Increasing the catalytic efficiency of Candida rugosa lipase for the synthesis of tert-alkyl butyrates in low-water media

Article abstract of DOI:10.3109/10242422.2011.615392

Reactions involving tert-alcohols and their esters are generally not catalyzed by lipases. Candida rugosa lipase is one of the few lipases which shows at least limited catalytic activity towards tert-alcohols and their esters. Using transesterification of tributyrin with tertiary butyl and amyl alcohols as a model reaction, it is shown that precipitation of lipase by a tertiary alcohol in the presence of a buffer with optimum concentration enhances the catalytic activity 7 fold as compared to rates obtained with lyophilized powders. Optimization of the ratio of triglyceride to tert-alcohols and medium engineering gave an initial rate which was 41 times higher than that obtained with lyophilized powders. Hence, use of a simple enzyme formulation, coupled with optimization of reaction conditions led to Candida rugosa lipase becoming a useful catalyst for catalyzing transesterification involving tertiary alcohols.

Full text of DOI:10.3109/10242422.2011.615392

Biocatalysis and Biotransformation, November–December 2011; 29(6): 238–245
ORIGINAL ARTICLE
Increasing the catalytic efficiency of Candida rugosa lipase for the
synthesis of tert-alkyl butyrates in low-water media
ABIR B. MAJUMDER & MUNISHWAR N. GUPTA
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
Abstract
Reactions involving tert-alcohols and their esters are generally not catalyzed by lipases. Candida rugosa lipase is one of the
few lipases which shows at least limited catalytic activity towards tert-alcohols and their esters. Using transesterification of
tributyrin with tertiary butyl and amyl alcohols as a model reaction, it is shown that precipitation of lipase by a tertiary
alcohol in the presence of a buffer with optimum concentration enhances the catalytic activity 7 fold as compared to rates
obtained with lyophilized powders. Optimization of the ratio of triglyceride to tert-alcohols and medium engineering gave
an initial rate which was 41 times higher than that obtained with lyophilized powders. Hence, use of a simple enzyme
formulation, coupled with optimization of reaction conditions led to Candida rugosa lipase becoming a useful catalyst for
catalyzing transesterification involving tertiary alcohols.
Keywords: Lipases, transesterification, tertiary alcohols, enzyme catalysis in low water media, tributyrin, enzyme precipitated
and rinsed with propanol
Introduction
shown at least low activity towards such substrates.
Among the enzymes used in low water media for
biotransformation, lipases occupy the most dominant
position. Esterification (Dandavate and Madamwar,
2007; Dave and Madamwar, 2008; Dave and
Madamwar, 2010), transesterification (Carrea and
Riva, 2000), interesterification (Bloomer et al., 1991),
kinetic resolution of alcohols and carboxylic acids
(Colton et al., 1995), are important types of reactions
catalysed by this versatile class of enzymes. One class
of compounds which are poor substrates for lipases
is tert-alcohols and their esters (O’Hagan and Zaidi,
1994; Boseley et al., 1997).There has been consider-
able interest in tertiary alcohols (and their deriva-
tives) as building blocks (Itoh et al., 1993; Forrat
et al., 2005; Forrat et al., 2008). Examples of natural
products containing t-alcohols as constituents include
Cinatrin C, Integerrimine, Herringtonine, Fostriecin,
Viridofungin and Zaraqozic acid (Raghavan and
Sreekanth, 2008). Few lipases e.g. porcine pancreatic
lipase (Zaks and Klibanov, 1994), Candida rugosa
lipase (CRL, formerly known as Candida cylindracea
lipase), (O’Hagan and Zaidi, 1994) and Candida ant-
arctica lipase A (CAL A) (Krishna et al., 2002) have
This is due to the presence of the “GGGX” loop and
other structural features creating enough space in the
binding pocket to accommodate bulky tertiary alco-
hols (Henke et al., 2002). In most of these cases,
either lyophilized powder or “straight from the bot-
tle” (as supplied by the vendor) forms of lipases have
been used. It is now well established that lyophiliza-
tion or drying under vacuum leads to structural
changes which are deleterious to the enzyme activity
and can not be reversed under low water conditions
(Lee and Dordick, 2002; Roy and Gupta, 2004a;
Hudson et al., 2005). Cross-linked enzyme aggre-
gates (CLEAs) (Lopez-Serrano et al., 2002; Shah
and Gupta, 2007), protein coated microcrystals
(PCMCs) (Kreiner et al., 2001; Shah and Gupta,
2007) and cross-linked protein coated microcrystals
(CLPCMCs) (Shah et al., 2008) have been shown to
be better forms for catalyzing reactions in low-water
media. In all such cases, “drying” (removal of excess
water) is carried out by precipitation with organic
solvents (Shah and Gupta, 2007). An even simpler
preparation obtained by precipitation with n-propanol
called EPRP (enzyme precipitated and rinsed with
Correspondence: Munishwar N. Gupta, Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. Tel: 91 011
2659 1503, Fax: 91 011 2658 1073. E-mail: munishwar48@yahoo.co.uk
(Received 10 January 2011; revised 5 May 2011; accepted 31 August 2011)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.615392

Synthesis of tert-alkyl butyrates 239
propanol) (Roy and Gupta, 2004b) gave higher rates
than lyophilized powders. Organic solvents other
than n-propanol have been used, so such prepara-
tions in general can be called enzyme precipitated
and rinsed with organic solvents (EPROS) (Shah and
Gupta, 2007). In earlier works, it has been shown that
EPROS are also more stereoselective than lyophilized
powders in low water media (Shah and Gupta, 2007).
In this present work, it is shown that one simple way
of catalyzing biotransformations involving tert-alcohols
(and their derivatives) quite effectively is by using
EPROS of CRL.This should pave the way for enzyme
based synthetic routes involving tert-alcohols and
their esters.
of water). Each rinsing step was followed by centrifuga-
tion at 9000 ꢂ g at 4°C for 5 min. Finally, the precipi-
tate was rinsed with tert-alcohols (1.5 mL, containing
3% water, w/w enzyme, which were going to be used
for alcoholysis) and were directly used for the reaction.
CLEAs (cross-linked enzyme aggregates)
CLEAs were made as mentioned earlier (Lopez-
Serrano et al., 2002) using 0.2 M phosphate buffer (pH
7.0). The precipitation followed by cross-linking was
done in dimethoxyethane or tert-butyl alcohol or in
tert-amyl alcohol by using 50 mM glutaraldehyde (20
μL of 25% aqueous solution) under constant shaking
on an orbital shaker at 300 rpm at 4°C for 17 h.
Materials and Methods
Enzymatic alcoholysis of tributyrin
Enzyme and chemicals
In screw-capped vials, tertiary alcohols were taken
in excess tributyrin such that, the reaction volume
was 1 mL. The concentration of alcohols in the
tributyrin (in excess and also playing the role of the
solvent) was 1 M.The alcohol was wetted with water
which was 3% (w/enzyme weight) i.e. 0.3 mg/10 mg
of enzyme (Zaks and Klibanov, 1994). Lipase for-
mulations, prepared from 10 mg commercial pow-
der, were added and the reaction was carried out on
an orbital shaker at 250 rpm at 20°C.
Candida rugosa lipase (Lipase AYS, Amano) was
obtained from Amano Enzymes Inc., Nagoya, Japan.
Anhydrous acetone (ꢀ99%), methyl tertiary butyl
ether (MTBE), cyclopentyl methyl ether (CPME) and
n-propanol (ꢀ99%) were purchased from Sigma-
Aldrich,St.Louis,USA.Tertiarybutylalcohol(ꢀ98%)
and tertiary amyl alcohol (ꢀ99%) were purchased
from Merck, Mumbai, India and Merck, Honenb-
runn, Germany respectively. Tributyrin (ꢀ99%) was
obtained from Himedia Laboratories Pvt. Ltd, Mum-
bai, India. Alcohols were distilled and dried over 3Å
molecular sieves overnight before use.
Gas chromatographic analysis
Aliquots from the reaction mixtures were taken at
different time intervals and analyzed by GC in an
Agilent 6890 N system fitted with a capillary column
EQUITY TM–5 (30 m ꢂ .32 mm ꢂ 0.25 μm film
thickness) from Supelco (Bellefonte, USA) with
flame ionization detection and programmed oven:
initial temperature 100°C, ramp 1 at 10°C/min up
to 150°C, ramp 2 at 15°C/min up to 250°C. Peaks
were assigned after running purified samples of the
products (see next section for details of purification
of the compounds). In GC analysis, the retention
times of the compounds were noted as: tributyrin
(11.16 min), dibutyrins (9.26 min and 9.29 min),
mono butyrin (6.65 min), tertiary butyl butyrate
(6.42 min), glycerol (3.39 min, in traces), butyric
acid (3.05 min) when tertiary butyl alcohol was the
chosen alcohol. In the absence of available authentic
compounds, it was not possible to identify the dibu-
tyrin peaks in terms of 1,3 and 2,3 dibutyrins (these
are not commercially available).These dibutyrins are
normally obtained by lipase catalysed hydrolysis of
triglycerides (Hou, 2005). However acyl migration
in lipase catalysed transesterification of tributyrin has
been extensively studied (Sjursnes and Anthonsen,
Preparation of different formulations of the biocatalyst
for low-water media
Lyophilized enzyme preparation. Candida rugosa lipase
powder (10 mg) was dissolved in 200 μL of 50 mM
phosphate buffer (pH 7.0), frozen at ꢁ20°C and was
lyophilized for 24 h.
EPROS (Enzyme precipitated and rinsed with organic
solvents). Candida rugosa lipase powder (10 mg) was
dissolved in 100 μL of phosphate buffer (50–250
mM, pH 7). Precipitation of the enzyme was done
by adding the enzyme solution into 1.5 mL ice-cold
anhydrous (dried with molecular sieves) organic sol-
vents viz. acetone, propanol, tert-alcohols in a 2 mL
eppendorf and was allowed to settle at 4°C. After
1 h the precipitate was collected by centrifugation at
9000 ꢂ g at 4°C for 5 min. The complete precipi-
tation of the enzyme was confirmed by protein as-
say of the supernatant after precipitation (Bradford,
1976). This precipitation was followed by repeated
rinsing (four times) with 1.5 mL of ice-cold precipitat-
ing solvent (anhydrous or containing desired amount

240 A. B. Majumder & M. N. Gupta
Scheme 1. Lipase catalyzed alcoholysis of tributyrin.
1994; Sjursnes et al., 1995).The focus of the current
work was to explore the possibility of making trans-
esterification with tert-alcohols more efficient for
obtainingcorrespondingtert-alkylbutyrate.However,
the product esters, the tributyrin and the mono
butyrin could be well separated from each other by
column chromatography (details given in the product
purification and characterization section) and the
GC peak positions of these compounds could be
confirmed. Similar pattern was observed in case of
tert-amyl alcohol with a slight shift in peak position
of the ester tert-amyl butyrate which was detected
at 6.5 min.
tyrins (0.43).The purified fractions were evaporated
in rotary evaporator and the identities of the prod-
ucts were confirmed by 1H-NMR analysis.The pres-
ence of 1,3- and 2,3- dibutyrins in the mixture of
dibutyrins were confirmed by 13C spectra, which
showed two carboxyl carbon peaks around δꢃ173
ppm in ∼1:2 ratio. These dibutyrins appear at 9.26
min and 9.29 min in the GC spectra with more
or less in the same ratio (Figure 5). C8H16O2
1
(tert-butyl butyrate, 24 mg, 17% yield) H-NMR
δ (CDCl3, 300 MHz, ppm): 0.9 (3 H, triplet, Jꢃ7.3
Hz), 1.3 (9 H, s), 1.7 (2 H, m, Jꢃ7.4, 7.3 Hz),
2.3 (2 H, triplet, Jꢃ7.4 Hz). C9H18O2 (tert-amyl
butyrate, 21 mg, 13% yield) 1H-NMR δ(CDCl3, 300
MHz, ppm): 0.9-1 (6 H, merged triplet, Jꢃ7.3,
7.2 Hz), 1.3 (6 H, s), 1.6 (2 H, quartet, Jꢃ7.2 Hz),
1.7 (2H, m, Jꢃ7.3, 7.4 Hz), 2.3 (2 H, triplet,
Jꢃ7.4 Hz).
Product purification and characterization
The products were purified by column chromatog-
raphy using a silica gel (200 mesh) column (10 cm
length ꢂ 3 cm diameter) and eluted with a mixture
of hexane, ethyl acetate and acetic acid taken in 80:
19:1 v/v ratio.The amount of the crude reaction mix-
ture loaded: 100 mg (∼100 micro litre), volume of
the eluent: 80 mL, flow rate 1 mL/ min.The fractions
were analysed by thin layer chromatogarphy using
hexane, ethyl acetate, acetic acid (in 80:20:1 v/v
ratio) and developed with iodine.The Rf values were:
tert-alkyl butyrate (0.83-0.9), tributyrin (0.75), dibu-
Results and Discussion
The earliest attempt at using lipases with t-alcohols
as substrates was transesterification of tributyrin with
tert-butyl alcohol (Scheme 1) (Zaks and Klibanov,
1994). Freeze-dried powder of Candida rugosa lipase
was used as the biocatalyst. In order to investigate
whether other enzyme forms could perform better,
Table I. Transesterification of tert-butyl alcohol with tributyrin under solvent free conditions.
C. rugosa lipase
formulations
Organic solvent used for
precipitation
Water added
(%, w/w, lipase)
Initial rates∗
(μM/mg/h)
Entry
1
2
3
4
5
6
7
8
FD
FD
–
–
0
3
0
3
0
3
0
3
0
16.6
2.2
68
2
90
EPROS†
EPROS
EPROS
EPROS
EPROS
EPROS
acetone
acetone
n-propanol
n-propanol
tert-butyl alcohol
tert-butyl alcohol
5
102
∗Synthesis of t-butyl butyrate: 1 mL reaction medium containing tert-alcohol (1M) in excess tributyrin (Zaks and Klibanov, 1994) was
incubated with 10 mg lipase formulations at 20°C under constant shaking at 250 rpm for 72 h. Initial rates were based upon conversions
obtained by GC analysis.
†50 mM phophate buffer (pH 7.0) was used during EPROS formation.

Synthesis of tert-alkyl butyrates 241
6
5
4
3
2
1
0
solvents and tert-butyl alcohol under identical condi-
tions. Therefore, 3% water originally used by Zaks
and Klibanov (1994) was used. Hence, no effort to
optimize water concentration was made at this stage.
The initial rates were based on the formation of the
tert-alkyl ester.These were calculated from the linear
part of the ester formation vs time plots (Figure 1).
All EPROS preparations gave higher initial rates
than the freeze-dried powder. Interestingly, tert-butyl
alcohol (as the precipitating and rinsing solvent)
gave the most catalytically efficient EPROS. This
may be due to some marginal “imprinting effect” of
the kind observed by Stähl et al. (1990) while pre-
cipitating an enzyme in the presence of a substrate.
To confirm that the observations were valid for
t-alcohols in general, alcoholysis of tributyrin with
tert-amyl alcohol was also carried out (Scheme 1,
Table II). The results obtained were similar to the
first reaction. A dry medium, without any added
water was found to produce very poor reaction rates
(Zaks and Klibanov, 1994); with EPROS some very
poor conversions were obtained while with the
freeze-dried powder no conversion occurred even
after 72 h. The role of small amounts of water in
enhancing protein flexibility and consequently initial
rates has been known for a long time (Zaks and
Klibanov, 1988). Addition of a small amount of
water (3%, w/w enzyme) caused a sharp rise in the
reaction rates and, under these low water conditions,
EPROS exhibited 5–6 times better rate than the con-
trols (Table I, II; entries 2, 8; Figure 1). Other water
concentrations between 0–3% or beyond 3% were
not examined. A completely anhydrous condition
was tried (0% water) and no reaction took place
(Table I). A higher amount of water is likely to cause
loss of the “imprinting effect”. Earlier results (Roy
and Gupta, 2004b) with EPRP, indicated that opti-
mization of the molarity of the buffer (from which
the enzyme is precipitated) leads to enhancement of
initial rates. Figure 2 shows change in initial rates
EPROS (t-amyl butyrate)
EPROS (t-butyl butyrate)
FD (t-amyl butyrate)
FD (t-butyl butyrate)
0
24
48
72
96
120
Time (h)
Figure 1. Formation of tert-alkylesters by different formulations
of C. rugosa lipase (prepared in 50 mM phosphate buffer). The
conversion (%): The ordinate parameter refers to formation of
t-alkyl esters only. Reaction conditions were similar to those in
Table I and II. Each reaction set was run in duplicate and
deviations between pairs of experimental data were within 2%.
the same reaction was investigated in the current
work. Table I shows that under the conditions (3%
w/w added water) used in Zaks and Klibanov (1994),
the CRL freeze-dried powder gave similar initial
rates. The purpose here was to compare the initial
rates obtained with freeze-dried preparations with
EPROS prepared by precipitation with two organic
Table II. Transesterification of tert-amyl alcohol with tributyrin under solvent free conditions.
C. rugosa lipase
formulations
Organic solvent used
for precipitation
Water added
(%, w/w, lipase)
Initial rates*
(μM/mg/h)
Entry
1
2
3
4
5
6
7
8
FD
FD
–
–
0
3
0
3
0
3
0
3
0
20
2
90
3
98
10
112
EPROS†
EPROS
EPROS
EPROS
EPROS
EPROS
acetone
acetone
n-propanol
n-propanol
tert-amyl alcohol
tert-amyl alcohol
∗Synthesis of t-amyl butyrate: 1 mL reaction medium containing t-alcohol (1M) in excess tributyrin (Zaks and Klibanov, 1994) was
incubated with 10 mg lipase formulations at 20°C under a constant shaking at 250 rpm for 72 h. Initial rates were based upon conversions
obtained by GC analysis.
†50 mM phophate buffer (pH 7.0) was used during EPROS formation.

242 A. B. Majumder & M. N. Gupta
Table III. Use of CLEAs for the synthesis of t-alkylbutyrates.
160
∗∗Total
Lipolytic
activity of the
precipitate
[U]
CLEA
activity
(%, free
enzyme)
Initial
rates†
(μM/
mg/h)
150
140
130
120
110
100
Entry
no
Precipitant∗
Acetone
n-propanol
DME
1
2
3
4
5
302
309
326
410
466
–
–
89
113
135
–
–
–
112
116
tert-butanol
tert-amyl alcohol
∗Three organic solvents which precipitated 100% or ꢀ 100%
activity with respect to the specific activity of the control (326
units/ mL/ 10 mg of commercial powder dissolved in phosphate
buffer, 200 mM, pH 7) were used for CLEA preparation.
∗∗The total Lipolytic activity in each precipitate was measured by
dissolving the precipitate completely in the assay buffer.
†These were initial rates of transesterification of the tributyrin
with the t-alcohol (used as the precipitant) under solvent free
conditions (and hence can be compared with corresponding
transesterification rates given in Table I and II).
90
80
EPROS (t-amyl alcohol)
EPROS (t-butanol)
better precipitants in this case. However while the
corresponding CLEAs exhibited better lipolytic
activity (Decker, 1977) in aqueous medium, when
they were used for synthesis they exhibited a lower
initial rate by about 30 μM/mg/h (Table III) com-
pared to the EPROs. These results indicate that a
more flexible enzyme structure is better for accom-
modating the bulky tertiary alkyl groups in the active
site. Presumably, rigidity introduced as a result of
cross-linking prevented accommodation of the tert-
alkyl group in the active site. Further experiments
were carried out with EPROS only.
0
50
100
150
200
250
300
350
Buffer conc (mM)
Figure 2. Effect of increase in buffer concentration on the initial
rates of EPROS. Reaction conditions were similar to those in
Table I and Table II. Each reaction set was run in duplicate and
deviations between pairs of experimental data were within 2%.
with a change in the molarity of the buffer. It shows
that with an increase in buffer molarity up to 150 mM
the initial rate increased sharply, reaching an opti-
mum at around 200 mM followed by a marginal
decrease. Use of this optimum buffer molarity led to
EPROS which gave initial rates of 134 μM mgꢁ1hꢁ1
for tert-butyl butyrate synthesis and 148 μM mgꢁ1hꢁ1
for tert-amyl butyrate synthesis. The increases in
rates over those observed without this optimization
(Table I and II) were significant, though not dramatic.
The conditions of enzyme precipitation with organic
solvents have been known to influence the resulting
enzyme activity for a long time, even using aqueous
solution (Niederauer and Glatz, 1992).
Effect of the relative molar ratio of the alcohol to the
acyl donor on the synthetic rate
It is expected that concentrations of substrate alcohol
should affect the enzyme activity and product yield
(as transesterifications are thermodynamically con-
trolled reactions) (Nordblad et al., 2008). Further-
more, alcohols including amyl alcohols are reported
to inhibit C. rugosa lipase (Bezbradica et al., 2006).
The concentration of alcohols will also govern the
medium polarity especially under the solvent free con-
ditions used in the present experiments. Figure 3
shows that a two times molar excess of tert-butyl alco-
hol (mol/mol tributyrin) and a four times molar excess
of tert-amyl alcohol (mol/mol tributyrin) were opti-
mum for t-butyl butyrate and t-amyl butyrate synthe-
sis respectively. It is interesting to note that, above
these optimum alcohol concentrations, the decrease
in initial rate was sharper for tert-butyl alcohol. Pre-
sumably, greater stripping of the essential layer of
water by the less polar alcohol was the determining
factor (Gupta, 1992; Carrea and Riva, 2000).
Use of CLEAs for synthesis
Cross-linked enzyme aggregates (CLEAs) of CRL
are another biocatalyst formulation used in organic
synthesis, which are known to exhibit higher initial
rates in low water media compared to the freeze-dried
formulations (Lopez-Serrano et al., 2002). CLEAs
involve use of organic solvents for precipitating the
enzyme as the first step.Table III shows that tertiary
butyl alcohol and tertiary amyl alcohol were also the

Synthesis of tert-alkyl butyrates 243
700
600
500
400
300
200
100
0
(2006) found that the initial rate of esterification was
better in amyl alcohol than in isooctane.
What led to such high rates with EPROS while
using the tert-alcohols in alcoholysis? To start with,
unlike most lipases,CRL shows some activity towards
t-alcohols (and their esters) since the GGGX loop
and some other structural features provide enough
space in the binding pocket for the quaternary car-
bon atom (Henke et al., 2002). Colton et al. (1995)
reported that “pre-treatment” of crude CRL with
2-propanol gave 2.3 to 25 times higher enantioselec-
tivity (towards seven esters) as compared to the
crude enzyme. The pre-treatment involved purifica-
tion and presumably change in the “closed to open
conformation”.The initial rates in anhydrous media
were not reported, so it is difficult to compare our
results with theirs. In the present work, precipitation
did not remove any contaminating protein (as all the
protein was found to precipitate with the organic
solvent), so no purification was involved. While
conversion to an open lid structure or a marginal
imprinting effect can not be ruled out, the probable
cause of obtaining an efficient catalyst was that the
enzyme was “dried” (removal of excess water) with-
out lyophilisation, thus preventing structural changes
(Lee and Dordick, 2002). Clearly, retaining flexibil-
ity would be necessary to accommodate the tert-
butyl group or tert-amyl group in the enzyme binding
pocket. Its importance can be appreciated by the fact
t-butyl alcohol
t-amyl alcohol
0
2
4
6
8
Mol: mol (t-alcohol : tributyrin)
10
12
Figure 3. Effect of increase in tert-alcohol concentration (x mols
tributyrin) on the initial rates of synthesis of tert-alkyl butyrates
under solvent free conditions. 1mL reaction medium containing
tertiary alcohol and tributyrin with molar ratios 0.3 (1M alcohol
in 3.2 M tributyrin) to 11.4 (0.7 M tributyrin in 8 M alcohol) was
incubated with 10 mg EPROS at 20°C under constant orbital
shaking at 250 rpm. Initial rate of tert-alkyl butyrate formation was
determined by GC. Each reaction set was run in duplicate and
deviations between pairs of experimental data were within 2%.
800
t-buty butyrate
700
600
500
400
300
200
100
0
t-amyl butyrate
Effect of the solvent on the initial rate
The previous reactions were carried out in solvent
free conditions. Here, alcohols predominantly dic-
tated the medium effects. In many earlier studies,
non polar media have been reported to give higher
reaction rates (Reslow et al., 1987).Thus using a non
polar solvent as a medium (instead of solvent free
conditions) was also tried. Figure 4 shows that while
all other solvents examined decreased the reaction
rate, the addition of octane led to a 1.5 fold increase
in the initial rate of t-butyl butyrate synthesis. Sig-
nificantly, in the case of tert-amyl butyrate synthesis,
mixing the t-amyl alcohol with other solvents includ-
ing octane did not improve the reaction rate to a
varying degree. This not only reflects the difference
in the two alcohols as nucleophiles but also as the
reaction medium, in accordance with earlier obser-
vations that tert-amyl alcohol is an unusually better
solvent based purely on the polarity of the solvents
as reaction media (Zaks and Klibanov, 1988; Shah
et al., 2008). Similar effects have been observed by
others. In Candida rugosa lipase catalyzed synthesis
of amyl butyrate by esterification. Bezbradica et al.
Solvent free Hexane Octane
MTBE
CPME
Figure 4. Effect of the presence of solvent on the initial rate of
synthesis of tert-alkyl butyrates. 2 mL reaction medium containing
1 mL of substrates (tert-butyl alcohol and tributyrin in 2:1 molar
ratio; tert-amyl alcohol and tributyrin in 4:1 molar ratio) and 1 mL
of added solvent was incubated with 20 mg lipase formulations
at 20°C under constant orbital shaking at 250 rpm. Initial rates
of the tert-alkyl butyrate formation were determined by GC. Each
reaction set was run in duplicate and deviations between pairs of
experimental data were within 2%.

244 A. B. Majumder & M. N. Gupta
Figure 5. Gas chromatographic analysis: formation of tert-butyl butyrate and dibutyrins. A) After 24 h, at 16–17% conversion of tributyrin
to the tertiary ester B) after 4 days, at ∼20% conversion. The conversion of tributyrin to dibutyrins were 20% after 24 h andꢀ50%
after 4 days. The peak positions were confirmed after running purified NMR grade samples (The results of the NMR of the purified
compounds are given in terms of J values in the text).
that when EPROS were cross-linked to form CLEAs,
the initial rates in fact decreased. In the case of tert-
butyl butyrate synthesis, with 1 M alcohol in excess
tributyrin, under solvent free conditions, the decrease
in initial rate observed was from 134 μM mgꢁ1hꢁ1
to 110 μM mgꢁ1hꢁ1 and for tert-amyl butyrate it
was from 148 μM mgꢁ1hꢁ1 to 116 μM mgꢁ1hꢁ1
when EPROS were cross-linked to form CLEAs
(Table III, Figure 2).
Thus while the freeze-dried CRL gave 16 μM
mgꢁ1hꢁ1 for tert-butyl butyrate formation, under the
optimized conditions, EPROS of the same enzyme
gave 671 μM mgꢁ1hꢁ1 as the corresponding rate.
This is an increase of 41 fold.
In the present study, the yields were 17% and 13%
(both after column purification) for t-butyl butyrate
and t-amyl butyrate respectively. The synthesis of
n-butyl butyrate by transesterification of tributyrin
with n-butanol using C.rugosa lipase is reported to give
92% yield (Chowdary and Prafulla, 2002).This shows
that there are still considerable challenges associated
with using lipases for reactions involving t-alcohols.
Conclusion
By combining improved “drying” the lipase along
with optimization of reaction conditions, C. rugosa
lipase can be a fairly efficient catalyst for alcoholysis
with two different commonly used tert-alcohols.
C. rugosa lipase is one of the few lipases which natu-
rally shows at least limited catalytic activity towards
tertiary alcohols and their esters. The present work
shows that precipitation of the lipase with a tertiary
alcohol in the presence of a buffer of optimum con-
centration enhanced the catalytic activity seven fold
compared to the similar rates obtained with lyo-
philized powders. Optimization of the ratio of trig-
lyceride to tertiary alcohols and medium engineering
enhanced the initial rate 41 fold.
The study also demonstrates that for the synthesis
of tertiary alcohol esters, a simple preparation like
EPROS was better than more complicated formula-
tions like CLEAs which, in many cases, have proved
very efficient biocatalysts in low-water media (Lopez-
Serrano et al., 2002; Majumder et al., 2008).

Synthesis of tert-alkyl butyrates 245
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Acknowledgement
The work was supported by the “Core group grant
for applied biocatalysis” Department of Science and
Technology (DST), India. Additional financial sup-
ports from the funds obtained from the Department
of Biotechnology (DBT), Govt. of India and UKI-
ERI are also gratefully acknowledged.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
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