10.1002/cbic.201900356
ChemBioChem
COMMUNICATION
product and starting material, affording excellent process yields
(90-95%).
95% A 5% B; 4.50 min 95% A 5% B. Injection volume 2 µL) at 40 °C with
a flow rate of 0.8 mL/min. The retention times in minutes were: 2-
phenylethylamine (2.0 min), 2-phenylacetaldehyde (2.3 min).
Isolated enzymes, when compared with partially purified
fractions or whole cells biotransformations, behave more
similarly to traditional catalysts and generally do not cause side
reactions, and of course never encounter permeability problems.
In particular transaminases, with respect to MAO or AASs, offer
a better control of the deamination step which does not generate
NH3 or H2O2 as the amino group is simply transferred onto a
sacrificial acceptor. Both NH3 or H2O2, if let to accumulate in the
reaction vessel, can cause pH increase as well as strong
oxidizing reaction environment, which could eventually inactivate
the enzyme involved in the biotransformations (even under flow
conditions). HEWT, when pyruvate is the amino acceptor, forms
ʟ-alanine that can be easily recovered via scavenging columns
as previously reported15. Notably HEWT is completely stable
under segmented flow (buffer/toluene), and the same packed
bed reactor was used to perform all the experiments without any
loss of the activity.
Flow reactions with immobilized HEWT and co-immobilized PLP
Continuous flow biotransformations were performed using a R2+/R4
Vapourtec flow reactor equipped with an Omnifit glass column (6.6 mm
i.d. × 100 mm length) filled with 1.3 g of imm-HEWT/Co-immobilized PLP
(5 mg/gresin 1 mM PLP). 40 mM sodium pyruvate in HEPES buffer (10
mM, pH 7.5) and 40 mM amino donor solutions were prepared. The two
solutions were mixed in a T-piece. A second junction for additional
supplement of toluene was installed before the packed enzyme column.
The resulting segmented flow stream (80:20 buffer/toluene) was directed
to the imm-HEWT/co-imm PLP (reactor volume: 1.8 mL, flow rate: 0.12
mL/min). An in-line acidification was performed by using an inlet of 1 N
HCl aqueous solution (flow rate: 0.1 mL/min) that was mixed to the
exiting reaction flow stream using a T-junction. After an in-line extraction
using a Zaiput liquid-liquid separator, the organic and aqueous phases
were analyzed by HPLC exploiting a calibration curve and the toluene
containing the desired product was evaporated to yield the aldehydes.
Analysis of biogenic aldehydes reactions
The flow biotransformations were analyzed by a Thermo Ultimate 300s
HPLC equipped with a Accucore™ C18 LC Column (2.6 µm, 4.6 mm x
150 mm). The mobile phase was composed by A: 0.05% formic acid in
water, B: Acetonitrile. The compounds were detected using a DAD
detector at 280 nm after a gradient run increasing the concentration of B
as follows: 0-5 min 10%, 5-10 min 50%, 10-11 min 10% (Injection
volume 10 µL) at 40 °C with and flow rate of 1 mL/min. The retention
times in minutes were: dopamine (2.7 min); DOPAL (3.4 min); 3,4-
methoxyphenetilamine (3.4 min); 3,4-methoxyphenylacetaldehyde (4.2
min); 4-hydroxyphenetilamine (2.9 min); 4-hydroxyphenylacetaldehyde
In summary a new strategy for the synthesis of aromatic
biogenic aldehydes was developed. HEWT was further
stabilized due to the co-immobilization with PLP producing a
self-sufficient heterogeneous biocatalyst for deamination
reaction without exogenous addition of cofactors. In addition,
with respect to previously reported data on amine conversion,17b
here the concentration of the starting material was doubled and
still yields of 90-95% were achieved. Side condensation
reactions typically present in the batch methods were never
observed.
(3.5
min);
4-methoxyphenetilamine
(3.2
min);
4-
methoxyphenylacetaldehyde (4.0 min); 3-methoxyphenetilamine (3.0
min); 3-methoxyphenylacetaldehyde (3.8 min). The isolated aldehydes
were further characterized by 1H-NMR spectra which corresponded to
those previously reported in literature.
The in-line extraction step allowed for the separation and
recovery of the pure biogenic aldehydes in the toluene stream,
while ʟ-alanine/ traces of unreacted amines remained in the
water phase, with no further work-up procedure making the
process fully automated.
Reaction rate comparison between batch and flow mode
Specific reaction rates in batch and continuous-flow systems were
calculated using the following equations:21
Equation 1.
rbatch = ƞp / t mb (µmol / min g)
The combination of continuous mode and biocatalysis not only
leads to significant reduction of the reaction times (15 min) and
where [ƞp] is the amount of product (expressed as µmol), t is the reaction
time (expressed as min), and mb [g] is the amount of biocatalyst
employed.
increased productivity but also has established
sustainable routes to an array of valuable products.
a highly
Equation 2.
rflow = [P]x f / mb (µmol / min g)
where [P] is the product concentration flowing out of the reactor
(expressed as µmol mL-1), f is the flow rate (expressed as mL min-1), and
mb [g] is the amount of biocatalyst loaded in the column.
Experimental Section
Characterization of the products
The purity of aldehydes was assessed by HPLC and 1H NMR. 1H NMR
Expression, purification, and immobilization of HEWT in E. coli
spectra were recorded with
a Varian Mercury 300 (300 MHz)
spectrometer. Chemical shifts (δ) are expressed in ppm, and coupling
constants (J) are expressed in Hz.
Protein expression and purification was performed following previously
reported protocols in Cerioli et al.20 Immobilization was conducted
according to the procedure reported by Benítez-Mateo et al.18 The
retained activity observed for the co-immobilized HEWT/PLP system was
>50% with respect to the free form with exogenously added cofactor (free
enzyme: 5 U/mg, immobilized system 2.6 U/mg).
3,4-Dihydroxyphenylacetaldehyde (DOPAL): NMR (300 MHz, DMSO-d6)
δ (ppm): 9.58 (t, J = 2.33 Hz, 1H ), 6.70 (d, J = 7.99 Hz, 1H), 6.61 (d, J =
2.13 Hz, 1H), 6.47 (dd, J = 7.99, 2.13 Hz, 1H), 3.52 (d, J = 2.40 Hz, 2H),
2.85 (t, J = 7.3 Hz, 2H), 1.93 (s, 3H).
Batch reactions with immobilized HEWT
3,4-Dimethoxyphenylacetaldehyde:NMR (300 MHz, CDCl3) δ (ppm): 9.75
(t, J = 2.45 Hz, 1H ), 6.89 (d, J = 8.15 Hz, 1H), 6.81 (dd, J = 8.15, 2.05
Hz, 1H), 6.73 (d, J = 2.0 Hz, 1H), 3.98 (s, 6H), 3.65 (d, J = 2.48 Hz, 2H).
Batch reactions using the imm-HEWT with co-immobilized PLP were
performed in 1.5 mL micro centrifuge tubes; 500 μL reaction mixture in
10 mM HEPES buffer pH 7.5, containing 20 mM pyruvate, 20 mM amino
donor substrate and 50 mg of imm-HEWT with co-immobilized PLP (5
mg/gresin, 1 mM PLP) was left under gentile shaking at 37 °C. 10 μL
aliquots were quenched with a 50:50 mixture of hydrochloric acid (HCl)
0.2%:acetonitrile solution every hour and then analyzed by LC-MS
equipped with a Waters X-Bridge C18 (3.5 µm, 2.1 mm x 30.0 mm). The
compounds were detected using a DAD detector at 250 nm after a 5 min
gradient run (A: 0.1% Ammonia in water, B: Acetonitrile. Gradient: 0 min
5% A 95% B; 3.10 min 0% A 100% B; 3.50 min 0% A 100% B; 3.51 min
4-Hydroxyphenylacetaldehyde: NMR (300 MHz, DMSO-d6) δ (ppm): 9.62
(t, J = 2.18 Hz, 1H ), 9.34 (s, 1H), 7.03 (m, 2H), 6.74 (m, 2H), 6.73 (d, J =
2.0 Hz, 1H), 3.98 (s, 6H), 3.61 (d, J = 2.20 Hz, 2H).
4-Methoxyphenylacetaldehyde: NMR (300 MHz, DMSO-d6) δ (ppm):
9.65 (t, J = 2.03 Hz, 1H ), 7.16 (m, 2H), 6.92 (m, 2H), 6.73 (d, J = 2.0 Hz,
1H), 3.74 (s, 3H), 3.68 (d, J = 2.09 Hz, 2H).
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