Development of lipase immobilization technique using sago as support for enzymatic esterification and transesterification
One of the major enzymes produced and used in industries is lipase. Lipases are very versatile catalyst and its applications expand to various industries like oil and fat, dairy, pharmaceutical, detergent, leather, cosmetics and paper. The hitch is enzymes are very sensitive to the change in the...
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Format: | Thesis |
Language: | English |
Published: |
2020
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Subjects: | |
Online Access: | http://psasir.upm.edu.my/id/eprint/105986/1/NUR%20SYAZWANI%20BINTI%20MOHTAR.pdf |
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Summary: | One of the major enzymes produced and used in industries is lipase. Lipases
are very versatile catalyst and its applications expand to various industries like
oil and fat, dairy, pharmaceutical, detergent, leather, cosmetics and paper. The
hitch is enzymes are very sensitive to the change in the environment like
temperature and pH. One way to resolve this problem is to immobilize the
desired enzyme on a support material. Enzyme immobilization does not only
stabilize the enzyme, but it also makes the enzyme suitable to be used for
reactions that are sensitive to water because the immobilized enzymes are often
in a dry form. This is very important for lipase, as the majority use of lipase in
the industry is for ester synthesis and fat modification (transesterification), which
only occur in a water-limiting condition. Therefore, the objectives of this research
are to develop a simple and effective method to immobilize lipase, using sago
as the support, and test the immobilized lipase for esterification and
transesterification activity. Sago is the starch extracted from the pith of
Metroxylon sagu palm trees. It is deemed suitable as the immobilization support
due to its inert nature, stable gel structure and excellent resistance to heat and
shear.
A thermostable lipase isolated from Geobacillus sp. ARM (ARM lipase) was used
in the immobilization study to develop a simple and efficient method to increase
the stability of the enzyme. Pure ARM lipase was used to minimize the
contaminant that might affect the results of the experiments. Two immobilization
techniques using different supports were tested, that is, adsorption on chitosan,
amberlite, and layered double hydroxide and entrapment in sago. The highest
lipase activity determined was by entrapment of the lipase in gelatinized sago
that subsequently spray-dried. The result was supported by the surface area and porosity analysis where the surface area, pore volume, and pore radius
decreased by 67%, 50%, and 10%, respectively, when lipase was immobilized
into the sago, which indicated that the space had been occupied by the enzyme.
This immobilization method has efficiently improved the thermal stability of the
enzyme, where the half-life of the enzyme at 80°C was 4 hours, in comparison
to the activity of the free enzyme, which dropped instantly from the very first
hour. This immobilization method has also successfully preserved the enzyme
for a longer shelf-life, which was 9 months at 10°C and 2 weeks at room
temperature. The immobilized ARM lipase maintained its optimum temperature
for enzyme activity at 70°C and a pH preference from pH7 to pH9. Overall, the
results of thermal and pH characterization have shown that the immobilization
of lipase on a natural support material such as sago is very promising for
industrial use, especially in food manufacturing applications.
Two model applications were done to test the lipase immobilized using the newly
developed method for synthesis activity in a water-free environment. The
immobilized enzyme was used for the synthesis of ethyl oleate by esterification
reaction and the synthesis of cocoa butter alternative by transesterification
reaction. The usage of dry immobilized lipase is very relevant for these
applications. For these experiments, a commercial enzyme, Rhizopus oryzae
lipase (ROL), which was immobilized using the newly developed method was
used to compare with the ARM lipase. The immobilized ROL showed an
outstanding result for the esterification (81.3% at 60°C) compared to ARM lipase
(9.9% at 60°C), therefore, it is used for further experiments. The highest
conversion of ethyl oleate obtained using the immobilized ROL from this
research was 97% with the incubation in a water bath shaker at 40°C and 50°C,
200 rpm shake, for 12 hours, with the substrate ratio of ethanol and oleic acid of
2:1. For the synthesis of cocoa butter alternative using the immobilized ROL in
sago, different ratios of palm mid-fraction, olive oil, and stearic acid were used
as the substrates. Based on the chemical composition analysis determined by
the high performance liquid chromatography (HPLC) and gas chromatographymass
spectrometry (GCMS), the product using substrate ratio 1:0:1 has the
closest reading to cocoa butter compared to the other ratios, with 21.77%
palmitoyl-oleoyl-palmitoyl glycerol, 27.71% palmitoyl-oleoyl-stearoyl glycerol,
13.39% stearoyl-oleoyl-stearoyl glycerol, the total saturated fatty acids of
66.07%, and unsaturated fatty acids of 33.93%. As for the thermal
characteristics, the product using substrate ratio 1:1:1 has the closest reading to
cocoa butter compared to the other ratios, with the slip melting point of 36.2°C;
the solid fat content of 7.73% and 2.02% at 30°C and 40°C respectively. The
DSC thermogram showed melting peaks at lower than 37°C (11.56°C and
33.41°C). In a nutshell, the newly developed method of enzyme immobilization
using sago as the supporting material is suitable for lipases from a bacterial and
a fungal source, for the forward and reverse reactions of lipase. |
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