Beilstein J. Org. Chem. 2012, 8, 186–191.
Conclusion
expressed in 2 L shaking flasks containing 400 mL TB expres-
In conclusion, the use of P450-monooxygenases in the hydroxy- sion media, which was supplemented with 400 µL trace
lation of n-alkanes, which proceeds advantageously at elements (0.64 µM CuSO4, 0.69 µM ZnSO4, 0.72 µM MnSO4,
decreased reaction temperatures, has been reported. In addition, 0.76 µM CoCl2, 59.43 µM Na2-EDTA, 61.79 µM FeCl3) and
a biocatalytic oxidation of n-butane, as a representative example the respective antibiotic (for E. coli DH5α, harbouring pCWori,
for a “liquid gas”, was carried out with enzymatic in situ 100 µg/mL ampicillin and for E. coli BL21 Gold (DE3) LacIQ1,
cofactor regeneration and without the need for high-pressure harbouring pALXtreme-1a, 50 µg/mL kanamycin). Cultivation,
running at an initial reaction temperature of 0 °C, led exclu- described [20]. E. coli cells were harvested by centrifugation
sively to the formation of 2-butanol as the only regioisomer, at a (10 min, 2900 g at 4 °C) and cell pellets were washed in 50 mM
product concentration of 0.16 g/L. The practical experimental KPi buffer (pH 7.0), and stored at −20 °C overnight. For prepar-
setup of this process allows the direct use of n-butane, which ation of crude cell extracts, frozen cell pellets were resus-
has a boiling point of −0.5 °C, in liquid form and can be easily pended in 50 mM KPi buffer (pH 7.0) prior to disruption with
applied in laboratories lacking high-pressure equipment. A an Avestin EmulsiFlex-C3 high-pressure homogenizer (Ottawa,
current challenge, addressed in our laboratories, is the further ON, Canada) by applying three cycles of 1500 bar. The lysate
improvement of process efficiency and volumetric productivity was centrifuged (30 min, 16000 g at 4 °C) in a Sorvall RC-6
of this enzymatic oxidation process. In addition, studies of Plus centrifuge (Thermo Scientific, Rockford, IL, USA) and
enzyme stability during this biotransformation process are further clarified by filtration through a 0.45 µm filter (Roth,
planned as future work.
Karlsruhe, Germany). Subsequently, crude cell extracts were
shock-frozen in liquid nitrogen, lyophilized for 48 h at −54 °C
in an Alpha 1-2 LD plus Freeze dryer (Christ, Osterode am
Harz, Germany) and stored at −20 °C until further use.
Experimental
Bacterial strains and expression vectors
pCWori P450 BM-3 F87V [16] and E. coli BL21 Gold (DE3)
lacIQ1 P450 BM-3 19A12 (this work).
Typical procedure for the hydroxylation of
n-octane with in situ cofactor regeneration
Construction of the P450-monooxygenase BM-3
19A12 expression system
variant [17] was purchased as synthetic DNA (Life Tech- the F87V-mutant of P450-monooxygenase BM-3 (3.82 U; refer-
nologies, Darmstadt, Germany) and subcloned into the ring to the activity of n-octane), n-octane (16.3 µL; 0.1 mmol),
pET28a(+)-derived pALXtreme-1a vector, harbouring the wild- D-glucose (90.1 mg; 0.5 mmol) and glucose dehydrogenase
18]. For the purpose of replacing the wild-type heme domain activity for D-glucose) were added. After 20 min of stirring at rt
of P450-monooxygenase BM-3, both the synthetic DNA and or 8 °C, the reaction was started by the addition of 2 mol %
vector were cut for 1 h with 1 U each of FastDigest® endonu- (1.48 mg) or 10 mol % (7.44 mg) of NADP+. The mixture was
cleases NcoI and MssI (Fermentas, St. Leon-Rot Germany) and stirred in a sealed flask for 24 h at rt, or for 8 h at 8 °C, fol-
purified by gel extraction (NucleoSpin® Gel Clean up, lowed by a slow warm-up to rt in the subsequent 16 h. Then,
Macherey-Nagel, Düren, Germany). A ligation reaction was pyridine (0.3 mmol) was added as an external standard to the
performed with the T4 DNA Ligase (Fermentas, St. Leon-Rot reaction mixture. For the work-up step the aqueous reaction
Germany) according to the manufacturer’s instructions. The mixture was subdivided into five portions. Each of them (1 mL)
competent E. coli BL21 Gold (DE3) lacIQ1 cells [18], which ylene chloride (1 mL; by shaking the vials in a thermomixer at
were prepared by following a standard protocol [19]. For the 25 °C for 30 min and removal of the aqueous layers with a
chemically competent cells a transformation efficiency of syringe). The work-up step was repeated twice. In total, the
1
× 107 cfu/µg pUC19 was determined.
aqueous layers were extracted three times with methylene chlo-
ride (3 × 5 mL). The organic and aqueous layers were sep-
arated by centrifugation for 30 min at 13.000 rpm. Afterwards
the aqueous layers were removed by a syringe and the
Expression and isolation of the P450-monooxygen-
ase BM-3 F87V and 19A12 variants
Both P450-monooxygenase BM-3 variants, P450-monooxygen- combined organic layers were evaporated at 900 mbar and
ase BM-3 F87V and P450-monooxygenase BM-3 19A12, were 40 °C for about 30 min under vacuum. The amount of the
189