Acidic proteinase from Aspergillus usamii catalyzed Michael addition of ketones to nitroolefins

Article abstract of DOI:10.1016/j.molcatb.2011.04.005

A new promiscuous activity of AUAP (acidic proteinase from Aspergillus usamii) was investigated in Michael addition of cyclic and acyclic ketones to aromatic and heteroaromatic nitroolefins in organic media in the presence of water. The yields were obtain

Full text of DOI:10.1016/j.molcatb.2011.04.005

Journal of Molecular Catalysis B: Enzymatic 71 (2011) 108–112
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Acidic proteinase from Aspergillus usamii catalyzed Michael addition of ketones
to nitroolefins
Ke-Ling Xu, Zhi Guan, Yan-Hong He∗
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new promiscuous activity of AUAP (acidic proteinase from Aspergillus usamii) was investigated in
Michael addition of cyclic and acyclic ketones to aromatic and heteroaromatic nitroolefins in organic
media in the presence of water. The yields were obtained from 38% to 84%.
© 2011 Elsevier B.V. All rights reserved.
Received 14 December 2010
Received in revised form 31 March 2011
Accepted 5 April 2011
Available online 13 April 2011
Keywords:
Biocatalyst
Michael addition
Acidic proteinase
Ketone
Nitroolefin
1. Introduction
acylase from Escherichia coli displayed a promiscuous activity to
catalyze the C–C bond formation reaction of 1,3-dicarbonyl com-
Biocatalysis as an efficient and green biotransformation tool in
organic synthesis has attracted much attention of chemists and bio-
chemists in recent years [1–3]. Especially, catalytic promiscuity in
biocatalysis which means using old enzymes to form new bonds
and follow new pathways, was greatly extended and expanded
rapidly [4–7]. On the other hand, the Michael addition is a pow-
erful, useful and atom economical reaction to form carbon–carbon
and carbon–heteroatom bonds [8–12]. In recent years, some ele-
gant works on enzyme catalyzed Michael-type reactions have been
reported. Some lipases and proteases have been applied in the
Michael-type addition to form the carbon-hetero bonds [13–29].
A few examples of enzyme-catalyzed C–C bond formations via
Michael addition have also been reported. For instance, Berglund
and co-workers gave the first example of C–C bond formation
by lipase-catalyzed Michael addition between 1,3-dicarbonyls and
␣,␤-unsaturated carbonyl compounds. They used lipase B from
Candida Antarctica as a scaffold and increased its reaction speci-
ficity for Michael additions by the substitution of one amino acid
(Ser105Ala) in the active site through rational design [8]. Recently
they found that the CAL-B Ser105Ala variant was able to cat-
alyze the Michael addition between methyl acrylate and acetyl
acetone in aqueous media instead of the native hydrolytic pro-
cess [30]. Lin and co-workers discovered that a zinc-dependent
pounds to methyl vinyl ketone in organic media [31,32]. Griengl
and co-workers investigated lipase-catalyzed Michael addition
with respect to the synthetic potential and stereochemistry of
the products [33]. Very recently, we reported the immobilized
lipase from Thermomyces lanuginosus catalyzed asymmetric C–C
Michael addition for a wide range of 1,3-dicarbonyl compounds and
cyclohexanone to aromatic, heteroaromatic nitroolefins and cyclo-
hexenone, and the enantioselectivities up to 83% ee were obtained
[34]. However, to the best of our knowledge, proteinase catalyzed
C–C bond formation via Michael addition has not been reported
yet. Herein, we wish to report the promiscuous activity of AUAP
(acidic proteinase from Aspergillus usamii No 537) in Michael addi-
tion of ketones to nitroolefins in organic media in the presence of
water.
2. Experimental
2.1. Materials
Acidic protease from A. usamii No 537 (50 U/mg), alkaline
protease from Bacillus licheniformis No 2709 (200 U/mg), neu-
tral protease from Bacillus subtilis A.S.1.398 (130 U/mg) and
trypsin from porcine pancreas (4 U/mg) were purchased from
Xuemei Enzyme Co. Ltd. (Wuxi, China). Pancreatin from porcine
pancreas (4 U/mg), papain from papaya latex (650 U/mg), nucle-
ase from Penicillium citrinum (5 U/mg), lysozyme from hen
egg white (20,000 U/mg), bromelain from pineapple peduncle
∗
Corresponding author. Fax: +86 23 68254091.
E-mail address: heyh@swu.edu.cn (Y.-H. He).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.04.005

K.-L. Xu et al. / Journal of Molecular Catalysis B: Enzymatic 71 (2011) 108–112
109
Table 1
70
The catalytic activities of different enzymes in Michael reactiona.
65
60
55
50
45
40
35
30
O
O
NO2
enzyme, 25°C
+
NO2
DMSO/H2O, 168 h
.
Entry Enzyme
Yield (%)b
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
1
2
3
4
5
6
AUAP (acidic proteinase from Aspergillus usamii No 537) 48
Water content (water/DMSO,v/v)
Trypsin from porcine pancreas
Pancreatin from porcine pancreas
Alkaline proteinase from Bacillus licheniformis No 2709
Bromelain from pineapple peduncle
Cellulase from Trichoderma
26
11
8
4
Fig. 2. Influence of water content in organic solvent on the yield of AUAP-catalyzed
Michael reaction. Conditions: AUAP (200 mg), cyclohexanone (10.0 mmol), trans-␤-
nitrostyrene (1.0 mmol), and DMSO (4 mL) at 30 ◦C for 144 h. Deionized water was
added from 0 to 0.35 (water/DMSO, v/v).
3
7
8
9
10
11
12
13
Nuclease from Penicillium citrinum
Papain from papaya latex
5
Trace
Trace
Trace
No reaction
Trace
Table 2
Neutral proteinase from Bacillus subtilis A.S.1.398
Lysozyme from hen egg white
None
Influence of temperature on the yield of the AUAP-catalyzed Michael additiona.
Entry
Temperature (◦C)
Time (h)
Yield (%)b
AUAP denatured with SDSc
AUAP inhibited with NBSd
Trace
1
2
3
4
5
6
30
35
40
45
50
55
120
120
120
120
120
120
60
63
68
75
69
65
a
All the reactions were carried out using trans-␤-nitrostyrene (1.0 mmol),
enzyme (200 mg), cyclohexanone (10.0 mmol), DMSO (5 mL) and deionized water
(0.75 mL) at 25 ◦C for 168 h.
b
Yield of the isolated product after chromatography on silica gel.
c
Pre-treated with SDS at 100 ◦C in DMSO (5 mL) in the presence of deionized
a
All reactions were carried out using trans-␤-nitrostyrene (1.0 mmol), cyclohex-
anone (10.0 mmol), AUAP (200 mg), deionized water (0.60 mL), DMSO (4 mL) at
specified temperature.
water (0.75 mL) for 24 h.
d
Pre-treated with NBS at 25 ◦C in DMSO (5 mL) in the presence of deionized water
(0.75 mL) for 24 h.
b
Yield of the isolated product after chromatography on silica gel.
2.3. Typical experimental procedure for enzyme-catalyzed
Michael reactions.
A mixture of nitroolefin (1.0 mmol), AUAP (200 mg) and ketone
(20 mmol) in DMSO (4 mL) and deionized water (0.60 mL) was
stirred for the specified time at 45 ◦C. The reaction was terminated
by filtration to remove the enzyme. CH2Cl2 was used to wash the fil-
ter paper to assure that products obtained were all dissolved in the
filtrate. Then the filtrate was washed three times with water. The
organic phase was dried over anhydrous Na2SO4, and the solvents
were removed under reduced pressure. The crude products were
purified by column chromatography with petroleum ether/ethyl
acetate as eluent.
Fig. 1. Effects of solvent on AUAP-catalyzed Michael addition of cyclohexanone and
trans-␤-nitrostyrene. Conditions: trans-␤-nitrostyrene (1.0 mmol), AUAP (200 mg),
cyclohexanone (10.0 mmol), deionized water (0.60 mL), solvent (4 mL) at 30 ◦C for
144 h.
3. Results and discussion
In our initial investigation, in order to ascertain the appropriate
enzyme for Michael addition of ketones to nitroolefins, the Michael
addition of cyclohexanone with trans-␤-nitrostyrene was used as
a model reaction, and 10 enzymes were screened. The results were
shown in Table 1. The best result of 48% yield was achieved by
using AUAP as a catalyst after 168 h (Table 1, entry 1). Trypsin,
pancreatin and alkaline proteinase also showed catalytic activities
which gave the product in yields of 26%, 11% and 8%, respectively
(Table 1, entries 2–4). Bromelain, cellulase and nuclease presented
low activities in this reaction (Table 1, entries 5–7). Moreover,
in the presence of papain, neutral proteinase or lysozyme only
trace product was observed on TLC (Table 1, entries 8–10). Just
as we expected, the Michael addition of cyclohexanone to trans-
␤-nitrostyrene in the absence of enzyme showed no adduct after
168 h (Table 1, entry 11). Besides, in order to prove this reaction
was catalyzed by APAU instead of the amino acids on the surface of
the enzyme, the experiments using denatured enzyme and adding
the inhibitor have been carried out. When the reactants were incu-
bated with SDS-denatured AUAP, only trace adduct was observed
(Table 1, entry 12). It suggested that the tertiary structure of AUAP
(500 U/mg) and cellulase from Trichoderma (10 U/mg) were pur-
chased from Pangbo Biological Engineering Co. Ltd. (Nanning,
China). Unless otherwise noted, all reagents were obtained
from commercial suppliers and were used without further
purification.
2.2. Analytical methods
All reactions were monitored by thin-layer chromatography
(TLC) with Haiyang GF254 silica gel plates. Flash column chro-
matography was carried out using 100–200 mesh silica gel at
increased pressure. The 1H NMR spectra were recorded with
TMS as internal standard on
a Bruker AMX-300 MHz spec-
trometer. Chemical shifts were expressed in ppm and coupling
constants (J) in Hz. All the known products were character-
ized by comparing the 1H NMR with those reported in the
literature.

110
K.-L. Xu et al. / Journal of Molecular Catalysis B: Enzymatic 71 (2011) 108–112
Table 3
Investigation of substrate scope in AUAP-catalyzed Michael additiona.
O
O
R
AUAP
DMSO/H2O, 45oC
NO2
NO2
+
R
.
Entry
1
Donor
Acceptor
Time (h)
120
Yield (%)b
80
dr (syn/anti)c
O
NO2
94:6 (93:7)d
O
O
NO2
2
3
120
120
79
78
80:20 (85:15)d
77:23 (85:15)d
NO2
OCH3
NO2
O
4
96
77
77:23
OCH3
O
O
NO2
5
6
132
108
80
81
81:19 (82:18)d
H3CO
NO2
NO2
94:6
Cl
O
7
120
78
92:8
Cl
O
O
O
O
NO2
8
72
72
72
48
80
78
77
79
92:8 (91:9)d
Cl
NO2
NO2
NO2
9
96:4 (89:11)d
93:7 (90:10)d
71:29 (80:20)d
Br
10
11
Cl
Cl
NC
NO2
O
O
12
13
72
77
69
79:21
87:13
NO2
NO2
O
120

K.-L. Xu et al. / Journal of Molecular Catalysis B: Enzymatic 71 (2011) 108–112
111
Table 3 (Continued)
Entry
Donor
Acceptor
Time (h)
96
Yield (%)b
84
dr (syn/anti)c
O
NO2
14
15
80:20 (81:19)d
NO2
NO2
O
O
108
108
47
38
–
16
85:15
a
All the reactions were carried out using nitroolefin (1.0 mmol), AUAP (200 mg), ketone (20.0 mmol), deionized water (0.60 mL) and DMSO (4 mL) at 45 ◦C.
Yield of the isolated product after chromatography on silica gel.
b
c
Determined by 1H NMR spectroscopy.
d
Numbers in parenthesis refer to the dr (syn/anti) obtained by chemocatalytic procedure using piperidine as a catalyst.
was essential in the process. Moreover, according to the literature
The temperature is another important factor for the enzyme-
catalyzed reactions, due to their effects on the enzyme stability
and reaction rate. We further studied the temperature effect on the
AUAP-catalyzed Michael addition using cyclohexanone and trans-
␤-nitrostyrene as a model reaction (Table 2). It was found that the
yield of the enzymatic reaction could be increased by raising the
temperature, and the highest yield of 75% was reached at 45 ◦C.
However, when the temperature was higher than 45 ◦C, the yield
of product decreased, probably due to the gradual denaturation of
the enzyme. Thus, the optimum temperature for the reaction was
45 ◦C.
Next, to further optimize the experimental conditions, we then
examined the effect of cyclohexanone stoichiometry on the reac-
tion. The best result was achieved by using 20 equivalents of
cyclohexanone. Thus, we chose 20 equivalents of cyclohexanone as
the optimum molar equivalent for the Michael addition (for details
see the Supplementary Information).
Finally, in order to explore the scopes and limitations of the
method, the reactions of cyclohexanone, cyclopentanone, acetone
and butan-2-one with different aromatic and heteroaromatic
nitroalkenes were carried out under optimal conditions (Table 3).
It can be seen that the AUAP-catalyzed Michael addition of cyclo-
hexanone with aromatic nitroalkenes gave the desired products
in good yields (77–81%) (Table 3, entries 1–12). The electron-
withdrawing groups on aromatic nitroalkenes could enhance the
reactivity of the substrates. For example, 4-cyano-␤-nitrostyrene
reacted with cyclohexanone to give the corresponding product
in satisfied yield after only 48 h (Table 3, entry 11). 4-Chloro,
4-bromo, 2,4-dichloro and 3-nitro substituted ␤-nitrostyrenes
gave good yields after 72 h (Table 3, entries 8–10, 12). On the
contrary, when there were electron-donating groups on the
aromatic nitroalkenes, the reactions were slow, and relatively
longer reaction times were required to reach the satisfied yields
(Table 3, entries 2–5). For instance, 4-methoxy-␤-nitrostyrene
reacted with cyclohexanone to give the product in yield of 80%
after 132 h. (Table 3, entry 5). Moreover, the largest electronic
effect was found for substitutents in the para position for both
electron-withdrawing and electron-donating groups. Besides,
the AUAP-catalyzed Michael reaction between heteroaromatic
nitroalkene and cyclohexanone was also performed, which gave
the corresponding product in moderate yield (Table 3, entry 13).
Furthermore, the enzymatic Michael addition of cyclopentanone
with trans-␤-nitrostyrene could obtain a good yield of 84% after
96 h (Table 3, entry 14). In addition, acyclic ketones as Michael
donors were also accepted by the enzyme. The reaction of ace-
tone with trans-␤-nitrostyrene gave a low yield of 47% (Table 3,
entry 15). Also, the reaction between butan-2-one and trans-␤-
[35] NBS (N-bromosuccinimide) is an inhibitor of AUAP. Thus, the
control experiment with NBS-inhibited AUAP was performed, and
only trace product was observed (Table 1, entry 13). The experi-
ment suggested that the Michael addition was catalyzed by APAU
instead of the amino acids on the surface of the enzyme. Thus, we
confirmed that AUAP catalyzed the Michael addition.
Next, the volume of solvent directly related to the concentration
of enzyme and substrates, and could influence the reaction yield. So
we examined the effect of volume of solvent on the AUAP-catalyzed
Michael addition. It was found that the best yield could be reached
at 4 mL of DMSO (with 0.60 mL H2O). Thus, we chose 4 mL solvent
(with 0.60 mL H2O) as the optimum solvent volume for the follow-
ing study of AUAP-catalyzed Michael addition (for details see the
Supplementary Information).
Solvents often play an important role in enzymatic reactions.
Enzymes not only work in anhydrous organic media, but they
acquire remarkable properties such as enhanced stability, altered
substrate and enantiomeric specificities, and the ability to catalyze
unusual reactions which are impossible in aqueous media [36–39].
Thus, a solvent screen was performed using cyclohexanone and
trans-␤-nitrostyrene as a model reaction. Some representative
organic solvents were screened and the results were shown in Fig. 1.
AUAP showed the good Michael addition activity in DMSO, and the
best yield of 63% was obtained at 30 ◦C for 144 h. The reaction in
DMF gave a yield of 37%, while both THF and ethanol provided the
yields less than 20%. In the other tested solvents, including DCM,
TBME (tert-butyl methyl ether), cyclohexane and H2O, the activ-
ity of reaction was very poor and the yields were less than 10%.
Hence, DMSO was chosen as the optimum solvent for the enzymatic
Michael addition.
At the same time, enzymes require a specific amount of water
bound to them to maintain activities, and water concentration
affects the activity of enzymes [40–43]. Therefore, it is impor-
tant to confirm the optimal water content in the reaction system.
We analyzed the effects of water content on the AUAP-catalyzed
Michael addition of cyclohexanone and trans-␤-nitrostyrene in
DMSO (Fig. 2). It was found that the yield of the enzymatic reac-
tion could be raised by increasing the concentration of water,
and the highest yield could be reached at water/DMSO = 0.15
(v/v). However, once the water content surpassed 0.15, the yield
decreased evidently. Thus, the optimum water content for the reac-
tion was 0.15 (water/DMSO, v/v), which was in coincidence with the
amounts of water addition we used in the experiments of enzyme
and solvent screen. The results indicated that water is obviously
essential for the enzyme-catalyzed Michael addition in organic
medium.

112
K.-L. Xu et al. / Journal of Molecular Catalysis B: Enzymatic 71 (2011) 108–112
nitrostyrene only gave 3-methyl-5-nitro-4-phenylpentan-2-one
in yield of 38% after 108 h (Table 3, entry 16), and a small amount
of 6-nitro-5-phenylhexan-3-one was obtained as well.
[4] U.T. Bornscheuer, R.J. Kazlauskas, Angew. Chem. Int. Ed. 43 (2004) 6032–6040.
[5] A. Ghanem, Tetrahedron 63 (2007) 1721–1754.
[6] V. Gotor-Fernandez, E. Busto, V. Gotor, Adv. Synth. Catal. 348 (2006) 797–812.
[7] K. Li, T. He, C. Li, X.W. Feng, N. Wang, X.Q. Yu, Green Chem. 11 (2009) 777–779.
[8] M. Svedendahl, K. Hult, P. Berglund, J. Am. Chem. Soc. 127 (2005) 17988–17989.
[9] H.X. Dai, S.P. Yao, J. Wang, Biotechnol. Lett. 28 (2006) 1503–1507.
[10] P.F. Li, S.G. Wen, F. Yu, Q.X. Liu, W.J. Li, Y.C. Wang, X.M. Liang, J.X. Ye, Org. Lett.
11 (2009) 753–756.
[11] B. Tan, X.F. Zeng, Y.P. Lu, P.J. Chua, G.F. Zhong, Org. Lett. 11 (2009) 1927–1930.
[12] A.D. Lu, P. Gao, Y. Wu, Y.M. Wang, Z.H. Zhou, C.C. Tang, Org. Biomol. Chem. 7
(2009) 3141–3147.
[13] T. Kitazume, T. Ikeya, K. Murata, J. Chem. Soc. Chem. Commun. 17 (1986)
1331–1333.
[14] T. Kitazume, K. Murata, Y. Kokusho, S. Iwasaki, J. Fluorine Chem. 39 (1988)
75–86.
[15] J. Kjellberg, N.G. Johansson, Nucleosides Nucleotides 8 (1989) 225–256.
[16] T. Kitazume, K. Murata, J. Fluorine Chem. 36 (1987) 339–349.
[17] J.M. Xu, S.P. Yao, W.B. Wu, D.S. Lv, X.F. Lin, J. Mol. Catal. B: Enzym. 35 (2005)
122–127.
[18] O. Torre, I. Alfonso, V. Gotor, Chem. Commun. 15 (2004) 1724–1725.
[19] P. Carlqvist, M. Svedendahl, C. Branneby, K. Hult, T. Brinck, P. Berglund, Chem-
BioChem 6 (2005) 331–336.
[20] Y. Cai, S.P. Yao, Q. Wu, X.F. Lin, Biotechnol. Lett. 26 (2004) 525–528.
[21] J. Priego, C. Ortiz-Nava, M. Carrillo-Morales, A. Lopez-Munguia, J. Escalante, E.
Castillo, Tetrahedron 65 (2009) 536–539.
It was also worthy to note that this enzymatic Michael addi-
tion had a moderate to excellent diastereoselectivity (syn/anti
71:29–96:4) in favor of the syn-isomers. In order to verify whether
the diastereoselectivity observed was contributed by the enzyme
or just the result of thermodynanmic control, chemocatalytic
Michael additions were also conducted using piperidine as a
catalyst. The similar results of diastereoselectivity were obtained
from the chemocatalytic procedure, which indicated that the
diastereoselectivity observed was the result of thermodynanmic
control (Table 3, entries 1–3, 5, 8–11 and 14). Finally, we examined
the ee value of the products by chiral HPLC, but to our disappoint-
ment, no enantioselectivity could be observed. Similar results
were reported for the enzymatic Michael addition catalyzed by
other enzymes [19,21]. The reason will be further investigated.
4. Conclusion
[22] Y. Cai, X.F. Sun, N. Wang, X.F. Lin, Synthesis 5 (2004) 671–674.
[23] S.P. Yao, D.S. Lv, Q. Wu, Y. Cai, S.H. Xu, X.F. Lin, Chem. Commun. 17 (2004)
2006–2007.
[24] O. Torre, V. Gotor-Fernanderz, I. Alfonso, L.F. Garicia-Alles, V. Gotor, Adv. Synth.
Catal. 347 (2005) 1007–1014.
[25] Y. Cai, Q. Wu, Y.M. Xiao, D.S. Lv, X.F. Lin, J. Biotechnol. 121 (2006) 330–337.
[26] C. Qian, J.M. Xu, Q. Wu, D.S. Lv, X.F. Lin, Tetrahedron Lett. 48 (2007) 6100–6104.
[27] J.L. Wang, J.M. Xu, Q. Wu, D.S. Lv, X.F. Lin, Tetrahedron 65 (2009) 2531–2536.
[28] Q. Wu, J.M. Xu, L. Xia, J.L. Wang, X.F. Lin, Adv. Synth. Catal. 351 (2009)
1833–1841.
[29] S.C. Wang, W.H. Johnson, C.P. Whitman, J. Am. Chem. Soc. 125 (2003)
14282–14283.
[30] M. Svedendahl, B. Jovanovic, L. Fransson, P. Berglund, ChemCatChem 1 (2009)
252–258.
[31] J.M. Xu, F. Zhang, Q. Wu, Q.Y. Zhang, X.F. Lin, J. Mol. Catal. B: Enzym. 49 (2007)
50–54.
[32] J.M. Xu, F. Zhang, B.K. Liu, Q. Wu, X.F. Lin, Chem. Commun. 20 (2007)
2078–2080.
In conclusion, we describe here the readily available AUAP
catalyzed Michael addition of ketones to nitroolefins in DMSO in
the presence of water. The reaction conditions including organic
solvents, solvent volume, water content, temperature, substrate
stoichiometry were optimized. The scope of the reaction was tested
by varying the nitroalkenes and ketones. For most of aromatic and
heteroaromatic nitroalkenes, satisfied yields were obtained. This
acidic proteinase catalyzed Michael reaction provides a novel case
of catalytic promiscuity and might be a useful synthetic method
for application.
Acknowledgement
[33] G.A. Strohmeier, T. Sovic, G. Steinkellner, F.S. Hartner, A. Andryushkova,
T. Purkarthofer, A. Glieder, K. Gruber, H. Griengl, Tetrahedron 65 (2009)
5663–5668.
Financial support from Natural Science Foundation Project of CQ
CSTC (2009BA5051) is gratefully acknowledged.
[34] J.F. Cai, Z. Guan, Y.H. He, J. Mol. Catal. B: Enzym. 68 (2011) 240–244.
[35] G.L. Guan, W.C. Dong, J.Y. Chen, H.X. Pan, A.Q. Kong, B. Hou, Acta Microbiol. Sin.
25 (1985) 255–261.
Appendix A. Supplementary data
[36] A.M. Klibanov, Trends Biochem. Sci. 14 (1989) 141–144.
[37] S. Tawaki, A.M. Klibanov, J. Am. Chem. Soc. 114 (1992) 1882–1884.
[38] K. Griebenow, A.M. Klibanov, J. Am. Chem. Soc. 47 (1996) 11695–11700.
[39] A.M. Klibanov, Nature 409 (2001) 241–246.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2011.04.005.
[40] C. Orrenius, T. Norin, K. Hult, G. Carrea, Tetrahedron: Asymmetry 6 (1995)
3023–3030.
[41] E. Wehtje, J. Kaur, P. Adlercreutz, S. Chand, B. Mattiasson, Enzyme Microb.
Technol. 21 (1997) 502–510.
[42] D. Costes, E. Wehtje, P. Adlercreutz, Enzyme Microb. Technol. 25 (1999)
384–391.
[43] E.P. Hudson, R.K. Eppler, J.M. Beaudoin, J.S. Dordick, J.A. Reimer, D.S. Clark, J.
Am. Chem. Soc. 131 (2009) 4294–4300.
References
[1] A. Schmid, J.S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt, Nature 409
(2001) 258–268.
[2] H.J. Lamble, M.J. Danson, D.W. Hough, S.D. Bull, Chem. Commun. 1 (2005)
124–126.
[3] X.W. Feng, C. Li, N. Wang, K. Li, W.W. Zhang, Z. Wang, X.Q. Yu, Green Chem. 11
(2009) 1933–1936.