Hydroxynitrile Lyase from Arabidopsis thaliana: Identification of Reaction Parameters
AtHNL and considering the enzyme concentration in the re-
spective immobilisate.
Example: When the same amount of immobilized enzyme
achieved 50% substrate conversion within 1/5 of time, rela-
tive to the precipitated enzyme, its relative activity is 500%.
temperature), before they were transferred into a dense
closable screw cap vial and stored at 48C. The activity was
finally assayed by following the hydrocyanation of 1a under
identical reaction conditions. All reactions were monitored
by chiral GC. Relative activities were calculated by compar-
ing the substrate conversion at a defined reaction time
(30 min). Water bound to celite-AtHNL was measured by
thermogravimetry using a Perkin–Elmer TGA7 thermogra-
vimetric analyzer. The measurements were performed under
nitrogen atmosphere in the range of 25–6258C at a heating
rate of 108CminÀ1. The initial sample mass was always in
the range 4–12 mg.
Recycling of Celite-AtHNL or Solgel-AtHNL
Celite or solgel containing 3–5 mg AtHNL was sealed into
an organic solvent-resistant, fine-woven nylon mesh “tea-
bag” [nylon net, pore size 0.4 mm; sealing unit: Polystar 100
GE-GS (Rische + Herfurth, Germany)] for easy removal
from the reaction medium by filtration. Five consecutive hy-
drocyanation reactions (substrate: 1a) were performed over
60 min with the celite-AtHNL “tea-bag” and the solgel-
AtHNL “tea-bag”, respectively, as described for immobi-
lized AtHNL. The “tea-bags” were washed between each re-
action cycle with pure MTBE or potassium phosphate
buffer (50 mM, pH 6.0) to remove remaining product and
refresh the immobilisate. Chiral GC was used to monitor
the reactions.
Acknowledgements
D.O. thanks Roger A. Sheldon for establishing contact with
the TU Delft – Biocatalysis & Organic Chemistry Group.
This work was financial supported by the Deutsche For-
schungsgemeinschaft in frame of the research training group
“BioNoCo” GK 1166 and NWO/ACTS (project no.
053.63.304). M.P. thanks Dr. Menno Sorgedrager for fruitful
discussion and the IBOS program of NWO/ACTS (project
no. 053.63.304) for financial support.
Investigation of Potential Leakage of Active AtHNL
from Immobilisates
Two identical celite-AtHNL or solgel-AtHNL samples,
shrink-warped in a nylon mesh “tea-bag”, were prepared.
Each sample was used for addition of HCN to benzaldehyde
1a as described in the synthesis protocol for immobilized
AtHNL. In one reaction the “tea-bag” containing the immo-
bilized AtHNL was inside the reaction medium for the
whole reaction time while it was removed from the reaction
medium after 8 min in the parallel reaction. Both samples
were monitored over 60 min by chiral GC. After removal of
the “tea-bag” the reaction should be aborted, when no
active enzyme leaks into reaction medium.
References
[1] a) M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M.
Kesseler, R. Stꢀrmer, T. Zelinski, Angew. Chem. 2004,
116, 806–843; Angew. Chem. Int. Ed. 2004, 43, 788–824;
b) R. Gregory, Chem. Rev. 1999, 99, 3649–3682; c) J.
Wang, W. Wang, W. Li, X. Hu, K. Shen, C. Tan, X. Liu,
X. Feng, Chem. Eur. J. 2009, 15, 11642–11659; d) M.
North, D. Usanov, C. Young, Chem. Rev. 2008, 108,
5146–5226.
[2] a) J. Holt, U. Hanefeld, Curr. Org. Synth. 2009, 6, 15–
37; b) L. Rosenthaler, Biochem. Z. 1908, 14, 238–253.
[3] a) J. N. Andexer, J. V. Langermann, U. Kragl, M. Pohl,
Trends Biotechnol. 2009, 27, 599–607; b) R. Hatti-Kaul,
U. Tçrnvall, L. Gustafsson, P. Bçrjesson, Trends Bio-
technol. 2007, 25, 119–124; c) D. Pollard, J. Woodley,
Trends Biotechnol. 2007, 25, 66–73.
[4] a) J. Andexer, J. von Langermann, A. Mell, M. Bocola,
U. Kragl, T. Eggert, M. Pohl, Angew. Chem. 2007, 119,
8833–8835; Angew. Chem. Int. Ed. 2007, 46, 8679–8681;
b) P. Carr, D. Ollis, Protein Pept. Lett. 2009, 16, 1137–
1148.
[5] J. Guterl, J. Andexer, T. Sehl, J. von Langermann, I.
Frindi-Wosch, T. Rosenkranz, J. Fitter, K. Gruber, U.
Kragl, T. Eggert, J. Biotechnol. 2009, 141, 166–173.
[6] J. von Langermann, J. Guterl, M. Pohl, H. Wajant, U.
Kragl, Bioprocess Biosyst. Eng. 2008, 31, 155–161.
[7] a) G. Lin, S. Han, Z. Li, Tetrahedron 1999, 55, 3531–
3540; b) E. Wehtje, P. Adlercreutz, B. Mattiasson, Bio-
technol. Bioeng. 1990, 36, 39–46.
[8] a) D. Costes, E. Wehtje, P. Adlercreutz, Enzyme
Microb. Technol. 1999, 25, 384–391; b) M. Paravidino,
M. Sorgedrager, R. Orru, U. Hanefeld, Chem. Eur. J.
2010, 16, 7596–7604.
Investigation of Storability of Celite-AtHNL and
Solgel-AtHNL
One batch containing an adequate amount of celite- or
solgel-immobilized AtHNL was prepared as described in the
standard protocols and transferred into a dense closable
screw cap vial. Over a period of 31 days the vial was stored
at 48C. From time to time equal amounts of immobilized
AtHNL were taken from the batch and residual activity was
assayed by measuring the hydrocyanation of 1a under iden-
tical reaction conditions. All reactions were monitored by
chiral GC. Relative activity was calculated by comparing the
substrate conversion at a defined reaction time (celite-
AtHNL: 25 min; solgel-AtHNL: 5 min).
Investigation of the Influence of Water Concentration
(w/w) on the Activity of Celite-AtHNL
One adequate batch of celite-AtHNL was prepared as de-
scribed above. The batch was split into 5 aliquots (A–E).
Sample A was directly transferred into a dense closable
screw cap vial and stored at 48C. Samples B–E were trans-
ferred into four desiccators and incubated over a saturated
salt solution of MgACHTNUTRGNE(UGN NO3)2 aw =0.54, MgCl2 aw =0.33, NaCl
aw =0.75 or K2SO4 aw =0.95 to adjust a specific humidity
[9] D. Costes, G. Rotcenkovs, E. Wehtje, P. Adlercreutz,
(water activity aw) in each desiccator for at least 16 h (room
Biocatal. Biotransform. 2001, 19, 119–130.
Adv. Synth. Catal. 2011, 353, 2399 – 2408
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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