VIETNAM GENERAL CONFEDERATION OF LABOUR

TON DUC THANG UNIVERSITY

FACULTY OF APPLIED SCIENCES

SEMINAR PROTEIN AND ENZYME TECHNOLOGY

EXTRACTION, ISOLATION AND

PURIFICATION OF LIPASE

Supervisor: Ph.D NGUYEN THI CAM VI

Student group: 09

HO CHI MINH CITY, 2024

 VIETNAM GENERAL CONFEDERATION OF LABOUR
TON DUC THANG UNIVERSITY
FACULTY OF APPLIED SCIENCES

SEMINAR PROTEIN AND ENZYME TECHNOLOGY

EXTRACTION, ISOLATION AND

PURIFICATION OF LIPASE

Supervisor: Ph.D NGUYEN THI CAM VI

Student group: 1/ DANG PHAN THAI HA - 622H0002
2/ NGUYEN QUAN VAN ANH - 622H0026
3/ QUAN THIEN PHUC - 622H0034

HO CHI MINH CITY, 2024

 i

ACKNOWLEDGEMENT

I would like to express my sincere gratitude to Ms. Nguyen Thi Cam Vi,
who has been an invaluable mentor throughout my thesis journey. Her
guidance and encouragement have been instrumental in helping me develop a
comprehensive understanding of this topic. Thanks to her insightful feedback, I
was able to refine my research questions and strengthen my arguments.
I also want to thank the school library for providing me with access to a
wealth of resources. Their support was essential in helping me find the
information I needed to complete my research.
I am thrilled to have finally completed this thesis. Once again, I would
like to express my deepest appreciation to everyone who has supported me.
We sincerely thank you!
Ho Chi Minh City, November 2024
 ii

CONTENTS

ACKNOWLEDGEMENT....................................................................................i
CONTENTS........................................................................................................ii
LIST OF TABLES..............................................................................................iv
LIST OF FIGURES.............................................................................................v
LIST OF ABBREVIATIONS............................................................................vi
CHAPTER 1: OVERVIEW.................................................................................1
1.1. Enzyme lipase........................................................................................1
1.1.1. What is the structure of lipase enzyme?.......................................1
1.2. Sources of lipase enzymes?....................................................................3
1.2.1. Plant lipases..................................................................................3
1.2.2. Animal lipases..............................................................................3
1.2.3. Bacterial lipase..............................................................................3
1.2.4. Fungal lipases...............................................................................4
1.3. Role of lipase enzyme............................................................................4
1.3.1. Types of lipases and their sites of action:.....................................5
1.3.2. Roles of lipase in the body............................................................5
1.4. Classification of lipases..........................................................................5
CHAPTER 2: EXTRACTION, ISOLATION AND PURIFICATION OF
LIPASE................................................................................................................7
2.1. Methods for extracting lipase from plants..............................................7
2.1.1. Preparation of Crude Plant Material.............................................7
2.1.2. Purification of Plant Lipases.........................................................8
2.2. Obtaining lipase from papaya latex........................................................9
2.2.1. Conditions for latex collection......................................................9
2.2.2. Time for latex collection.............................................................10
2.2.3. Procedure for collecting papaya latex.........................................10
2.2.4. Procedure for Crude Lipase Extraction from Papaya Latex.......10
 iii

2.3. Extraction and Purification of Lipase from Crude Papaya Latex........11

2.3.1. Extraction of lipase using a buffer solution containing SLS......12
2.3.2. Fractional Precipitation of Lipase with Ammonium Sulfate......12
2.3.3. Ion Exchange Chromatography for Lipase Purification.............13
2.3.4. Electrophoretic method...............................................................14
2.4. Activity of purified lipase....................................................................16
2.4.1. Determination of lipase molecular weight..................................16
2.4.2. Determine the kinetic parameters Km and Vmax...........................17
CHAPTER 3: APPLICATION..........................................................................19
3.1. Application in food industry................................................................19
3.1.1. Lipase in dairy industry..............................................................20
3.2. Lipase in detergent industry.................................................................22
3.3. Lipase in pharmaceutical......................................................................22
3.4. Lipase in biodiesel industries...............................................................23
REFERENCES..................................................................................................25
Vietnamese........................................................................................................25
English...............................................................................................................25
 iv

LIST OF TABLES

Table 2.1 : Components for preparing a 15% polyacrylamide gel (for 12 cm x 10

cm glass plates)..................................................................................................16
Table 3.2 : Lipase applications in the food industry [17].....................................19
Table 3.3 : Examples of lipase in cheese production............................................20
 v

LIST OF FIGURES

Figure 1.1 : Lipase enzyme in living cells..............................................................2

Figure 1.2 : Structure of lipase enzyme.................................................................2
Figure 1.3 : Enzyme lipase.....................................................................................5
Figure 2.1 :Immature papaya with a smooth surface............................................10
Figure 2.2 : Flowchart for crude lipase extraction from dried papaya latex.........11
Figure 2.3 : Dialysis to remove (NH4)2SO4 salt from protein precipitate.............13
Figure 2.4 : Column chromatography process......................................................14
Figure 2.5 : Image from SDS-PAGE....................................................................17
Figure 2.6 : The Lineweaver-Burk plot was used to determine the kinetic
parameters, Km and Vmax, of papaya latex lipase................................................18
Figure 3.1 : Applications of lipase in industries...................................................19
 vi

LIST OF ABBREVIATIONS

A
P Ammonium persulfate
S
A
Ammonium sulfate
S
C 3-[(3-
H Cholamidopropyl)dimet
A hylammonio]-1-
P propanesulfonate
S hydrate
S
D sodium dodecyl sulfate
S
S
L Sodium Lauryl Sulfate
S
 1

CHAPTER 1: OVERVIEW

1.1. Enzyme lipase

Lipase, an ester hydrolase, specializes in catalyzing the hydrolysis of

lipid molecules. This enzyme is indispensable for the digestive process,
transporting, and processing dietary fats such as triglycerides in most living
organisms, including some viruses. Lipases typically act on specific sites (A1,
A2, or A3) of the glycerol backbone of lipid substrates, primarily within the
small intestine. Human pancreatic lipase (HPL), a prime example, is the key
enzyme that breaks down triglycerides in human digestion, yielding
monoglycerides and free fatty acids.
Lipase catalyzes the hydrolysis of triacylglycerol into diacylglycerol and
a fatty acid anion. It belongs to the hydrolase family, catalyzing the breakdown
of fats. This enzyme is involved in the digestion of fats in our body. It is
present in the glands of the mouth and pancreas. This enzyme is also found in
gastric juice and lysozyme. There are two types of lipases: acid lipases and
alkaline lipases. Acid lipases include lingual lipase and gastric lipase, while
alkaline lipases include pancreatic lipase. Lingual lipase is thought to be the
first step in lipid digestion. It is mainly active in infants because pancreatic
enzymes are not fully developed.

1.1.1. What is the structure of lipase enzyme?

Lipase is a subfamily of esterase. Lipases play essential roles in the

digestion, transport, and processing of dietary fats in most organisms. They are
distinguished from esterases by the fact that they catalyze the hydrolysis of
water-insoluble substrates (such as triglycerides). The main structure of lipase
usually includes an alpha/beta hydrolase fold, a structure consisting of eight
parallel beta sheets surrounded by alpha helices on both sides. This is the
region responsible for the catalytic activity of the enzyme [12].
2

Figure 1.1: Lipase enzyme in living cells

Figure 1.2: Structure of lipase enzyme

 3

1.2. Sources of lipase enzymes?

1.2.1. Plant lipases

Plant lipases have been isolated from leaves, oils, stems and seeds of oil-
bearing plants and cereals. These enzymes are of interest due to their easy
availability, low cost and diversity. However, the substrate specificity of plant
lipases for industrial applications has not been fully determined due to a lack of
research on the production of plant lipases. Direct applications using crude
lipase extracts from seed oils have opportunities in the biofuel field as it does
not require purification and immobilization steps, reducing production costs
[13].

1.2.2. Animal lipases

Animal lipases used in industrial processes include pancreatic and

forestomach lipases. The main application of pancreatic lipase is the production
of industrial phospholipase A2 (PLA2) used to produce lysolecithin, a natural
emulsifier for food, cosmetics and the pharmaceutical industry. Crude porcine
pancreatic lipase is one of the most widely used enzymes because it is cheaper
than other animal lipases. However, the semi-pure form of this lipase is very
expensive.
Currently, the main commercial source of lipase is bacteria, yeast and
fungi. This change may be due to the fact that lipases and phospholipases from
natural sources do not always meet the requirements of industrial biocatalysis
in terms of activity, stability and specificity [13].

1.2.3. Bacterial lipase

Bacterial lipases are primarily extracellular enzymes and their

production is significantly influenced by the composition of the culture
medium. Agricultural by-products are low-cost raw materials for lipase
 4

production and address the waste problem. Bacillus sp., Pseudomonas sp. and
Burkholderia sp. are the most common bacterial species widely used for lipase
production, mainly due to their high enzyme activity.
In general, Bacillus lipases are easy to produce and can function in
organic solvents, having advantages in the synthesis of esters, serving the food,
cosmetics and biodiesel production industries. Many lipases maintain activity
at high temperatures and pH, in the presence of surfactants, hydrogen peroxide,
sodium hypochlorite, so they can be applied in detergent formulations [13].

1.2.4. Fungal lipases

Fungi have the ability to produce lipases that can grow in a number of
habitats, including used vegetable oils, oil-contaminated soil as well as spoiled
food and milk. The fungal genera of greatest interest for lipase production are
Aspergillus, Rhizopus, Penicillium, Mucor, Geotrichum and Fusarium.
Filamentous fungi are also considered a good source of extracellular lipases for
large-scale industrial production [13].

1.3. Role of lipase enzyme

Lipase is an enzyme that plays an important role in the process of fat

digestion in our body. It acts as a "biological scissor", cutting large fat
molecules into smaller ones for the body to easily absorb.
Main functions of lipase:
 Fat digestion: Lipase breaks down triglycerides (a type of fat) into fatty
acids and glycerol. These substances are then absorbed by the body and
used to provide energy or storage.
 Fat absorption: The breakdown products of fats after being processed by
lipase become more water-soluble, helping the body absorb them through
the small intestine wall.
 Fat transport: Fatty acids and glycerol, after being absorbed, are transported
to different cells in the body for use or storage.
 5

1.3.1. Types of lipases and their sites of action:

Lingual lipase: The first lipase to come into contact with food, starting
the process of fat breakdown in the mouth.
Gastric lipase: Continues the process of fat breakdown in the stomach.
Pancreatic lipase: This is the most important type of lipase, secreted
from the pancreas and acts strongly in the small intestine.

1.3.2. Roles of lipase in the body

Providing energy: Fats are an important source of energy for the body.
Lipase helps the body convert fats into energy.
Absorbing fat-soluble vitamins: Vitamins like A, D, E, and K need fat to
be absorbed. Lipase helps this process to happen effectively.
Protecting internal organs: A layer of fat surrounds the internal organs,
protecting them from external impacts.[14]

1.4. Classification of lipases

There are different types of lipases, classified based on where they are
produced:

Figure 1.3: Enzyme lipase

 6

Pancreatic lipase: This is the most important lipase in humans when it

comes to digestion. It is produced in the pancreas and secreted into the small
intestine, where it acts on dietary fats.
Hepatic lipase: Produced in the liver, hepatic lipase helps regulate the
amount and type of lipids in the body. It is important for lipoprotein
metabolism, transporting fat through the blood.
Lingual lipase: Produced in the salivary glands, lingual lipase initiates
fat digestion in the mouth. It continues to function in the stomach due to its
acid stability.
Gastric lipase: Secreted by the chief cells in the stomach, gastric lipase
also contributes to fat digestion in the stomach.
Endothelial lipase: This lipase is mainly found in blood vessels, where it
is involved in the metabolism of high-density lipoprotein cholesterol (HDL).
[15]
 7

CHAPTER 2: EXTRACTION, ISOLATION AND

PURIFICATION OF LIPASE

2.1. Methods for extracting lipase from plants

Plant lipases are often used in the form of crude extracts or partially
purified extracts, or as pure lipases obtained through extraction methods.
Several methods for extracting lipase from plants are described below. Each
method typically involves several different steps depending on the plant part
used or the desired degree of purity. The biomass processing steps commonly
used to extract lipase include: preparing the crude plant extract and purifying
the lipase.

2.1.1. Preparation of Crude Plant Material

The preparation of crude plant material typically involves two steps:

grinding the biomass and removing lipids from the ground biomass.
Except for latex, plant enzyme extraction usually begins by grinding
different parts of the raw material. Grinding breaks down the plant biomass
into smaller particles, increasing the surface area exposed to the external
environment and enhancing the enzyme's ability to interact with the extraction
solvent [6].
Removing lipids from the crude plant material using organic solvents is
a necessary step. Lipids present in the biomass, primarily fatty acids and
triacylglycerols, can interfere with the extraction process and affect lipase
activity. Lipid removal creates favorable conditions for aqueous lipase
extraction, increasing extraction yield and lipase activity. Research by
Kapranchikov and colleagues showed that removing lipids before enzyme
extraction improved lipase activity from wheat germ by 1.8 times. Kazim Kose
also used acetone to remove lipids from rice bran before lipase extraction [8].
 8

The most effective solvents for removing lipids from plants are diethyl
ether, acetone, and n-hexane. The choice of solvent in this step is crucial as
some organic solvents can inhibit the catalytic activity of enzymes. In many
cases, n-hexane often gives the best results, with negligible lipase loss, and n-
hexane is highly volatile. According to Hassanien, cold acetone (20°C) can also
be a good solvent for lipid removal. This is because acetone can preserve lipase
activity [8]."

2.1.2. Purification of Plant Lipases

The purification of plant lipases aims to obtain pure lipase, facilitating

research on their properties and structure. Typically, lipase purification
techniques involve the following processes: enzyme extraction with buffer
solution, precipitation, and column chromatography. This process is applicable
to water-soluble lipases. For water-insoluble lipases, such as those from papaya
latex, an initial pre-extraction step using a solution containing surfactants is
required to extract the lipase from the papaya latex structure.
Several research efforts to isolate lipase from papaya latex have been
unsuccessful. This can be explained by the covalent bonding of lipase to
isoprene polymers in the latex. Therefore, Abdelkafi and colleagues used a
Tris-HCl pH 8 buffer solution containing 20 mM CHAPS surfactant to
solubilize papaya latex and partially isolate the enzyme. However,
characterization results showed that this enzyme exhibited more esterase
activity than lipase activity [2].
CHAPS, chemically known as 3-[(3-
Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, is a
zwitterionic surfactant used to solubilize proteins. SLS, or Sodium N-Lauroyl
Methyl Glycinate, is composed of the sodium salt of a fatty acid and the amino
acid Sarcosine (Methyl Glycinate), and is an anionic surfactant. This surfactant
can extract proteins from the polymer structure of papaya latex.
 9

Based on these findings, a Tris-HCl pH 8 buffer solution containing the

surfactant SLS was selected in this study to extract lipase from the papaya latex
structure.
The next step in the lipase extraction process is precipitation. The most
common technique used to precipitate lipase proteins is ammonium sulfate
(AS) fractional precipitation. Lipase from papaya seeds and peel has also been
extracted and precipitated using AS [90], and lipase from oat seeds precipitated
with 50% saturated AS had an activity 1.97 times higher than that of the crude
lipase [4].
Research by several authors has shown that the highest lipase activity is
often achieved in precipitate fractions with AS saturation concentrations
ranging from 60% to 90%. To effectively recover lipase after the precipitation
step, the AS salt must be removed by dialysis.
In addition to AS precipitation, acetone precipitation can also be used at
appropriate ratios. Organic solvents are generally used less frequently as they
can cause irreversible denaturation of lipase proteins [7].
Chromatography is the most widely applied method in the purification
of plant lipases after the precipitation step (pre-purification). Chromatography
separates different proteins contained in the precipitate based on their affinity
for the stationary phase. The most commonly used techniques are ion exchange
chromatography and hydrophobic interaction chromatography. Lipase from
castor bean has been purified using ion exchange chromatography with a
Diethylaminoethyl (DEAE) stationary phase [115]. Lipase (EpLL) from cactus
pear latex has also been purified by anion exchange chromatography on a
DEAE-Cellulose column with a 12.57-fold purification [3].

2.2. Obtaining lipase from papaya latex

2.2.1. Conditions for latex collection

Latex is collected from green fruits with a smooth skin and a weight of
0.3-0.5 kg. Overly young or old fruits yield a small amount of latex that is not
viscous and has low enzyme activity. To obtain the highest enzyme activity
 10

product, latex should be collected from fruits that are approximately 10 weeks
old [1].

Figure 2.4:Immature papaya with a smooth surface

2.2.2. Time for latex collection

Latex collection should begin between 6 and 8 am when the humidity is

high. Papaya latex is collected at the beginning of September.

2.2.3. Procedure for collecting papaya latex

Use a razor blade to make several vertical cuts along the fruit at its
largest diameter, with the cuts spaced 3-5 cm apart (do not cut deeper than 2
mm, otherwise water and starch from the fruit will mix with the latex and
reduce the quality of the collected latex).
Collect the flowing latex in a plastic bottle for 4-6 minutes. After
collecting the latex, seal the lid tightly and store it immediately in a
refrigerator. Then, transfer it to a laboratory for storage at -20°C [1].

2.2.4. Procedure for Crude Lipase Extraction from Papaya Latex

Papaya latex collected from the orchard was frozen and freeze-dried at -
40°C for 16 hours to obtain dry papaya latex.
Diagram of crude lipase extraction from papaya latex:
 11

Figure 2.5: Flowchart for crude lipase extraction from dried papaya latex

Step 1: Add cold distilled water to the dry latex and stir for 5 minutes.
Step 2: Centrifuge the sample at 6000 rpm for 20 minutes at 4°C, and remove
the supernatant.
Step 3: Repeat steps 1 and 2 three more times.
Step 4: The obtained precipitate was freeze-dried at -40°C for 16 hours. The
sample was finely ground, and the resulting crude lipase preparation was stored
in a tightly sealed glass vial [10], [11].

2.3. Extraction and Purification of Lipase from Crude Papaya Latex

Lipase in papaya latex is considered a natural immobilized lipase, and

extracting this lipase from the latex structure presents numerous challenges.
Many scientists have researched ways to extract lipase from papaya latex but
have yet to achieve complete success. The aim of this study is to use the
surfactant SLS to extract lipase from the papaya latex structure and to use the
techniques of fractional precipitation with ammonium sulfate, ion exchange
 12

chromatography, and electrophoresis to isolate the lipases present in the crude

preparation.

2.3.1. Extraction of lipase using a buffer solution containing SLS

2.3.1.1. Method for solubilizing lipid-free lipase

1g of crude enzyme from papaya latex was weighed and added to 50 ml
of a solution containing n-hexane: 2-propanol. The mixture was shaken for 30
minutes at 4°C to remove lipids from the latex. It was then centrifuged at 7500
rpm at 4°C for 20 minutes. The insoluble pellet was collected and rotary
evaporated to remove the solvent. The solid residue was then dissolved in 50
ml of a solution containing 0.1 M Tris-HCl pH 8, containing 0.5% (w/v) SLS.
The solution was shaken for 30 minutes and then centrifuged at 7500 rpm at
4°C for 20 minutes to obtain the liquid and solid fractions.

2.3.1.2. Method for solubilizing lipid-containing lipase

1g of crude lipase enzyme from papaya latex was dissolved in 100 ml of
a solution containing 0.1 M Tris-HCl pH 8, containing 0.5% SLS. The mixture
was shaken for 30 minutes and then centrifuged at 7500 rpm at 4°C for 20
minutes.

2.3.2. Fractional Precipitation of Lipase with Ammonium Sulfate

2.3.2.1. Principle:
When a salt is added to a protein solution, the salt molecules (NH4)2SO4
dissociate into ions. These ions bind to water molecules, causing the protein to
lose its hydration shell, leading to protein-protein interactions and the
formation of a precipitate. The precipitating effect of salt ions is directly
proportional to their ionic strength. Therefore, each type of protein (enzyme) in
the mixture will precipitate maximally at a specific concentration of a particular
salt.

2.3.2.2. Procedure:
 13

The protein solution obtained after dissolving the crude lipase was used
for precipitation with AS salt.
Step 1: Use a graduated cylinder to determine the volume of the protein
solution.
Step 2: Add AS to the protein solution to reach 40% saturation at 40°C, and let
stand for 30 minutes.
Step 3: Centrifuge to collect the precipitate and supernatant.
Step 4: The supernatant is then subjected to steps 1 and 2 to reach 50%
saturation and step 3 is performed.
Step 5: Repeat step 4 to reach 60%, 70%, 80%, and 90% saturation.
Step 6: The precipitates obtained at different saturated salt fractions were
redissolved in 1 ml of 0.1 M Tris-HCl buffer, pH 8. The dissolved solution was
then dialyzed against distilled water to remove salt using a cellophane
membrane for 48 hours.
Finally, the lipase activity of the dialysate was determined by
spectrophotometry for each saturated salt fraction [9].

Figure 2.6: Dialysis to remove (NH4)2SO4 salt from protein precipitate

2.3.3. Ion Exchange Chromatography for Lipase Purification

2.3.3.1. Principle
Ion exchange chromatography is based on the ion exchange reaction
between charged proteins and ions on the ion exchange resin. A positively
charged group acts as an exchanger for negative ions and is thus called an
anion exchanger, and vice versa.
 14

2.3.3.2. Procedure
The sample from the 50-60% AS precipitation fraction was desalted by
dialysis against a cellophane membrane for 48 hours and then loaded onto an
ion exchange chromatography column. A HiTrap Q Sepharose Fast Flow
column (5.0 ml, 1.6 cm × 2.5 cm) was used in a fast protein liquid
chromatography system (GE Akta Purifier 100, Sweden). The column was first
equilibrated with 0.01 M Tris-HCl buffer at pH 9. The sample was eluted with
a linear gradient of NaCl from 0 to 1 M at a flow rate of 0.1 M NaCl/min. The
flow rate was set at 1 ml/min. The eluted fractions were collected in 1 mL tubes
and the absorbance was measured at 280 nm. The activity of the purified lipase
in each collected fraction was then determined by spectrophotometry.

Figure 2.7: Column chromatography process

2.3.4. Electrophoretic method

2.3.4.1. Principle
The principle of polyacrylamide gel electrophoresis (PAGE) was first
introduced by Weintraub and colleagues in 1959. The primary component of
the gel is acrylamide (CH2=CH-CO-NH2), which polymerizes in the presence
of free radicals (generated by ammonium persulfate - APS) and is stabilized by
TEMED (N,N,N',N'-tetramethyl-ethylenediamine). When bisacrylamide (N,N'-
methylene-bisacrylamide) is added, cross-linking occurs between the chains,
forming a gel. The porosity of the gel is determined by the chain length and the
degree of cross-linking.
 15

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a technique

used to separate a mixture of proteins based on their molecular weight. SDS
binds to proteins at a ratio of 1.4 g SDS per 1 g protein, denaturing the proteins
and imparting a uniform negative charge. This disrupts the tertiary and
quaternary structures of proteins by breaking hydrogen bonds and unfolding the
molecules. Disulfide bonds between cysteine residues are cleaved by reducing
agents such as β-mercaptoethanol or dithiothreitol. The SDS-bound
polypeptide chains, now linear and negatively charged, migrate through the gel
based solely on their size. Under the influence of an electric field, the
molecules move towards the positive electrode at a rate inversely proportional
to their molecular weight. Smaller molecules migrate faster, while larger
molecules migrate slower, resulting in a separation of proteins based on size.
[21]
2.3.4.2. Procedure
Preparation of a 15% polyacrylamide gel, equipment, and chemical
preparation. Clean the glass plates with ethanol, then rinse thoroughly with
distilled water. Assemble the glass plates into the cassette. Test the seal with
distilled water; if water leaks from the seal, repeat. Then, pour the gel and place
the cassette in the electrophoresis chamber, filling it with running buffer (1X
Electrophoresis buffer). Be careful not to trap air bubbles under the glass plate.
Use a pipette to remove excess gel from the wells. Run a prerun for
approximately 5 minutes at 80-100 V to provide ions for the electric field.
Next, use a Hamilton syringe to load 20 μL (30 mg protein) of each antigen
protein sample, mixed with 5 μL of loading dye (5X), into separate wells in the
gel. However, the protein molecular weight marker is loaded into a separate
well (approximately 6 μL). The protein molecular weight marker used is "SDS-
PAGE Molecular Weight Standards" from BioBase, consisting of 9 fragments
ranging from 10 to 170 kDa. Connect the electrophoresis system to the power
supply, and the antigen proteins will migrate from the cathode to the anode.
Run electrophoresis at 60-100 V for 2.5-4.5 hours until the dye front reaches
the end of the gel (time may vary depending on the sample). Remove the gel
 16

from the electrophoresis chamber and place it in a staining tray containing gel
staining solution (containing Coomassie Blue R250). Gently shake at
approximately 25 rpm for 10-15 minutes, then wash the gel with gel destaining
solution on a shaker at low speed. Change the washing solution every 10
minutes until complete, and then image the gel.[21]
Table 2.1: Components for preparing a 15% polyacrylamide gel (for 12 cm x 10 cm glass plates)

Compound Separating gel 15% Stacking gel 5%

DW 1,25 mL 0,88 ml
Acrylamide 2,5 mL 260 μL
Separating buffer 1,25 mL -
Stacking buffer - 380 μL
TEMED 7,5 μL 2μL
APS 10% 45 μL 12 μL
Vortex approximately 1 Vortex approximately 1
- 2 minutes minute

2.4. Activity of purified lipase

2.4.1. Determination of lipase molecular weight

The purified lipase solution obtained from the chromatography column

was further analyzed by SDS-PAGE to determine its molecular weight. The
electrophoresis results are shown in Figure 2.5:
 17

Figure 2.8: Image from SDS-PAGE

M: protein standards (10-170 kDa); 1-3: replicates of the precipitate fraction at

50-60% AS.
The results in Figure 2.5 show two protein bands in the sample,
suggesting the presence of two proteins. One protein has an estimated size of
35-40 kDa, and the other is approximately 40-55 kDa. These results are
consistent with the chromatography data. It can be concluded that there are two
types of lipase with molecular weights ranging from 35-55 kDa in the
precipitate fraction at 50-60% AS.
The molecular weight of papaya latex lipase is similar to that of
microbial and plant lipases, such as the purified lipase from Microbacterium
sp., which has a molecular weight of 40 kDa, or lipase from Tunisian
Euphorbia peplus latex. In contrast, the molecular weight of lipase from Raphia
mesocarp is 35 kDa.[20]

2.4.2. Determine the kinetic parameters Km and Vmax

To investigate the affinity between the enzyme and substrate, the kinetic
parameters of lipase on p-NPP substrate were determined. The Km and Vmax
values of lipase with p-NPP substrate were calculated from the construction of
the Lineweaver-Burk plot using the lipase activity as a function of substrate
 18

concentration. The Lineweaver-Burk plot showed a linear relationship,

indicating that the hydrolysis of p-NPP by lipase followed Michaelis-Menten
kinetics, as presented in Figure 2.6.

Figure 2.9: The Lineweaver-Burk plot was used to determine the kinetic parameters, K m and
Vmax, of papaya latex lipase.

The calculated Km and Vmax values of the purified papaya latex lipase
from the Lineweaver-Burk plot were 1.12 mM and 1.2 x 10^ mM/min/mL,
respectively. The Km value is influenced by the type of substrate and
environmental conditions such as pH, temperature, ionic strength, and polarity.
A lower Km value indicates a higher affinity between the enzyme and substrate,
resulting in a faster reaction rate. The purified papaya latex lipase exhibited a
higher affinity for p-NPP compared to lipase from Microbacterium sp. with a
Km of 3.2 mM as reported by Ritu R, et al. However, the ability of papaya latex
lipase to form a complex with p-NPP is lower compared to lipases from palm
fruit, thermophilic Bacillus stearothermophilus MC 7, and Acinetobacter sp.
AU 07, with Km values of 0.01 mM, 0.33 mM, and 0.51 mM, respectively.[20]
 19

CHAPTER 3: APPLICATION

Lipases are one of the important groups of biocatalysts used in

biotechnological applications. Lipases have been isolated from many species of
plants, animals, bacteria, fungi, and yeast. Lipases extracted from
microorganisms are used in various industries such as dairy, food, detergents,
textile, pharmaceutical, cosmetic and biodiesel industries. It is also used for
synthesis of fine chemicals, agrochemicals and new polymeric materials.
Research on microbial lipases, has increased due to their great commercial
potential. Lipases are added to detergents such as household and industrial
laundry and also in household dishwashers, where their function is removal of
fatty residues and cleaning clogged drains. Their applications in our daily are
increasing day by day [15].

Figure 3.10: Applications of lipase in industries

3.1. Application in food industry

Table 3.2: Lipase applications in the food industry [17]

Food Action Product of application

industry
Dairy Hydrolysis of milk fat, cheese Development of flavouring
foods ripening, modification of butter agents in milk, cheese and
fat. butter.
Bakery Flavour improvement Shelf-life propagation.
foods
Beverages Improved aroma Alcoholic beverages, e.g.
sake, wine.
Food Quality improvement Mayonnaise, dressings and
dressings whippings.
 20

Health Transesterification Health foods.

foods
Meat and Flavour development Meat and fish product, fat
fish removal.
Fats and Transesterification, hydrolysis Cocoa butter, margarine, fatty
oils acids, glycerol, mono and
diglycerides.

3.1.1. Lipase in dairy industry

For the hydrolysis of milk fat, to modify the fatty acid chain lengths and
to boost the flavour of cheeses lipases are widely used in the dairy industry.
Currently, it is also applicable in the speeding up the ripening of cheese and
lipolysis of fat, butter and cream. By the action of lipases on milk fat various
products particularly soft cheeses with specific flavour characteristics
generated with free fatty acids [16].
Lipases are extensively used in the dairy industry for the hydrolysis of
milk fat. The dairy industry uses lipases to modify the fatty acid chain lengths,
to enhance the flavours of various cheeses. Current applications also include
the acceleration of cheese ripening and the lipolysis of butter, fat and cream.
The free fatty acids generated by the action of lipases on milk fat endow many
dairy products, particularly soft cheeses with their specific flavour
characteristics. The traditional sources of lipases for cheese flavour
enhancement are animal tissues, especially pancreatic glands (bovine and
porcine) and pre-gastric tissues of young ruminants (kid, lamb and calf). A
whole range of microbial lipase preparations have been developed for the
cheese manufacturing industry from M. miehei, A. niger, A. oryzae and several
others. Enzyme modified cheese (EMC) is produced when cheese is incubated
in the presence of enzymes at elevated temperature in order to produce a
concentrated flavour by lipase catalysis for use as an ingredient in other
products, such as dips, sauces, soups and snacks.
Table 3.3: Examples of lipase in cheese production

Cheese type Lipase source

Romano Kid/lamb pre-gastic
Domiati Mucor miehei
Feta
 21

Camembert Penicillium camemberti

Mazarella Calf/kid pre-gastic
Parmesan
Provolone
Fontina Mucor miehei
Ras
Romi
Roque fort Penicillium roqueforti
Cheddar Aspergillus oryzae/A.niger
Manchego
Blue
Gastric lipases have been used to accelerate ripening and flavour
development of many cheese types, including cheddar, provolone and ras
cheeses. Lipase addition enhances the rate of fatty acid liberation, which
accelerates flavour development relative to control. These studies indicated that
liberated fatty acid profiles of the accelerated process were identical to the
control and the total quantities of short-chain liberated fatty acids (C4 to C6)
were important for the development of typical cheddar cheese flavour during
ripening. The addition of calf lipase and increasing the ripening temperature
(from 7° to 53°C) result in a significant increase in the liberation of fatty acids.
The disadvantage with this is that the lipase continues to be active after
ripening and can cause the development of strong rancid flavour.
Lipases are usually added to Italian cheese, viz. paramesan, provolone,
and romano, to intensify their flavour 76. During ripening, there is a steady
increase in the concentration of liberated fatty acids and total soluble nitrogen.
Lipases release the fatty acids from triglycerides, thereby triggering the
development of cheese flavour.
Cocoa butter contains palmitic and stearic acids and has a melting point
of approximately 37°C, leading to its melting in mouth which results in a
cooling sensation. In 1976, Unilever filed a patent describing a mixed
hydrolysis and synthesis reaction to produce a cocoa butter substitute using an
immobilized lipase. This technology was commercialized by Quest-Lodrs
Croklaan, based on immobilized R. miehei lipase, which carries out a
 22

transesterification reaction replacing plamitic acid with stearic acid to give the
desired stearic–oleic–stearic triglyceride [17].

3.2. Lipase in detergent industry

After the great commercial success of proteases as deter-gent additives,

the enzyme industry undertook major efforts to introduce lipases as a second
group of detergent enzymes. The functional importance of lipases in the
detergent industry is that of improving the washing capacity of detergents and
removing fatty and proteinaceous food stains in laundry, which are difficult to
remove under mild washing condition. Besides the removal of fatty residues in
laundry and dishwashers, lipases can be used in formulations for cleaning of
clogged drains[18]. In addition, to assess the compatibility of lipase. The use of
enzymes is found in all detergent, formulations which are presently common in
developing countries, containing enzymes. There are different brands of
laundry that claim different qualities of laundry detergent are very common on
market.
Genus Pseudomonas spp.: This is the most widely used bacterial source
for lipase production. Species like Pseudomonas aeruginosa are notable for
their tolerance to high temperatures and alkaline pH levels, making them ideal
for detergents. Products such as Lumafast and Lipomax utilize lipase derived
from these bacteria.
Thermomyces lanuginosus: Novozymes' Lipolase is a leading lipase
enzyme employed in the detergent industry. It is highly effective in breaking
down fats and oils within alkaline detergent formulations [19].

3.3. Lipase in pharmaceutical

Microbial lipases are used to enrich PUFAs from animal and plant
lipids, and their mono and diacylglycerides are used to produce a variety of
pharmaceuticals44. PUFAs are increasingly used as food additives,
pharmaceuticals and nutraceuticals because of their metabolic benefits. Many
 23

PUFAs are essential for normal synthesis of lipid membranes and

prostaglandins.
Considerable effort is being made to obtain optically, pure compounds,
which are pharmacologically more active than its antipode. Profens, a class of
nonsteroidal anti-inflammatory drugs, are active in the (s)-enantiomer form.
Lee et al and Xie et al synthesized pure (s)-ibuprofen using lipase-catalyzed
kinetic resolution via hydrolysis and esterification, respectively.
Nutraceuticals are food components that have health benefits beyond
traditional nutritional value. Novel biotechnology tools, like immobilization,
have also been applied for the isolation and incorporation of such food
components in ordinary foods. Successful synthesis of nutraceuticals has been
reported by employing immobilized lipases, such as those from C. antarctica
and Lactobacillus ruteri.
Lipases are also used in the synthesis of the artificial sweetener sucralose by
regioselective hydrolysis of octaacetylsucrose [18].

3.4. Lipase in biodiesel industries

Biocatalyst based biotechnological applications are receiving increasing

attention. Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3) are the
important biocatalysts because of their excellent biochemical and physiological
properties. Lipases are the hydrolytic enzymes that can be used in various
industrial applications for alcoholic’s, acidosis’s, aminolysis and hydrolysis
reactions. Biodiesel production in present of biocatalyst as lipase is a stunning
application. Lipase catalyzed biodiesel production was reported at 2012.
Lipase-catalyzed trans esterification which involves two steps. Firstly, it
involve in hydrolysis of the ester bond and secondly on esterification with the
second substrate (Fan et al., 2012). Although lipases from different sources are
able to catalyze the same reaction, bacterial and fungal lipases are mostly used
in biodiesel production such as A.niger, C.antarctica, C.rugosa, C. viscosum,
M. miehei, P.cepacia, P.fluorescens, P. lipolyticum, Rhizopusoryzae,
Streptomyces sp., T. lanuginose, and C. rugosa, obtained from yeast, is the
 24

most commonly used microorganism used for the production of lipase (Sharma
et al.,2001). Recently, Streptomyces sp. was investigated as a potent lipase
producing microbe for biodiesel production and found applicable in the field of
biodiesel (Cho et al., 2012) [19].
.
 25

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