Immobilization of Isolated Lipase From Mouldy Copra
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Lipase is a very important enzyme in modern biotechnology. Many industries use
enzyme as biocatalysis. Lipase is well known to have high activity in hydrolytic reaction and
chemical compound synthesis. Lipase can be a biocatalysis for hydrolytic reaction,
esterification, alcoholysis, acidolysis, and aminolysis. The enzyme catalysis capacity for
chemical reaction can be described through its activity. The catalytic reaction level by
enzyme correlates indirectly to its enzyme activity. Several microbes can be used to produce
lipase: Candida, Aspergillus and Rhizopus2.
The commercial lipase is very expensive due to its difficult long production process.
The use of dissolved lipase enzyme as a biocatalizator is not very economical compared to
dissolved lipase enzyme (immobile lipase). Dissolved enzyme is relatively unstable and
cannot be used again and again (reusable)3. The use of enzyme is limited to once use only so
that each initial processing must use new enzyme. This is not efficient and costly. These
defects can be overcome through enzyme immobility to increase its stability.
Immobile enzyme is an enzyme both physically and chemically are not free to move so
that it can be controlled and managed when the enzyme must contact with substrate.
Immobilization prevents enzyme diffusion into reaction mixture and it is easy to retrieve
from product flow by separation of simple solid-liquid, so that the enzyme can be reusable4.
In order to immobilize enzyme, observation to support material used is necessary. The
supports commonly used are polystyrene latex and EP400 accurel5, Hp-206, eupergit7 and
silica micro-particle8. In this study lipase immobilization was conducted by adsorption using
silica gel as a support. The use of silica gel as a support material is due to its surface
adsorption ability and good intra-molecule and has interlocking capacity to provide large
surface for the media9.
Experimental
The culture (isolated Aspergillus oryzae) was grown under optimal conditions for lipase
production. The initial volume of the culture was 500 mL containing pepton 0.5 g/100 mL,
KH2PO4 0.1 g/ 100 mL, FeSO47H2O 0.001 g/100 mL and olive oil 1 mL/100 mL.
0
Temperature and pH were controlled at 37 C and 7.0. After incubation for 8 days, the
culture broth was centrifuged at 3500 rpm for 30 min. The clear supernatant containing
the lipase was used for future studies.
Enzyme activity assay
Lipase enzyme activity is determined by using Vorderwulbecke et al.10 modified with the
following procedures: As much as 0.1 mL of lipase enzyme solution added with 0.89 mL
borate buffer with concentration 0.05 M (pH 8.2). The reaction began by adding 0.01 mL
substrate p-nitrophenilbutirate 0.1 M (dissolver dimetilsulfoxide) and rapidly shaken and
0
then the reaction mixture was incubated for 10 minutes at 45 C temperature, then the
reaction mixture was measured its adsorption at the wave length 410 n m. The lipase enzyme
activity was measured based on p-nitrophenol formed from lipase enzyme hydrolysis result
to substrate p-nitrophenilbutirate.
Partial purification of enzyme
To 2180 mL of the culture supernatant, ammonium sulphate was added (70% saturation)
0
at 4 C over night. The precipitate was collected by centrifugation at 10.000 rpm at 4 0C for
20 min and dissolved in 3 mL 0.05 M borate buffer (pH 8.2). The lipase activity and the
protein concentration were determined11.
Dali, Seniwati
