60
N.L.D. Nyari et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 52–61
Table 2
immobilized enzyme to the reaction medium, in this case, favored
Reaction yield obtained in the catalyzed reactions by immobilized CALB lipase in
polyurethane.
used in future applications employing a continuous flow rate at
different temperatures. The results obtained in the current work
are superior to those in previous reports in the literature by
When the immobilized enzyme was stored in the reaction
medium (oleic acid and ethanol) for 150 days, the enzyme remained
80, 71 and 63% of the original activity at 40, 60 and 80 ◦C, respec-
tively (Fig. 5).
In this way, exposure of the immobilized enzyme to the reaction
enzyme can be used in future applications employing a continuous
flow rate at different temperatures. The results of this study are
better than those reported by Guncheva et al. [39], Hernandez and
Fernandez-Lafuente and [44] Fernandes et al. [40] these authors
also highlighted that this process could preserve enzyme activity
at higher temperatures, and was even twice as active as the native
enzyme.
Enzymatic synthesis
Yield (%)
Molar ratio (acid/alcohol)
(1/3)
(3/1)
(1/1)
Geranyl oleate
Geranyl propionate
Ethyl oleate
10.3
96.6
96.6
26.5
97.3
98.0
33.9
97.7
99.9
3.5. Application of immobilized CALB in catalytic syntheses
reactions
The immobilized CALB was used in esterification reactions,
since it presented high activity, stability and recyclability in these
reactions. The results of conversions obtained in the enzymatic syn-
thesis reactions of geranyl oleate, geranyl propionate and ethyl
oleate esters. The enzymatic synthesis of geranyl oleate, geranyl
of geranyl oleate, at a molar ratio of 1:1, was 33.9%. Geranyl pro-
pionate presented conversion of around 97%, at all molar ratios,
conversion (Table 2).
3.3. Effect of pH on the immobilized lipase activity
The results of the current study demonstrate the potential appli-
cation of this immobilized enzyme in synthesis reactions, especially
for geranyl propionate and ethyl oleate. Similar results were found
by Paroul et al. [25,26] and Nicoletti et al. [34]. Considering the
high conversion and low cost of citronella oil, it can be confirmed
by results obtained in this work that the substitution of commer-
cial alcohols by essential oils can contribute to make the process
economically viable.
Immobilized CALB was stored at different pH levels (3–10) at
ambient temperature (10–25 ◦C) to evaluate the changes in lipase
interaction with the environment. The residual activity was mea-
sured after 30 days, showing 68, 68, 100, 88, 75, 55, 59 and 74%
residual activity at pH 3, 4, 5, 6, 7, 8, 9 and 10, respectively (Fig. 6).
Dhake et al. [41] showed that a change in pH had no significant
the range of 5–8. It was also shown that the immobilized enzyme
been well documented that the optimum pH range of yeast lipases
is generally between pH 5 and 8, with a few exceptions of lower pH
optima of 2.0 [42–44].
4. Conclusions
The immobilization process resulted in a 535% increase in
enzyme activity compared to the free enzyme. This result sug-
gests a beneficial effect of immobilization on enzyme activity. The
immobilized enzyme application in geranyl propionate and ethyl
oleate synthesis resulted in 97 and 83% conversion, respectively.
This study indicates that immobilization CALB is a promising tech-
nique, especially given the simplicity of the process, its low cost
and the ability of supported applications in biocatalysis reactions
of interest to the food industry.
The results obtained in the current work are superior to those
in previous reports in the literature by Liu et al. [45].
Another important parameter in enzyme-catalyzed reactions is
the evaluation of the biocatalyst reuse, in order to decrease the
cost of the process. Fig. 7 shows the behavior of the immobilized
CALB submitted to washing with and without hexane in a continu-
ous cycle. The results obtained after continuous recycling, with and
without hexane washes, presented distinct behavior, since after six
cycles, the immobilized enzyme washed with hexane had 49% of
its initial activity. On the other hand, a completely different behav-
resisted 36 continuous cycles with 51% of the initial activity. This
fact can be associated with the non capacity of hexane modifying
the active site of the enzyme, leading to an increase in enzyme
activity, as already reported by [46].
Fig. 8a shows the behavior of the immobilized enzyme stored in
dry ambient conditions at different temperatures (room: 10–25 ◦C,
refrigeration: 2–8 ◦C and 40 ◦C) after each cycle of use Fig. 8b.
ature and 40 ◦C, respectively. The results obtained in the current
work are superior to those in previous reports in the literature by
Bustamante-Vargas et al. [32], Cadena et al. [47], Guncheva et al.
[48] and Barbosa et al. [49].
Acknowledgments
The authors would like to thank URI-Campus de Erechim, CNPq,
CAPES and FAPERGS for the infra-structure and financial support
for this research.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
References