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I Dewa Gde Mayun Permana
Lutfi Suhenda
Presented at International Conference on Science, Technology and Humanities
The Patra Jasa Bali Resort and Villas
22-23 October 2018
Background
The need for lipase in the world continues to increase and the price is
expensive
Microbes are one source of lipase fast growing
Coconut pulp Waste
Lauric acid (C-12) : 46,64-48,03%
Lipases from microbes that
grow in different media have
different characters
Characteristic of Lipase ???
(Temperature and pH) ??
Lipase is an enzyme that catalyzes the fat hydrolysis reaction
Lipase is much needed by the food industries, cosmetics, medicine
and other industries
Microbial growth media
The Purpose of the Research :
1. Obtain the optimum incubation time to obtain lipase production
which has the highest activity
2. Obtain the optimum temperature and pH reaction of lipases from
microbe in coconut pulp media
Coconut pulp
0,15 M Phosphat buffer 0,6 M Sucrosa, 1mM EDTA, 10
mM KCl, MgCl2 1mM pH 7,5
Incubation
(Optmization of time incubation)
Homogenization
(Vortex 5 menit)
Centrifugation
(40 menit, 10.000 rpm, 4 0C)
Separation
Supernatant
Activity assay (Hydrolysis) :
Optimization of pH and Temperature
using RSM (CCD)
- Sediment
- Lipid layer
Methods
1. Incubation Time
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
1 2 3 4 5 6 7
Act
ivit
y (
U/
ml)
Incubation time (day)
Aktivitas lipase
2. The Central Point of Temperature
0.00
0.10
0.20
0.30
0.40
0.50
0.60
30 35 40 45
Act
ivit
y (
U/m
l)
Temperature (0C)
Aktivitas lipase
3. The Central Point of pH
0.00
0.50
1.00
1.50
2.00
2.50
3.00
4 5 6 7 8
Act
ivit
y (
U/g
bah
an
)
pH
Aktivitas lipase
Central Composit Design
pH (code) Temperature
(code) pH Temperature Activity (Unit/ml)
-1 -1 4 35 2.016
1 -1 6 35 2.354
-1 1 4 45 1.403
1 1 6 45 1.909
-1,44 0 3,6 40 1.710
1,44 0 6,1 40 2.277
0 -1,44 5 33 1.786
0 1,44 5 47 2.231
0 0 5 40 3.396
0 0 5 40 3.749
0 0 5 40 3.856
0 0 5 40 3.366
0 0 5 40 2.890
Source DF Adj SS Adj MS F-
Value P-Value
Model 5 7.20658 1.44132 10.66 0.004
Linear 2 0.36152 0.18076 1.34 0.322
pH 1 0.33850 0.33850 2.50 0.158
Suhu 1 0.02302 0.02302 0.17 0.692
Square 2 6.83795 3.41897 25.29 0.001
pH*pH 1 3.90465 3.90465 28.88 0.001
Suhu*Suhu 1 3.82515 3.82515 28.29 0.001
2-Way Interaction 1 0.00711 0.00711 0.05 0.825
pH*Suhu 1 0.00711 0.00711 0.05 0.825
Error 7 0.94649 0.13521
Lack-of-Fit 3 0.36883 0.12294 0.85 0.534
Pure Error 4 0.57767 0.14442
Total 12 8.15307
Equation of lipase activity based on the RSM method
Activity (Unit/ml) = -61,6 + 7,36 pH + 2,320 Temp – 0,749 pH*pH – 0,02966 Temp*Suhu + 0,0084 pH*Temp
0
1
2
45
45
40
53
6
3
)lm/tinU( sativitkA
uhuS
Hp
pH
Su
hu
6.05.55.04.54.0
45.0
42.5
40.0
37.5
35.0
>
–
–
–
–
–
< 0.5
0.5 1.0
1.0 1.5
1.5 2.0
2.0 2.5
2.5 3.0
3.0
(Unit/ml)
Aktivitas
Response Surface of Lipase activity
Contour of Lipase activity
D-Optimaly of Lipase Activity
RSM analysis obtained optimum lipase temperature of 39.790C and optimum pH of 5.13 with a theoretical activity of 3.47 U / ml or specific activity of 1.54 U / mg
Activity validation was obtained at 3.40 U / ml or 97,87% of the RSM calculation
CONCLUSION
1. The optimum incubation time is obtained for 5 days.
2. The center point of the reaction temperature is obtained at
400C and pH at 5
3. RSM analysis obtained optimum lipase temperature of
39.790C and optimum pH of 5.13 with a theoretical activity
of 3.47 U /ml or specific activity of 1.54 U / mg
4. Activity validation was obtained at 3.40 U / ml or 97,87%
of the RSM calculation
Thank you to LPPM-Unud for providing research funding in 2018