Elsevier
Sequoia/Printed
in The Netherlands
L16
250 g NaC1, 20 g MgS04 - 7H20, 3 g trisodium citrate. 2H20, 2 g KCl, 10 g peptone, 1000 ml distilled water at pH 7.0. Incubation was carried out at 40 + 1 “C under illumination. E. coli NCL 2065 ATCC was obtained from National Chemical Laboratory, Poona (India) and was made salt-tolerant (20% NaCl). Enrichment of E. coli was done in a medium containing nutrient broth + 20% NaCl. Photobiological studies were carried out at 40 f 1 “C in a reaction vessel wherein the system for the reduction of ketone, cyclohexanone, by hydrogen thus contains 1 mg (wet weight) of H. halobium MMTa2 and 1 mg (wet weight) of salt-tolerant E. coli suspended in a 25 ml nutrient medium with 500 mg (0.2 M) of cyclohexanone. The reaction vessel was illuminated with a light intensity of 0.3 mW cm-‘. The hydrogen evolved by the coupled system of H. hulobium MMTzz and E. coli was utilized for the reduction of the ketone. Samples were taken out at various time intervals and were made free of bacterial cells by centrifugation. The cell-free suspension was extracted with ether and the ether fraction analysed by GC and NMR. Gas chromatographic analysis was done on Shimadzu GC-9A with Porasil B column. The other conditions of GC analysis were: carrier gas Na, flow rate 60 ml min-l, column temperature 200 “C, injection temperature 175 “C, TCD temperature 200 “C, current 50 mA and detector used was a TCD. The products were characterised by 13C NMR at 24.99 MHz and ‘H NMR at 99.55 MHz frequencies, using JEOL FT-100 Fl’ NMR. ‘H NMR in DMSO-d, and 13C NMR were carried out in a 10 mm NMR tube using CO axial D20 capillary for lock. Samples for NMR studies were prepared by extracting the reaction mixture with diethyl ether. The ether extract was dried over anhydrous sodium sulphate and the ether removed. The residue was used for taking both ‘H and 13C NMR spectra. Characterisatti of products The percentage of the products cyclohexanol, cyclohexane and unchanged
cyclohexanone was determined by the area normalisation method (GC analysis) by measuring the areas of the peaks in GC and comparing them with the areas of the peaks of the authentic samples at a particular concentration. The products were also confirmed by standard ‘H and 13CNMR spectra. Results
and discussion
Evolution
of hydrogen
Bacteriorhodopsin present within the purple membrane of H. halobium functions as a light-driven proton pump which creates an electrochemical gradient across the plasma membrane. The purple membrane is oriented in such a manner that release of protons occurs into the surrounding medium. The absorption of a photon results in the deprotonation of the Schiff base linkage between retinal and lysine, and the apoprotein undergoes a conformational change which allows the unidirectional migration of a proton from the opposite side of the membrane by the ionisation of water. The
L17
protons thus released, when coupled to the electrons donated by the hydrogenase of E. co& result in the evolution of hydrogen. Reduction
of cyclohexanone
by hydrogen
Regeneration of NADH and ketone hydrogenation by hydrogen with the combination of hydrogenase and alcohol dehydrogenase is reported [17]. Ketones such as 2-pentanone, 2-butanone, 2-cyclohexanone and cyclohexanone were easily hydrogenated by hydrogen gas; after 65 h, the conversion of 2-butanone was 87% [ 171. H. halobium MMTZ2 under photobiological conditions reduced cyclohexanone to cyclohexanol and further to cyclohexane. The rate of reduction of cyclohexanone is 3.40X 10m3 M min-‘, equal to the rate of formation of cyclohexanol which is 3.42 X 10e3 M min-‘. Formation of cyclohexane starts after the build up of cyclohexanol concentration in 15 mins at the rate of 6.90 x 10e4 M ruin-‘, which is approximately five times slower than the rate of formation of cyclohexanol. Figure 1 and Table 1 show the time dependence of cyclohexanol and cyclohexane formation. It is apparent that a steady state concentration of cyclohexanone is achieved after about 50% conversion. Its further reduction to cyclohexanol stops because of the toxicity of cyclohexane to microbial cells. Scheme 1 represents the mechanism of ketone hydrogenation.
Time (mid
1. Amount of products formed through reduction of cyclohexanone catalysed by I-I. h&&km MMTz2 and E. coli. (M) cyclohexanone, (A-A) cyclohexanol, (n--O) cyclohexane. Fig.
L18 TABLE 1 Reduction of cyclohexanone to cyclohexanol and cyclohexane by Halobacterium
halobium
m22.
Products (mol)
cyclohexanone (substrate) cyclohexanol cyclohexane
PURPLE
0
10 min
15 min
30 min
45 min
0.192
0.158
0.138
0.134
0.134
0.0 0.0
0.029 0.0
0.052 0.0
0.039 0.012
0.004 0.046
COMPLEX
/ INTERMEDIATE(S)
\ INTERMEDIATE
t 42 nm COMPLEX
k 2H;
Ze-( HYDKOGENASE)
I HZ I CYCLOHEXANONE-+CYCLOHEXANX;fiCYCLOHEXANE
W-h,CO -$&+C&COH Cyclohexanone
Cyclohexanol
+/$,H,,
+ Hz0 Cyclohexane
Scheme 1. Mechanism of ketone hydrogenation
The system is very efficient in the reduction of ketoneqwith a conversion of 50% within 15 min. The turnover rates are 3.4 mol alcohol per mg of the wet weight of bacteria per minute. References 1 2 3 4
5 6 7 8 9 10
C. H. Wong, G. M. Whitesides et al., J. Am. Chem. Sot., 103 (1981) 6227. C. H. Wong and G. M. Whitesides, J. Org. CYt.em.,47 (1982) 2816. Z. Shaked, J. J. Barber and G. M. Whitesides, J. Org. Cti. 46 (1981) 4101. R. Dicosimo, C. H. Wong, L. Daniels and G. M. Whitesides, J. Org. Chem., 46 (1981) 4622. D. Mandier and I. Wiliner, J. Am. Chem. Sot., 106 (1984) 5352. P. Cuendet and M. Gratzel, Photo&em. PhotobioZ.,39 (1984) 609. R. Weinkamp and E. Steckhan, Angew. Ch.em. Int. Ed. En&, 22 (1983) 497. H. Simon, J. Bader, H. Gunther, S. Nevmann and J. Thanos, Angew. Chem. ht. Ed. En.&, 24 (1985) 539. C. H. Wong and G. H. Wbitesides, J. Am. C&m. Sot., 103 (1981) 4890. Z. Shaked and G. M. Whltesides, J. Am. Chem. Sot., 102 (1980) 7104.
LlQ 11 12 13 14 15 16 17
K. Kalyanasundamm,Coo&. Chem. Rev., 46 (1982) M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Iti. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen M. M. Taqui Khan and J. P. Bhatt, Int. J. Hydrogen K. Otsuka, S. Aono, I. Okura and F. Hasumi, J. Mol.
159. Energy, 14 (1989) 643. Energy, 15 (1990) 473. Energy, 15 (1990) 477. Energy, in press. Energy, in press. Cutal., 51 (1989) 35.