Biocatalytic reduction of alkenes in micro-aqueous organic solvent catalysed by an immobilised ene reductase

Biocatalytic asymmetric reduction of alkenes in organic solvent is attractive for enantiopurity and product isolation, yet remains under developed. Herein we demonstrate the robustness of an ene reductase immobilised on Celite for the reduction of activated alkenes in micro-aqueous organic solvent. Full conversion was obtained in methyl t-butyl ether, avoiding hydrolysis and racemisation of products. The immobilised ene reductase showed reusability and a scale-up demonstrated its applicability.


General information
Chemicals: NADPH and NADP + were purchased from Prozomix.All other chemicals were purchased from Sigma-Aldrich, abcr GmbH or TCI Europe at the highest purity available and used as received.Enzyme: GDH-101 was obtained from Johnson Matthey (London, England), as a lyophilised powder with an activity of 20.5 U/mg.Celite: Celite® R-632, R-633 and R-648 were received via Dr. Y. Guiavarc'h from Imerys (France) (Table S1).Celite™ 545 was purchased from Thermo Scientific™.

TsOYE production and purification
TsOYE (from Thermus scotoductus SA-01, accession number B0JDW3) was recombinantly produced in E. coli BL21(DE3) cells with the plasmid pET-22b(+)-tsoye.A 100 mL pre-culture of LB medium containing 100 μg mL -1 of ampicillin was inoculated with a glycerol stock of E. coli BL21(DE3)-pET-22b(+)-tsoye and incubated overnight at 37 °C and 180 rpm.500 mL of TB medium supplemented with 100 μg mL -1 of ampicillin in a 2 L shake flask was inoculated with the pre-culture (5% v/v) and incubated at 37 °C and 180 rpm until the OD600 reached 0.6 (approximately 2 h 30 min), 0.1 mM of IPTG was added for induction, and the cell culture was incubated overnight at 30 °C and 180 rpm.
Cells were harvested by centrifugation at 17,500 × g for 30 min at 4 °C.The resulting cell pellet was washed and re-suspended in a 20 mM MOPS-NaOH buffer at pH 7.0 supplemented with a spatula tip of DNase I, MgCl2, and one tablet of EDTA-free Complete TM protease inhibitor.The cell pellet was resuspended in buffer and lysed using a Multi-Shot Cell Disruption System (Constant Systems Ltd, Daventry, UK) over two cycles.Cell debris were separated from the crude extract by centrifugation at 17,500 × g for 30 min at 4 °C.The supernatant was filtered.
Heat purification was performed by incubating the supernatant in 50 mL Greiner tubes for 1 h 30 min in a water bath at 70 °C.Precipitated proteins were removed by centrifuging at 38,500 × g for 30 min at 4 °C two times.A clear and bright yellow solution of TsOYE was obtained, supplemented with flavin mononucleotide (FMN) and incubated on ice for 30 min.The protein solution was concentrated using an Amicon® Ultra-15 Centrifugal Filter Device (molecular cut-off 30 kDa) and washed with 20 mM MOPS-NaOH pH 7.0 buffer until the flow-through was colourless.The resulting heat-purified TsOYE was flash frozen in liquid nitrogen and stored at -80 °C until later use.

Enzyme concentration and activity
Total protein concentration was measured with a Bradford assay using bovine serum albumin (BSA). 1 The purified TsOYE stock solution used for immobilisation was determined to be 152 µM.
Concentration of flavin-bound TsOYE was determined by UV-Vis absorbance following standard protocol for flavoproteins (using FMN extinction coefficient at 446 nm ε446 = 12.2 mM -1 cm -1 ), 2 with the following compounds and concentrations: 1 mL buffer 20 mM MOPS-NaOH pH 7.0; TsOYE (amount to reach an absorbance between 0.1 and 0.2), and 20 μL sodium dodecyl sulfate (SDS, 0.2% w/v final, from a stock solution of 10% w/v in MilliQ).Enzyme purity was assessed by SDS-PAGE and estimated to be >90% pure.
The specific activity of TsOYE for cyclohexenone was 9 U/mg, measured following the consumption of NADPH at 340 nm (ε = 6.22 mM -1 cm -1 ) as described previously. 3,4 he assay mixtures contained final concentrations of 50 mM MOPS-NaOH buffer pH 7.0, 10 mM cyclohexenone, 0.2 mM NADPH, at 22.6 °C, 1 mL in volume, all components were thermostated prior to measurements.

Enzyme immobilisation
Enzyme immobilisation on celite was performed according to a previous protocol with slight modifications. 5In this work, 200 mg of Celite carrier were washed three times with buffer 50 mM MOPS-NaOH pH 7.0.Then, 500 µL of the enzyme TsOYE were added to each of the washed carrier.An aliquot of 70 µL was taken at t = 0 h for reference.The enzyme-carrier mixture was slowly shaken for 5 h at 20 °C.After 5 h, each sample was centrifuged, the supernatant was removed, and the immobilised enzyme was frozen.Enzyme concentration before and after immobilisation was determined by UV-Vis absorbance as described above. 2

Biotransformations
Biotransformations were performed with 1 mL reaction volume in 2 mL microcentrifuge tubes.

Free enzyme
For reactions with free TsOYE (Figure 2, Table S2), the reactions were set up with the corresponding organic solvent, 1.4 µM TsOYE, 0.2 mmol NADPH, 10 U/mL BsGDH, 20 mmol glucose, 10 mmol cyclohexenone, 1 mL in volume, at 30 °C and 900 rpm in an Eppendorf ThermoMixer C for 24 h.As a control, buffer saturated MTBE, using 50 mM MOPS-NaOH pH 7.0, was used in the reaction conditions described above.

Immobilised enzyme
For reactions with 15 mg of immobilised TsOYE on Celite 545 (Table S3 entries 1-3), the reactions were set up with the corresponding organic solvent, 0.2 mmol NADPH, 10 U BsGDH, anhydrous glucose, 10 mmol cyclohexenone 1a, 1 mL in volume, at 30 °C with the specified conditions shown in Table S3.

Control experiments
Control experiments (Table S4) were performed with cyclohexenone as substrate in absence of enzymes.Tubes were shaken for 6, 8 or 24 h at 30 °C and 900 rpm in an Eppendorf ThermoMixer C. Aliquots (100 µL) were taken from the organic supernatant, diluted in 50 µL of EtOAc supplemented with 5 mM of tridecane as internal standard, dried with anhydrous MgSO4, centrifuged, and transferred into GC vials for analysis.To determine whether GDH-101 could reduce either the substrate cyclohexenone or product cyclohexanone under the same reaction conditions, control experiments were performed: 1) 10 mmol of cyclohexenone, 1 mmol NADP + , 27.7 mmol glucose and 2 mg GDH-101 in MTBE.No conversion was observed.

Scale-up
Scale up reactions with 50 mmol 2-methyl-N-phenylmaleimide were set up with MTBE solvent, 9.3 mg substrate, 50 mg immobilised TsOYE on Celite R-633 and R-648, 2 mmol of NADP + , 2 mg GDH-101 and 60 mmol of glucose, 1 mL in volume.The vials were shaken for 24 h at 30 °C and 180 rpm on an incubator shaker (New Brunswick Scientific Excella E24 Incubator Shaker Series).Aliquots (100 µL) were taken from the organic supernatant, diluted in 50 µL of EtOAc supplemented with 5 mM of tridecane as internal standard, dried with anhydrous MgSO4, centrifuged, and transferred into GC vials for analysis.For Table 2 entries 19 and 20, the pure product was separated from the organic solvent by evaporation of the solvent and the solid product was obtained in 91% isolated yield in both cases, and analysed by NMR in DMSO-d6 (Figures S17 and S18 for the R-633-TsOYE reaction).Spectra are in agreement with literature. 6

Immobilised enzyme recovery
Enzyme reusability was determined through several cycles of cyclohexanone synthesis.The reaction mixture contained: 10 mmol cyclohexenone, 1 mmol NADP + , 27.7 mmol solid anhydrous glucose, 2 mg GDH-101, 25 mg of salt pairs (Na2HPO4•12H2O/Na2HPO3•5H2O, 1:1 w/w), 50 mg of immobilised TsOYE on Celite R-633 or Celite 545, and 0.98 mL MTBE.The reaction mixture was incubated and shaken at 900 rpm and 30 °C for 24 h.At the end of the reaction, the liquid mixture was separated from the immobilised enzyme by centrifugation and a new reaction mixture was prepared for the next operational cycle.

Table S1 .
Physical properties of Celite carriers

Table S4 .
Reaction conditions for control reactions without TsOYE in MTBE. a