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