A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents

Article abstract of DOI:10.1016/j.bmcl.2011.03.059

Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best pr

Full text of DOI:10.1016/j.bmcl.2011.03.059

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2934–2936
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Bioorganic & Medicinal Chemistry Letters
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A chemically modified lipase preparation for catalyzing the
transesterification reaction in even highly polar organic solvents
⇑
Kusum Solanki, Munishwar Nath Gupta
Department of Chemistry, 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:
Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino
groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It
was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and com-
plete activity. PCMC (protein coated microcrystals), CLPCMC (crosslinked protein coated microcrystals),
EPROS (enzyme precipitated and rinsed with organic solvents) and pH tuned preparations of modified
and unmodified lipase were prepared and used for carrying out transesterification reaction with n-octane
and dimethyl formamide (DMF) as reaction medium. In n-octane, among all the preparations, CLPCMC of
Received 2 February 2011
Revised 11 March 2011
Accepted 16 March 2011
Available online 21 March 2011
Keywords:
Chemical modification
À1
À1
modified lipase gave highest rate (1970 nmol min mg ) as compared to unmodified pH tuned lipase
Pyromellitic dianhydride
Pseudomonas cepacia lipase
Low water organic media
Transesterification reactions
Crosslinked protein coated microcrystals
À1
À1
(
128 nmol min mg ). In DMF, with both 1% (v/v) and 5% (v/v) water content, CLPCMC showed highest
À1
À1
initial rate of 0.72 and 7.2 nmol min mg , respectively. Unmodified pH tuned lipase showed no activity
at all in DMF with both 1% and 5% (v/v) water content.
Ó 2011 Elsevier Ltd. All rights reserved.
Lipases have found large number of applications in catalyzing
transesterification/esterification reactions in nearly anhydrous or-
of excess water) is carried out by precipitation rather than lyoph-
1
2
ilization. Also, in that case, the preparation was found to shift
for giving maximum initial rate from 0.97 to 0.33 in acetoni-
1
,2
ganic solvents. While enzymes, in general and lipases in particu-
lar work well for organic synthesis and kinetic resolution in such
a
w
1
2
trile. These results with a-chymotrypsin indicated that the addi-
3
,4
media, two key issues remain. Firstly, the initial rates are not
as high as the catalytic efficiency of enzymes in more conventional
tional charges placed on the enzyme as a result of chemical
modification did enable the enzyme to hold on to essential water
molecules when the enzyme was placed in a reasonably polar
5
–8
water rich media.
Secondly, most of the enzymes show even fur-
1
2
ther poor activity in the presence of polar organic solvents like
and water soluble organic solvent like acetonitrile. In the present
8
,9
DMSO or DMF. The accepted view is that essential water layer
work, a two-pronged strategy based upon the following has been
5
,8
13,14
around the enzyme is stripped off by these highly polar solvents.
attempted (a) modifying the enzyme with PMDA
(b) drying
This is important for organic chemists as many organic compounds
of interest do not have appreciable solubility in highly nonpolar
solvents like n-octane (a solvent often used to measure initial rates
the enzyme by precipitation with organic solvents with or without
the presence of additives and also followed by subsequent chemi-
1
5–21
cal crosslinking with glutaraldehyde.
It is shown that as a re-
in nonaqueous enzymology6 ). Rees et al. modified
,8
10
a-chymo-
sult of this strategy Pseudomonas cepacia lipase can catalyze
transesterification reaction even in highly polar organic solvents
like DMF.
11
trypsin with pyromellitic dianhydride (PMDA) to confer large
number of additional negative charges on the protein surface in
an attempt to enable the protein to compete better for water with
organic solvents (Fig. 1). The lyophilized powders of the modified
enzyme, in fact gave lower rates than even those of unmodified
lyophilized powder. In an insightful analysis, the changes in the io-
nic state during lyophilization was found to be responsible for this
unexpected result. A later work from our laboratory showed that
2
2
TNBS assay showed that 5 out of 7 free lysine amino acids
were modified with PMDA. The degree of modification of free ami-
2
3
no groups was calculated to be 72%. Different high performance
1
6
21
19
biocatalyst preparations (PCMC
CLPCMC , EPROS
and pH
tuned lyophilized powder (lyophilized from a solution in aqueous
buffer at pH 7.0, which is the optimum pH for this enzyme in aque-
5
,24
the same modification with
a
-chymotrypsin did indeed result in
ous buffers )) of modified lipase were prepared and its transeste-
2
5–27
a biocatalyst preparation which gave high transesterification activ-
ity in low water media, provided ‘drying’ of enzyme (the removal
rification activity (1-hexanol with tributyrin)
was measured
with n-octane and DMF as reaction media.
2
8
Figure 2 shows the activity recovery and amount of protein
2
9
precipitated when different organic solvents were used as pre-
cipitants. 1,2-dimethoxyethane was found to be the best precipi-
tant and hence for the preparation of all high performance
⇑
Corresponding author. Tel.: +91 11 26591503/6568; fax: +91 11 2658 1073.
E-mail addresses: munishwar48@yahoo.co.uk, appliedbiocat@yahoo.co.in (M.N.
Gupta).
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.059

K. Solanki, M. N. Gupta / Bioorg. Med. Chem. Lett. 21 (2011) 2934–2936
2935
O
O
COO-
R
HN CO
H NR
2
O
O
-OOC
COO-
O
O
Protein
PMDA-modified derivative
Pyromellitic dianhydride
Figure 1. Chemical modification of protein with Pyromellitic dianhydride (PMDA).
tents (1% (v/v) and 5% (v/v)) (Table 2). Again, the performance of
all the three preparations (EPROS, PCMC and CLPCMC) exceeded
well beyond the pH tuned lyophilized powders of lipase (which
showed no activity at all). Also, the modified lipase preparations
were better than unmodified ones. With both, 1% (v/v) and 5% (v/
v) water content, CLPCMC of modified lipase performed best
among all the preparations. With 1% (v/v) water content in DMF,
CLPCMC of modified and unmodified lipase gave initial rates of
1
40
20
00
1
1
8
0
6
0
4
0
À1
À1
0
.72 and 0.05 nmol min mg , respectively (Table 2). At same
2
0
0
water content, PCMC and EPROS of both modified and unmodified
lipase showed very low catalytic activity. With 5% (v/v) water con-
tent, CLPCMC of modified and unmodified lipase gave initial rates
of 7.2 and 2.5 nmol min mg , respectively (Table 2). It is inter-
esting to note that% (v/v) water which was found better (out of
Activity
Protein
À1
À1
1
% and 5%) was different for various enzyme preparations. PCMC
of both unmodified lipase and modified lipase worked better with
% (v/v) water. In all other cases, 5% v/v water gave better catalytic
Precipitating solvents
1
Figure 2. Effect of different precipitating solvents on PMDA modified P. cepacia
lipase. Lipase was precipitated with different precipitating solvents. Then the lipase
hydrolytic activity and protein precipitated were determined after re-dissolving the
precipitate in an aqueous buffer.
rates. This means that water partitioned away from the organic
solvent by the enzyme did not meet its optimum hydration level
for maximum activity with 1% (v/v). With 5% (v/v) water content,
PCMC of both modified and unmodified lipase showed very less
À1
À1
activity (<0.001 nmol min mg ). That is understandable as at
5% (v/v) water, it is likely that salt core would start dissolving. It
is interesting to observe that crosslinking in case of CLPCMC seems
to have at least partially avoided this problem. The initial rates ob-
biocatalyst preparations, 1,2-dimethoxyethane was chosen as the
precipitating solvent.
Table 1 summarizes the catalytic performances of pH tuned
lyophilized powder, EPROS, PCMC and CLPCMC of unmodified
and modified lipase. Much of the early work on enzyme catalysis
in nearly anhydrous media was carried out using such pH tuned
lyophilized powders. Hence, this preparation has been included
as a ‘control’ to compare with. The initial rates obtained during
the assay (Table 1) showed that CLPCMC was the best catalyst fol-
lowed by PCMC > EPROS > pH tuned in n-octane. For all the prepa-
rations, biocatalyst formulations of modified lipase were better
than corresponding formulation of unmodified lipase. The initial
rates showed by CLPCMC of modified lipase (1970 nmol min
À1
À1
served with modified EPROS were 0.42 nmol min mg and with
À1
À1
unmodified EPROS were 0.19 nmol min mg
.
To sum up, this work shows that by using the synergy between
enhancing changes in the enzyme surface (by chemical method)
and ‘drying’ (removal of excess water) enzymes by precipitation
in the presence of additives and subsequent cross linking yields
biocatalyst preparations which work better than lyophilized pow-
ders in nearly anhydrous media. In this work, P. cepacia lipase has
been chosen as this is known to be among the best lipase for cat-
3
0
alyzing transesterification reaction. The possibility of using li-
pases in organic solvent like nearly anhydrous DMF opens up
further opportunities for using lipases in organic synthesis and ki-
netic resolution. Right from early days of nonaqueous enzymology,
this has been a much sought after goal. Early efforts using protein
À1
À1
mg ) were 15.4 times higher than the initial rates showed by
À1
À1
pH tuned unmodified lipase (128 nmol min mg ).
After this, these preparations were evaluated for catalyzing the
transesterification reaction in DMF with two different water con-
Table 1
Table 2
Transesterification reaction of tributyrin with n-hexanol catalyzed by different
biocatalyst formulations of P. cepacia lipase with n-octane as a reaction medium
Transesterification reaction catalyzed by different biocatalytic formulations of PMDA
modified P. cepacia lipase with DMF as a reaction medium
Lipase formulation
Initial rates
Fold
increase
À1
À1
Biocatalyst preparation
Initial rates (nmol min mg )
À1
À1
(
nmol min mg
)
1
0
% (v/v) water
5% (v/v) water
pH tuned (unmodified)
128
240
620
974
513
472
1150
1970
1
EPROS of unmodified lipase
PCMC of unmodified lipase
CLPCMC of unmodified lipase
pH tuned (modified)
EPROS of PMDA modified lipase
PCMC of PMDA modified lipase
CLPCMC of PMDA modified lipase
1.9
4.8
7.6
4.0
3.7
9.0
15.4
pH tuned
0
EPROS of unmodified lipase
EPROS of modified lipase
PCMC of unmodified lipase
PCMC of modified lipase
CLPCMC of unmodified lipase
CLPCMC of modified lipase
<0.001
<0.001
0.01
0.02
0.05
0.19
0.42
<0.001
<0.001
2.5
0.72
7.2
Initial rates were calculated from the aliquots taken within 0.25–1 h. The per-
centage conversions during these time periods were in the range of 1–25% and were
in linear range. Enzyme used was biocatalyst formulation made from 2 mg solid
enzyme (in 2 ml of reaction volume) in each case.
Initial rates were calculated from the aliquots taken within 1–5 h. The percentage
conversions during these time periods were in the range of 1–20% and were in
linear range. Enzyme used was biocatalyst preparation made from 2 mg solid
enzyme (in 2 ml of reaction volume) in each case.

2
936
K. Solanki, M. N. Gupta / Bioorg. Med. Chem. Lett. 21 (2011) 2934–2936
engineering31 and lately through directed evolution32 have been
made to obtain enzyme mutants which can function in the aque-
ous cosolvent mixtures containing high percentage of DMF. In that
context, CLPCMC, is a simple biocatalyst preparation for any organ-
ic chemist to convert and use any commercially available lipases
for catalyzing reactions in highly polar organic solvents.
15. Kreiner, M.; Moore, B. D.; Parker, M. C. Chem. Commun. 2001, 1096.
16. Preparation of PCMC of lipase: Five milligram of P. cepacia lipase was dissolved
in 50 mM phosphate buffer, pH 7.0 (100 l) followed by addition of 0.3 ml of
l
saturated solution of potassium sulfate. The remaining procedure was same as
that reported for PCMCs of proteases,15 except instead of n-propanol, 1,2-
dimethoxyethane was used as precipitating and rinsing agent. The biocatalyst
preparation was then rinsed thrice with corresponding dry organic solvent or
organic solvent containing various amounts of water.
1
1
1
7. Roy, I.; Gupta, M. N. Bioorg. Med. Chem. Lett. 2004, 14, 2191.
8. Shah, S.; Gupta, M. N. Biorg. Med. Chem. Lett. 2007, 17, 921.
Acknowledgments
9. Preparation of EPROS of lipase: In case of P. cepacia lipase, the enzyme (5 mg)
solution was made in 6 ml of 100 mM phosphate buffer, pH 7.0 and was cooled
to 4 °C. 1,2-Dimethoxyethane (DME) was used as a precipitating and rinsing
agent instead of n-propanol. The remaining procedure was same as for
K.S. thanks University Grant Commission (UGC) for the Senior
Research Fellowship. The support provided by Department of Sci-
ence and Technology in the form of ‘Core group’ grant and Depart-
ment of Biotechnology, both Govt. of India organizations are also
acknowledged.
17
preparation of EPRP of proteases. The biocatalyst formulation of P. cepacia
18
lipase thus formed has been named as EPROS (enzyme precipitated and
rinsed with organic solvent).
2
2
0. Shah, S.; Sharma, A.; Gupta, M. N. Biocatal. Biotransform. 2008, 26, 266.
1. Preparation of CLPCMC of lipase: The PCMC of lipase obtained were dispersed in
5
00 ll of 1,2-dimethoxyethane followed by addition of 10 ll of glutaraldehyde
References and notes
(25% v/v in water). The mixture was kept at 4 °C for 1 h with constant shaking
at 300 rpm. The CLPCMC thus formed was then rinsed thrice with 1,2-
dimethoxyethane followed by rinsing thrice with corresponding dry organic
solvent or organic solvent containing various amounts of water and again
centrifuged at 5000 g for 5 min at 4 °C to remove the organic solvents.
1
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26. Transesterification reaction catalyzed by P. cepacia lipase: This transesterification
reaction is same as used by several people working with lipases.25 The
transesterification reaction of 1-hexanol and tributyrin (250 mM each) was
catalyzed by P. cepacia lipase in organic solvent in a total volume of 2 ml. The
reaction was started by adding enzyme formulation to the reaction medium
1
0. Rees, D. G.; Gerashchenko, I. I.; Kudryashova, E. V.; Mozhaev, V. V.; Halling, P. J.
Biocatal. Biotransform. 2002, 20, 161.
1
1. Mozhaev, V. V.; Šikšnis, V. A.; Melik-Nubarov, N. S.; Galkantaite, N. Z.; Denis, G.
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Biochem. 1988, 173, 147.
25
and incubated at 30 °C with constant shaking at 200 rpm. Samples were
taken at appropriate time intervals and analyzed by gas chromatography (GC).
27. GC analysis: Transesterification reaction products formed after catalysis with P.
cepacia lipase, were analyzed on Agilent Technologies 6890 N network GC
systems, USA, with a flame ionization detector. The capillary column of length
30 m, internal diameter of 0.25 mm with nitrogen as the carrier gas at a
1
1
2. Solanki, K.; Shah, S.; Gupta, M. N. Biocatal. Biotransform. 2008, 26, 258.
3. n-Octane, DMF and 1,2-diemthoxyethane (anhydrous grade with water
content less than 0.001% (v/v)) were obtained from Sigma Chemical (St.
Louis, MO, USA). n-Propanol and tris(hydroxymethyl)aminomethane were
obtained from Merck (Mumbai, India). PMDA was a product of Eastman Kodak
Company (Rochester, NY, USA). P. cepacia lipase was a gift from Amano Enzyme
Inc. (Nagoya, Japan). Tributyrin (>99%) was obtained from Himedia
laboratories Pvt. Ltd. (Mumbai, India). Glutaraldehyde (25% w/v aqueous
solution) was purchased from Merck (Hohenbrunn, Germany). All solvents
were dried over molecular sieves before using. All other chemicals used were
of analytical grade.
À2
constant pressure of 4 kg cm was used. The column oven temperature was
programmed with an initial temperature of 150 and was increased thereafter
À1
to 250 °C at the rate of 10 °C min with injector and detector temperature at
25
240 and 250 °C, respectively.
28. Jain, P.; Jain, S.; Gupta, M. N. Anal. Bioanal. Chem. 2005, 381, 1480.
29. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
1
4. Modification of lipase with PMDA: The acylation of lipase with PMDA was
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W.; Kojic-Prodic, B. Eur. J. Biochem. 2001, 268, 3964.
carried out as described.1 PMDA solution prepared in dimethylsulfoxide
1
(
2 mM, 1 ml) was added dropwise at 4 °C to 0.04 mM enzyme solution (10 ml,
31. Wong, C. H.; Chen, S. T.; Hennen, W. J.; Bibbs, J. A.; Wang, Y.-F.; Liu, J. L.-C.;
Pantoliano, M. W.; Whitlow, M.; Bryan, P. N. J. Am. Chem. Soc. 1990, 112, 945.
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2005, 22, 21.
prepared in 100 mM Tris–HCl buffer, pH 7.0) with stirring. The pH of the
reaction was maintained at 7.0 using 20 mM NaOH. The reaction mixture was
stirred for 2 h followed by its dialysis against 10 mM Tris–HCl buffer (pH 7.0).