Microbial Lipases and Their Industrial Applications: Review
Microbial lipases (triacylglycerol acyl-hydrolases, EC 126.96.36.199) catalyze both the hydrolysis and synthesis of long-chain acylglycerols. They are currently given much attention with the rapid development of enzyme technology. The chemo-, regio- and enantio-specific characteristics of lipase tends to be a focus research area for scientists and industrialists. Compared to plants and animals, microorganisms have been found to produce high yields of lipases. This review describes various industrial applications of microbial lipases in the area of food industry, oil and fat industry, detergent industry, pulp and paper industry, leather industry, textile industry, in organic synthesis, production of cosmetics and biodiesel production. This makes lipases the most widely used class of enzymes in different industrial activities through the application of bioprocess technology. The aim of this review is not to discuss every lipase described in the literature but rather to present recent information on the production, characterization and industrial application of lipases in our daily activities in order to improve our life styles.
February 24, 2012; Accepted: April 10, 2012;
Published: July 03, 2012
Human beings have been using enzyme for different purposes starting from ancient
civilizations. Today, nearly 4000 enzymes are known and of these, nearly 200
are in commercial use. At least 75% of all industrial enzymes (including lipases)
are hydrolytic and the majority of the industrial enzymes are of microbial origin
(Godfrey and West, 1996; Wilke, 1999).
Enzymes or microbial cells are used as biological catalysts due to their high
specificity and economic advantages without any environmental impact. This is
because; (1) Enzymes work better at gentle and available temperatures and other
environmental conditions. It is possible to use enzymes instead of harsh conditions
and harsh chemicals, as the result it could be helpful to save energy and prevent
pollution. In the process, it does not use expensive corrosive-resistant equipment.
(2) Enzymes are highly specific thus, the production of unwanted by products
is avoided and there is no need to have extensive downstream processes. (3)
Enzymes can be immobilized and can therefore be reuse several times. (4) Enzymes
can also be used to treat waste consisting of harmful compounds. (5) Normally
enzymes able to decompose naturally by help of decomposers, so the entire chemical
components of the enzymes are readily recycled back to nature (Gennari
et al., 1998). Furthermore, because of the many different bio-transformations
that enzymes can catalyze, the numbers of enzymes used in commercial scale highly
increased (Sharma et al., 2001a).
When we compare microbial enzymes with that of enzymes originated from plants
or animals, microbial enzymes have variety of catalytic activities, high production
capacity within a short period of time and ease for genetic manipulation. However,
microbial enzymes can be produced at any time and do not affected by seasonal
fluctuations. The other advantage of microorganisms over that of plants and
animals is that they can grow rapidly on inexpensive media. Microbial enzymes
are more stable interms of activity (Wiseman, 1995).
Only about 2% of the worlds microorganisms have been tested as enzyme
sources of which bacteria isolates take higher share than yeasts (Frost
and Moss, 1987) and tend to have neutral or alkaline pH optima and are often
thermostable. Currently, genetic and environmental manipulation techniques help
to increase the yield of cells (Demain, 1971), through
the conversion of inducible enzyme into constitutive type or it is also possible
to increase the enzyme activity by inducing it or modifying the enzyme under
utilization (Betz et al., 1974) using microbial
cells. This is because microorganisms have short generation times, relatively
simple nutritional requirements and since screening procedures for the desired
characteristic are comparatively easier than higher organisms.
Enzymes are widely applied in the field of scientific research, cosmetic production,
medical diagnostics and chemical analyses, therapeutic applications and industrial
catalysis in the special syntheses (Sharma et al.,
2001b). Of those well known significant enzymes, lipases have unique characteristics
that they can carry out reactions at the interface between aqueous and non-aqueous
media. This is primarily due to their ability to utilize relatively a wide spectrum
of substrates, high stability towards extremes of temperature, pH and organic
solvents, do not require cofactors. The enantioselective and regioselective
nature of lipases have been utilized for the resolution of chiral drugs, fat
modification, synthesis of coca butter constituents, biofuels and for synthesis
of personal care products and flavour enhancers (Jaeger
and Reetz, 1998). Therefore, the chemo-, regio- and enantio-specific characteristics
of these enzymes has caused tremendous interest among scientists and industrialists
(Saxena et al., 2003).
Based on three-dimensional structure of various lipases, all have been classified
as serine hydrolases (Winkler et al., 1990; Jaeger
et al., 1993). This is because of the active site that composed of
the catalytic triad Ser-Asp (Glu)-His is similar to serine proteases (Derewenda
and Sharp, 1993; Brumlik and Buckley, 1996). However,
the natural substrates of lipases are triglycerides having very low solubility
in water. Under normal conditions, lipases catalyze the hydrolysis of ester
bonds of triglycerides to glycerol and free fatty acids at the interface between
an insoluble substrate phase and the aqueous phase in which the enzyme is dissolved.
That means, in contrast to esterases, lipases are activated only when adsorbed
to an oil-water interface (Martinelle et al., 1995)
and could not hydrolyze dissolved substrates in the bulk fluid. In contrast,
esterases show normal Michaelis-Menten kinetics in aqueous solution. A true
lipase will split emulsified esters of glycerine and long-chain fatty acids
(Fig. 1). As it has been investigated at experimental conditions,
such as, they are capable of reversing the reaction in the absence of water.
||The catalytic action of lipases: A triglyceride can be hydrolyzed
to form glycerol and fatty acids, or the reverse (synthesis) reaction can
combine glycerol and fatty acids to form the triglyceride
This reverse reaction form glycerides from fatty acids and glycerol through
esterification reaction. Lipases are also used to catalyze the transesterification
reaction using alcohol together with fats, oils and free fatty acids to produce
alkyl esters (biodiesel). The use of lipases to catalyze the transesterification
of fatty acids to alkyl esters for use as biodiesel has not yet been commercially
applied. However, the process has been investigated experimentally (Fig.
Because of their wide-ranging significance, lipases remain a subject of intensive
study (Bornscheuer, 2000). Research on lipases is focused
particularly on structural characterization, general characterization of performance
(Bornscheuer, 2000) and industrial applications. In comparison
with this effort, relatively little work has been done on development of lipase
bioreactor systems for commercial use.
Development of lipase-based technologies for the synthesis of novel compounds
is rapidly expanding the uses of these enzymes (Liese et
al., 2000). An increasing number of lipases with suitable properties
are becoming available and efforts are underway to commercialize biotransformation
and synthesis based on lipases (Liese et al., 2000).
In conclusion, lipases catalyze both the hydrolysis and the synthesis of esters
formed from glycerol and long-chain fatty acids (Fig. 1).
These reactions usually proceed with high region- and /or enantioselectivity,
making lipases an important group of biocatalysts in organic chemistry. The
reasons for the enormous biotechnological potential of microbial lipases include
the facts that they are (1) stable in organic solvents, (2) do not require cofactors,
(3) possess broad substrate specificity (4) act over a wide range of pH and
temperature and (5) exhibit a high enantioselectivity. Currently, lipases are
produced by animals, plants and microorganisms. The most commonly animal lipase
is produced from pancreatic gland. With regard to plant, papaya latex, oat seed
land castor seed can serves source of lipase (Akoh et
al., 2007). Microorganisms have been found to produce high yields of
lipases compare to the animal and plants. This is because their production,
commercialization and application at industrial scale is more simple than animal
and plant ones (Akoh et al., 2007; Antczak
et al., 2009). Lipases produced from microorganisms such as bacterial
and fungal are widely applied in the field of biotechnology and organic chemistry.
As the result, most commercially produced lipases have been produced from fungi
and bacteria. Among lipase producing organisms, Table 1 lists
those microbes that appear to be the most widely used in biotechnology (Jaeger
and Reetz, 1998).
The high-level production of microbial lipases requires not only the efficient
over expression of the corresponding genes but also a detailed understanding
of the molecular mechanisms governing their folding and secretion. The optimization
of industrially relevant lipase properties can be achieved by directed evolution.
Furthermore, novel biotechnological applications have been successfully established
using lipases for the synthesis of biopolymers and biodiesel, the production
of enantio-pure pharmaceuticals, agrochemicals and flavour compounds (Jaeger
and Eggert, 2002). Some of the industrially important chemicals manufactured
from fats and oils by chemical processes could be produced by lipases with greater
rapidity and better specificity under mild conditions (Vulfson,
1994). The chemo-, regio- and enantiospecific behavior of these enzymes
has caused tremendous interest among scientists and industrialists (Saxena
et al., 2003).
Lipases from a large number of bacterial, fungal and plant and animal sources
have been purified to homogeneity (Saxena et al.,
2003). Lipases isolated from different sources have a wide range of properties
depending on their sources with respect to positional specificity, fatty acid
specificity, thermostability, pH optimum, etc. (Huang, 1984).
The objective of this review is not to discuss every lipase and lipase related
points in the literature but rather to present information on selected concepts
of lipases and their applications at industrial scale. Therefore, the paper
reviews the fundamental knowledge available on lipases, with particular emphasis
on isolation, production, purification, three-dimensional structure, immobilization
and thermostability of lipases. Moreover, it focuses on the industrial applications
of lipases in the area of food industry, oil and fat industry, detergent industry,
pulp and paper industry, leather industry, textile industry, in organic synthesis,
production of cosmetics and biodiesel production.
WHAT EXACTLY IS A LIPASE?
Currently, there is no inclusive answer to this simple statement question.
Until recently, two criteria have been used to classify a lipolytic enzyme as
a true lipase (EC 188.8.131.52): (a) it should be activated by the presence
of an interface, that is, its activity should sharply increase as soon as the
triglyceride substrate forms an emulsion. This is commonly known as interfacial
activation (Sarda and Desnuelle, 1958). (b) It should
contain a lid which is a surface loop of hydrophobic oligo-peptide
covering the active site of the enzyme and moving away on contact with the interface
and immediately the substrate enter the binding pocket (Van
Tilbeurgh et al., 1993). However, these obviously suggestive criteria
are not suitable for classification, mainly because of the presence of a number
of exceptions. Some lipases have a lid but not exhibiting interfacial activation
(Verger 1997). Even there are microbial lipases lacking
a lid that covers the active site in the absence of lipid-water interfaces.
Therefore, lipases are simply defined as carboxyl-esterases catalyzing the hydrolysis
(and synthesis) of long-chain triglycerides (Ferrato et
al., 1997). However, there is no strict definition available for the
term long-chain, but glycerol esters with an acyl chain length of
greater than 10 carbon atoms can be regarded as lipase substrates, with triglyceride
being the standard substrate. One lipase type differs from the others in length
and architecture of the binding domain of the α/β-hydrolase fold proteins.
That is why lipases have wide range of substrate diversity.
BASIC CHARACTERISTICS OF LIPASES
The lipases produced by organisms can be widely used for different activities
in different form, such as extracellular, intracellular, immobilized and regiospecific.
Extracellular lipase refers to the use of the enzyme that has been previously
extracted from the producing organism and purified using different techniques.
On the other hand, intracellular lipase refers to the use of the enzyme while
it is still contained in the producing organism (Robles-Medina
et al., 2009). Both extracellular and intracellular lipase could
be immobilized using a solid support (Jegannathan et
al., 2008). They can also be regiospecific by nature which means they
only act on specific bonds of the triglyceride molecule (Robles-Medina
et al., 2009).
Extracellular lipase: Microbial lipases are mostly extracellular which
can be produced by submerged fermentation or solid state fermentation. The fermentation
process is usually followed by purification process in order to increase the
degree of purity thereby improve the biocatalyst activity of the enzyme (Balaji
and Ebenezer, 2008; Barberis et al., 2008).
The important purification step for producing extracellular lipase is a complex
process and it depends on the origin and structure of the lipase (Saxena
et al., 2003). The large scale production of extracellular lipases
should be economical, fast, easy and efficient. Unfortunately, the cost of novel
purification technologies is higher than production (Joseph
et al., 2008). The majority of immobilized lipases which are commercially
available are entirely extracellular (Robles-Medina et
al., 2009). The most commonly used immobilized lipases are: Novozym
435, Lipozyme RM IM and Lipozyme TL IM which are secreted from Candida antarctica,
Rhizomucor miehei and Thermomyces lanuginosus, respectively (Robles-Medina
et al., 2009).
Intracellular lipase: the cost for purification of extracellular lipases
is high and the alternative method used to solve this problem is the use of
the whole cells as biocatalysts. Rather than using enzyme lipase, the use of
compact cells as it is for intracellular production of lipases or fungal cells
immobilized within porous biomass support particles as a whole biocatalyst represents
an attractive process for bulk production of biodiesel and polyesters (Iftikhar
et al., 2008). The utilization of lipase found in the cells is referred
to as intracellular lipase (Robles-Medina et al.,
2009). Some microorganisms used as source of lipase are able to be spontaneously
immobilized on certain supports. This reduced or eliminates the costly purification
step and the need for an extended immobilization process which is necessary
in comparison with the extracellular lipase (Fukuda et
Isolation and screening of lipase-producing microorganisms: Lipases
are produced by many microorganisms and higher eukaryotes. Most commercially
useful lipases are of microbial origin. Lipase-producing microorganisms have
been found in diverse habitats such as industrial wastes, vegetable oil processing
factories, dairies, soil contaminated with oil, oilseeds and decaying food (Sztajer
et al., 1988), as well as from compost heaps, coal tips and hot
springs (Wang et al., 1995). Some of the lipase-producing
microorganisms are listed in Table 1.
Lipase-producing microorganisms belong to bacteria, fungi, yeasts and actinomyces.
The lipases found among these microbial sources are quite diverse and typically
vary from one another in physical, chemical and biological properties. Even
although a large number of lipolytic enzymes are known in microorganisms, not
all such enzymes are suitable for commercial utilization. Such factors as pH
range, tolerance of emulsification and surfactants, temperature tolerance, storage
capability and the like are important considerations in the selection and development
of a commercially useful product (Sharma et al.,
A simple and reliable method for detecting lipase activity in microorganisms
has been described by Sierra (1957). This method uses
the surfactant Tween 80 in a solid medium to identify a lipolytic activity.
The formation of opaque zones around the colonies is an indication of lipase
producing organisms. Modifications up on this assay have been carried out, like
the use of various Tween surfactants in combination with Nile blue or neatsfoot
oil and Cu2+ salts. On the other hand, screening of lipase producers
on agar plates is frequently done by using tributyrin as a substrate (Cardenas
et al., 2001) and clear zones around the colonies indicate production
of lipase. Screening systems making use of chromogenic substrates have also
been described (Yeoh et al., 1986). The most
widely used substrates are tributyrin and triolein which are emulsified mechanically
in various growth media and poured into a plate. Lipase production is indicated
by the formation of clear halos around the colonies grown on tributyrin-containing
agar plates (Atlas, 1996) and orange-red fluorescence
visible on irradiation with a conventional UV hand lamp at 350 nm on triolein
plates with rhodamine B are widely used (Kouker and Jaeger,
Lipase activity in bacterial culture supernatants is determined by hydrolysis
of p-nitrophenylesters of fatty acids with various chain lengths and spectrophotometric
detection of p-nitrophenol at 410 nm. A more laborious but reliable method for
identifying a true lipase is the determination of fatty acids liberated
from a triglyceride by titration (Jensen, 1983).
Currently, lipases are not only used to hydrolysis of substrates but also important
for synthesis of different substances from monomers. A standard reaction is
the lipase-catalyzed esterification of an alcohol with a carboxylic acid (Reetz
and Jaeger, 1998).
|| Lipases producing microorganisms
The first speed of ester formation could be determined by gas chromatography.
Currently, there is no standard or single method used to determine the enantioselectivity
of a lipase-catalyzed organic reaction. As a result of this, the enantioselectivity
of product formation is determined either by gas chromatography or High Performance
Liquid Chromatography (HPLC), with chirally modified columns.
Production and media development for lipase: Microbial lipases are produced
mostly by submerged culture (Ito et al., 2001),
but solid state fermentation methods (Chisti, 1999a)
can be used also. The Solid State Fermentation (SSF) is an interesting alternative
for microbial enzyme production due to the possibility of using residues and
by-products of agro-industries as nutrient sources and support for microorganism
development. The use of by-products as substrates for lipase production, adds
high value and low-cost substrates may reduce the final cost of the enzyme (Menoncin
et al., 2008, Rodriguez et al., 2006).
Many studies have been carried out to define the optimal culture and nutritional
requirements for lipase production by submerged culture. However, production
of lipase through submerged fermentation needs, large space, complex media and
also needs complex machinery, equipment and control systems. Moreover, submerged
fermentation for production of lipase at large scale demands high energy demand,
higher capital and recurring expenditure (Satyanarayana,
Immobilized cell culture has been applied in a few cases (Hemachander
et al., 2001). In several cases, immobilization of microbial cells
producing lipases increase the extent of reaction and facilitate the downstream
processing. This is because it avoids washout of the cells at dilution rates,
it helps to increase cell concentration in the reactor and easy separation of
cells from the product containing solution (Gunasekaran
and Das, 2005).
Generally, lipase production is influenced by the type and concentration of
carbon and nitrogen sources, the culture pH, the growth temperature and dissolved
oxygen concentration (Elibol and Ozer, 2001). Lipidic
carbon sources seem to be generally essential for obtaining a high lipase yield;
however, a few authors have produced good yields in the absence of fats and
oils (Shimada et al., 1992).
Purification of lipases: Purification of lipase is essential for industries
of fine chemicals, pharmaceuticals and cosmetics. It is also significant to
investigate and understand the 3-D structure of the enzyme and their structure-function
relationships (Saxena et al., 2003). Currently
many lipases have been widely purified and characterized without losing their
activity and stability profiles depending to pH, temperature and effects of
metal ions and chelating agents. The methods used for purification of lipases
are nonspecific techniques, some of which are extraction, precipitation, hydrophobic
interaction, chromatography, gel filtration, crystallization and ion exchange
chromatography. Affinity chromatography is significant to reduce most purification
steps needed (Woolley and Peterson, 1994). If the objective
is production of lipase for industrial use, the purification technique should
be inexpensive, rapid, high-yielding and liable to large-scale operations. The
degrees of quality of the products are entirely to depend on the purpose and
the economic point of view. For instance, the lipase for synthetic reactions
in pharmaceutical industry needs further purification (Koblitz
and Pastore, 2006).
Shear tolerance of lipases: The application of strong mechanical agitation
and emulsifiers can improve interfacial area in the bioreactors. However, a
combination of interface and agitation sometimes affect the activity and stability
of lipases. Extreme agitation and liquid-liquid interfaces are frequently common
in lipase-assisted hydrolysis (Rooney and Weatherley, 2001).
Shear-associated inactivation of many enzymes (Chisti, 1999b)
including lipases (Lee and Choo, 1989; Mohanty
et al., 2001) at gas-liquid and liquid-liquid interfaces have been
The rate of interfacial lipase denaturation is directly proportional to the
increment of temperature (Lee and Choo, 1989) and turbulence
in the fluid (Chisti, 1999b). That means, the denaturation
rate constant depends on the specific power input in the reactor and the amount
of gas-liquid interface present (Mohanty et al.,
2001). To avoid or reduce the rate of denaturation, addition of polypropylene
glycol is significant (Lee and Choo, 1989). In this
case, interfacial denaturation is mostly common by losing the 3-dimentional
structure lipase rather than molecule breakage into multiple peptides (Lee
and Choo, 1989).
Three-dimensional structure of lipases: The determination of the three-dimensional
structures and the factors that determine their regiospecificity and enantiospecificity
are essential to make suitable lipases for specific applications. From 1990
to 1998 the three dimensional structure of 12 types of lipases from different
sources have been studied which, with the exception of pancreatic lipases, are
all of microbial origin (Jaeger and Reetz, 1998 (Sharma
et al., 2001a). The molecular weights of these enzymes range from
19 to 60 kDa.
With regard to lipases structure, all had very similar folds despite a lack
of amino acid sequence similarity (Cygler et al.,
1992; Smith et al, 1992). All have almost
the same three-dimensional structure characterized as α/β-hydrolase
folding (a specific sequence of α-helices and β-strands) (Balkenhohl
et al., 1997) with most of them containing a helical segment called
the lid that covers the active site when the enzyme is in the so-called closed
conformation. When lipid aggregates are available, the lid opens immediately
and the enzyme activity is going to increased, a condition is termed as interfacial
activation. Moreover, the lipase core is composed of a central β sheet
that is composed of eight different β strands (β1-β8)
connected by up to six α helices (A-F). Canonical fold of α/β-hydrolase
is an ideal example for this case. The canonical α/β-hydrolase fold
consists of a central, mostly parallel β sheet of eight strands with the
second strand anti-parallel. The parallel strands range from β3
to β8 are connected by α helices which pack on either side
of the central β sheet. The β sheet has a left-handed super-helical
twist such that the surface of the sheet covers about half a cylinder and the
first and last strands cross each other at an angle of 900. The curvature
of the β sheet may differ significantly among the various enzymes and also,
the spatial positions of topologically equivalent α helices may vary considerably.
They differ substantially in length and architecture, in agreement with the
large substrate diversity of these enzymes (Jaeger et
al, 1999). The active site of the α/β-hydrolase fold enzymes
composed of three catalytic residues which are referred as nucleophilic residue
(serine, cysteine, or aspartate), a catalytic acid residue (aspartate or glutamate)
and a histidine residue, always in this order in the amino acid sequence (Ollis
et al, 1992). The order of the residues is different from that observed
in any of the other proteins that contain catalytic triads. In lipases, the
nucleophile has been found to be a serine residue, whereas the catalytic acid
is either an aspartate or a glutamate residue. The nucleophilic Ser residue
is located at the C-terminal end of strand β5 in a highly conserved pentapeptide
GXSXG, forming a characteristic β-turn-α motif which is known as nucleophilic
elbow. The hydrolysis of the substrate is started with a nucleophilic attack
by the catalytic-site-Ser oxygen on the carbonyl carbon atom of the ester bond,
leading to the formation of a tetrahedral intermediate stabilized by hydrogen
bonding to nitrogen atoms of main chain residues that belong to the so-called
oxyanion hole. An alcohol is produced and released from an acyl-lipase
complex which is finally hydrolyzed with the production of the fatty acid and
regeneration of the enzyme.
Immobilization of lipases: Enzyme immobilization increases the number
of enzyme molecules per unit area increasing the efficiency of the reaction.
Like with other enzymes, the advantages of immobilizing lipases include the
repetitive use of a given batch of enzyme, better process control, enhanced
stability, enzyme-free products (Rahman et al., 2005),
increased stability of polar substrates, shifting of thermodynamic equilibria
to favour ester synthesis over hydrolysis, reduction of water dependent side
reactions such as hydrolysis, elimination of microbial contamination and the
potential for use directly within a chemical process. In the presence of organic
solvents, immobilized lipase has been showed enhanced activity (Ye
et al., 2005).
Currently, many methods have been used to immobilize lipases, including adsorption
or precipitation onto hydrophobic materials (Wisdom et
al., 1984), covalent attachment to functional groups (Shaw
et al., 1990), entrapment in polymer gels (Telefoncu
et al., 1990), adsorption in macroporous anion exchange resins (Rizzi
et al., 1992), microencapsulation in lipid vesicles (Balcao
et al., 1996) and sol-gel entrapment (Krishnakant
and Madamwar, 2001). Of those immobilized approaches, enzyme entrapment
by help of inorganic matrixes such as silica gel makes more efficient (Shtelzer
et al., 1992). In this regard, Candida antarctica B (Novozym
435) was immobilized on mesoporous silica with octyltriethoxysilane and it retained
its activity even after 15 reaction cycles (Blanco et
al., 2004). Calcium carbonate was found to be the most suitable adsorbent
when crude Rhizopus oryzae lipase was immobilized on different supports
and it exhibited long-chain fatty acid specificity (Ghamguia
et al., 2004). The lipase from Pseudomonas cepacia was gel-entrapped
by polycondensation of hydrolysed tetramethoxysilane and isobutyltrimethoxy
silane and was subjected to repeated use without losing much of its activity
(Noureddini et al., 2005).
Thermophilic and psychrophilic lipases of microbial origin: The optima
activity of lipases obtained from conventional sources range from 30 and 60°C.
However, currently, lipases were obtained from extremophiles, i.e., organisms
adapted to life in high temperature, with maximum activity over 70°C (Bacillus
thermocatenulatus) or with high activity at low temperature as is the case
for enzymes produced by Antarctic bacteria, such as Pseudomonas and Moraxella
sp. Such extreme and unusual features open the possibility to apply these
enzymes without further modification using molecular engineering approaches
to adapt them for use in reactions carried out at high temperatures or, conversely
low temperature processes such as that of detergents (low temperature washes)
or in food processing (Demiorijan et al., 2001).
Generally, lipases are further divided into three based on their degree of temperature
stability; namely psychrophilic, mesophilic and thermophilic. Thermostable enzymes
can be obtained from mesophilic and thermophilic organisms; even psycrophiles
have some thermostable enzymes (Adams et al., 1995).
Currently, lipases from thermophilic and psychrophilic organisms have been proved
to be more useful for biotechnological applications (Imamura
and Kitaura, 2000). Therefore, this review only focuses on thermophilic
and psychrophilic organisms.
Thermophilic lipases: The demand of thermostable lipases for different
applications has been growing rapidly. Most of the studies were carried out
to produce lipases from mesophilic microorganisms. Many lipases from mesophiles
are stable at elevated temperatures (Sugihara et al.,
1991). Proteins from thermophilic organisms have also been proved to be
more useful for biotechnological applications than similar proteins from mesophiles
due to their stability at high temperature (Imamura and
Kitaura, 2000). Enzymes with high thermostability are important to have
higher reaction rate at higher operation temperature. This is because higher
temperature can increase solubility of substrates and also help to lower substrate
viscosity and thereby avoid environmental contamination (Mozhaev,
Recently, biotechnologically significant enzymes were produced from hyperthermophilic
archaebacteria, such as Pyrococcus furiosus and Thermotoga sp.
(Adams et al., 1995) (Table 2).
Thermostable lipases from such microbial sources are highly advantageous for
biotechnological applications, since they can be produced at low cost and exhibit
improved stability at high extreme temperature (Handelsman
and Shoham, 1994). Currently, there has been a great demand for thermophilic
and thermostable enzymes in various industrial fields. Thus, thermostable lipases
from various sources have been purified and characterized using appropriate
procedures (Sugihara et al., 1991). Lipases operating
chemical reaction at elevated temperatures have the following advantages. (1)
A higher diffusion rates. (2) Increased solubility of lipids and other hydrophobic
substrates in water. (3) Decreased substrate viscosities. (4) Increased reactant
solubility. (5) Higher temperature faster reaction rates. (6) Reduced risk of
microbial contamination (Hasan et al., 2006) Thermophile
microorganisms are a valuable source of thermostable lipase with desired properties
usually associated with stability in solvents and detergents for potential biotechnological
and industrial applications (Haki and Rakshit, 2003).
These enzymes have been applied to synthesis biopolymers, pharmaceutical chemicals,
agrochemicals, cosmetics, flavours and biodiesel (Haki and
Currently, thermostable lipases have been isolated from many sources, including
Pseudomonas fluorescens (Kojima et al., 1994);
Bacillus sp. (Wang et al., 1995); B.
coagulans and B. cereus (El-Shafei and Rezkallah,
1997); B. stearothermophilus (Kim et al.,
1997); Geotrichum sp. and Aeromonas sobria (Lotrakul
and Dharmsthiti, 1997; Macedo et al., 1997)
and P. aeruginosa (Sharon et al., 1998).
The enzyme from P. aeruginosa was significantly stabilized by Ca2+
and was inactivated by EDTA. This inactivation could be overcome by adding CaCl2,
suggesting the existence of a calcium-binding site in P. aeruginosa lipase.
|| Thermophilic and Psychrophilic lipase producing microorganisms
One of the more notable thermostable enzymes was isolated by Wang
et al. (1995) from a Bacillus strain. This enzyme had maximum
activity at 60°C and retained 100% of the original activity after being
held at 75°C for 30 min. The half-life of the enzyme was 8 h at 75°C
(Wang et al., 1995). The enzyme retained at least
90% of the original activity after being incubated at 60°C for 15 h (Wang
et al., 1995). Other highly thermostable lipases have been reported
(Gao and Breuil, 1995; Kim et
al., 1998; Lee et al., 1999).
Thermal stability of a lipase is clearly related with its structure (Zhu
et al., 2001). Thermostability is also influenced by environmental
factors such as pH and the presence of metal ions. At least in some instances,
thermal denaturation appears to occur through intermediate states of unfolding
of the polypeptide (Zhu et al., 2001). Mutations
in the lid region of the lipase can significantly affect heat stability
(Zhu et al., 2001). Attempts are being made to
protein engineer lipases for improved thermal stability. Compared to the native
enzyme, thermal and operational stability of many lipases can be significantly
enhanced by immobilization (Arroyo et al., 1999;
Hiol et al., 2000). For instance, C. antarctica
lipase B could be thermally stabilized by immobilization (Arroyo
et al., 1999).
Psychrophilic lipases: Cold adapted lipases are largely distributed
in microorganisms existing at low temperatures nearly 5°C. Although a number
of lipase producing organisms are available, only a few bacteria and yeast were
exploited for the production of cold adapted lipases (Joseph,
2006). Attempts have been made from time to time to isolate cold adapted
lipases from these microorganisms having high activity at low temperatures.
Various studies showed that a high bacteria count has been recorded as high
as 105 and 106 per ml water column and in the sea ice,
respectively (Delille, 1993). Cold adapted bacterial
strains were isolated mostly from Antarctic and Polar regions which represent
a permanently cold (0±2°C). A marine bacterium Aeromonas hydrophila
growing at a temperature range between 4 and 37°C was found to produce
cold active lipolytic enzyme (Pemberton et al., 1997).
Few bacterial genera have been isolated and characterized from deep-sea sediments
where temperature is below 3°C. They include Aeromonas sp. (Lee
et al., 2003), Pseudoalteromonas sp. and Psychrobacter
sp. (Zeng et al., 2004) and Photobacterium
lipolyticum (Ryu et al., 2006). Permanently
cold regions such as glaciers and mountain regions are another habitat for psychrophilic
lipase producing microorganisms (Joseph et al., 2007).
The soil and ice in Alpine region also harbor psychrophilic microorganisms which
produces cold active lipases.
Even though many psychrophilic and psychrotrophic bacteria produce lipases,
it is clear that only a few lipolytic fungus was reported to produce cold active
lipases (Table 2). An extensive research has been carried
out in the cold active lipase of Candida antarctica compared to the other
psychrophilic fungi. Candida lipolytica, Geotrichum candidum and Penicillium
roqueforti have also been isolated from frozen food samples and reported
to produce cold active lipases (Alford and Pierce, 1961).
Psychrotrophic lipolytic moulds such as Rhizopus sp. and Mucor sp.
were grown on milk and dairy products and soft fruits (Coenen
et al., 1997).
Cold active lipases have lately attracted attention of communities as a result of their increasing use in the organic synthesis of chiral intermediates. Due to their low optimum temperature and high activity at very low temperatures which are favorable properties for the production of relatively frail compounds. Cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists. The present review describes various industrial applications of cold active microbial lipases in the medical and pharmaceuticals, fine chemical synthesis, food industry, domestic and environmental applications.
With increasing interest in psychrophiles and their applications, cold active
lipases will represent a larger share of industrial enzyme market in the coming
years. The cold active lipases offer novel opportunities for biotechnological
exploitation based on their high catalytic activity at low temperature. The
cold enzymes along with the producing microorganisms cover a broad spectrum
of biotechnological applications. Their current application include additives
in deter-gents (cold washing), additives in food industries (fermentation, cheese
manufacture, bakery, meat tenderizing), environmental bioremediations (digesters,
composting, oil degradation or xenobiotic biology applications and molecular
biology applications), bio-transformation and heterologous gene expression in
psychrophilic hosts to prevent formation of inclusion bodies (Feller
et al., 1996). A number of relatively straightforward reasons for
applications of cold active enzymes in biotechnology have been mentioned by
various authors (Cavicchioli et al., 2002).
In summary, Enantioselective interesterification and transesterification have
great significance in pharmaceutical for selective acylation and deacylation
(Stinson, 1995). Some of the industrially important
chemicals manufactured from fats and oils by chemical processes could be produced
by lipases with greater rapidity and better specificity under mild conditions
(Vulfson, 1994). In the food industry, reaction needs
to be carried out at low temperature in order to avoid changes in food ingredients
caused by undesirable side-reaction that would otherwise occur at higher temperatures.
Lipases have become an integral part of the modern food industry. The use of
enzymes to improve the traditional chemical processes of food manufacture has
been developed in the past few years. The use of cold active lipase in the formulation
of detergents would be of great advantage for cold washing that would reduce
the energy consumption and wear and tear of textile fibers (Feller
and Gerday, 2003). The industrial dehairing of hides and skin at low temperature
using psychrophilic lipase together with protease or keratinase would not only
save energy but also reduce the impacts of toxic chemicals used in dehairing.
This is because they have no negative impact on sewage treatment processes and
do not present a risk to aquatic life. The other common commercial application
of lipase as detergent includes in dish washing, clearing of drains clogged
by lipids in food processing or domestic/industrial effluent treatment plants
(Bailey and Ollis, 1986). As determined by Buchon
et al. (2000), cold adapted lipases have great potential in the field
of wastewater treatment, bioremediation in fat contaminated cold environment
and active compounds synthesis in cold condition.
INDUSTRIAL APPLICATIONS OF LIPASES
Microbial lipases constitute an important group of biotechnologically valuable
enzymes, mainly because of the versatility of their applied properties and ease
of mass production. Microbial lipases are highly diversified in their enzymatic
properties and substrate specificity which make them very attractive for industrial
applications. Next to proteases and carbohydrases, lipases are considered to
be the third largest group based on total sales volume. The commercial use of
lipases is a billion-dollar business that comprises a wide variety of different
applications (Jaeger et al., 1999). The majority
of the enzymes used for industrial applications are of microbial origin and
are produced in conventional aerobic submerged fermentations which allows greater
control of the conditions of growth than solid-state fermentations (Cheetham,
1995). Currently lipases have received increased attention, evidenced by
the increasing amount of information about lipases in the current literature.
Lipases are valued biocatalysts because they act under mild conditions, are
highly stable in organic solvents, show broad substrate specificity and usually
show high regio- and/or stereoselectivity in catalysis (Snellman
et al., 2002). The usefulness of bacterial lipase in commerce and
research drives from its physiological and physical properties. A large amount
of purified lipase could become available, i.e. ease of mass production. Bacterial
lipases are generally more stable than animal or plant lipases. Lipases are
active under ambient conditions and the energy expenditure required to conduct
reactions at elevated temperatures and pressures is highly reduced and thereby
the destruction of labile reactants and products are also highly reduced. Thermophilic
microorganisms and enzymes stable at high temperatures and adverse chemical
environments are of advantage in industrial uses. Due to high degree of specificity
of enzymes, unwanted side products that normally appear in the waste stream
are also reduced or eliminated. The use of enzymes can decrease the side reactions
and downstream problems. One the unique characteristics of lipases is that they
remain active in organic solvents in field of industrial application. When immobilized
lipases are used under typical industrial conditions, reactor temperatures
as high 70°C are possible for prolonged periods.
Lipases are widely used in the processing of fats and oils, detergents and
degreasing formulations, food processing, the synthesis of fine chemicals and
pharmaceuticals, paper manufacture and production of cosmetics (Rubin
and Dennis, 1997). Lipase can be used to accelerate the degradation of fatty
waste materials (Masse et al., 2001) and a synthetic
plastic (polyurethane) (Takamoto et al., 2001).
Some of industrial important microorganisms are presented on Table
Lipases in food industry: Fats and oils are essential constituents of
|| Industrial applicable lipase produced from different microorganisms
The nutritional and sensory value and the physical properties of a triglyceride
are greatly influenced by factors like position of the fatty acid in the glycerol
backbone, the chain length of the fatty acid and its degree of unsaturation.
Lipases allow us to modify the properties of lipids by altering the location
of fatty acid chains in the glycerol and replacing one or more of the fatty
acids with new ones (Pabai et al., 1995; Undurraga
et al., 2001).
Microbial lipases which are regiospecific and fatty acid specific are of enormous
importance and could be exploited for industrial vegetable oils. Cheap oils
could also be upgraded to synthesize nutritionally important structured triacylglycerols,
low calories triacylglycerols and oleic acid enriched oils. Lipase mediated
modifications are highly significant in oil industry for the production of structured
lipids since enzymatic modifications are specific and can be carried out at
moderate reaction conditions (Gupta et al., 2003).
Lipases have also been widely used in food industry to modify flavour by synthesis
of esters of short chain fatty acids and alcohols which are known flavour and
fragrance compounds (Macedo et al., 2003). Generally,
microbial lipases are widely applied in different fields including flavour development
for dairy products (cheese, butter, margarine, alcoholic beverages, milk chocolate
and sweets), achieved by selective hydrolysis of fat triglycerides to release
free fatty acids. These fatty acids can serve as either flavours or flavour
Lipases are also used to remove fat from meat and fish products (Kazlauskas
and Bornscheuer, 1998) to produce lean meat. The fat is removed during the
processing of the fish meat by adding lipases and this procedure is called bio-lipolysis.
The lipases also play a significant role in the fermentative process of sausage
manufacture and to determine changes in long-chain fatty acid liberated during
ripening. Earlier, lipases of different microbial origin have been used for
refining rice flavour, modifying soybean milk and for improving the aroma and
accelerating the fermentation of apple wine (Seitz, 1974).
Cold active lipase from Pseudomonas strain P38 is widely used in non-aqueous
biotransformation for the synthesis of n-heptane of the flavoring compound butyl
caprylate (Tan et al., 1996). Immobilized lipases
from C. antarctica (CAL-B), C. cylindracea AY30,
H. lanuginosa, Pseudomonas sp. and Geotrichum candidum were
used for the esterification of functionalized phenols for synthesis of lipophilic
antioxidants in sunflower oil (Buisman et al., 1998).
Lipases in oil and fat industry: The use of enzymes in the oils and
fats industry is new, providing several solutions to both the industry problems
and the key to produce novel oils and fats. Lipases can catalyze reactions under
mild conditions (i.e., the industrial hydrolysis of fats and oils or the manufacture
of fatty acid amides), permitting high specificity; they can therefore be used
to obtain high-value chemicals for food and industrial uses at competitive production
costs. For example, cocoa butter fat required for chocolate production is often
in short supply and the price can fluctuate widely. However, lipase catalyzed
transesterification of cheaper oils can be used, for example to produce cocoa
butter from palm mid-fraction. The lipase catalyzed transesterification in organic
solvents is an emerging industrial application such as production of cocoa butter
equivalent, human milk fat substitute, pharmaceutically important Polyunsaturated
Fatty Acids (PUFA) and production of biodiesel from vegetable oils (Nakajima
et al., 2000). Therefore, lipase-based technology involving mixed
hydrolysis and synthesis reactions which are widely used in commercial activity
to upgrade some of the less desirable fats to cocoa butter substitutes (Undurraga
et al., 2001). One of the application of lipase-based technology
is used the immobilized Rhizomucor miehei lipase for the transesterification
reaction that replaces the palmitic acid in palm oil with stearic acid. Similarly,
a lipase-catalyzed interesterification of butter fat was used to decrease the
long-chain saturated fatty acids and a corresponding increase in C18:0 and C18:1
acid at position 2 of the selected triacylglycerol (Pabai
et al., 1995).
Another example is the use of lipases to enrich polyunsatured fatty acids (PUFAs)
from animal and plant lipids. Free PUFAs and their mono-and diglycerides are
subsequently used to produce a variety of pharmaceuticals (anti-inflammatories,
thrombolytics, etc.) (Jaeger and Reetz, 1998; Belarbi
et al., 2000). Because of their metabolic effects, PUFAs are increasingly
used as pharmaceuticals, nutraceuticals and food additives (Belarbi
et al., 2000). Many of the PUFAs are essential for normal synthesis
of lipid membranes and prostaglandins. Microbial lipases are used to obtain
PUFAs from animal and plant lipids such as menhaden oil, tuna oil and borage
oil. In addition, the flavour development for dairy products (cheese, butter,
margarine, bakery products, alcoholic beverages, milk chocolate and sweets)
is achieved by selective hydrolysis of fat triglycerides to release free fatty
acids which act as flavour precursors (Jaeger and Reetz,
Immobilized M. miehei lipase in organic solvent catalyzed the reactions
of enzymatic interesterification for production of vegetable oils such as; corn
oil, sunflower oil, peanut oil, olive oil and soybean oil containing omega-3
polyunsaturated fatty acids. Lipases are important to hydrolyze lipids so as
to obtain fatty acids and glycerol, both of which have important industrial
applications. For instance, fatty acids are used in soap production (Hoq,
1985) and glycerol is widely served as raw material for pharmaceutical industries.
Lipases in the detergent industry: The most commercially important field
of application for hydrolytic lipases is their addition to detergents which
are used mainly in household and industrial laundry and in household dishwashers.
The cleaning power of detergents seems to have peaked; all detergents contain
similar ingredients and are based on similar detergency mechanisms. To improve
detergency, modern types of heavy duty powder detergents and automatic dishwasher
detergents usually contain one or more enzymes, such as protease, amylase, cellulase
and lipase (Ito et al., 1998). Detergent lipases
are especially selected to meet the following requirements: (1) a low substrate
specificity, i.e., an ability to hydrolyze fats of various compositions; (2)
ability to withstand relatively harsh washing conditions (pH 10-11, 30-60°C);
(3) ability to withstand damaging surfactants and enzymes (e.g., linear alkyl
benzene sulfonates and proteases) which are important ingredients of many detergent
formulations. Moreover, the trend towards lower washing temperatures has made
the removal of grease spots a bigger problem, particularly for cotton and polyester
clothes. Some specific lipases are capable of removing greasy stains such as
lipstick, frying fats, butter, sauces, etc. (Jaeger and
Reetz, 1998). Enzymes can also reduce the environmental load of detergent
since they are biodegradable, leaving no harmful residues; have no negative
impact on sewage treatment processes and do not present a risk to aquatic life.
Particularly, the use of cold active lipase in the formulation of detergents
would be of great advantage for cold washing that would reduce the energy consumption
and wear and tear of textile fibers (Feller and Gerday,
2003).The use of cold active lipase as a liquid leather cleaner and as an
ingredient in bleaching composition (Nakamura and Nasu, 1990)
has been reported. Similarly its use in decomposition of lipid contaminants
in dry-cleaning solvents (Abo, 1990), contact lens cleaning
(Bhatia, 1990), degradation of organic wastes on the
surface of exhaust pipes, toilet bowls, etc., (Moriguchi
et al., 1990) have been reported.
The most important lipase in the market was originally obtained from Humicola
lanuginosa. It is produced in large scale by Aspergillus oryzae host
after cloning the Humicola gene into this organism. Lipolase which originated
from the fungus T. lanuginosus was also expressed in A. oryzae. Lipases
isolated from Pseudomonas mendocina, Pseudomonas alcaligenes were also
used in detergent industry. Alkaline lipase produced by Acinetobacter
radioresistens had an optimum pH of 10 and was stable over a pH range of
6-10; therefore have great potential for application in the detergent industry
(Chen et al., 1998). Currently, lipases with
the desired properties are obtained through a combination of continuous screening
(Yeoh et al., 1986; Wang
et al., 1995; Cardenas et al., 2001)
and protein engineering (Kazlauskas and Bornscheuer, 1998).
Lipases in pulp and paper industry: Pitch is a term used
to collectively describe the hydrophobic components of wood (triglycerides and
waxes). Pitch and related substances which are usually creating problems are
common in paper mills ((Jaeger and Reetz, 1998). These
problems appear as sticky deposits in the paper machines and can cause holes
and spots in the final paper. However, lipases are used to remove the pitch
from the pulp produced during paper making processes (Jaeger
and Reetz, 1998). Lipases hydrolyze up to 90% of triglycerides in the pitch
into glycerol/ monoglycerides and fatty acids which are far less sticky and
more hydrophilic (easy to wash) (Jaeger and Reetz, 1998).
Nippon Paper Industries, in Japan, have developed a pitch control method that
uses the Candida rugosa fungal lipase to hydrolyze up to 90% of the wood
triglycerides (Jaeger and Reetz, 1998).
Generally, the enzymatic pitch control method using lipases have been in use
in a large-scale paper-making process as a routine operation since early 1990s
(Bajpai, 1999). Lipases in paper industry can generally
increase the pulping rate of pulp, increase whiteness and intensity, decrease
chemical usage, prolong equipment life, reduce pollution level of waste water,
save energy and time and reduce composite cost. The addition of lipase from
Pseudomonas species (KWI-56) to a deinking composition for ethylene oxide-propylene
oxide adduct stearate improved whiteness of paper and reduced residual ink spots
(Fukuda et al., 1990).
Lipases in leather industry: Hides and skins contain proteins and fat
in the collagen fibres. Before the hides and skins are going tanned, these substances
must be partially or totally removed. The first treatment is soaking. This step
serves to remove the common salt and free the hide from blood and dirt. At the
same time, non-fibril proteins which hold the fibres together have to be eliminated
by the action of proteases. Proteolytic enzymes facilitate both the emulsification
of natural fat by hydrolyzing the wall of the fat cells and the soaking operation.
Lipases specifically degrade fat and do not damage the leather itself. Lipases
represent the method of removing fat in the degreasing process with the lowest
environmental impact. For bovine hides, lipases allow tensile to be completely
replaced. For sheepskins, the use of solvents is very common, but it can also
be replaced by lipases and surfactants.
Lipases in organic synthesis: Now days, of those industrially important
enzymes, lipases are widely used for organic reactions. They are used to catalyze
a wide variety of regioselective and stereoselective transformations (Kazlauskas,
1994; Berglund and Hutt, 2000). Currently, most of
lipases used as catalysts in organic chemistry are of microbial origin. These
enzymes work at hydrophilic-lipophilic interface and tolerate organic solvents
in the reaction mixtures. Use of lipases in the synthesis of enantiopure compounds
has been reported by Berglund and Hutt (2000). For instance,
Pseudomonas lipases are widely used in industry, especially for the production
of chiral chemicals which serve as basic building blocks in the synthesis of
pharmaceuticals, pesticides and insecticides. These enzymes show distinct differences
in regioselectivity and enantioselectivity, despite a high amino acid sequence
Generally, lipases have become one of the most important groups of enzymes
for its applications in organic syntheses. Lipases are used as biocatalyst in
the production of significant biodegradable compounds. Trimethylolpropane esters
were synthesized as lubricants. Lipases can catalyze ester syntheses and transesterification
reactions in organic solvent systems has opened up the possibility of enzyme
catalyzed production of biodegradable polyesters. Aromatic polyesters can also
be synthesized by lipase biocatalysis (Bailey and Ollis,
Lipases in textile industry: Lipases are widely used in the textile
industry to remove size lubricants and thereby to provide a fabric with greater
absorbency for improved levelness in dyeing. It is also used to reduce the frequency
of streaks and cracks in the denim abrasion systems. Lipases together with alpha
amylase are used for the desizing of denim and other cotton fabrics at commercial
scale (Rowe, 2001).
In the textile industry, polyester has certain key advantages such as it increases
strength, soft hand, stretch resistance, stain resistance, machine wash ability,
wrinkle resistance and abrasion resistance. Synthetic fibers have been processed
and modified by the action of enzymes for the use in the production of yarns,
fabrics, textiles, rugs and other consumer items. It relates to modification
of the characteristics of a polyester fiber as the result that such polyesters
are more susceptible to post-modification treatments. The use of poly-esterase
(closely related to lipase) can improve the ability of a polyester fabric to
uptake chemical compounds, such as cationic compounds, fabric finishing compositions,
dyes, anti-static compounds, anti-staining compounds, antimicrobial compounds,
antiperspirant compounds and/or deodorant compounds (Rowe,
Lipases in the production of cosmetics: Some cosmetic industries are currently produced isopropyl myristate, isopropyl palmitate and 2-ethylhexyl palmitate for use as an emollient in personal care products such as skin and sun-tan creams, bath oils etc. In this case, immobilized Rhizomucor miehei lipase was widely used as a biocatalyst. The use of the enzyme instead of the commonly used acid catalyst gives products of much higher quality with minimum downstream refining process.
Wax esters (esters of fatty acids and fatty alcohols) have similar applications
in personal care products and are also being manufactured by the action of lipase
produced from C. cylindracea using batch bioreactor. The overall production
cost in this way is slightly higher than that of the conventional method used,
however the cost is compensated by the improved quality of the final product.
Water-soluble retinol derivatives were prepared by catalytic reaction of immobilized
lipase (Maugard et al., 2002). Lipases have been
used in hair waving preparation (Saphir, 1967). Lipases
have also been used as a component of topical anti-obese creams (August,
1972) or as oral administration (Smythe, 1951).
The role of lipases in medical and pharmaceutical application: Currently,
lipases are widely used in medical and pharmaceutical industry. For instance,
enantioselective interesterification and transesterification reaction by the
help of lipases have great significance in pharmaceutical industry for selective
acylation and deacylation reaction (Stinson, 1995).
Lipases play a prime role in production of specialty lipids and digestive aids
(Vulfson, 1994). The alteration of temperature during
the esterification reaction drastically changes the enantiomeric values and
also the stereo-preference. Lipases play an important role in modification of
monoglycerides for use as emulsifiers in pharmaceutical applications (Sharma
et al., 2001a).
Lipase from Candida rugosa has been used to synthesize lovastatin, a
drug that lower serum cholesterol level. S. marcescens lipase was widely
used for the asymmetric hydrolysis of 3-phenylglycidic acid ester which is a
key intermediate in the synthesis of diltiazem hydrochloride (Matsumae
et al., 1993).
The application of lipases in synthesis of fine chemicals: Some of the
industrially important chemicals manufactured from fats and oils by chemical
processes could be produced by lipases with greater rapidity and better specificity
under mild conditions (Vulfson, 1994). The use of industrial
enzymes allows the technologists to develop processes that more closely approach
the gentle, efficient process in nature. Some of the technological processes
using cold active lipase from C. antarctica have been patented by chemical,
food industries and pharmaceutical industries.
Lipases in biodiesel production: Biodiesel is a liquid biofuel which
are esters of long chain fatty acids and short chain alcohols. Biodiesel molecules
are synthesized through direct transesterification of vegetable oils and fats
with short chain alcohols (such as methanol and ethanol) in the presence of
suitable catalysts (Fig. 2) (Vicente et
Transesterification is the displacement of alcohol from an ester by another
alcohol in a process similar to hydrolysis, except that an alcohol is employed
instead of water (Srivastava and Prasad, 2000). Among
short chain alcohols, methanol and ethanol are usually used, especially, because
of its low cost and physicochemical advantages, methanol is used frequently.
This process has been widely used to reduce the viscosity of triglycerides,
thereby enhancing the physical properties of renewable fuels to improve engine
performance (Clark et al., 1984).
The main factors affecting transesterification are molar ratio of triglycerides
to alcohol, catalysts, reaction temperature and time, the contents of free fatty
acids and water in oils and fats (Freedman et al.,
1986). Biodiesel catalysts are currently, classified as alkali, acid, or
enzyme. This review is only focused on the production of biodiesel using lipase
For the production of biodiesel, an alkali-catalysis process has been established that gives high conversion levels of oils to methyl esters. However, it has several drawbacks, including the difficulty of purifying glycerol and the need for either removal of the catalyst or wastewater treatment. In particular, several steps such as the evaporation of methanol, removal of saponified products, neutralization and concentration, are needed to recover glycerol as an added value product.
To overcome these drawbacks which may limit the availability of biodiesel fuel;
enzymatic processes using lipase have recently been developed. Since the cost
of lipase production is the main hurdle to the commercialization of the lipase-catalyzed
process, the use of intracellular lipase or cell-surface-displayed lipase as
a whole-cell biocatalyst through the application of immobilization techniques
(Ban et al., 2002) has been considered as an
effective way to lower the lipase production cost. Unlike in the case of extracellular
lipase, these whole-cell biocatalysts can be prepared by simple cultivation
and recovered easily.
However, to utilize these whole-cell biocatalysts for industrial application,
a repeated methanolysis reaction cycle is required in order to produce high
methyl ester content of 90-95%. One potential solution is the use of a whole-cell
biocatalyst possessing a non-specific lipase from a source such as Candida
antarctica (Shimada et al., 1999) or
Pseudomonas cepacia (Kaieda et al., 2001)
within the cell or on the cell-surface, since these lipases realize methyl ester
content of more than 95%. Such a system could offer a promising prospect of
realizing industrial biodiesel fuel production. However, bodiesel production
by lipase is not yet commercialized.
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