10.1002/cctc.201600806
ChemCatChem
COMMUNICATION
Extraction of 6-Hydroxyhexanoic Acid
The system was calibrated with molecular weight standards of polystyrene
(MP 66,000; 28,000; 12,600; 9,130; 6,100; 4,920; 3,470; 2,280; 1,250, 162
g/mol) (PSS Polymer Standards Service GmbH, Germany).
The 6-HHA generated from the whole reaction cascade was obtained by
acidifying the reaction volume with trifluoroacetic acid and extraction with
equal volumes of methyl tert-butyl ether (MTBE) 3–4 times. Extraction
volumes were pooled and MTBE removed under reduced pressure. To
isolate 6-HHA generated from a preparative hydrolysis reaction the
reaction volume was first acidified with hydrochloric acid. The volume was
reduced under heat and reduced pressure by evaporating water until
sodium chloride precipitated. It was then resuspended in MTBE and
filtered to remove insoluble sodium chloride. MTBE was removed under
reduced pressure.
A detailed description of bacterial strains, plasmids, cultivation procedure,
enzyme activity assays, analytical procedures and experimental setup is
given in the supporting information.
Acknowledgements
We are grateful to Deutsche Bundesstiftung Umwelt (DBU) for
financial support (AZ 13268) and all cooperation partners. We
also thank Fermenta Biotech Ltd. for support finding an
appropriate CALB preparation.
Polymersation
Polymerisation of 6-HHA obtained from preparative hydrolysis of ECL
(30.61 g, 286 mmol) in water (30.27 g) was performed with 1 g CALB
(cLEcta, 3707 PLU) under solvent free conditions in a stirred tank reactor
at 70 °C under reduced pressure (80 mbar) to remove the water. Samples
were diluted with chloroform and immobilised catalyst separated by
Keywords: -Caprolactone • Oxidoreductases • Polymerisation •
Candida antarctica lipase B • Enzymatic cascade
filtration through
atmospheric conditions.
a 0.2 µm nylon filter. Samples were dried under
[1]
[2]
A. J. Willetts, C. J. Knowles, M. S. Levitt, S. M. Roberts, H. Sandey, N. F.
Shipston, J. Chem. Soc., Perkin Trans. 1 1991, 1608.
a) H. Mallin, H. Wulf, U. Bornscheuer, Enzyme Microb. Technol. 2013,
53, 283–287; b) H. Wulf, H. Mallin, U. Bornscheuer, Enzyme Microb.
Technol. 2012, 51, 217–224.
HPLC Analytics
Samples of -caprolactone and 6-hydroxyhexanoic acid for HPLC analytic
were performed by taking 50 µL of aqueous sample and added to 950 µL
water. In the case of a soluble enzyme (e.g., CALA lyophilisate), samples
were diluted in acetonitrile: water (1:1 v/v) mixture. Analytics was
performed with an Agilient 1100 Series device on a LiChrospher 100 RP-
18 (5 µm) LiChroCART 250-4 column at 30 °C. 50 % (v/v) Methanol in
water was used as the eluent with a flow rate of 1 mL/min (isocratic).
[3]
[4]
S. Staudt, U. T. Bornscheuer, U. Menyes, W. Hummel, H. Gröger,
Enzyme Microb. Technol. 2013, 53, 288–292.
a) J. H. Sattler, M. Fuchs, F. G. Mutti, B. Grischek, P. Engel, J. Pfeffer, J.
M. Woodley, W. Kroutil, Angew. Chem. Int. Ed. 2014, 53, 14153–14157;
b) J. H. Sattler, M. Fuchs, F. G. Mutti, B. Grischek, P. Engel, J. Pfeffer, J.
M. Woodley, W. Kroutil, Angew. Chem. 2014, 126, 14377–14381.
A. Bornadel, R. Hatti-Kaul, F. Hollmann, S. Kara, ChemCatChem 2015,
7, 2442–2445.
[5]
[6]
[7]
A. Bornadel, R. Hatti-Kaul, F. Hollmann, S. Kara, Tetrahedron 2015,
(available online) DOI: 10.1016/j.tet.2015.11.054.
GC Analytics
a) S. Schmidt, C. Scherkus, J. Muschiol, U. Menyes, T. Winkler, W.
Hummel, H. Gröger, A. Liese, H.-G. Herz, U. T. Bornscheuer, Angew.
Chem. Int. Ed. 2015, 54, 2784–2787; b) S. Schmidt, C. Scherkus, J.
Muschiol, U. Menyes, T. Winkler, W. Hummel, H. Gröger, A. Liese, H.-G.
Herz, U. T. Bornscheuer, Angew. Chem. 2015, 127, 2825–2828.
S. Schmidt, H. C. Büchsenschütz, C. Scherkus, A. Liese, H. Gröger, U.
T. Bornscheuer, ChemCatChem 2015, 7, 3951–3955.
a) R. Brenneis, B. Baeck, Biotechnol Lett 2012, 34, 1459–1463; b) S.
Kobayashi, H. Uyama, S. Namekawa, Polym. Degrad. Stabil. 1998, 59,
195–201; c) S. Namekawa, S. Suda, H. Uyama, S. Kobayashi, Int. J. Biol.
Macromol. 1999, 25, 145–151; d) J. Müller, M. A. Sowa, B. Fredrich, H.
Brundiek, U. T. Bornscheuer, ChemBioChem 2015, 16, 1791–1796.
Detection of cyclohexanol, cyclohexanone and -caprolactone was
performed by extraction of aqueous samples with chloroform. 200 µL of
aqueous sample or standard was transferred into a 1 mL tube, 10 µL
acetophenone (23.8 mg/mL DMSO) was added as an internal standard
and 300 µL chloroform was added. After 1 min of vortex and phase
separation by centrifugation 200 µL of chloroform phase was transferred
to a GC glass vial with a micro-inlet. Analysis was performed on an Agilent
Technologies 7890B (Santa Clara, CA, USA) device with a CP-Chirasil-
Dex CB (Agilent), 25 m x 0.25 mm x 0.25 µm column. Oven temperature
profile started from 150 °C for 3 min with a ramp to 167 °C (20 °C/min),
190 °C (30 °C/min) and held for 1.4 min. For calibration a mixture of
cyclohexanol (204.9 mg, 2.0 mmol), cyclohexanone (215.8 mg, 2.2 mmol)
and -caprolactone (221.7 mg, 1.9 mmol) in 10 mL water was used for a
serial dilution.
[8]
[9]
[10] A. Arbaoui, C. Redshaw, Polym. Chem. 2010, 1, 801.
[11] a) M. Labet, W. Thielemans, Chem. Soc. Rev. 2009, 38, 3484; b) H.
Uyama, K. Takeya, S. Kobayashi, Bull. Chem. Soc. Jpn. 1995, 68, 56–
61; c) D. Knani, A. L. Gutman, D. H. Kohn, J. Polym. Sci. A Polym. Chem.
1993, 31, 1221–1232.
[12] P. Howe, P. Watts, Concise international chemical assessment
document. Tin and inorganic tin compounds, World Health Organization,
Geneva, 2005, Vol. 65.
GPC Analytics
[13] a) S. Kobayashi, H. Uyama, S. Kimura, Chem. Rev. 2001, 101, 3793–
3818; b) S. Kobayashi, Proc. Jpn. Acad., Ser. B 2010, 86, 338–365.
[14] R. A. Gross, A. Kumar, B. Kalra, Chem. Rev. 2001, 101, 2097–2124.
[15] P. Inprakhon, P. Panlawan, T. Pongtharankul, E. Marie, L. O. Wiemann,
A. Durand, V. Sieber, Colloids and Surfaces B: Biointerfaces 2014, 113,
254–260.
Dried polymer samples were diluted in 997 µL tetrahydrofurane (THF) and
3 µL toluene as an internal flow standard. GPC was performed with THF
and 1 mL/min flow rate and polymers were separated on a GMHHR-L
Mixed Bed column (Viscotek, Houston, TX, USA) in series to a PLgel 3 µm
MIXED-E column (Agilent Technologies, Santa Clara, CA, USA). A
refractive index (RI) detector (K2301, KNAUER Wissenschaftliche Geräte
GmbH, Berlin, Germany) was used for monitoring the course of separation.
[16] A. Mahapatro, A. Kumar, R. A. Gross, Biomacromolecules 2004, 5, 62–
68.
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