Experimental Section, Materials and
Methods
General Information
All solvents, reactants and starting materials were received from
commercial suppliers (Sigma-Aldrich, ChemPur, Thermo Fischer Scientific
(Acros Organic)) and used as received. Ultrapure water (UPW,
0.06 µS⋅cm-1) was produced with an Ultra Clear
Reinstwassersystem by SG Water (now Evoqua, Guenzburg, Germany) and used
throughout this study. All experiments were carried out at atmospheric
conditions. An IKA HS 260c shaker (IKA-Werke, Staufen, Germany) tempered
with a Huber CC-K6 thermostat (Peter Huber Kältemaschinenbau AG,
Offenburg, Germany) was used throughout all experiments for tempering
and shaking of the reaction vessels at 180 rpm if not mentioned
otherwise. Recombinant (S )-selective IRED fromPaenibacillus elgii B69 was transformed, cloned and overproduced
in E. coli BL21 (DE3) as previously described .
Gaschromatography
The Conversion of all reactions was measured by gas chromatography with
a Trace 1310 gas chromatograph from Thermo Scientific (Dreieich,
Germany) with a flame ionization detector equipped with a HP-5 column
from Agilent Technologies (30 m x 0.250 mm, 0.25 µm, 19091J-433,
SN: USF724723H). Helium was used as the carrier gas (purity: 99.999%)
with a flow rate of 0.75 mL⋅min‑1 was used for all
measurements. Temperatures of the injector and detector were set to
250 °C. Temperature program: 40 °C for 10 min, followed by a heating
rate of 125.0 °C⋅min-1 to 250 °C and a hold time for
5 min. The sample was injected within split-mode with a split flow of
7.5 mL⋅min-1 and a purge flow of
3 mL⋅min‑1. Authentic reference material was used for
external quantification (see Figure S 1 to 4 for further information).
All measurements were carried out in triplicate and the calculated
standard deviations are shown.
General Procedure for
Biotransformations
50 mg lyophilized whole E. coli cells containing the
corresponding overproduced enzyme were rehydrated in 400 µL of a
100 mmol⋅L-1 sodium phosphate buffer
(NaPi) pH 7.5 and shaken at 30 °C and 180 rpm in a
TS-100 thermos shaker from Biosan. After 30 min, 100 µL of a 2‑MP
substrate stock solution (250 mmol·L-1 in
NaPi) and 50 mg of d‑glucose were added
successively to the cell suspension (100 mmol⋅L-1final concentration of the substrate and 500 mmol·L-1of d‑glucose). A blank sample without the whole cell
biocatalyst was prepared to ensure the absence of undesired reactions.
The vials were tightly sealed and shaken at 30 °C and 180 rpm for 24 h.
After 24 h the reaction vessels were centrifuged for 5 min at 14000 rpm
and 100 µL of the supernatant was transferred to 900 µL DCM together
with 30 µL of a 10 m NaOH. The basified extraction was promoted
by vigorous shaking, 250 µL of the DCM phase were transferred to GC
Vials together with 25 µL of a solution containing n -heptane as a
standard and afterwards analyzed by gas chromatography.
For the evaluation of the maximal substrate concentration, desired
volumes of the substrate either from stock solution or the pure
substance were mixed together with buffer and d‑glucose in a
500 µL-scale as mentioned above. Samples for GC were taken to
investigate the reaction system at definite time intervals (conversion
vs. time) and the final concentration of the substrate was set to
150 mmol·L-1.
Adsorption- and
Desorption-Studies
The specific characteristics of the commercially available resins are
shown in Table S1 (see SI). The exchange resins were used as received in
their native ionic form.
To evaluate the capability for a DSP of the case studied biocatalytic
IRED-catalyzed reaction, the capacity of the resins with respect to
substrate and product were determined. A 100 mmol·L-1test solution of product and substrate in 100 mmol·L-1NaPi buffer solution together with
500 mmol·L-1 of d‑glucose was prepared for
the adsorption experiments. 4 mL of this test solution were then added
to 0.1 g, 0.5 g and 1.0 g of each resin, respectively. The shaking‑flask
assays were performed at 30 °C and 180 rpm for 30 min. The adsorption of
the substrate and the product were determined by analyzing the residual
in the solution before and after adding the adsorbent resins via GC.
100 µL of the supernatant were transferred to 900 µL DCM together with
30 µL of a 10 m NaOH and further processed for the GC as
mentioned above. For calculations of the adsorption, the measured
concentration of the prepared test solution was set at 100% and
compared to the amount of substrate after the adsorption.
After the successful adsorption of 2-MP and 2-MPN desorption experiments
were executed with Amberlite IR-120(H), Dowex Monosphere 650C and
Marathon MSC(H) cation exchange resins. Therefore, 0.5 g of each resin
was loaded with the substrate and the product as mentioned above and
after a filtration-step rinsed in 4 mL of a two phase system composed of
5% NaOH and cyclopentyl methyl ether (CPME) at a volumetric ratio of
1:1 at 30 °C and 180 rpm for 30 min. Afterwards, the concentrations of
the substrate and the product in the ether phase were determined via GC
as mentioned above.
Preparative Scale
Experiment
For the preparative scale experiment, 15 g lyophilized whole E.
coli cells containing the corresponding overproduced enzyme were
rehydrated in 150 mL of a 100 mmol·L-1 sodium
phosphate buffer (NaPi) pH 7.5 and stirred at 30 °C.
After 30 min, 2.13 mL of 2‑MP and 15 g of d‑glucose were added
successively to the cell suspension (150 mmol·L-1final concentration of the substrate and 500 mmol·L‑1of d‑glucose). The reaction mixture was stirred at 30 °C for
24 h. The conversion of the reaction was tracked after {0; 6; 17 and
24} h by removing 500 µL from the reaction broth and centrifuging it at
14000 rpm for five minutes. 100 µL of the supernatant were then
proceeded and analyzed via GC as mentioned above.
After reaching a maximum conversion, 30 g Dowex Monosphere 650C cation
exchange resin (0.2 g·mL-1) were added to the reaction
broth and stirred for 30 min at 30 °C. A 500 µL-sample was taken and
analyzed as mentioned before to check the full adsorption onto the
resin. The cation exchange resin beads were filtered out of the reaction
broth, and the remaining resins were desorbed in 75 mL of 5% NaOH and
75 mL of CPME at 30 °C for 30 min. To promote the phase separation, the
reaction mixture was centrifuged for 5 min at 4000 rpm. The ether phase
was dried with Na2SO4 for 2 h.
Afterwards, the amine was oiled out with in situ generated HCl
gas as amine hydrochloride and the remaining solvent CPME was removed by
evaporation at 80 mbar. The purity of the viscous oil was checked by NMR
(see SI).