196
N. Li et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 190–197
previous study [23], the enzymatic reaction showed higher product
optical purity than that of the whole cell bioreduction (99.7% e.e. at
2.5 g/l of OPBE; 87.5% e.e. at 20 g/l of OPBE).
Further investigations on the exploitation of this enzyme along
with construction of recombinant strain for desired preparative
scale reactions are in progress in our laboratory.
4. Conclusions
A highly active carbonyl reductase from a yeast strain C. kru-
sei SW 2026 has been purified for up to 304-fold with a yield of
5.9%. The carbonyl reductase of C. krusei SW 2026 catalyzed the
reduction of OPBE to (R)-HPBE in high enantioselectivity of more
than 99.9% e.e. This is so far, to our knowledge, the only carbonyl
reductase with almost absolute enantioselective in the reduction of
OPBE. The enzyme exhibited optimal pH and temperature at 6.0 and
30 ◦C, and was relatively stable over an acidic pH range of 4.5–7.0
and temperature range of 10–40 ◦C, respectively. The process can
further be improved by cloning and coexpression of carbonyl reduc-
tase of C. krusei and cofactor regeneration system in a heterologous
host, which could potentially be used in the biocatalytic reaction to
produce the important intermediate of ACE inhibitors, (R)-HPBE.
Fig. 6. The time courses of the enzymatic reduction of OPBE in aqueous medium.
(ꢁ) Yield. (ꢀ) e.e. value. Reaction conditions: 10 mM OPBE, 10 mM NADPH, and
approximate 0.2 U of purified enzyme in phosphate buffer (100 mM, pH 6.0) at 30 ◦C
(200 rpm) for 6 h.
The enzymatic transformation of hydrophobic compounds in
aqueous solution is generally limited by its low solubility. The puri-
fied carbonyl reductase of C. krusei could catalyze the reduction of
hydrophobic OPBE in aqueous medium with good efficiency. The
tolerance of carbonyl reductase against different organic solvents,
such as dimethyl sulfoxide, dimethyl formamide, isopropanol,
tetrahydrofuran, ethanol, and pyridine, was also investigated (data
not shown). It was observed that, in the presence of organic sol-
observed with ethanol, exhibiting residual enzyme activity of
approximately 77.9%. Similar operational instability in organic sol-
vent was reported for carbonyl reductases from C. parapsilosis DSM
70125 [38], R. erythropolis DSM 743 [39], C. viswanathii MTCC 5158
[34], R. ruber DSM 44541 [33] and Rhodotorula sp. AS 2.2241 [44].
According to our result, heterocyclic organic solvents, pyridine
for example, significantly affected the enzyme activity, in which
only 15.0% residual activity was observed. It is hypothesized that
pyridine might have competitive inhibition effect on the enzyme
activity due to the structural similarity between heterocycle of pyri-
dine and benzene ring of OPBE.
Acknowledgments
We are grateful to the National Natural Science Foundation of
China (30900030), Research Fund for the Doctoral & Youth Schol-
ars Program of Higher Education of China (20090093120008), the
National High Technology Research and Development Program of
China (2007AA02Z226), for the financial support of this research.
Supplementary data associated with this article can be found, in
References
[1] H. Fujita, K. Yokoyama, M. Yoshikawa, J. Food Sci. 65 (2000) 564–569.
[2] M.S. Weinberg, A.J. Weinberg, D.H. Zappe, J. Renin-Angio-Aldo. S. (2000)
217–233.
[3] G. Iwasaki, R. Kimura, N. Numao, K. Kondo, Chem. Pharm. Bull. 37 (1989)
280–283.
Different detergents like Tween 80, Triton X-100 and SDS were
also tested for their effect on carbonyl reductase activity. All these
detergents had deleterious effect on the enzyme activity, especially
SDS (data not shown).
Taken together, our results indicate that certain functional
structures, such as intact thiol group, disulfide linkage, imidazole
group of histidine, and substrate/cofactor binding site, have impor-
tant roles in the catalytic activity of carbonyl reductase of C. krusei.
This study also demonstrates that the activity of the enzyme was
strongly inhibited in the presence of some heavy metal ions, strong
metal ion chelating agent, heterocyclic organic solvents, and pro-
tein denaturant.
[4] D.L. Coffen, P. Kataritis, J.J. Patridge, European Patent 0325971 (1989).
[5] A.G. Flynn, European Patent 0280285 (1992).
[6] X.-H. Li, C. Li, Catal. Lett. 77 (2001) 251–254.
[7] P. Herold, A.F. Indolese, M. Studer, H.P. Jalett, U. Siegrist, H.U. Blaser, Tetrehe-
dron 56 (2000) 6497–6499.
[8] Y. Inada, S. Oda, Japanese Patent 10304894 (l999).
[9] A. Liese, U. Kragl, H. Kierkels, B. Schulze, Enzyme Microb. Technol. 30 (2002)
673–681.
[10] S.-H. Huang, S.-W. Tsai, J. Mol. Catal. B: Enzym. 28 (2004) 65–69.
[11] Y.-L. Bai, S.-T. Yang, Biotechnol. Bioeng. 92 (2005) 137–146.
[12] I. Kaluzna, A.-A. Andrew, M. Bonilla, M.R. Martzen, J.D. Stewart, J. Mol. Catal. B:
Enzym. 17 (2002) 101–105.
[13] P.S.B. de Lacerda, J.B. Ribeiro, S.G.F. Leite, M.A. Ferrara, R.B. Coelho, E.P.S. Bon, E.L.
da Silva Lima, O.A.C. Antunes, Tetrahedron: Asymmetry 17 (2006) 1186–1188.
[14] Y.-Z. Chen, H. Lin, X.-Y. Xu, S.-W. Xia, L.-X. Wang, Adv. Synth. Catal. 350 (2008)
426–430.
[15] M.K.S. Vink, R. Weis, S. Kambourakis, I.A. Kaluzna, R. Keledjian, J.D. Rozzell,
Tetrahedron: Asymmetry 16 (2005) 3682–3689.
[16] I. Kira, N. Onishi, J. Biosci. Bioeng. 107 (2009) 116–118.
[17] R.N. Patel, Stereoselective Biocatalysis, Marcel Decker, New York, 2000, p. 362.
[18] K. Nakamura, R. Yamanaka, T. Matsuda, T. Haradab, Tetrahedron: Asymmetry
14 (2003) 2659–2681.
[19] H.E. Schoemaker, D. Mink, M.G. Wubbolts, Science 299 (2003) 1694–1697.
[20] J. He, Z. Sun, W. Ruan, Y. Xu, Process Biochem. 41 (2006) 244–249.
[21] J. He, X. Mao, Z. Sun, P. Zheng, Y. Ni, Y. Xu, Biotechnol. J. 2 (2007) 260–265.
[22] F. Zhang, Y. Ni, Z. Sun, P. Zheng, W. Lin, P. Zhu, N. Ju, Chin. J. Catal. 29 (2008)
577–582.
3.2.6. Time course of enzymatic reduction of OPBE to (R)-HPBE
Optical pure (R)-HPBE was used as an important intermediate
for the synthesis of several angiotensin-converting enzyme (ACE)
was carried out to confirm the catalytic activity of this enzyme
(Fig. 6). As expected, the purified enzyme produced (R)-HPBE in
an enantiomeric excess of more than 99.9% as determined by chi-
ral GC (supplementary Fig. 2). After 4 h of reaction, approximately
84.0% yield and nearly 100% e.e. were achieved. Compared with our
[23] W. Zhang, Y. Ni, Z.-H. Sun, P. Zheng, W.-Q. Lin, P. Zhu, N.-F. Ju, Process Biochem.
44 (2009) 1270–1275.
[24] M. Bradford, Anal. Biochem. 72 (1976) 248–254.