JOURNALOF BIOSCIENCE AND BIOENGINEFSING

Vol.
95,No.5,466-469.2003

Regeneration of Immobilized Candida antarctica

Lipase for Transesterification
JECH-WE1 CHEN’ AND WEN-TENG WU’*

Department of Chemical Engineering, National Xng Hua University Hsinchu 30043, Taiwan, R. 0. C‘.

Received 7 October 2002iAccepted 16 January 2003

Immobilized lipase from Cundida antarctica was employed to convert triglycerides to biodiesel
using alcohol. Immobilized lipase is frequently deactivated by lower alcohols with deactivation
being caused by the immiscibility between triglycerides and methanol or ethanol. When the lower
alcohol is adsorbed to the immobilized enzyme, the entry of triglycerides is blocked, which causes
the reaction to stop. An alcohol with three or more carbon atoms, preferably 2-butanol or tert-
butanol, can regenerate the deactivated immobilized enzyme. The present work established that
the activity of immobilized lipase could be significantly increased when such alcohols were used
for an immersion pretreatment of the enzyme. The activity of the commercially available immobi-
lized enzyme, Novozyme 435, increased about tenfold in comparison to the enzyme not subjected
to any pretreatment. Following complete deactivation of the enzyme by methanol, washing with
2-butanol and terl-butanol successfully regenerated the enzyme and restored it to about 56% and
75% of its original activity level, respectively.

[Key words: biodiesel, lipase, transesteritication, Candida antarctica]

It is known that there is a finite amount of fossil fuels
which will last for only a few more decades. Developing re-
newable sources of energy is therefore necessary. Among
the renewable resources for the production of alternative
fuels, fats and oils have attracted much attention. Methyl
esters of fatty acids, which are collectively known as bio-
diesel, can be produced by the methanolysis of tryglycerides
such as animal fats, used edible oil, and plant oils (1, 2).
Most industrial processes producing biodiesel use chemi-
cal methods. However, chemical processes in general re-
quire relatively high temperatures and the processing of raw
materials which contain a high content of impurities is often
prohibitively costly and complicated. The impurities mainly
consist of moisture and free fatty acids, commonly found in
cheap feedstock. The existence of these impurities causes
the generation of many undesirable by-products (e.g., soap)
when chemical processes are used. These by-products and
glycerol are difficult to separate from the biodiesel and thus,
chemical processes require additional procedures for the
separation and purification of biodiesel. Therefore, enzy-
matic methods are preferred over chemical methods.
There are two major problems in using lipase to produce
biodiesel. The first one is that the activity of lipase is rela-
tively low. Watanabe et al. (3), reported on the production
of biodiesel for which it took about 36 h to complete the
reaction. It is obvious that the reaction time is much longer
than that when chemical processes are used due to the low
lipase activity. The other problem is that the immobilized
enzyme is liable to be deactivated by a lower alcohol. Sev-
eral researchers have tried to overcome this effect and im-
* Corresponding author. e-mail: wtwu@mx.nthu.edu.tw
phone: +886-3-5742509 fax: +886-3-5742509
prove the lipase activity. For example, Nelson et al. (4) used
hexane as a diluent to prevent the deactivation by a lower
alcohol. Samukawa et al. (5) tried to maintain a very low
concentration of methanol during the reaction. However,
these methods have some inherent problems. Using a dilu-
ent decreases the reaction rate and the precise control neces-
sary to maintain the methanol concentration at a very low
level is not an appropriate approach for the production of
biodiesel on a large scale. From an economical point of
view, a continuous reaction process without the use of any
organic solvent is needed for the industrial production of’
biodiesel.
The aim of this work was to establish a method suitable
for enhancing the activity of immobilized lipase and regen-
erating the deactivated enzyme when its activity becomes
lower than a set value. Our results show that the immobi-
lized Candida antarctica lipase is a suitable catalyst for the
methanolysis of oils without using an organic solvent.
MATERIALS AND METHODS
Materials Soybean oil was purchased from the Taiwan Sugar
Corp. (Taipei, Taiwan) and used as a source oftriglycerides. Meth-
anol, ethanol, n-propanol, isopropanol, 1-butanol, 2-butanol, iso-
butanol, rert-butanol and hexane were obtained from RDH (Seelze,
Germany). All the standards for the estimation of lipids and fatty
acid methyl esters (FAMES) were purchased from Supelco (Belle-
fonte, PA, USA). The enzyme used was Novozyme 435 (immobi-
lized C. antarctica lipase) which was supplied by Novo Nordisk
(Bagsvaerd, Denmark).
Enzyme pretreatment For pretreatment, 0.3 g of the immo-
bilized enzyme was placed in different test tubes, and then various
solvents (i.e., soybean oil, methyl ester, isopropanol, 2-butanol,
tert-butanol, and n-hexane) were added to the tubes to swell the

466
VOI.. 95, 2003 REGENERATION OF IMMOBILIZED LIPASE 467

TABLE I. Effect of pretreatment on transesterification under different immersion conditions

Type of pretreatment Yield of methyl ester (%, w/w)

None 2.5
Immersion in soybean oil for 4 h 8.6
Immersion in soybean oil overnight 10.0
Immersion in methyl ester for 0.5 h and then soybean oil for a further 4 h 4.1
Immersion in methyl ester for 1 h and then soybean oil for a further 4 h 9.5
Immersion in methyl ester for 1.5 h and then soybean oil for a further 4 h x.9
Immersion in methyl ester for 2 h and then soybean oil for a further 4 h 9.1
Immersion in methyl ester for 1 h and then soybean oi1 overnight 11.1
Immersion in hexane for 1 h and then soybean oil overnight 3.5
Immersion in isopropanol for 1 h and then soybean oil for a further 1 h 16.8
Immersion in 2-butanol for I h and then soybean oil for a further 1 h 17.6
Immersion in [err-butanol for 1 h and then sovbean oil for a further 1 h 24.5

enzyme particles. Another test tube containing the enzyme without
any pretreatment was used as a control. The immersion conditions
are shown in Table 1. Each of the immersed enzymes, as well as
the control, was mixed with 5.7 g of soybean oil and 0.26 ml of
methanol to carry out the transesterification reaction. The reaction
was carried out for 30 min in an incubator at 30°C with shaking at
200 rpm. Upon completion of the reaction, 0.1 g of the sample was
withdrawn and the amounts of methyl ester and unreacted oil were
analyzed using HPLC. The activity of the immobilized enzyme
was determined on the basis of the initial rate of the reaction.
Enzyme deactivation The enzyme was deactivated by add-
ing different alcohols to the tubes containing enzyme and incubat-
ing the enzyme with the alcohol for a period of time.
Enzyme regeneration For investigation of enzyme regener-
ation, 0.3 g of the pretreated enzyme in different test tubes contain-
ing 5.7 g of soybean oil was treated with various amounts of meth-
anol. The mole ratios of the oil to methanol were 8 : 1, 5 : 1, 3 : 1,
3 :2, 1: 1,2 : 3, 1: 2 and I : 3. Each of the test tubes was designated a
serial number from t, to t,. The mixtures were shaken at 200 ‘pm
for 30 min at 30°C. The enzymes were partially or completely de-
activated and the deactivated enzymes were washed according to
the following scheme. (i) Oil washing: The enzyme was washed
with 10 ml of soybean oil three times, and immersed in soybean oil
in an incubator at 30°C overnight. (ii) Isopropanol washing: The
enzyme was washed with 10 ml of isopropanol three times and
then the isopropanol was washed off with soybean oil. Finally the
enzyme was immersed in soybean oil in an incubator at 30°C over-
night. (iii) 2-Butanol washing: The enzyme was washed with 10 ml
of 2-butanol three times and then the 2-butanol was washed off
-_
I Ml% I
with soybean oil. Finally, the enzyme was immersed in soybean oil
in an incubator at 30°C overnight. (iv) tert-Butanol washing: The
enzyme was washed with 10 ml of tert-butanol three times and
then the tert-butanol was washed off with soybean oil. Finally, the
enzyme was immersed in soybean oil in an incubator at 30°C over-
night.
To each of the test tubes containing 0.3 g of the regenerated en-
zyme 5.7 g of soybean oil and 0.26 ml of methanol were added and
the reaction allowed to proceed in an incubator at 30°C with shak-
ing at 200 rpm for 30 min. Upon completion, 0.1 g of sample was
removed and then analyzed using HPLC to measure the contents of
methyl ester and unreacted oil.
Analysis The concentrations of FAMES in the reaction
mixture were determined using HPLC with a UV detector at 205
nm. The analytical HPLC system consisted of two Shimadzu LC-
9A pumps and a SPD-IOA variable-wavelength UV detector
(Shimadzu, Kyoto). For this analysis, a LiChroCART RP-Cl8
analytical column of 250x4 mm (Merck, Darmstadt, Germany)
was employed. The mobile phase consisted of three different com-
ponents: hexane, isopropanol and methanol. Reservoir A contained
methanol and reservoir B contained a mixture of isopropanol and
hexane (5 : 4, v/v). The gradient went from 100% A to 50% A +
50% B linearly over 30 min. The flow rate of the mobile phase was
1 mlimin and the sample injection volume was 10 ~1. This non-
aqueous RP-HPLC method was modified from the method re-
ported by Holcapek et al. (6).
The extent of the transesterification of soybean oil was deter-
mined using the modified non-aqueous RP-HPLC method. Figure
1 shows a typical separation curve of a mixture of soybean oil and
biodiesel. The individual peaks of methyl esters of linolenic,
linoleic and oleic acids were separated from each other (retention
times of 4-7 min). The peaks that appear from 20 to 30 min repre-
sent the mixture of triglycerides. The peaks representing diglyc-
erides and monoglycerides were between the peaks of methyl
esters and triglycerides. At the beginning of the reaction there were
no other peaks except the peaks representing triglycerides. When
the transesterification reaction was complete, the major group of
peaks moved from the right to the left. The extent of the reaction
was determined on the basis of the sum of the peak areas of methyl
esters, diglycerides, monoglycerides, and triglycerides.

RESULTS AND DISCUSSION

Pretreatment Table 1 shows the effect of pretreat-

t,
0 5 10 15 20 25 30 ment on the activity of the immobilized enzyme. Immobi-
Time (min) lized lipase without any pretreatment exhibited very low
activity and the yield of methyl ester was 2.5% (w/w) after
FIG. 1. A typical separation curve for a reaction mixture of soy-
bean oil after methanolysis using HPLC. DGs, Diglycerides; MEs, me- 30 min of reaction. When the enzyme was immersed in
thyl esters; MGs, monoglycerides; TGs, triglycerides. higher alcohols (e.g., isopropanol, 2-butanol, or tert-bu-
468 CHEN AND WU
0 I I ’
0 al 40 60
Methanol concentration (g/P)
FIG. 2. The initial reaction rate ofthe transesterification versus the
concentration of methanol. The enzymes were pretreated with isopro-
panol (closed squares), soybean oil (open squares), 2-butanol (closed
triangles), and kr&butanol (closed diamonds).
tanol), the yield of methyl ester was about 7 to 10 times
higher than that for the immobilized enzyme without pre-
treatment. The activity of the lipase immersed in the alcohol
was found to be higher than that of the enzyme immersed in
methyl ester. When n-hexane was used for immersion pre-
treatment, the activity of the pretreated immobilized en-
zyme was close to that of the immobilized enzyme without
any pretreatment. This indicated that n-hexane is not a suit-
able solvent for the pretreatment of C. antarctica lipase.
Figure 2 shows the initial reaction rate of the transesterifica-
tion with various concentrations of methanol and the same
amount of the enzyme and soybean oil. When the immobi-
lized enzyme was appropriately pretreated, not only the ac-
tivity increased significantly, but also the ability of the en-
zyme to resist deactivation by methanol was higher. At high
concentrations of methanol, the immobilized enzyme pre-
treated with alcohol exhibited higher activity, particularly if
the pretreatment was with isopropanol or 2-butanol.
Deactivation of the enzyme Different types of alco-
hols were employed to test the deactivation effect on the en-
zyme. Both linear alcohols such as methanol, ethanol, pro-
pan01 and butanol and branched alcohols such as isopro-
panol, 2-butanol and isobutanol were used. The experimen-
o-.,.,.,., ,,,,,I,.,.
0 20 40 60 80 loo 120 140
Concentration of linear alcohols (g/l)
FIG. 3. The initial reaction rate of the transesterification versus the
concentration of various linear alcohols. Symbols: squares, methanol;
circles, ethanol: triangles, n-propanol; inverted triangles, n-butanol.
t - _--T-T------l---
a3 1M , 20 40 60 80 100 120 140 160 180 200
Concentration of branched alcohols (s/f)
FIG. 4. I’he initial reaction rate ofthe transesterilication versus the
concentration of variousbranched alcohols. Symbols: squares, isopro-
panol; triangles, 2-butanol; circles, isobutanol.
tal results are shown in Figs. 3 and 4. All of the linear alco-
hols were toxic to the immobilized enzyme in varying de-
grees, as shown in Fig. 3. The degree of deactivation was
found to be inversely proportional to the number of carbon
atoms in the linear lower alcohols. Figure 4 shows the deac-
tivation effect of the branched chain alcohols on the immo-
bilized enzyme. The degree of deactivation by the branched
alcohols was demonstrated to be lower than that by the lin-
ear alcohols. The curves for isopropanol and 2-butanol in
Fig. 4 are almost horizontal, i.e., isopropanol and 2-butanol
do not significantly deactivate the enzyme.
It was also observed that when the immobilized lipase
was deactivated by methanol or ethanol, the immobilized
enzyme particles underwent a conspicuous change in ap-
pearance, changing from the original transparent golden
color to an opaque gray color, accompanied by swelling and
caking. Furthermore, methanol and ethanol also exhibited
poor miscibility with soybean oil. When more than one
ninth of the theoretical mole number for methanol or etha-
nol required for the reaction was added to the oil. the re-
sulting mixture became an emulsion. Small droplets of the
alcohol were observed and the enzyme was significantly de-
activated. The concentration of alcohol at which an emul-
sion state was observed was identical to the concentration
causing substantial deactivation of the immobilized enzyme.
Higher alcohols exhibited better miscibility with soybean
oil. Alcohols with more than three carbon atoms were com-
pletely miscible with soybean oil at a concentration below
the theoretical mole ratio required for this reaction (ix..
alcohol : oil = 3 : 1). The experimental results indicated that
one of the main causes of deactivation of the enzyme was
due to the immiscibility between triglycerides and methanol
or ethanol. In addition, the material used for immobilizing
the enzyme was acrylic resin which could adsorb polar
compounds such as methanol and ethanol. When the con-
centration of lower alcohols in the mixture was high, the
alcohols formed small droplets which attached to the resin
160 180 ml
particles. As the alcohol was adsorbed to the immobilized
enzyme, the entry of triglycerides was blocked, causing the
reaction to stop.
Enzyme regeneration Figure 5 shows that the activ-
ity of the enzyme, which was deactivated in the test runs of
Var.. 95. 2003
Run number
FIG. 5. Activity of the regenerated enzyme deactivated by eight
different concentrations of methanol and washed with soybean oil or
isopropanol. Symbols: squares, oil washing; triangles, isopropanol wash-
ing.
Run number
FIG. 6. Activity of the regenerated enzyme deactivated by eight
different concentrations of methanol and washed with soybean oil,
2-butanol or rert-butanol. Symbols: squares, oil washing; triangles, 2-bu-
tanol washing; inverted triangles, tert-butanol washing.
t, through to t,, was regenerated by oil washing and isopro-
pan01 washing. The activity of the regenerated enzyme was
based on the conversion of soybean oil to methyl esters. It
can be seen from Fig. 5 that the enzyme in runs t, to t, was
not deactivated. However, in the case of runs t,, t,, and t,,
higher concentrations of methanol were added for deacti-
vation. Washing with soybean oil and isopropanol could not
effectively regenerate the enzymes deactivated in these
runs. On the other hand, washing the enzyme in these runs
with 2-butanol and tert-butanol gave better results. As shown
in Fig. 6, the activity of the enzyme in runs t6, t,, and t, was
significantly regenerated. Even in run t,, washing with 2-bu-
tanol and tert-butanol regenerated the enzyme to 56% and
75% of the original activity level, respectively. The reason
is that the methanol adsorbed on the immobilized enzyme
can be dissolved in 2-butanol or tert-butanol. Hence, wash-
ing with 2-butanol or tert-butanol is an efficient way to re-
generate the deactivated immobilized lipase.
Since the deactivated enzyme could be successfully re-
generated, a long-term experiment was carried out to test
the regeneration efficiency of the deactivated enzyme. A
continuous stirred tank reactor with a working volume of 5 1
REGENERATION OF IMMOBILIZED LIPASE 469
o,-------
0 10 20 30 40 50 60 0
Reaction time (d)
FIG. 7. Enzyme activity in a continuous stirred tank reactor. The
arrows denote the time of regeneration.
containing 250 g of pretreated enzyme was employed. The
reaction temperature was maintained at 30°C and the im-
peller speed was 100 rpm. Feeding of soybean oil together
with three molar equivalents of methanol was carried out at
a constant flow rate of 20 mUmin. The conversion of methyl
ester was set at 70%. When the conversion dropped below
70%, the deactivated enzyme was regenerated using tert-
butanol. There are three enzyme regenerations shown in
Fig. 7. The 3rd regeneration at around 48 d of operation was
carried out to test the regeneration capability after the en-
zyme was substantially deactivated. Figure 7 demonstrates
that the deactivated enzyme was successfully regenerated
and the conversion can be maintained above 70% for more
than 70 d.
Enzymatic methods commonly have problems of low
activity and easy deactivation. In previous studies, such as
those of Watanabe et ~2. (3), Nelson et al. (4) and
Samukawa et al. (5) these problems could not be solved.
The method proposed here substantially improved the activ-
ity of and successfully regenerated deactivated lipase.
REFERENCES
1. Fukuda, H., Kondo, A., and Noda, H.: Biodiesel fuel pro-
duction by transesterification of oils. J. Biosci. Bioeng., 92,
406-416 (2001).
2. Ma, F. R. and Hanna, M. A.: Biodiesel production: a review.
Bioresour. Technol., 70, l-l 5 (1999).
3. Watanabe, Y., Shimada, Y., Sugihara, A., Honda, H.,
Fukuda, H., and Tominaga, Y.: Continuous production of
biodiesel fuel from vegetable oil using immobilized Candida
antarctica lipase. J. Am. Oil Chem. Sot., 77,355-360 (2000).
4. Nelson, L. A., Foglia, T. A., and Marmer, W. N.: Lipase-
catalyzed production of biodiesel. J. Am. Oil Chem. Sot., 73,
1191-1195 (1996).
5. Samukawa, T., Kaieda, M., Matsumoto, T., Ban, K.,
Kondo, A., Shimada, Y., Noda, H., and Fukuda, H.: Pre-
treatment of immobilized Candida antarctica lipase for bio-
diesel fuel production from plant oil. J. Biosci. Bioeng., 90,
180-183 (2000).
6. Holcapek, M., Jandera, P., Fisher, J., and Prokes, B.: Ana-
lytical monitoring of the production of biodiesel by high-per-
formance liquid chromatography with various detection meth-
ods. J. Chromatogr. A, 858, 13-3 1 (1999).