Enhancing the catalytic efficiency of subtilisin for transesterification by dual bioimprinting

Article abstract of DOI:10.1016/j.tetlet.2015.05.101

Bioimprinting is a technique in which an aqueous solution of a protein molecule along with the imprint molecule is dried to remove bulk water. Subtilisin was dissolved in an aqueous buffer with a substrate analog and precipitated with the substrate alcoho

Full text of DOI:10.1016/j.tetlet.2015.05.101

Tetrahedron Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Enhancing the catalytic efficiency of subtilisin for transesterification
by dual bioimprinting
Joyeeta Mukherjee a, Munishwar N. Gupta b,
⇑
a Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
b Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioimprinting is a technique in which an aqueous solution of a protein molecule along with the imprint
molecule is dried to remove bulk water. Subtilisin was dissolved in an aqueous buffer with a substrate
analog and precipitated with the substrate alcohol to obtain a dually bioimprinted enzyme precipitate.
Precipitation was found to be a better bioimprinting technique than freeze drying used so far. The pre-
cipitation also enabled simultaneous bioimprinting with the alcohol substrate reinforcing the existing
imprint with the substrate analog. The dually bioimprinted subtilisin was found out to be 65-fold more
Received 4 May 2015
Revised 25 May 2015
Accepted 26 May 2015
Available online xxxx
Keywords:
Subtilisin
Bioimprinting
Transesterification
Enzyme precipitated and rinsed with
organic solvents
efficient than the freeze dried non-imprinted powder of subtilisin and could transesterify N-acetyl-
phenylalanine with n-propanol up to 82% in 5 h.
L-
Ó 2015 Published by Elsevier Ltd.
Introduction
much more active preparation called enzyme precipitated and
rinsed with propanol (EPRP) or more generally as enzyme precipi-
tated and rinsed with organic solvents (EPROS).17,30–32 Many years
back, Mosbach’s group showed that bioimprinting of a protease
alpha chymotrypsin could be carried out by precipitation (rather
than freeze drying).33
In the present work, we show that bioimprinting can be carried
out with either ester or even the alcohol substrate. It is, in fact,
much better if the enzyme is bioimprinted with both the ester
and alcohol substrates simultaneously. As the alcohols used were
water miscible short chain alcohols, this dual bioimprinting could
be carried out in a facile manner by simply precipitating the
enzyme in the presence of an excess amount of ester from the
aqueous mixture into the substrate alcohol. This dual bioimprint-
ing was shown to result in highly efficient biocatalysts in nearly
anhydrous organic solvents (bioimprinted EPROS) in the case of
subtilisin which is a well characterized protease and proteases
are frequently used in such media for organic synthesis.15,34,35
Using proteases in low water containing organic solvents
enables synthesis of esters, amides and peptides by esterification/
transesterification/transamidation reactions.1–5 Esters are valuable
organic compounds which find diverse applications as fragrances,
flavours, chemical intermediates, biodiesel and biolubricants.6–11
Proteases are useful for the synthesis of esters (and amides) of
amino acids as well as sugars.12–15 Essentially, this synthetic
approach represents reversal of the hydrolytic reactions which
these enzymes catalyse in aqueous buffers.1–4 Enzymes are
used as heterogeneous catalysts (in insoluble form) in such
media.1–4,16 In general enzymes show much less catalytic
efficiency under these nearly anhydrous conditions.
A large
number of efforts have been directed towards obtaining enzyme
preparations which are more efficient catalysts than ‘straight from
the vendor’ or freeze-dried preparations.3,15,17–24
Bioimprinting enzymes by freeze drying their aqueous solutions
along with their substrates/substrate analogs have given enzyme
preparations which upon use in organic solvents result in a consid-
erable increase in reaction rates.25–29 The bioimprinted enzymes
have the ‘frozen substrate analog induced conformations’ and
hence are more active than the unimprinted enzymes. We have
shown in numerous cases that ‘drying’ (removal of bulk water)
enzymes by precipitation rather than freeze drying results in a
Results and discussion
The use of bioimprinting with substrates to enhance transester-
ification efficiency has mostly been limited to substrate analogs
(esters or amides).25,27,33,36 Transesterification in the case of alco-
holysis involves two substrates—an ester and an alcohol which acts
as a nucleophile. Hence, the nucleophile alcohol should also be able
to bioimprint the enzyme. In fact, Kampp (alcohol) in the case of
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.tetlet.2015.05.101
0040-4039/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Mukherjee, J.; Gupta, M. N. Tetrahedron Lett. (2015), http://dx.doi.org/10.1016/j.tetlet.2015.05.101

2
J. Mukherjee, M. N. Gupta / Tetrahedron Letters xxx (2015) xxx–xxx
alcoholysis by subtilisin have been estimated many years back.37
Dordick’s group has used nucleophiles sucrose and thymidine for
bioimprinting subtilisin successfully.27,38 We have tried bioim-
printing with short chain alcohols by simply using these to precip-
itate subtilisin. Table 1 shows the initial rates of transesterification
Table 2
Transesterification of N-acetyl-L-phenylalanine ethyl ester with n-butanol in anhy-
drous n-hexane using the EPROS of subtilisin Carlsberg (SC)
Precipitating
agent
Initial rates of transesterification with n-butanol
(nmol minÀ1 mgÀ1
)
n-Propanol
n-Butanol
tert-Butanol
Ethanol
Methanol
Acetone
61
75
45
36
32
28
12
of N-acetyl-L-phenylalanine ethyl ester with n-propanol catalysed
by subtilisin bioimprinted by n-propanol, n-butanol, tert-butanol,
ethanol, methanol, acetone and acetonitrile. As different organic
solvents precipitate enzymes to different extents,39 the initial rates
by definition are normalized with respect to the amount of subtil-
isin precipitated. Both n-propanol and n-butanol were found to be
equally good bioimprinting agents. This is understandable as n-
propanol and n-butanol are structurally not very different. On
the other hand shorter chain alcohols ethanol and methanol were
less successful in bioimprinting for the reaction. The much lower
rates observed when the enzyme was precipitated in acetone and
acetonitrile indicated that the results with alcohols were not
artifacts.
Table 2 shows the initial rates of transesterification of the
amino acid ester with n-butanol with a similar set of bioimprinted
EPROS. Significantly, n-butanol was the best bioimprinting agent.
These initial rates were about half of what were observed with
the first transesterification reaction. Hence it may be concluded
that subtilisin seems to prefer n-propanol as a nucleophile as com-
pared to n-butanol. So, bioimprinting had a greater impact and
bioimprinting with either n-butanol or n-propanol made a differ-
ence during transesterification with n-butanol.
The particle size of the precipitate is likely to play an important
role because of the mass transfer constraints.40 Light scattering
measurements of the subtilisin precipitated/bioimprinted with
various organic solvents (Fig. 1) showed that all the precipitates
had particle size in a narrow range of 350–480 nm diameters.
The precipitates obtained with propanol/butanol consisted parti-
cles of higher sizes than those with methanol/ethanol. This clearly
established that the effect on initial rates (Tables 1 and 2) were due
to bioimprinting and not mass transfer constraints.
The use of CD (with a spinning cell accessory) has been
described recently to look at the closeness of the structure of
enzymes in insoluble form to the native structure.32 It was shown
that the closeness to the native structure correlated well with the
activities observed with various enzyme preparations in organic
media.32 Figure 2 shows the far UV CD spectra of the various sub-
tilisin EPROS. The corrected CD spectra show that the order of the
molar ellipticity was native subtilisin in buffer > n-propanol bioim-
printed subtilisin > n-butanol bioimprinted subtilisin > methanol
bioimprinted subtilisin P tert-butanol bioimprinted subtil-
isin P ethanol bioimprinted subtilisin > acetonitrile precipitates
of subtilisin. The CD spectra of the EPROS obtained with acetone
could not be recorded as acetone absorbs strongly in the range of
190–280 nm. The EPROS are reported to show a great shift in the
Acetonitrile
SC (1 mg) was dissolved in 50 mM Tris–HCl buffer, pH 7.8. EPROS was prepared and
the transesterification reaction was set up as mentioned in the experimental sec-
tion. The experiment was done in duplicate and the error between each set was
within 3%.
secondary structure as compared to that of native subtilisin in
aqueous buffers.32 However, among the EPROS prepared with the
various organic solvents, the ones with n-propanol and n-butanol
change signs of the CD spectra closest to the native spectra
(Fig. 2) as compared to the other spectra which change signs much
farther away. This indicates that the secondary structure of the
EPROS with n-propanol and n-butanol are better as compared to
the other EPROS. This is also in line with the activity data (Table 1).
The next obvious step was to try simultaneous/dual bioimprint-
ing with a substrate analog, for example, an amide (used by Russell
and Klibanov25) and the alcohols. Table 3 shows the effect of
bioimprinting in various cases on the initial rates of transesterifica-
tion of N-acetyl-L-phenylalanine ethyl ester with n-propanol. On
the basis of the results presented in Tables 1 and 2, we can assume
that precipitation of subtilisin with acetone did not lead to any
bioimprinting. So, just the enzyme precipitated with acetone
resulted in much higher rates than those reported for bioimprinted
subtilisin when the bioimprinting with ester analog was carried
out by freeze drying.25 Bioimprinting with N-acetyl-
L-tyrosinamide
gave a further 1.4Â enhancement in the initial rates. With both
ethanol and propanol, dual bioimprinting resulted in a more effi-
cient biocatalyst preparation. Again, n-propanol, the substrate in
the chosen transesterification reaction, was the best bioimprinting
molecule along with N-acetyl-L-tyrosinamide. It was interesting to
check whether the better initial rates also translated into the better
conversions. Figure 3 shows the use of biocatalyst preparations
with dual bioimprinting with the right alcohol and the amide
resulted in the highest conversion. The unimprinted subtilisin
(precipitated with acetone) was the least efficient biocatalyst.
While the present work was in progress, we came across a
report by Yan et al.,41 in which a crude preparation of lipase was
bioimprinted by drying its mixture with oleic acid and methanol
in aqueous organic co-solvent by freeze drying. In order to improve
the catalytic efficiency, these authors tried to synergize the
approach with immobilization, lecithin coating and salt activa-
tion.42 The best increase in catalytic efficiency reported was
19-fold over the unimprinted enzyme. We show that dual bioim-
printing was successful even in the case of a protease, subtilisin.
Table 1
Transesterification of N-acetyl-L-phenylalanine ethyl ester with n-propanol in anhy-
drous n-hexane using the EPROS of subtilisin Carlsberg (SC)
Precipitating
agent
Initial rates of transesterification with n-propanol
(nmol minÀ1 mgÀ1
)
Conclusions
n-Propanol
n-Butanol
tert-Butanol
Ethanol
Methanol
Acetone
129
130
100
96
95
72
The removal of bulk water (‘drying’) results in rigidification of
the conformation of the protein molecule. Use of this dried prepa-
ration in nearly anhydrous organic solvents ensures that this rigid
conformation is preserved.15,42 Hence, any induced conformation
by a ligand in aqueous buffer is preserved during ‘drying’. This is
the basis of bioimprinting proteins.25 The results described in the
current work exploit the earlier observations that precipitation
with organic solvents can be used for drying enzymes and in fact
generally gives more active enzyme preparations for use in organic
Acetonitrile
25
SC (1 mg) was dissolved in 50 mM Tris–HCl buffer, pH 7.8. EPROS was prepared and
the transesterification reaction was set up as mentioned in the experimental sec-
tion. The experiment was done in duplicate and the error between each set was
within 3%.
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J. Mukherjee, M. N. Gupta / Tetrahedron Letters xxx (2015) xxx–xxx
3
Figure 1. Determination of the sizes of the EPROS of subtilisin prepared in various organic solvents by light scattering.
media.17,30,31 Hence precipitation with a suitably chosen alcohol in
the presence of an amide/ester led to a highly active subtilisin
preparation for catalysing transesterification reactions in organic
media.
We recently reported that the same transesterification reaction
(with n-propanol) catalysed by the lyophilized subtilisin under
identical conditions showed an initial rate of 3.2 nmol minÀ1 mgÀ1
protein.15 In the current work, the dually bioimprinted subtilisin
catalysed the reaction with an initial rate of 207 nmol minÀ1 mgÀ1
protein (Table 3). Hence a simple precipitation based dual bioim-
printing led to a 65-fold increase in the initial rates over the initial
rates shown by the unimprinted lyophilized powders of subtilisin.
Use of enzyme catalysis in organic media is fast emerging as a
useful strategy in organic synthesis.13,14,16 Transesterification/
Figure 2. Far UV CD spectra of subtilisin EPROS prepared in various organic
solvents. A special spinning cell accessory was used to record the spectra. The
suspensions were taken and the spectra recorded at a scan speed of 50 nm/min and
an average of 4 scans was used. Each spectrum was corrected for the solvent
spectra.
Table 3
Effect of dual bioimprinting with alcohol as well as ester analog
Precipitant
Imprint molecule
None
Initial rates (nmol minÀ1 mgÀ1
)
n-Propanol
132
207
N-Acetyl-
L-tyrosinamide
L-tyrosinamide
L-tyrosinamide
Ethanol
Acetone
None
98
140
N-Acetyl-
Figure 3. Time course for the transesterification of N-acetyl-L-phenylalanine ethyl
ester with n-propanol in anhydrous n-hexane using the dual imprinted EPROS of
subtilisin Carlsberg (SC). SC (1 mg) was dissolved 50 mM Tris–HCl buffer, pH 7.8
None
72
102
N-Acetyl-
SC (1 mg) was dissolved in 50 mM Tris–HCl buffer, pH 7.8 containing 10 mM N-
acetyl- -tyrosinamide wherever mentioned. EPROS was prepared and the transes-
terification reaction was set up as mentioned in the experimental section. The
experiment was done in duplicate and the error between each set was within 3%.
containing 10 mM N-acetyl-L-tyrosinamide wherever mentioned. EPROS was pre-
L
pared and the transesterification reaction was set up as mentioned in the
experimental section. The experiment was done in duplicate and the error between
each set was within 3%.
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4
J. Mukherjee, M. N. Gupta / Tetrahedron Letters xxx (2015) xxx–xxx
esterification reactions have proved especially valuable in this con-
text.3,5,12 Hence, the results described here may be of considerable
utility in organic synthesis including kinetic resolution of racemic
mixtures.13,14,16,43
scans at the rate of 20 nm/min and a data pitch of 1 nm. The spec-
tra were corrected for the background by subtracting the spectrum
of the solvent, that is, 50 mM Tris–HCl buffer, pH 7.8 for native SC
and the respective organic solvents for the EPROS. 1 mg mLÀ1
native SC solution in the aqueous buffer was used for recording
the spectrum. In the case of the EPROS, after the CD spectra had
been recorded, the suspensions were collected from the cell and
redissolved in Tris–HCl buffer, pH 7.8 by gentle vortexing and
the clear supernatant containing the dissolved protein was used
to find the enzyme concentration by measuring its absorbance at
280 nm. CD data were expressed as mean residual ellipticity in
Experimental section
Materials
Subtilisin Carlsberg (Protease from Bacillus licheniformis Type
VIII), N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tyrosi-
deg cm2 dmolÀ1
.
namide were procured from Sigma–Aldrich Co. (St. Louis, USA).
Solvents like n-propanol, n-hexane, acetonitrile and acetone were
obtained from Sigma–Aldrich Co. (St. Louis, USA) and they con-
tained less than 99.8% water. The anhydrous solvents were further
stored over oven-dried (140 °C) molecular sieves (3 Å) at least 24 h
prior to use. tert-Butanol and n-butanol were obtained from
Fischer Scientific, Mumbai, India. Ethanol was procured from
Merck, Hohenbrunn, Germany. All other reagents and solvents
were obtained from Fisher Scientific and were of the highest grade
commercially available.
Size determination by DLS
The EPROS of SC prepared in various solvents were analyzed by
DLS on MALVERN Zetasizer Nano ZS instrument. The suspensions
were taken right after their preparation in the organic solvent in
a glass cuvette and equilibrated for 20 s and run for 15 scans to
get the average hydrodynamic diameter. The intensity average size
distribution was thus obtained for all the EPROS preparations.
Acknowledgements
Methods
We acknowledge financial support from the Department of
Science and Technology (DST) [Govt. of India] [Grant No.:
SR/SO/BB-68/2010] and Department of Biotechnology (DBT)
[Govt. of India] [Grant No.: BT/PR14103/BRB/10/808/2010]. J.M.
thanks the Council of Scientific and Industrial Research [Govt. of
India] for the Senior Research Fellowship. We thank Prof.
Prashant Mishra (Department of Biochemical Engineering and
Biotechnology, IIT Delhi, India) for his interest in this work and sev-
eral useful research discussions.
Preparation of EPROS of subtilisin Carlsberg44
Subtilisin Carlsberg (SC) (1 mg) was dissolved in 50 mM Tris–
HCl buffer, pH 7.8 (0.5 mL) with or without the addition of
10 mM N-acetyl-L-tyrosinamide. It was precipitated in various
organic solvents (6 mL) at 4 °C and 200 rpm. In the case of precip-
itation in n-butanol, the precipitation was carried out at 25 °C as
the miscibility of water in n-butanol is low at lower temperatures.
The miscibility of water in n-butanol at 25 °C is 20% which is well
within the precipitation conditions. The precipitation was carried
out for 30 min after which it was centrifuged, the supernatant
was removed and the precipitate (EPROS) was washed five times
with the precipitating solvent. Finally the EPROS was washed with
the reaction medium before carrying out the transesterification
reaction.
References and notes
1. Mattiasson, B.; Adlercreutz, P. Trends Biotechnol. 1991, 9, 394.
2. Gupta, M. N. Eur. J. Biochem. 1992, 203, 25.
3. Methods in Non-Aqueous Enzymology; Gupta, M. N., Ed.; Birkhauser: Basel, 2000.
4. Halling, P. J. Curr. Opin. Chem. Biol. 2000, 4, 74.
5. Fessner, W. D.; Anthonsen, T. Modern Biocatalysis: Stereoselective and
Environmentally Friendly Reactions; Wiley-VCH: Weinheim, 2008.
6. Common Fragrance and Flavor Material; Bauer, K., Garbe, D., Sueburg, H., Eds.;
VCH Publishers: New York, 1990.
7. Chang, S. W.; Shaw, J. F.; Shieh, C. J. Food Technol. Biotechnol. 2003, 41, 237.
8. Antczak, M. S.; Kubiak, A.; Antczak, T.; Bielecki, S. Renew. Energy 2009, 34, 1185.
9. Banerjee, A.; Singh, V.; Solanki, K.; Mukherjee, J.; Gupta, M. N. Sustain. Chem.
Process. 2013, 1, 14.
Protein estimation
The EPROS prepared according to the method described above
were dissolved in aqueous buffer (1 mL) and protein concentration
was determined by recording the absorbance at 280 nm. The sub-
tilisin used in this work is 99.8% pure as mentioned in Sigma–
Aldrich, hence absorbance at 280 nm was found to be a reliable
method to estimate protein during these investigations.
10. Gryglewicz, S.; Muszynski, M.; Nowicki, J. Ind. Crop. Prod. 2013, 45, 25.
11. Malhotra, D.; Mukherjee, J.; Gupta, M. N. J. Biosci. Bioeng. 2015, 119, 280.
12. Applied Biocatalysis; Straathof, A. J. J., Adlercreutz, P., Eds.; Harwood Academic
Publishers: Amsterdam, 2000.
13. Carrea, G.; Riva, S. Angew. Chem., Int. Ed. 2000, 39, 2226.
14. Carrea, G. Riva, S. In Asymmetric Organic Synthesis with Enzymes; Gotor, V.,
Alfonso, I., Garcia-Urdiales, E., Eds.; Wiley-VCH: Weinheim, 2008; pp 3–20.
15. Mukherjee, J.; Mishra, P.; Gupta, M. N. Tetrahedron Lett. 2015, 56, 1976.
16. Adlercreutz, P. Chem. Soc. Rev. 2013, 42, 6406.
17. Roy, I.; Gupta, M. N. Bioorg. Med. Chem. Lett. 2004, 14, 2191.
18. Hudson, E. P.; Eppler, R. K.; Clark, D. S. Curr. Opin. Biotechnol. 2005, 16, 637.
19. Shah, S.; Sharma, A.; Gupta, M. N. Anal. Biochem. 2006, 351, 207.
20. Majumder, A. B.; Mondal, K.; Singh, T. P.; Gupta, M. N. Biocatal. Biotransform.
2008, 26, 235.
21. Kreiner, M.; Moore, B. D.; Parkar, M. C. Chem. Commun. 2001, 1096.
22. Shah, S.; Sharma, A.; Gupta, M. N. Biocatal. Biotransform. 2008, 26, 266.
23. Minteer, S. D. Enzyme Stabilization and Immobilization: Methods and Protocols;
Humana Press: New York, 2011.
24. Mukherjee, J.; Gupta, M. N. Chem. Cent. J. 2012, 6, 133.
25. Russell, A. J.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 11624.
26. Mingarro, I.; Abad, C.; Braco, L. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3308.
27. Rich, J. O.; Dordick, J. S. J. Am. Chem. Soc. 1997, 119, 3245.
28. Fishman, A.; Cogan, U. J. Mol. Catal. B 2003, 22, 193.
Transesterification reaction
The transesterification reaction was carried out as described
earlier.45 The reaction mixture contained 20 mM N-acetyl-
L-pheny-
lalanine ethyl ester and 1 M n-propanol/n-butanol in anhydrous
n-hexane. The enzyme preparation as prepared above was added
to the reaction mixture and shaken at 200 rpm at 25 °C. The
product was assayed by high performance liquid chromatography
(HPLC) using a Zorbax C-18 reversed phase column (150 mm
 4.6 mm  5
l) at a flow rate of 1 mL/min (eluent used was
water:acetonitrile:glacial acetic acid = 55:40:5 v/v) at 258 nm,
and the initial rate was obtained from the linear portion of the
reaction trajectory.45
Circular dichroism measurements
Far UV CD spectra of the EPROS of SC were measured on a JASCO
J-815 spectropolarimeter using an in house fabricated spinning
cylindrical sample cell holder as described previously.32,46 The
spectra were recorded from 195 nm to 250 nm as an average of 4
29. Kahveci, D.; Xu, X. Biotechnol. Lett. 2011, 33, 2065.
30. Solanki, K.; Gupta, M. N. Chem. Cent. J. 2008, 2, 1.
31. Majumder, A. B.; Gupta, M. N. Biocatal. Biotransform. 2011, 29, 238.
32. Solanki, K.; Gupta, M. N.; Halling, P. J. Bioresour. Technol. 2012, 115, 147.
33. Stahl, M.; Mansson, M.-O.; Mosbach, K. Biotechnol. Lett. 1990, 12, 161.
Please cite this article in press as: Mukherjee, J.; Gupta, M. N. Tetrahedron Lett. (2015), http://dx.doi.org/10.1016/j.tetlet.2015.05.101

J. Mukherjee, M. N. Gupta / Tetrahedron Letters xxx (2015) xxx–xxx
5
34. Clapes, P.; Adlercreutz, P.; Mattiasson, B. J. Biotechnol. 1990, 15, 323.
41. Yan, J.; Yan, Y.; Yang, J.; Xu, L.; Liu, Y. Process Biochem. 2009, 44, 1128.
42. Dordick, J. S. Biotechnol. Prog. 1992, 8, 259.
43. Patel, R. N. Stereoselective Biocatalysis; Marcel-Dekker: New York, 2000.
44. Shah, S. Biochim. Biophys. Acta 2007, 1770, 94.
35. Reslow, P.; Adlercreutz, P.; Mattiasson, B. Eur. J. Biochem. 1988, 177, 313.
36. Stahl, M.; Jeppsson-Wistrand, U.; Mansson, M. O.; Mosbach, K. J. Am. Chem. Soc.
1991, 113, 9366.
37. Chatterjee, S.; Russell, A. J. Biotechnol. Bioeng. 1992, 40, 1069.
38. Rich, J. O.; Bedell, B. A.; Dordick, J. S. Biotechnol. Bioeng. 1995, 45, 426.
39. Schoevaart, R.; Wolbers, M. W.; Golubovic, M.; Ottens, M.; Kieboom, A. P.; van
Rantwijk, F.; van der Wielen, L. A.; Sheldon, R. A. Biotechnol. Bioeng. 2004, 87,
754.
45. Ru, M. T.; Dordick, J. S.; Reimer, J. A.; Clark, D. S. In Enzymes in Nonaqueous
Solvents Methods and Protocols; Vulfson, E. N., Halling, P. J., Holland, H. L., Eds.;
Humana Press: New Jersey, 2001; pp 3–11.
46. Ganesan, A.; Price, N. C.; Kelly, S. M.; Petry, I.; Moore, B. D.; Halling, P. J. Biochim.
Biophys. Acta 2006, 1764, 1119.
40. Halling, P. J. In Enzyme Catalysis in Organic Solvents: A Comprehensive Handbook;
Drauz, K., Waldman, H., Eds.; Wiley-VCH: Weinheim, 2002; pp 259–285.
Please cite this article in press as: Mukherjee, J.; Gupta, M. N. Tetrahedron Lett. (2015), http://dx.doi.org/10.1016/j.tetlet.2015.05.101