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1. Introduction
Lipases (triacylglycerol hydrolase EC. 3.1.1.3) are enzymes that
hydrolyze triacylglycerols to diacylglycerol, monoacylglycerol, fatty
acids, and glycerol. Lipases are produced in several microorganisms, plants, and animals. They also catalyze synthesis reactions,
such as aminolysis, alcoholysis, and interesterification (1).
Lipases from microorganisms are the most studied lipases; however, other sources of lipases could be plants and animals (2). In the
case of plants, lipases are mostly present in oilseeds and laticifers
(secretory cells in the leaves and/or stems of plants that produce
latex and rubber). Plant lipases have two principal functions: to
provide energy by hydrolyzing the oils stored in the seeds (3) and
protection (e.g., as antifungic (phytopathogen) agents), like in the
case of laticifer lipases and some lipases from Arabidopsis (4, 5).
Plant lipases have interesting biochemical properties: pH
ranges between 4 and 9 and remains active at temperatures between
37 and 80C (3, 6). The isolation and purification of plant lipases
are carried out with relatively simple techniques; nevertheless, only
Georgina Sandoval (ed.), Lipases and Phospholipases: Methods and Protocols, Methods in Molecular Biology, vol. 861,
DOI 10.1007/978-1-61779-600-5_7, Springer Science+Business Media New York 2012
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Lipases are found in oleaginous plants due to their high triacylglycerol content. Some examples of this kind of plants are corn,
sunflower, rapeseed, castor bean, or sesame beans. In oilseeds,
triacylglycerols function as energy reserve source. They are stored
in the form of oil bodies, used in the production of energy, and
quickly consumed during the germination process. The first step
involves the mobilization of fatty acids, which is controlled by
different lipolytic enzymes. In fact, the lipolytic activity in seeds
normally appears during the germination (11, 12). In seeds, lipases
also can be found in subcellular compartments, in which they may
be free or associated with organelles (11).
1.1.2. Cereals
1.1.3. Laticifers
Other source of plant lipases are laticifers. Latex produced by laticifers is a milky fluid constituted by different compounds, like proteins, alkaloids, resin, sugars, oils, etc., all of which disperse in an
aqueous medium. Most of the lipolytic activity in the latex is related
to its insoluble fraction (this is the case for many laticifers, especially in Caricaceas). In addition, because lipolytic enzymes are
mostly trapped in the insoluble fraction, they are considered as
naturally immobilized enzymes (14). The presence of lipolytic
activity in the latex in several plant families, including Asclepciadaceae, Moraceae, Apocynaceae, Euphorbaceae, Caricacea, and
Bromeliacea was reported as early as 1935 (8). The function of
these enzymes has not been fully elucidated; however, it is known
that they are involved in the metabolism of terpenes and plant
defense against external agents.
Babaco (Carica
pentagona)
Carica papaya
(25)
(6, 23)
(34, 35)
(36)
(37)
(33)
References
Not performed
DEAE-cellulose column
By ion exchange chromatography
Solvent extraction
TG hydrolysis
Oat shelf-life studies
Hydrolysis and esterification
Using octyl-sepharose
Purification
Phospholipid hydrolysis
Biodiesel synthesis
Esterification of fatty acids and
glycerol
Applications
TG triglycerides, PC phosphatidylcholina
a
Optimal working conditions (pH, T) and selectivity (R regioselectivity, CL chain-length preference, SD fatty acid saturation degree preference)
pH 7.5, 37C
pH 4.5, R: sn-1 and sn-2, CL: short
and medium, SD: unsaturated
pH 8.5, 3040C, R: sn-1,3
Biochemical characteristicsa
Laticifers
Euphorbia characias
Wheat
Corn
Oat
Cereals
Rice (bran)
Coconut
Oleaginous plants
Jatropha curcas
Castor bean
Source
Table 1
Biochemical characteristics and purification of some plant lipases
7
Plant Lipases: Partial Purification of Carica papaya Lipase
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1.2. Purification of
Lipases from Laticifers
The extraction of lipases from the latex polymer matrix has not
been easy. Classically, the first step for the enrichment of the lipase
activity present in the latex is the removal of soluble proteins in
the mixture or a mix of water and a solvent in order to eliminate
lipids present in the sample and achieve the extraction (2426).
Other strategy that has been employed is the use of some nonionic and zwitterionic detergents (CHAPS, Triton TX-100, etc.).
In the case of Euphorbia characias lipase, Fiorillo et al. (25) studied various detergents for extraction and combined them with
various physical and chemical processes, including the use of a
zwitterionic detergent (CHAPS) which improved the extraction
yield.
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2. Materials
2.1. Partial Purification
of CPL
3. Cutter.
3. Methods
3.1. Partial Purification
of CPL
3.1.1. Latex Collection
3.1.2. Latex Purification
(see Note 3)
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I. Rivera et al.
10. Dry the pellet in a speed-vac or by freeze drying and test the
lipase activity by your usual activity assays.
4. Notes
1. Alternatively, commercial C. papaya latex could be used, but
lipolytic activity is higher in freshly collected samples.
2. Reagent concentrations and pH were previously optimized.
3. The entire procedure should be carried out on ice or in a cold
room in order to avoid sample degradation.
4. The smaller the samples, the better the results in washing. The
same for solvent and NLS treatment. A good approach is to
weigh 20 mg of latex in 1.5-mL microtubes and adding 1 mL
of water, solvent, or extraction solution, but for great quantities of latex it is better to perform more washes.
5. Supernatant contains papain (protease) and other water-soluble enzymes. If desired, recover these enzymes.
6. Five cold-water washes assure the protease elimination in our
samples, but preferably check protease activity in the
supernatant.
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Acknowledgments
This work was supported by CONACYT (Mexico) grant CB-2008104429 and ECOS (France)ANUIES/CONACYT (Mexico)
grant M08-A03.
References
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(2000) Customizing lipases for biocatalysis: a
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2. Hasan F, Shah AA, Hameed A (2006) Industrial
applications of microbial lipases. Enzym Microb
Technol 39:235251
3. Mala Pahoja VS, Ali M (2002) A review of
enzymatic properties of lipase in plants, animals
and microorganisms. Pak J Appl Sci 2:474484
4. Kwon SJ, Jin HC, Lee S et al (2009) GDSL
lipase-like 1 regulates systemic resistance associated with ethylene signaling in Arabidopsis.
Plant J 58:235245
5. Lee DS, Kim BK, Kwon SJ et al (2009)
Arabidopsis GDSL lipase 2 plays a role in
pathogen defense via negative regulation of
auxin signaling. Biochem Biophys Res Com
379:10381042
6. Bhardwaj K, Raju A, Rajasekharan R (2001)
Identification, purification, and characterization of a thermally stable lipase from rice bran.
A new member of the (Phospho) lipase family.
Plant Physiol 127:17281738
7. Villeneuve P (2003) Plant lipases and their
applications in oils and fats modification. Eur J
Lipid Sci Tech 105:308317
8. Paques FW, Macedo GA (2006) Plant lipases
from latex: properties and industrial applications. Quim Nova 29:9399
9. Mukherjee KD (1994) Plant lipases and their
application in lipid biotransformations. Prog
Lipid Res 33:165
10. de Sousa JS, Cavalcanti-Oliveira EdA, Aranda
DAG et al (2010) Application of lipase from
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29.
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