Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007

Enzymes, chemicals and media

Pfu⁺-polymerase was obtained from Roboclon (Berlin, Germany) and dNTPs from Roth (Karlsruhe, Germany). Restriction enzymes were obtained from New England Biolabs (Beverly, MA, USA). For SDS-PAGE analysis, the prestained PAGE ruler plus from Fermentas (St.Leon-Rot, Germany) was used. All other chemicals were purchased from Fluka (Buchs, Switzerland), Sigma-Aldrich (Munich, Germany) or Acros Organics (Geel, Belgium). For DNA-purification from PCR, the MinElute PCR-purification Kit by Qiagen (Hilden, Germany) was used. Furthermore the Miniprep Kit from Qiagen was used for plasmid purification. HisTrap 5 mL FF columns and Sephadex G25 were obtained by GE Healthcare (Uppsala, Sweden). The plasmid pET-28b(+) was from Novagen (Darmstadt, Germany). The BCA kit was purchased from Interchim (Montluçon, France).

Amplification and cloning

Amplification of the 2,5-DKCMO gene was performed with chromosomal DNA containing the CAM-plasmid with oligonucleotides supplemented with restriction sites for Nde I at the N-terminus and Xho I at the C-terminus (Nde I_2,5-DKCMO_fw: 5'- GGAATTCATATGAAATGCGGATTTTTCCATACCCC-3'; 2,5-DKCMO_Xho I_rv: 5'- CCGCTCGAGTCAGCCCATTCGAACCTT-3'). After initial denaturation for 5 min at 95°C, the cycling program was followed for 25 cycles: 45 s, 95°C denaturation, 45 s, 58°C primer annealing, 70 s, 72°C elongation. The final elongation step was performed over 10 minutes at 72°C. The resulting 1092 kb fragment was digested with Nde I and Xho I and ligated into pET-28b digested with the same enzymes. The resulting plasmid with a N-terminal His-tag fusion was called pET-28_2,5-DKCMO (Figure 3).

Bacterial strains and culture conditions

P. putida NCIMB 10007 (equivalent to ATCC 17453) was purchased from the German National Resource Center for Biological Material (DSMZ). For cultivation of P. putida, basal salt medium without antibiotics as described previously was used (Gagnon et al. 1994). E. coli cells were cultivated in terrific broth (TB) medium (12 g tryptone, 24 g yeast, 4 g glycerol in 1 L buffer autoclaved separately). Overnight cultures were grown in Luria Bertani (LB) medium (10 g tryptone, 5 yeast, 5 g NaCl in 1 L dest H₂O). LB and TB media were supplemented with 100 μg/mL kanamycin.

Transformation of E. coli strain BL21-DE3 (Novagen, genotype: [95 F- ompT hsdSB (rB-mB-) gal dcmrne131 (DE3)]) with pET-28_2,5-DKCMO was carried out by the heat shock method described by Chung et al. (1989). Expression of recombinant 2,5-DKCMO in E. coli BL21 was performed by cultivation at 37°C to an OD₆₀₀ of 0.5, than addition IPTG to a final concentration of 0.1 mM and shifting the culture to 20°C and 200 rpm for another 16 h of cultivation.

Gene expression analysis

Gene expression analysis was performed with crude cell extract. Samples standardized to cell amount were taken during cultivation. Cells were harvested by centrifugation and resuspended in sodium phosphate buffer (50 mM, pH 7.5). Cell disruption was performed by FastPrep (40 s, 4 m/s; MP Biomedicals, Solon, OH, USA). For SDS-PAGE analysis, the supernatant was substituted with Laemmli buffer (Laemmli 1970). SDS-PAGE was carried out on 12% resolving gels. Proteins were stained with a Coomassie R250/G250 solution.

Enzyme purification

Cells were harvested by centrifugation and resuspended in sodium phosphate buffer (50 mM, pH 7.5). Cell disruption was performed by a single passage through a French pressure cell. Recombinant 2,5-DKCMO was purified by affinity chromatography via N-terminal His-tag on an automated Äkta purifier system. After centrifugation of disrupted cells for 45 min at (10,000 × g), the supernatant with recombinant 2,5-DKCMO was added to the column. A 5 mL HisTrap FF crude column with bound Ni²⁺ was equilibrated with sodium phosphate buffer (100 mM, pH 7.5) supplemented with 300 mM NaCl and 30 mM imidazole. After passing through of the crude extract, the column was washed with three column volumes of sodium phosphate buffer (100 mM, pH 7.5) supplemented with 300 mM NaCl and 30 mM imidazole followed by two column volumes of sodium phosphate buffer (100 mM, pH 7.5) supplemented with 300 mM NaCl and 60 mM imidazole to remove unspecific bound proteins. Elution was performed by adding three column volumes of 300 mM imidazole in sodium phosphate buffer (100 mM, pH 7.5) supplemented with 300 mM NaCl. Fractions of washing and elution steps were collected to analyze purity by SDS-PAGE. In order to remove imidazole and NaCl from the eluate, the pooled elution fractions were loaded to a 60 mL size exclusion column (Sephadex G25 matrix), which was equilibrated with sodium phosphate buffer (50 mM, pH 7.5) before. Proteins fractions were recognized via online absorption measurement at 280 nm and collected. Determination of protein content of purified and desalted protein as well as crude extract was carried out with the BCA-kit and a standard curve of BSA in the same buffer in a range of 2-0.005 mg/mL was used. Samples were measured in triplicates in three different dilutions.

Biocatalytic reactions and GC analysis

For biocatalysis, His-tag purified 2,5-DKCMO, crude extracts of E. coli BL21 pET28_2,5-DKCMO cultivations and resting cells were used. Reactions were carried out in sodium phosphate buffer (50 mM, pH 7.5) Substrates were used in concentrations from 0.5-2 mM, the cofactor FMN was used at a final concentration of 0.3 mM. NADH was used in equimolar amounts to the substrate. Purified 2,5-DKCMO was employed in concentrations of 1.5-2 mg/mL, crude extracts in concentrations of 12-15 mg/mL. Incubation was performed in 24-well MTP at 800-1000 rpm. Sample volume was 1 mL. Extraction of substrates and products was performed by vortexing of samples with 600 μl and 400 μl of ethyl acetate subsequently. Samples were dried over anhydrous sodium sulfate. Separation of aqueous and organic phase was done by centrifugation. The organic solvent was evaporated in a vacuum centrifuge. 120 μL of fresh EtOAc was added, and samples were analyzed by GC-MS on a QP 2010 (Shimadzu Europa GmbH, Duisburg, Germany) with a BPX5 column (5% phenyl-/95% methylpolysilphenylene siloxane, SGE GmbH, Darmstadt, Germany). Injection temperature was set to 220 °C. Detection temperature for (+)-1, (-)-1, 13, 14 and 15 was 60°C for 5 min followed by a gradient of 10°C/min to 180°C maintained for 3 min. Detection temperature for 16 was 120°C. For 17, 240°C for 5 min followed by a gradient of 2°C/min to 270°C was used and maintained for 5 min. 6-8 were analyzed at 60°C. 9 and 10 were detected isothermal at 160°C. Detection temperature for 11 was 90°C and for 12 100°C.

Specific activity is given in units per milligram (U/mg) protein. One unit is defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of substrate per minute.

Cloning, expression and purification of 2,5-diketocamphane 1,2-monooxygenase

The 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) from Pseudomonas putida NCIMB 10007 is encoded on the CAM operon on the transmissible 230 kb CAM plasmid (Rheinwald et al. 1973). First chromosomal and plasmid DNA were isolated from the P. putida strain NCIMB 10007 cultivated with camphor as sole carbon source. The gene was then amplified by gradient PCR using gene specific primers derived from the corresponding gene [GenBank: AY450285]. The PCR product was afterwards ligated into the expression vector pET-28b fused to the N-terminal His-tag to allow a functional expression of the 2,5-DKCMO in E. coli and easy protein purification by affinity chromatography. The N-terminal tag was favored compared to the C-terminal tag, because our experience with 4-hydrocyacetophenonemonooxygenase (HAPMO) from P. putida JD1 indicated that BVMO-expression is decreased by the use of C-terminal tags (Rehdorf et al. 2009).

The utilization of E. coli BL21(DE3) as expression host yielded primarily soluble 2,5-DKCMO protein after 16 h cultivation at 20°C in TB medium, while cultivation at 30°C yielded in insoluble inclusion bodies (data not shown). SDS-PAGE analysis of crude cell extract led to a clear band at approx. 40 kD shown in Figure 4, which corresponds to the theoretical estimated molecular weight of 42.9 kD of the His-tagged protein.

Substrate specificity of 2,5-DKCMO

To determine the substrate specificity of 2,5-DKCMO a variety of compounds representing different classes of BVMO-substrates were investigated in biocatalysis experiments using the crude enzyme extract (Figure 5). Only bicyclic ketones were converted under the chosen conditions by the crude extract containing 2,5-DKCMO (Table 2). For all monocyclic ketones (6-8), aromatic ketones (9-11), the aliphatic 2-decanone (12) tested as well as for 1-indanone (16) and progesterone (17) no conversion could be determined. The biocatalysis with substrates, which were converted was further investigated using the pure enzyme and specific activities were determined in biocatalysis experiments in 1 mL scale with 2 mM of substrates at 25°C for 15 h (Table 2).

Interestingly, in our study (-)-1 was also converted by the 2,5-DKCMO, although purified enzyme isolated from wild-type strain cultivation was claimed to be specific for the (+)-enantiomer (Jones et al. 1993). As we have recombinantly produced the BVMO in the E. coli host, which does not have its own BVMO and the conversion of (-)-camphor was observed with crude cell extract as well as His-tag purified protein, we can only speculate whether the purified protein described by Jones et al. 1993 was indeed homogenous. Norcamphor (13) and (±)-cis-bicyclo [3.2.0] hept-2-en-6-one (14) were better accepted as substrates than camphor in general, and furthermore (R,R)-bicyclo [2.2.1] heptane-2,5-dion (15), which is structurally similar to the natural substrate 2,5-diketocamphane (3), is also converted. In addition, the conversion of 14 was performed with resting cells expressing 2,5-DKCMO, where 11% conversion could be observed after 6 h of biocatalysis at 0.5 mM substrate concentration.