Biocatalysis and Biotransformation

By Lais P.Dill, Debora M. Kochepka, Nadia Krieger & Luis P. Ramos

(27 Feb 2018): Synthesis of fatty acid ethyl esters with conventional and
microwave heating systems using the free lipase B from Candida antarctica,
Biocatalysis and Biotransformation

By
Thanwarat Boonkerd B6229993
Phirom Boun B6234546
Advisor
Asst.Prof.Dr Atthaphon Maneedaeng

1
Outlines
03
01 02 Objectives
Introduction Background

04 05 06
Procedure Conclusion
Results &
Discussion

2
Introduction

Triglyceride 04

Product

01 Keywords 03
Technique

02
Free Candida
antarctica lipase B
(CALB L)

3
Background

Rudolf Diesel
4
Background

5
 Objectives

➢ To understand ethanolysis of soybean oil

carried out by using the free lipase B in a
solvent-free system.
➢ To compare conventional and microwave
heating systems for FAEE production.
➢ To study the conversion of lipase with a
microwave heating system for FAEE
production.

6
Materials

Free Candida
antarctica lipase B Commercial Ethanol (99.5 wt.%) Tetrahydrofuran
(CALB L) refined soybean oil (THF, HPLC grade)

7
 Proceduce

01 Ethanolysis
03 Analysis of
reaction products

Characterization
of soybean oil 02 CALB L reuse in
soybean oil 04

8
 Procedure
Characterization of soybean oil

Titration using NaOH to Coulometric titration to Gas chromatography to

determine acid content determine water content determine total ester content
9
 Procedure
Ethanolysis Reaction

10
 Procedure
Ethanolysis with CHS & MHS

11
 Experimental
CALB L reuse in soybean oil ethanolysis

After the reaction, the phases were separated by decantation and the aqueous phase,
which contains CALB L, was used directly in a new reaction cycle.

12
 Procedure
Analysis of reaction products

● Determination of fatty acid ethyl esters

2 phases were separated at the end of the reaction
➔ Aqueous phase (containing the enzyme and glycerol)
➔ Organic phase (containing free fatty acids, esters and unreacted glycerides).

● Quantification of free fatty acids

MA is the molar mass of oleic acid divided by 10

V is the volume (L) of NaOH solution spent during titration
C is the concentration of the NaOH solution (mol L"1)
m is the sample mass (g).
13
Results and Discussion

14
 Results and Discussion

- CHS obtains 85.5% conversion at 4h

- MHS obtains 80.7% conversion at 0.25h

Reaction reached equilibrium in 0.25 h for MHS Each heating system required different
and in 4 h for CHS, which achieving a total reaction conditions for optimal performance
ester content of 65 wt.%. MHS = 16 times faster then CHS
15
 Conclusion

● Candida antarctica B lipase was used to convert soybean oil to FAEE using
both CHS and MHS.
● MHS reached equilibrium in a reaction time 16 times shorter than CHS.
● High ethanol:oil molar ratios have a negative effect on FAEE synthesis
with both CHS and MHS due to the partial inactivation of the enzyme.
● The recovery and reuse of CALB L was demonstrated for the first time in a
microwave-assisted FAEE synthesis.

16
 References

● Dill, L. P., Kochepka, D. M., Krieger, N., & Ramos, L. P. (2019). Synthesis of fatty acid ethyl
esters with conventional and microwave heating systems using the free lipase B from
Candida antarctica. Biocatalysis & Biotransformation, 37(1), 25-34.
doi:10.1080/10242422.2018.1443079

● Queiroz, M. L. B., Boaventura, R. F., Melo, M. N., Alvarez, H. M., Soares, C. M. F., Lima, Á.
S., . . . Fricks, A. T. (2015). MICROWAVE ACTIVATION OF IMMOBILIZED LIPASE FOR
TRANSESTERIFICATION OF VEGETABLE OILS. Química Nova, 38(3), 303-308.
doi:10.5935/0100-4042.20150031

● Yonghong, M., Guili, W., Na, Y., Zhiqi, Z., Yuejuan, L., Xiaomei, L., . . . Jilun, L. (2011).
Two-step synthesis of fatty acid ethyl ester from soybean oil catalyzed by Yarrowia lipolytica
lipase. Biotechnology for Biofuels, 4(1), 6-14. doi:10.1186/1754-6834-4-6

● https://www.sciencedirect.com/topics/chemistry/ethanolysis

17