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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
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.
0 20 40 60 80 1000
15
30
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90 Passium fruit Corn Sunflower Castor bean
Hydr
olyt
ic Ac
tivity
(IU/
g cr
ude
extra
ct)
Germination (h)Zymogram of hydrolysis of ester catalyzed by VEED (lanes 1, 2 and 3) and by VEEG (lines 4, 5 and 6).
Dormant physic nut
Germinatedphysic nut
Seed Oil Lipases – pH
4 5 6 7 80
20
40
60
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100
Castor bean Sunflower Corn Passion fruit
Rela
tive
activ
ity (%
)
pH
• Lipase from germinated Physic nut seeds: pH 8.0 (Santos et al., 2010).
• Lipase from germinated rape seeds: pH 7.0 (Sana et al., 2004).
• Lipase from germinated peanut seeds: pH 8.5 (Sanders and Pattee, 1975).
• Lipase from germinated soybean seeds: pH 8.0 (Gadge et al., 2011).
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 • Lipase from germinated Physic nut
seeds: pH 40°C (Santos et al., 2010).
• Lipase from germinated rape seeds: 37°C (Sana et al., 2004).
• Lipase from germinated soybean seeds: 24°C (Gadge et al., 2011).
30 35 40 45 50 55 60
40
60
80
100
Corn Castor bean Sunbflower Passion fruit
Rela
tive
activ
ity (%
)
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 0.5% (v/v) NaClO
Endosperm tissue removal
Trituration (e.g. knife-mill)
Delipidation with cold acetone
Filtration
Ratio 1:2 (%m/v)
Cold acetone (20% m/v)
Ratio 1:5 (%m/v)
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 oil:buffer, 37◦C, 2.0% m/m of crude lipase extract and 100 mM sodium acetate pH 4.5.
• Babassu and palm oils: 15.9% m/m oil:buffer, 43.5◦C, 3.0% m/m of crude lipase extract and 100 mM sodium acetate pH 4.5.
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 .
0 20 40 60 800
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Babassu oil Palm oil Soybean oil
Hydr
olys
is (%
)
Reaction time (min)
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
A.P.P. Bressani, K.C.A. Garcia, D.B. Hirata, A.A. Mendes. Bioprocess Biosyst. Eng. 38 (2015) 287–297 .
0 20 40 60 80 100 120 1400
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40
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35% m/m 40% m/m 46.8% m/m
Hydr
olys
is (%
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Reaction time (min)
• Hydrolysis: 35◦C, 6.0% m/m of crude lipase extract from dormant castor bean seeds, pH 4.5 and stirring of 1000 rpm (stirred-tank reactor).
• Esterification reaction: Esterification of purified FFA with hexanol catalyzed by immobilized Thermomyces lanuginosus lipase on poly-hydroxybutyrate particles (conversion ≈92% after 15 min of reaction).
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)
• Experimental
conditions: Reaction temperature of 40°C, reaction time of 4 h, 20% m/m of castor bean lipase (dormant) previously incubated at pH 4.0 by 4 h, and molar ratio glycerol:FFA of 1:3.
• Maximum ester conversion: 52.37% TAG.
M. Tüter. J. Am. Oil Chem. Soc. 75 (1998) 417–420.
• Several esters
• Experimental conditions: 37°C, castor bean lipase (germinated seeds after 1 day), molar ratio alcohol:FFA (2:1) in hexane medium after 6 h of reaction.
• Maximum ester conversion: ˃90% using several alcohols (C1-C4) and stearic/oleic acids.
E.R. Su, Y. Zhou, P.Y. You, D.Z. Wei. J. Shangai 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 medium.
J.S. Sousa, A.G. Torres, D.M.G. Freire. Grasas y Aceites 66 (2015) e071.
ICO: Interesterified coconut oil. IPO: Interesterified palm oil. IOO: Interesterified olive oil.
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.
