M. Kotik et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 41–48
47
Fig. 5. Preparative resolution of racemic epoxide 10 using the EH Kau2 at high substrate concentration.
ery. The use of PCR with degenerate consensus primers and genome
walking may help us mine this untapped resource.
Acknowledgments
This work was supported by the Czech Science Foundation
(grant 204/06/0458) and by the Institutional Research Concept No.
AV0Z50200510. We also acknowledge support from the Academy
Fig. 6. Preparative enantioconvergent biohydrolysis of racemic epoxide 11 using
the EH Kau2.
Appendix A. Supplementary data
system with 2.4 g of epoxide 10 and thawed cells, 1.15 g of (1R,2R)-
1
0 with an ee of 99.0%, and 1.22 g of (1R,2S)-10a with an ee of 99.8%
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2010.01.016.
were isolated with yields of 48% and 45%, respectively. These exper-
iments clearly showed that the Kau2 biocatalyst can be applied in
biphasic biotransformation reactions with high epoxide concen-
trations of 80 g/L (600 mM). Three reaction cycles with recycled
Kau2 biocatalyst were also performed, using the same preparative-
scale biotransformation conditions as described above. A decrease
of 30% in enzymatic activity was observed for the second cycle. Con-
sequently, a longer reaction time of 95 min was needed to obtain
ee-values of ≥99.0% for (1R,2R)-10 and (1R,2S)-10a. The enzymatic
activity in the third cycle was determined to be 45% of the original
activity, and the reaction time increased to 160 min. Future experi-
ments will show if a better operational stability for repeated batch
reactions may be achieved through enzyme immobilization. Using
the same biocatalyst, a preparative-scale enantioconvergent pro-
cess was carried out with 1.0 g of substrate 11 using an epoxide
concentration of 13 g/L (97 mM) in the reaction mixture (Fig. 6).
The biotransformation afforded an excellent yield (1.1 g, 97%) of
the diol (1R,2R)-11a in nearly enantiopure form (ee 98.3%). The
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