Proof of concept In vitro approach of a carboxylate reductase

Performed reaction

• : 3-OH benzoic acid

• : carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) (purified)

• : 3-OH benzaldehyde

• Cofactors: NADPH and ATP

• Coenzymes for regeneration:

-Polyphosphate kinase from Meiothermus ruber

-SmPPK – Polyphosphate kinase from Sinorhizobium meliloti

-EcPPase – Pyrophosphatase from Escherichia coli

Materials (Stock Solutions)

Buffer:

• 4x 400 mM MOPS buffer

Cofactors:

• 100 mg Sodium polyphosphate (solubilized in 170 µL 1M KOH and 1.83 mL ddH2O)

• ATP 50 mM

• NADPH 50 mM pH indicator:

• Bromothymol blue Materials (Stock solutions)

Coenzymes:

• MrPPK 200 µg/mL

• SmPPK 80 µg/mL

• EcPPase 50 µg/mL

• GDH 0.2 µg/mL

• NoCAR 100 µg/mL

Substrate:

• 10 mM of 3-OH benzoic acid solubilized in 250 mM KOH

Stock concentration Key components Conc. in reaction (mM) Stocks (mM) MOPS buffer pH 7.5 100 Buffer (MOPS pH 7.50) 400 Substrate 10 aromatic acid 250 NADP(H) 0,5 NADP(H) 50 ATP 1 ATP 50 MgCl2 12.5 MgCl2 50 Sodium polyphosphate 8 mg/mL Sodium polyphosphate 50 mg/mL β-D-(+)-glucose 100 β-D-(+)-glucose varies

conc/ reaction (µg/ mL) MrPPK 200 SmPPK 80 EcPPase 50 NoCAR 100 conc./reaction (ug/ mL) conc./reaction (U/mL) GDH 0.2 GDH 50 Preparation of stocks and amounts CCE liquid stock

Stock coenzy- Conc.* purity (µg/ Volume/rxn from Corrected vo- 9 reactions mes Conc. (µg/mL) mL) stock (µL) lume/rxn (µL) (µL) MrPPK 6100.00 3050.00 16.39 16.4 147.60 SmPPK 8490.00 4245.00 4.71 4.8 43.20 EcPPase 9670.00 4835.00 1.29 1.3 11.70 GDH-105 50 50 1.00 1 9.00 Total 23.40 23.5 211.50 Procedure

1. Set up a coenzyme mix for all cofactor regeneration reactions and adapt needed amounts according to the amount of reaction vials.

2. Weigh in 3-OH benzoic acid with the wanted molarity and dissolve in 250 mM KOH.

3. Dissolve 100 mg Sodium polyphosphate (Grahams salt) solubilized in 170 µL KOH

(1M) and 1.83 mL H2O.

4. Set up the MOPS buffer and adjust pH to 7.5.

5. Weigh in the needed amount of ATP and NADPH and solubilize in according amount of double distilled water.

6. Prepare a quenching solution with pure acetonitrile (ACN) and pure formic acid in a ratio of 19:1 (ACN:Formic.).

7. Pre heat a thermoblock to 30°C (850 rpm) and a centrifuge (15,000g) to 4°C.

8. Prepare glass vials for the reaction.

9. Pipette all components in the following amounts into each reaction vial in the follow- ing order:

Mastermix for all reactions 10 mM substrate Components 3 reactions (µL) Double distilled water 86.5 259.5 Ready buffer 4x 62.5 187.5 Polyphosphate (50 mg/mL) 40 120 NADP(H) (50 mM) 2.5 7.5 ATP (50 mM) 5 15 Coenzyme stock 23.50 70.5 Bromothymol blue 5 15 Total 225 675 Procedure

10. As the last steps add the substrate and main enzyme:

Reaction vial/eppi 10 mM subst. Mastermix 225 Substrate (250 mM) 10 NoCAR 10 Total 245

11. Place vials into the preheated thermoblock and start it at 850 rpm.

12. Prepare as many Eppendorf tubes as you have glass vials with 180 µL quenching solu- tion.

13. After the first 30 minutes add 5 µL 1 M KOH into each vial to adjust the pH.

14. Perform the first sampling step. After the 30 min pipet 100 µL of the reaction solution out of the glass vial into the Eppendorf tube with the quenching solution.

15. Centrifuge these Eppendorf tubes at 15,000 g, 4°C for 5 min.

16. As a final step, pipet the supernatant of the Eppendorf tubes into a HPLC glass vial and measure the composition.

17. Perform these sampling steps (13-15) and repeat these each 30 min for 2 hours and once overnight.

18. Perform HPLC measurements. The HPCL should be washed two times with ACN be- fore you measure the samples and should be washed with ACN after you finished you measurements.

Hints

• Always create the mastermixes for wanted amounts of reaction vials + 1 to always have sufficient solution for pipetting.