Page 3 of 4
Please d Co h ne omt Ca do j mu s mt margins
Chemical Communications
COMMUNICATION
+
DOI: 10.1039/C7CC04465H
Table 1: H
2
-driven biocatalysis in flow using CNCs modified with hydrogenase and NAD reductase, with or without an NADH-dependent enzyme.
a
Entry
NADH-dependent enzyme
Substrate
NAD (0.5 mM)
acetophenone (8.7 mM)
pyruvate (2 mM)
pyruvate (12.5 mM)
Product
NADH
(S)- 1-phenylethanol
Alanine
% conversion
Total Turnover Number
+
1
2
3
4
-
67
30
90
40
1,800
24,000
19,600
ADH
LAlaDH
LAlaDH
Alanine
54,000
a
+
+
Total turnover number: molecules of product generated per NAD reductase. Experimental conditions: H
2
-saturated solution containing NAD , H
2
flowing through the
-
1
headspace above the solution reservoir, flow rate ca 25 µL min . Substrate concentrations are recorded in the Table. For reductive amination reactions, ammonium
chloride (150 mM) was used as the nitrogen source.
change in absorbance at 340 nm. LAlaDH has specific activity of
A further flow experiment for H -driven biotransformation of
2
-
1
>
20 U mg (Sigma). Experiments were set up under anaerobic
acetophenone to 1-phenylethanol was conducted with lower
cofactor and acetophenone concentrations such that the
concentration of acetophenone was approximately equivalent
conditions in a glove box (Glove Box Technologies, Ltd, <1 ppm
O ). The CNCs were modified by passing a pre-mixed solution of
2
the required enzymes into the column and leaving them to
o
to the solubility of H in water (1 mM). This showed some adsorb over an hour at 4 C. The columns were washed by
2
flowing through ca 0.5 mL buffer to remove unadsorbed
conversion (8 %) in a single pass through the CNC (see ESI
Figure S8).
+
enzyme. The H -saturated reaction mixture containing NAD
2
(
Prozomix) and either acetophenone or pyruvate (Sigma) was
The results presented here represent a modular approach
then cycled through the column using a peristaltic pump or an
Asia syringe pump (Syrris). For experiments with in-line UV-
to H -driven biocatalytic hydrogenation reactions in flow. The
2
+
ability to reduce NAD to NADH using H gas in continuous visible sampling, the solution was pumped from the CNC, into a
2
low volume quartz cuvette (path length, 1 cm, Hellma) before
being pumped back into the CNC. For hydrogenation reactions,
the reaction mixture was cycled from the CNC and into a sealed
flow provides a platform for ‘plugging in’ various NADH-
dependent enzymes. Overall, this tackles three of the
challenges in biocatalysis: enzyme immobilisation, cofactor
recycling and simple translation into continuous processing.
vessel with H flowing through the headspace at atmospheric
2
pressure. HPLC protocols for acetophenone to 1-phenylethanol
[
9]
This research was supported financially by the European conversion have been described previously.
Further
experimental detail and raw data is provided in the ESI. There
are no conflicts of interest to declare.
Research Council (EnergyBioCatalysis-ERC-2010-StG-258600 to
K.A.V.) and INSPIRE award EP/J015202/1 to N.G. and K.A.V..
H.A.R., L.A.T. and K.A.V. are supported by Engineering and
Physical Sciences Research Council (EPSRC) IB Catalyst award
EP/N013514/1. N. G. is supported by the Royal Society,
European Research Council (DEDIGROWTH-ERC-2009-StG-
[
[
1] R. Ciriminna, M. Pagliaro, Org. Process Res. Dev. 2013, 17,
479–1484.
2] P. J. Cossar, L. Hizartzidis, M. I. Simone, A. McCluskey, C. P.
1
Gordon, Org. Biomol. Chem. 2015, 13, 7119–7130.
2
40500,
PoC2015-680559), and the European Commission (CONTACT-
FP7-PEOPLE-ITN-2008-238363, BCN-Tubes-FP6-2004-NMP-TI- [4] W. A. van der Donk, H. M. Zhao, Curr. Opin. Biotechnol. 2003,
4, 421–426.
5] J.-M. Laval, C. Bourdillon, J. Moirouxt, J. Am. Chem. SOC 1984,
06, 4701–4706.
DEVICE-ERC-2011-PoC-309786,
CONDUCT-ERC- [3] M. Irfan, T. N. Glasnov, C. O. Kappe, ChemSusChem 2011, 4,
300–316.
1
4
-033350). J.Q. and T.H.L. were supported by EPSRC DTA
[
awards EP/J500495/1 and EP/L505031/1, respectively. We are
grateful to Dr Oliver Lenz and Dr Lars Lauterbach (Technical
1
+
[6] B. Siritanaratkul, C. F. Megarity, T. G. Roberts, T. O. M.
Samuels, M. Winkler, J. H. Warner, T. Happe, F. A. Armstrong,
Chem. Sci. 2017, 8, 4579–4586.
University, Berlin) for providing samples of NAD reductase
and soluble hydrogenase, to Prof Fraser Armstrong and Ms
Elena Nomerotskaia (University of Oxford) for samples of E.
coli hydrogenase 2 and to Dr Beatriz Dominguez (Johnson
Matthey Catalysis and Chiral Technologies) for providing ADH
[
[
[
7] R. W. Coughlin, M. Aizawa, B. F. Alexander, M. Charles,
Biotechnol. Bioeng. 1975, 17, 515–526.
8] S. K. Yoon, E. R. Choban, C. Kane, T. Tzedakis, P. J. A. Kenis, J.
Am. Chem. Soc. 2005, 127, 10466–10467.
9] H. A. Reeve, L. Lauterbach, O. Lenz, K. A. Vincent,
ChemCatChem 2015, 7, 3480–3487.
1
05.
Notes and references
[10] C. Jimenez-Gonzalez, P. Poechlauer, Q. B. Broxterman, B.-S.
Yang, D. Am Ende, J. Baird, C. Bertsch, R. E. Hannah, P. Dell
’orco, H. Noorman, et al., Org. Process Res. Dev. 2011, 15,
‡
Preparation of carbon nanotube columns is described in the
[17]
ESI. + The hydrogenase (Escherichia coli hydrogenase 2)
and
900–911.
NAD reductase (I64A variant of the soluble hydrogenase from
Ralstonia eutropha, in which the inherent hydrogenase moiety is [11] W. Feng, P. Ji, Biotechnol. Adv. 2011, 29, 889–895.
[
18]
inactivated),
were isolated and purified following published [12] L. Wang, H. Zhang, C.-B. Ching, Y. Chen, R. Jiang, Appl.
protocols. The alcohol dehydrogenase (ADH 105, Johnson
Matthey Catalysis and Chiral Technologies) was used as
supplied. The LAlaDH (from Bacillus subtilis, A7189, Sigma) was
also used as supplied. Stock solutions of ADH 105 or LAlaDH
Microbiol. Biotechnol. 2012, 94, 1233–1241.
13] H. A. Reeve, P. A. Ash, H. Park, A. Huang, M. Posidias, C.
Tomlinson, O. Lenz, K. A. Vincent, Biochem. J. 2017, 474, 215–
[
230.
-
1
were prepared at a concentration of 10 mg mL in buffer
solution (throughout, pH 6.0, 50 mM Bis-Tris). The activity of the
[
14] D. Zhao, K. Ding, ACS Catal. 2013, 3, 928–944.
[15] A. K. Holzer, K. Hiebler, F. G. Mutti, R. C. Simon, L. Lauterbach,
O. Lenz, W. Kroutil, Org. Lett. 2015, 17, 2431–2433.
16] B. Tomaszewski, R. C. Lloyd, A. J. Warr, K. Buehler, A. Schmid,
-
1
ADH 105 was determined to be 0.5 U mg for NADH-linked
acetophenone reduction in solution assays monitored by a
[
This journal is © The Royal Society of Chemistry 20xx
Chem. Commun., 2016, 00, 1-3 | 3
Please do not adjust margins