PROPERTIES AND

BIOTECHNOLOGICAL
APPLICATIONS OF LIPASES
FROM SEED OILS

Adriano Aguiar Mendes

Instituto de Química, Universidade Federal de Alfenas

Alfenas/MG - Brazil
 Lipases
• Hydrolysis of ester bonds in tri-, di-, and
monoacylglycerols to glycerol and free fatty acids.

• Organic media: esterification, transesterification

and interesterification.

• Industrial applications: production of

pharmaceuticals, agrochemicals, structured
lipids, biolubricants, sugar esters, resolution of
racemates, biopolymers, biodiesel, cosmetics and
flavors.

A.A. Mendes, P.C. Oliveira, H.F. de Castro. J. Mol. Catal. B: Enzym. 78 (2012) 119–134.
 Lipases: Interfacial activation

R. Fernández-Lafuente. J. Mol. Catal. B: Enzym. 62 (2010) 197–212.

 Lipase Sources

• Microorganisms: bacteria and fungi

(filamentous and yeasts).

• Tissue animals: porcine pancreas.

• Several plants: seed oils, cereals and latex.

• The most plant lipases used in biotransformation

reactions – seed oils and latex.

R.W.M. Mounguengui , C. Brunschwig, B. Baréa , P. Villeneuve, J. Blin. Progress Energy Combust. Sci. 39 (2013)
441–456.
 Plant lipases – Advantages
• They are available from natural sources (easy
accessibility).

• Low cost and high substrate specificity.

• No requirement of immobilization step (naturally

“immobilized” onto solid materials from seeds).

• Interesting alternative for commercial exploitation

as industrial enzymes.

P. Villeneuve. Eur. J. Lipid Sci. Technol. 105 (2003) 308–317.

 Plant Lipases
• Seed oils: castor bean (Ricinus communis), physic
nut (Jatropha curcas L.), Rape (Brassica napus),
sunflower (Helianthus annuus) soybean (Glycine
max), etc.

• Cereals: rice bran (Oryza sativa), wheat germ

(Triticum aestivum L.), barley (Hordeum vulgare),
corn (Zea mays), etc.

• Latex: papaya (Carica papaya) and babaco

(Vasconcellea × heilbornii; syn. Carica pentagona).

R.W.M. Mounguengui , C. Brunschwig, B. Baréa , P. Villeneuve, J. Blin. Progress Energy Combust. Sci. 39 (2013)
441–456.
 Plant Lipases
• These enzymes are found in dormant and
germinated seeds.

A.-L. Quettier, P.J. Eastmond. Plant Physiol. Biochem. 47 (2009) 485–490.

 Seed oil Lipases: Germination
Passium fruit
Hydrolytic Activity (IU/g crude extract)

90 Corn
Sunflower
Castor bean
75

60

45

30

15
Dormant Germinated
physic nut physic nut
0
0 20 40 60 80 100 Zymogram of hydrolysis of ester catalyzed by VEED
(lanes 1, 2 and 3) and by VEEG (lines 4, 5 and 6).
Germination (h)

K.C. Santos, D.M.J. Cassimiro, M.H.M. Avelar, D.B. Hirata, H.F. de Castro, R. Fernández-Lafuente, A. A. Mendes. Ind.
Crops Prod. 49 (2013) 462– 470 .
J.S. Sousa, E.D.C. Oliveira, D.A.G. Aranda, D.M.G. Freire. J. Mol. Catal. B: Enzym. 65 (2010) 133–137.
 Seed Oil Lipases – pH
• Lipase from germinated Physic nut
100
seeds: pH 8.0 (Santos et al., 2010).

80
• Lipase from germinated rape seeds:
Relative activity (%)

pH 7.0 (Sana et al., 2004).

60

• Lipase from germinated peanut

40
seeds: pH 8.5 (Sanders and Pattee,
1975).
20 Castor bean
Sunflower
Corn
Passion fruit • Lipase from germinated soybean
0
4 5 6 7 8
seeds: pH 8.0 (Gadge et al., 2011).
pH

N.K. Sana, I. Hossin, E.M. Haque, R.K. Shaha. Pakistan J. Biol. Sci. 7 (2004) 246.–252.
T.H. Sanders, H.E. Pattee. Lipids 10 (1975) 50–64.
P.P. Gadge, S.D. Madhikar, J.N. Yewle, U.U. Jadhav, A.D. Chougale, V.P. Zambare, M.V. Padul. Am. J. Biochem.
Biotechnol. 7 (2011) 141–145.
 Seed Oil Lipases – Temperature
100 • Lipase from germinated Physic nut
seeds: pH 40°C (Santos et al.,
2010).
80
Relative activity (%)

• Lipase from germinated rape seeds:

37°C (Sana et al., 2004).
60

• Lipase from germinated soybean

Corn seeds: 24°C (Gadge et al., 2011).
40 Castor bean
Sunbflower
Passion fruit
30 35 40 45 50 55 60

Temperature (°C)

K.C. Santos, D.M.J. Cassimiro, M.H.M. Avelar, D.B. Hirata, H.F. de Castro, R. Fernández-Lafuente, A. A. Mendes.
Ind. Crops Prod. 49 (2013) 462– 470 .
 Hydrolytic activity of plant lipases

K.C. Santos, D.M.J. Cassimiro, M.H.M. Avelar, D.B. Hirata, H.F. de Castro, R. Fernández-Lafuente, A. A. Mendes.
Ind. Crops Prod. 49 (2013) 462– 470 .
 Substrate specificity – Castor bean
lipase (Hydrolysis)

P.J. Eastmond. J. Biol. Chem. 279 (2004) 45540– 45545 .

 Preparation of Delipidated Extracts
Sterilization with Ratio 1:2 (%m/v)
0.5% (v/v) NaClO

Endosperm tissue
removal

Trituration
Cold acetone
(e.g. knife-mill) (20% m/v)

Delipidation with cold

Ratio 1:5 (%m/v)
acetone

Filtration

Particle size
classification
K.C. Santos, D.M.J. Cassimiro, M.H.M. Avelar, D.B. Hirata, H.F. de Castro, R. Fernández-Lafuente, A. A. Mendes. Ind.
Crops Prod. 49 (2013) 462– 470 .
Biotechnological Applications –
Hydrolysis of Triacylglycerols
• Free fatty acids (FFA): important class of compounds for
the production of esters of industrial interest (biodiesel,
biolubricants, sugar esters, plasticizers, flavor esters, additives
for diesel fuels, etc.).
 Hydrolysis of Canola Oil
• Biocatalyst: 2% m/v of
enzymatic crude extract
from dormant castor bean
seeds.
• Reaction time: 2 h.
• Stirring: 1000 rpm
(stirred-tank reactor).
• pH: 4.5
• Reaction temperature:
37.5°C.

M.H.M. Avelar, D.M.J. Cassimiro, K.C. Santos, R.C.C. Domingues, H.F. de Castro, A. A. Mendes. Ind. Crops Prod. 44
(2013) 452– 458 .
 Hydrolysis of Soybean, Palm and
Babassu oils
• Soybean oil: 44.1% m/m
100 oil:buffer, 37◦C, 2.0% m/m
of crude lipase extract and
80 100 mM sodium acetate
pH 4.5.
Hydrolysis (%)

60
• Babassu and palm oils:
40 15.9% m/m oil:buffer,
43.5◦C, 3.0% m/m of crude
20 Babassu oil lipase extract and 100 mM
Palm oil
Soybean oil
sodium acetate pH 4.5.
0
0 20 40 60 80

Reaction time (min)

K.C. Santos, D.M.J. Cassimiro, M.H.M. Avelar, D.B. Hirata, H.F. de Castro, R. Fernández-Lafuente, A. A. Mendes. Ind.
Crops Prod. 49 (2013) 462– 470 .
 Ester synthesis - Hydroesterification

Hydrolysis • Triacylglicerols + 3 H2O ↔ 3 FFA + Glycerol

Esterification • FFA + Alcohol ↔ Ester + H2O

A.P.P. Bressani, K.C.A. Garcia, D.B. Hirata, A.A. Mendes. Bioprocess Biosyst. Eng. 38 (2015) 287–297 .
E.C.G. Aguieiras, E.D. C. Oliveira, A.M. de Castro, M.A.P. Langone , D.M.G. Freire. Fuel 135 (2014) 315–321.
 Biolubricant Synthesis – Hydrolysis
Step of Macaw Palm Oil
• Hydrolysis: 35◦C, 6.0% m/m
100 of crude lipase extract from
dormant castor bean seeds, pH
80 4.5 and stirring of 1000 rpm
(stirred-tank reactor).
Hydrolysis (%)

60
• Esterification reaction:
Esterification of purified FFA
40
with hexanol catalyzed by
35% m/m immobilized Thermomyces
20 40% m/m lanuginosus lipase on poly-
46.8% m/m hydroxybutyrate particles
0 (conversion ≈92% after 15 min
0 20 40 60 80 100 120 140 of reaction).
Reaction time (min)

A.P.P. Bressani, K.C.A. Garcia, D.B. Hirata, A.A. Mendes. Bioprocess Biosyst. Eng. 38 (2015) 287–297 .
 Biodiesel Synthesis – Hydrolysis Step
of Macaw Palm Oil
• Hydrolysis: Complete hydrolysis of macaw palm oil at 50% m/v after 6 of
reaction catalyzed by crude lipase extract from dormant castor bean seeds (7.8
U/g of oil), pH 4, 30°C under continuous stirring.

• Esterification reaction: Esterification of purified FFA with ethanol (molar

ratio ethanol:FFA 2:1) catalyzed by several lipases at 40°C (conversion ≈91%
after 8 h of reaction).

E.C.G. Aguieiras, E.D. C. Oliveira, A.M. de Castro, M.A.P. Langone , D.M.G. Freire. Fuel 135 (2014) 315–321.
 Biodiesel Synthesis – Hydrolysis Step
of Physic Nut Oil
• Hydrolysis: 97%
hydrolysis using 10% m/m
of germinated physic nut
seeds at pH 8.0, 10% m/m
of oil at 40°C after 2 h of
reaction.

• Esterification reaction:
Esterification of purified
FFA with
methanol/ethanol (molar
ratio alcohol:FFA 3:1)
catalyzed by niobic acid at
200°C (conversion ≈97%
after 2 h of reaction).

J.S. Sousa, E.D.C. Oliveira, D.A.G. Aranda, D.M.G. Freire. J. Mol. Catal. B: Enzym. 65 (2010) 133–137.
 Esterification – Germinated Rape
Seeds
• Flavor and fragrance esters: used in food, beverage, cosmetic,
pharmaceutical and personal care industries.

• Experimental conditions: Esterification reaction at 40 ◦C for 48 h

in hexane medium containing 0.25 M of alcohols and 0.25 M caproic
acid, and 50 g/L of rape seed lipase (4-6 days of germination).

• Maximum ester conversion: 1-butanol (88%)

isoamyl alcohol (93%)
(Z)-3-hexen-1-ol (88%)

M. Liaquat. J. Mol. Catal. B: Enzym. 68 (2011) 59–65.

 Esterification – Castor Bean Lipase
• Structured Lipids (SLs) • Several esters

• Experimental • Experimental
conditions: Reaction conditions: 37°C, castor
temperature of 40°C, bean lipase (germinated
reaction time of 4 h, 20% seeds after 1 day), molar
m/m of castor bean lipase ratio alcohol:FFA (2:1) in
(dormant) previously hexane medium after 6 h of
incubated at pH 4.0 by 4 h, reaction.
and molar ratio
glycerol:FFA of 1:3. • Maximum ester
conversion: ˃90% using
• Maximum ester several alcohols (C1-C4) and
conversion: 52.37% TAG. stearic/oleic acids.

M. Tüter. J. Am. Oil Chem. Soc. 75 (1998) 417– E.R. Su, Y. Zhou, P.Y. You, D.Z. Wei. J. Shangai
420. Univ. 1 4 (2010) 137–144.
 Interesterification – Germinated
Physic Nut Seeds
• Structured Lipids (SLs):
Enriched of vegetable oils with
PUFAs (EPA and DHA) from
fish oil (acidolysis).

• Experimental conditions:
Reaction temperature of 40°C,
reaction time of 24 h, 10% m/m
of biocatalyst, molar ratio
oil:FFA of 1:3 in hexane
ICO: Interesterified coconut oil.
medium.
IPO: Interesterified palm oil.
IOO: Interesterified olive oil.

J.S. Sousa, A.G. Torres, D.M.G. Freire. Grasas y Aceites 66 (2015) e071.
Future Perspectives

• Extraction, Purification and Immobilization by Different

Protocols: Modulation of their catalytic properties (stability,
selectivity and catalytic activity).

• Molecular Genetic Technology: use of bacteria and yeasts as

host organisms to provide sufficient amounts of the enzyme.
 Muchas Gracias

emails: adriano.mendes@unifal-mg.edu.br
adriano_amendes@yahoo.com.br