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Chapter 7

Plant Lipases: Partial Purification of Carica papaya Lipase


Ivanna Rivera, Juan Carlos Mateos-Daz, and Georgina Sandoval
Abstract
Lipases from plants have very interesting features for application in different fields. This chapter provides
an overview on some of the most important aspects of plant lipases, such as sources, applications, physiological functions, and specificities. Lipases from laticifers and particularly Carica papaya lipase (CPL) have
emerged as a versatile autoimmobilized biocatalyst. However, to get a better understanding of CPL biocatalytic properties, the isolation and purification of individual C. papaya lipolytic enzymes become necessary. In this chapter, a practical protocol for partial purification of the latex-associated lipolytic activity from
C. papaya is given.
Key words: Lipase, Laticifer, Seeds, Oleaginous plants

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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a few plant lipases have been purified to homogeneity due to the


complexity of the purification process.
Plant lipases have a large amount of potential applications at
laboratory scale (7, 8). As an example, plant lipases have proven to
be highly specific for fatty acids present within the plant (3); this
feature can be exploited in biotechnological applications, like the
production of biodiesel and tailored lipids (9, 10).
Lipases are used in different industries, like pharmaceutical,
food, production of renewal energy, and detergents (2). However,
the application of plant lipases at industrial scale is complicated due
to the difficulty to extract and obtain a sufficient lipase quantity
from plant material (7, 10). In order to overcome this problem,
cloning and expressing them in an adequate host is one alternative
to be explored.
1.1. Plant Lipase
Sources
1.1.1. Oleaginous Plants

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

Cereals are members of the Poaceae or Gramineae plant families.


They are mostly cultivated to provide food energy, but they are
also a source of lipases. In cereals, grains contain until 10% of lipids, normally located in the embryo and aleurone (12). In wheat,
the majority of the lipolytic activity (7580%) is located in the bran
and 2025% in the germ (13).

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.

pH 8, 50C, aw 0.38, R: sn-1,3, CL:


short, SD: unsaturated
pH 9, 50C, R: sn-3

Babaco (Carica
pentagona)
Carica papaya

Hydrolysis of TG and synthetic


monoesters
Alcoholysis of sunflower oil,
Naproxen resolution
Lipids modification, asymmetric
resolutions

(22, 38, 39)


(14, 23, 27, 28)

Detergent extraction, functional


proteomics

(25)

(6, 23)
(34, 35)
(36)
(37)

(33)

(10, 1921, 31, 32)

(10, 29, 30)

References

Not performed

Detergent (CHAPS) extraction

DEAE-cellulose column
By ion exchange chromatography
Solvent extraction

TG hydrolysis
Oat shelf-life studies
Hydrolysis and esterification

Using octyl-sepharose

Partial by precipitation and


ultrafiltration
By proteomic analysis and expression in Escherichia coli
Not performed

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 5, 60C, CL: short and medium

pH 11, 80C, R: nonspecific on TG,


sn-2 on PC
CL: Long, SD: unsaturated
75C (EII isoenzyme) and 65C
(EIII isoenzyme)
pH 5.5, 3237C

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

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I. Rivera et al.

The main biochemical characteristics and applications of some


plant lipases and its purification methods are given in Table 1.
1.1.4. Carica papaya Lipase

In 1935, lipolityc activity in papaya latex was reported and is known


as C. papaya lipase (CPL). However, it was not until 1991 that the
interest in studying this enzyme started. An sn-3 stereoselectivity
of CPL was observed in interesterification reactions (15) while an
sn-1,3 regioselectivity was observed in the production of structured lipids resembling human milk fat, where CPL had similar
activity to those of the commercial lipases tested (16). In the
production of wax esters from the esterification with oleyl alcohol,
papaya latex is able to esterify long-chain fatty acids (17). CPL has
also been applied in the sitostanol esterification using canola oil
and oleic acid as acyl donor group (18, 19). CPL has also been
used to resolve naproxen and 2-aryl propionic esters, fenoprofen
and ketoprofen (2022). This demonstrates the ability of CPL to
accept a wide variety of substrates (14, 23).

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.

1.2.1. Purification of Carica


papaya Latex Lipase

Most of the lipolytic activity is associated to the latex-insoluble


fraction. Until recently, attempts to isolate latex-associate lipolytic
enzymes were successful. In 2009, Abdelkafi et al. (27) reported
the extraction of 10% of the lipase activity, corresponding to a
GDSL-motif carboxylester hydrolase. As recently as this year
(2011), the first sequence of triacylglycerol lipase from C. papaya
latex was determined by functional proteomics by Dhouib et al.
(28). This enzyme shows 35% identity and 51% similarity to the
castor bean acid lipase, which suggests that it is responsible for an
important part of the lipolytic activity present in the latex.
At this moment, it is unclear which of the lipolytic enzymes in
the latex are responsible for the catalytic activity observed in the
many applications of CPL. Therefore, isolation, purification, cloning, and expression of individual lipolytic enzymes present in
C. papaya become necessary. In this chapter, a practical protocol for
partial purification of the latex-associated lipolytic activity from C.
papaya is given.

Plant Lipases: Partial Purification of Carica papaya Lipase

119

2. Materials
2.1. Partial Purification
of CPL

1. Papaya trees with unripe fruits.

2.1.1. Collection of CPL


Latex (see Note 1)

3. Cutter.

2.1.2. Latex Purification

1. 2-Methyl-2-propanol (terbutyl alcohol).

2. 50-mL Falcon tubes or similar containers.


4. Bucket with ice.

2. n-Hexane technical grade.


3. Solvent solution: 1:1 (v/v) 2-methyl-2-propanol:n-hexane.
4. Extraction solution: 0.1 M TrisHCl, 1 M NaCl, 0.5%
N-lauroyl sarcosine (NLS), pH 8.0 (see Note 2).
5. Bucket with ice.
6. Cold (4C) distilled water.
7. Vortex.
8. Microfuge.
9. Speed-vac or freeze dryer.

3. Methods
3.1. Partial Purification
of CPL
3.1.1. Latex Collection
3.1.2. Latex Purification
(see Note 3)

1. On unripe fruits, perform longitudinal cuts of 23-mm deep


and collect the latex in suitable containers (see Fig. 1). While
collecting or for immediate use, keep the content on ice and
freeze at 20C for later use.
1. Add 50 mL of cold water to 7.5 g of fresh latex and stir vigorously in vortex for 3 min (see Note 4).
2. Centrifuge the sample at 5,000 g (in a microfuge) and 4C
for 20 min and discard the supernatant (see Note 5).
3. Repeat steps 1 and 2 four more times (see Notes 4 and 6).
4. Treat the drained or freeze-dried latex with the solvent solution (50 mL for 1 g of latex) and vortex (see Note 4).
5. Centrifuge at 15,500 g and 4C for 10 min and discard the
supernatant (see Note 7).
6. Repeat steps 4 and 5 at least two more times (see Note 4).
7. Dry the pellet in a speed-vac or by freeze drying.
8. Resuspend the pellet in the extraction solution (50 mL for 1 g
of pellet) and vortex (see Note 4).
9. Centrifuge at 15,500 g, 4C, for 10 min. Discard the supernatant (see Note 8).

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I. Rivera et al.

Fig. 1. Collection of Carica papaya latex from unripe fruits.

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.

Plant Lipases: Partial Purification of Carica papaya Lipase

121

7. Solvent treatment extracts lipids from the latex. This is an


optional step required for biochemical studies (because lipids
could interfere with some activity analysis). Solvent-treated
latex becomes less stable and loses some activity.
8. This supernatant contains soluble esterases. To recover them,
filter the supernatant through a 0.45-m membrane and concentrate by ultrafiltration or in a freeze dryer.

Acknowledgments
This work was supported by CONACYT (Mexico) grant CB-2008104429 and ECOS (France)ANUIES/CONACYT (Mexico)
grant M08-A03.
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