Microbial asymmetric CH oxidations of simple hydrocarbons: A novel monooxygenase activity of the topsoil microorganism Bacillus megaterium

Article abstract of DOI:10.1002/1099-0690(200008)2000:16<2923::AID-EJOC2923>3.0.CO;2-L

A Bacillus megaterium strain was isolated from topsoil by a selective screening procedure with allylbenzene as xenobiotic substrate. It is demonstrated for the first time, from analytical-scale experiments, that this microorganism hydroxylates a variety of simple n-alkanes (hexane through nonane) and cycloalkanes (cyclohexane and cyclooctane) to afford optically active alcohols in up to 99% enantiomeric excess (ee). In the case of the n-alkanes, the ω-1, ω-2 and ω-3 regioisomers were obtained. This enzymatic activity is unprecedented for Bacillus megaterium strains and is generally rarely observed in bacteria.

Full text of DOI:10.1002/1099-0690(200008)2000:16<2923::AID-EJOC2923>3.0.CO;2-L

FULL PAPER
Microbial Asymmetric CH Oxidations of Simple Hydrocarbons: A Novel
Monooxygenase Activity of the Topsoil Microorganism Bacillus megaterium
Waldemar Adam,[a] Zoltan Lukacs,*[a] Chantu R. Saha-Möller,[a] Bernhard Weckerle,[b] and
Peter Schreier[b]
Keywords: Alkanes / Asymmetric oxidation / Hydroxylation / Monooxygenase / Biocatalysis / Bacillus megaterium /
Biotransformations
A Bacillus megaterium strain was isolated from topsoil by a
afford optically active alcohols in up to 99% enantiomeric ex-
selective screening procedure with allylbenzene as xenobi- cess (ee). In the case of the n-alkanes, the ω-1, ω-2 and ω-3
otic substrate. It is demonstrated for the first time, from ana- regioisomers were obtained. This enzymatic activity is un-
lytical-scale experiments, that this microorganism hy- precedented for Bacillus megaterium strains and is generally
droxylates a variety of simple n-alkanes (hexane through
rarely observed in bacteria.
nonane) and cycloalkanes (cyclohexane and cyclooctane) to
Introduction
capsulatus Bath and Methylosinus trichosporium OB3b, or
by purified methane monooxygenases.[15,16] However, with
these enzymes the highest efficiency is obtained with meth-
ane as substrate, while for substrates with increased chain
length, the yields are significantly reduced.[16] Furthermore,
only two species of microorganisms are known which pro-
duce P450-dependent alkane hydroxylases. These are the
Rhodococcus rhodochrous ATCC 19067 and the Acinetob-
acter calcoaceticus EB104, both of which are ω hy-
droxylases and, therefore, not suitable for asymmetric ox-
idation.[17]
CH oxidations are some of the most versatile reactions
in organic chemistry, since potentially useful oxyfunctional-
ized synthons may be obtained from readily accessible hy-
drocarbons. The currently available synthetic methods re-
quire harsh reaction conditions and yield racemic prod-
ucts.[1] Therefore, much effort has been expended in the de-
velopment of efficient and selective catalytic oxidations of
hydrocarbons.[2Ϫ5] For instance, metal-catalyzed asymmet-
ric CH oxidations have recently been carried out with chiral
auxiliaries[6] and optically active oxidants[7Ϫ8] to yield en-
antiomerically enriched products; however, in most cases,
only low enantioselectivities have been obtained for unac-
tivated alkanes. Moreover, biomimetic studies have been
conducted to gain insight into the catalytic mechanism of
the CH insertion and to improve its stereoselectivity.[9Ϫ11]
Despite these efforts, the development of a broadly applic-
able and effective catalytic asymmetric hydroxylation of un-
functionalized hydrocarbons remains a challenge.
As an alternative, microorganisms have been successfully
employed for the selective oxyfunctionalization of unactiv-
ated CH bonds in a variety of substrates.[12] So far, most
of this work has focused on the hydroxylation of steroids,
terpenes and other complex natural products.[13,14] In the
case of simple alkanes, the substrates of interest in the pre-
sent work, most of the hydroxylations have been performed
by methanogenous microorganisms, like Methylococcus
To obtain a more efficient biocatalyst for the asymmetric
hydroxylation of unactivated hydrocarbons, a selective
screening procedure with allylbenzene (5Ϫ25 µL) as sub-
strate was applied to soil samples of different origin. Previ-
ously, we have reported on the oxyfunctionalization of aryl-
alkanes with microorganisms isolated by this newly de-
veloped screening protocol.[18] In this work, we present our
results on the hydroxylation of simple hydrocarbons by a
Bacillus megaterium strain isolated from topsoil. This strain
is known to hydroxylate long-chain fatty acids, fatty amides
[17,19]
and fatty alcohols predominantly in the ω-1 position;
however, the hydroxylation of simple alkanes by B. megater-
ium appears not to have been reported to date. We demon-
strate herein the unprecedented asymmetric biocatalytic ox-
idation of the unactivated alkanes 1a؊f to the correspond-
ing alcohols 2 and 3, by B. megaterium, in high enantiom-
eric excess.
[a]
Institute of Organic Chemistry, University of Würzburg,
Results and Discussion
Am Hubland, 97074 Würzburg, Germany
Fax: (internat.) ϩ49(0)-931/888-4756
E-mail: adam@chemie.uni-wuerzburg.de
http://www-organik.chemie.uni-wuerzburg.de
To assess the feasibility of the biocatalytic asymmetric
oxidation of simple alkanes with the topsoil microorganism
B. megaterium, the biotransformations were carried out on
an analytical scale. For this purpose, cultures of the bacteria
(75 mL) were grown overnight in an open system in min-
[b]
Institute of Food Chemistry, University of Würzburg,
Am Hubland, 97074 Würzburg, Germany
Fax: (internat.) ϩ49(0)-931/888-4756
E-mail: adam@chemie.uni-wuerzburg.de
http://www-organik.chemie.uni-wuerzburg.de
Eur. J. Org. Chem. 2000, 2923Ϫ2926
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0816Ϫ2923 $ 17.50ϩ.50/0
2923

W. Adam, Z. Lukacs, C. R. Saha-Möller, B. Weckerle, P. Schreier
FULL PAPER
imum media and then incubated with 0.8 mmol of the re- center of the enzyme, and thus different regioselectivities
spective hydrocarbon for 17 h at 30 °C. Subsequently, the are observed.
bacterial broth was centrifuged and the clear medium was
n-Heptane (1b) was converted into the regioisomeric al-
extracted with a pentane/dichloromethane mixture (2:1) for cohols heptan-2-ol (2bα), heptan-3-ol (2bβ) and heptan-4-
24 h to recover the products and unchanged starting mat- ol (2bγ) in a ratio of 34:21:15 with excellent enantiomeric
erial from the bacterial medium. The organic solvent was excesses [Ͼ 99% ee for the S enantiomers of 2bα and 2bβ
carefully removed by distillation over a Vigreux column to (entry 2)]. Similarly, octan-2-ol (2cα) and octan-3-ol (2cβ)
prevent further loss of the highly volatile products, and the are obtained as major products from n-octane (1c) with ee
resulting residue was submitted to chiral GC analysis. Our values Ͼ 99% for the S enantiomers, while octan-4-ol (2cγ)
results confirmed that the strain isolated from topsoil hy- is formed in small amounts and only in 6% ee (entry 3).
droxylates the n-alkanes 1a؊d and the cyclic derivatives 1e,f The reason for this low enantioselectivity at the γ-methylene
enantioselectively to the alcohols 2 and 3 (Scheme 1). This site of n-octane (1c) is presumably the consequence of poor
demonstrates a novel enzymatic activity for Bacillus megat- differentiation by the enzyme between the two sterically
erium which has not been observed before.
similar n-propyl and n-butyl substituents. For n-nonane
Scheme 1. Asymmetric hydroxylation of alkanes 1 by Bacillus megaterium; the respective ketones of these alcohols are formed as minor
products (cf. Table 1)
For this biocatalytic process, the time of substrate addi-
tion plays a crucial role with regard to the product forma- Table 1. Regio- and enantioselectivities of the oxygen-atom inser-
tion into the CϪH bonds of n-alkanes 1a؊d and cycloalkanes 1e,f
by Bacillus megaterium cultures
tion, since the addition of hydrocarbon during the early log
and stationary phases results in an approximately fourfold
decrease of product formation over the addition during the
mid-log phase (data not shown). This indicates that the sub-
strate affects enzyme expression when added during the
most pronounced growth phase, and thus induces monoox-
ygenase activity. Consequently, the optimized biotrans-
formation conditions were applied to the hydroxylation of
the alkanes 1a؊f. The regio- and enantioselectivities of
these CH oxidations by B. megaterium are given in Table 1.
n-Hexane (1a) yielded hexan-3-ol (2aβ) as major and the
α-hydroxy alcohol 2aα as minor products in an enantiom-
eric excess (ee) of 54% and 93%, respectively (Table 1, entry
1). The diminished ee value for hexan-3-ol (2aβ) is not un-
expected as the molecule is more symmetric than 2aα and,
therefore, stereodifferentiation becomes worse. Low
amounts of the corresponding ketones of the alcohols 2aα
Selectivity [%]
Regio-[b]
Entry[a]
Alkane
Alcohol
Enantio-[c]
1
2
1a
1b
2aα
2aβ
2bα
2bβ
2bγ
2cα
2cβ
2cγ
2dα
2dβ
2dγ
3e
19 (11)
57 (13)
34 (17)
21 (13)
15
53 (19)
17 (5)
6
34 (27)
23 (9)
7
79 (21)
35 (65)
93
54
Ͼ99 (S)
Ͼ99 (S)
[d]
3
4
1c
1d
Ͼ99 (S)
Ͼ99 (S)
6
Ͼ99
98
[e]
-
[d]
[d]
5
6
1e
1f
3f
[a] Substrate (0.8 mmol) added at mid-log phase; 17 h incubation at
[b]
30 °C, 120 rpm. Ϫ Ratio of regioisomeric alcohols; the product
distribution is normalized to 100% and the amounts of the corres-
and 2aβ were also observed. However, the enantiomeric ex- ponding ketones are given in parentheses; error Ϯ2% of stated
cess, in particular for 2aβ, cannot be reconciled in terms of
values; conversions and yields are not given since starting materials
and products are extremely volatile and evaporated in the course
[c]
kinetic resolution of the respective alcohol through enanti-
oselective oxidation to the ketone because the amount of
the latter is too small to affect the enantioselectivity of the
biotransformation. Therefore, the observed ee values are the
result of direct asymmetric hydroxylation of n-hexane by
the microorganism. In stark contrast to n-hexane (1a), the
of the open-system experiments of these bioconversions. Ϫ En-
antiomeric excesses (ee values) were determined by multi-dimen-
sional gas chromatography on a cyclodextrin column; absolute con-
figurations, when given, were determined by comparison with au-
thentic reference samples, by lipase-catalyzed kinetic resolution of
the racemic alcohols, or by comparison with literature data; error
Ϯ2% of stated values. Ϫ [d] Alcohol does not possess a chiral center.
Ϫ
[e]The enantiomeric excess of the minor product was not deter-
hydroxylation of the other n-alkanes 1b؊d predominantly mined.
affords the α-hydroxy alcohols 2bα-dα (entries 2Ϫ4). Pre-
sumably, steric effects force the long-chain alkanes (chain (1d), the results are comparable to those of n-heptane (1b)
length Ͼ C6) to position themselves differently in the active and n-octane (1c), since mainly nonan-2-ol (2dα) and
2924
Eur. J. Org. Chem. 2000, 2923Ϫ2926

Microbial Asymmetric CH Oxidations of Simple Hydrocarbons
FULL PAPER
trometer coupled with a Fisons GC 8000 (Fisons, Mainz-Kastel,
Germany).
nonan-3-ol (2dβ) are obtained, while nonan-4-ol (2dγ) is
found only as a minor product (entry 4).
Gas-chromatographic analysis on cyclodextrin columns
demonstrated that the α and β alcohols 2 of the alkanes
1b؊d are formed enantiomerically pure. For all entries, the
respective ketones are observed only for the α and β re-
gioisomers; however, in the case of n-nonane (1d), the
chemoselectivity is diminished, especially for the α-hydroxy
alcohol 2dα, since higher amounts of ketones are produced.
The cyclic alkanes cyclohexane (1e) and cyclooctane (1f)
are also readily hydroxylated by B. megaterium to yield the
nonchiral alcohols 3e and 3f (entries 5 and 6). As with the
n-alkanes, overoxidation to the ketones significantly in-
creases, more so for cyclooctane (65% ketone) than for
cyclohexane (21% ketone). This clearly demonstrates that
the oxidative activity becomes more prevalent with in-
creased ring size. A similar trend is apparent for the oxida-
tion of n-hexane (1a) through n-nonane (1d) and implies
that hydrocarbons with longer alkyl chains and larger rings
serve as better substrates for enzymatic oxidation by bac-
terial cells.
General Procedure for the Biotransformation of the Hydrocarbon:
Liquid minimal media (75 mL) were prepared according to Dwor-
kin et al.,[20] 5 mL/L of a mineral salt solution[21] were added and
autoclaved (121 °C, 16 min). The culture was maintained under
sterile conditions during the addition of glucose (5 g/L). For inocu-
lation of the liquid media, cultures were taken from freshly grown
plates. In an open system and under sterile conditions, ca. 0.8 mmol
of the hydrocarbon substrate was added and the liquid culture was
allowed to grow for ca. 18 h at 30 °C. Control experiments without
bacteria were carried out to verify the stability and authenticity of
the starting material; no oxidation products were observed under
these conditions. After ca. 17 h, the culture was worked up by
sonication of the bacterial broth for 15 mins. and centrifugation at
15000g for 20 min. The supernatant solution was extracted with
120 mL pentane/dichloromethane (2:1) by liquid-liquid extraction
for 24 h. The extract was dried over Na2SO4 and distilled over a
Vigreux column (40 °C, 990 mbar) to prevent loss of volatile com-
pounds by evaporation. The products were submitted to gas chro-
matographic analysis, coupled with mass spectrometry, and their
identity was verified by comparison with authentic reference
samples. Conversions were not determined due to the volatility of
the substrates, which evaporated during the course of the bioconv-
ersion. The enantiomeric excess of the alcohols was determined by
multidimensional gas chromatography (MDGC) on the following
columns:
Conclusions
a) 2,3-O-Diacetyl-6-O-tert-butyldimethylsilyl-β-cyclodextrin col-
umn for 2aα, 2aβ, 2bα, 2bβ, 2cβ, 2cγ, 2dα
b) 2,3-O-Diethyl-6-O-tert-butyldimethylsilyl-β-cyclodextrin column
for 2cα, 2dβ
Our present analytical-scale experiments show that the
nonpathogenic, topsoil bacterium Bacillus megaterium
readily hydroxylates a variety of unactivated simple alkanes
stereoselectively to the corresponding regiomeric alcohols
in high enantiomeric excesses (up to 99% ee). This is the Assignment of the absolute configuration was done by lipase-cata-
lyzed kinetic resolution of the alcohols 2 and, wherever possible,
confirmed by comparison with authentic enantiomerically pure ref-
erence samples.
first demonstration that Bacillus megaterium may oxidize
unfunctionalized hydrocarbons enantioselectively. The ex-
cellent stereoselectivity observed for the n-alkanes studied
here makes further investigations of this microorganism
worthwhile. Isolation and overexpression of the pertinent
monooxygenase enzyme in the present B. megaterium strain
should significantly improve the biocatalytic efficiency and
facilitate preparative applications of this biotransformation
for the asymmetric CH oxidation of simple hydrocarbons.
Acknowledgments
We express our gratitude to Dr. U. Rdest (Institute of Microbio-
logy, University of Würzburg, Germany) for her valuable help and
advice. Furthermore, we thank the Deutsche Forschungsgemein-
schaft (SFB 347 ‘‘Selektive Reaktionen Metall-aktivierter Mole-
küle’’) and the Fonds der Chemischen Industrie for their generous
financial support.
Experimental Section
[1]
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HP 5890A (HewlettϪPackard, Palo Alto, CA, USA). Multidimen-
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a ‘‘live-T’’ switching device (Siemens, München, Germany) or with
a moving-column stream-switching system (MCSS) coupling device
on two Fisons GC 8000 instruments (Fisons, Mainz-Kastel, Ger-
many). Mass spectrometry was conducted on a MD 800 mass spec-
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