10.1002/cbic.201600447
ChemBioChem
FULL PAPER
Each organic solvent (0.75 mL) ethyl acetate (EA), diethyl ether
(DEE), methyl-t-butyl ether (MTBE), 2-isopropyl ether (DIPE), dibutyl
ether (DBE), and hexane (HEX) was mixed with equal volumes of
400 mM citrate-phosphate buffer (0.75 mL; pH 4.0) and equilibrated
with shaking at 1500 rpm for 60 min. Native or purified recombinant
PeHNLs were added to the citrate-phosphate buffer phase, gently
mixed without disturbing the interface between the aqueous and
organic phases. The enzyme activity at time zero were measured
under the satuated solvent in aqueouse phase and then incubated
for 12 h at 30°C with shaking at 1500 rpm. Remaining enzyme
activities were analyzed as described above.
To investigate transcyanation reactions in biphasic systems, organic
phases containing benzaldehyde (250 mM) were mixed with 400 mM
citrate buffer (pH 4.0) containing 2.34 U of each partially purified
PeHNL in a total volume of 1.5 mL in 2.0-mL micro-tubes. Reactions
were initiated by adding acetone cyanohydrin (900 mM), and the
mixture was incubated at 10°C with shaking at 1500 rpm in an
incubator shaker (BioShaker M-BR-022UP, Taitec Corporation,
Tokyo, Japan). Aliquots of sample (50 µL) were collected from the
organic phase at various times and analyzed using HPLC with a
chiral column as described above, and ee were determined
according to relative concentrations (mM) of the two enatiomers
using Eq. (1):
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ee (%) = [푅 −[푆] × 100
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[
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푅 +[푆]
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Acknowledgments
The financial support given to A. Nuylert from Ministry of Education,
Culture, Sport, Science and Technology (MEXT) of Japan is deeply
appreciated. This research was funded in part by a grant-in-aid for
Scientific Research B from The Japan Society for Promotion of
Sciences (No. 26292041) to Y. Asano. We would like to thank Dr.
Atsutoshi Ina, who helped with the protein mass spectrometry
analysis, and Dr. Masashi Nakata, the chairman of the Botanic
Gardens of Toyama: Botanic Gardens of Toyama, Toyama, Japan,
for providing us with leaves of P. edulis used in parts of this study.
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Keywords: Hydroxynitrile lyase • Passiflora edulis • enzyme
catalysis • glycosylation • enzyme stability
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