390

Lipases for biotechnology

Karl-Erich Jaeger and Thorsten Eggert
Lipases constitute the most important group of biocatalysts for
biotechnological applications. The high-level production of
microbial lipases requires not only the efficient overexpression
of the corresponding genes but also a detailed understanding
of the molecular mechanisms governing their folding and
secretion. The optimisation 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 enantiopure pharmaceuticals,
agrochemicals, and flavour compounds.
Addresses
Institute for Molecular Enzyme Technology, Heinrich-Heine-Universitt
Dsseldorf, Forschungszentrum Jlich, D-52425 Jlich, Germany;
e-mail: karl-erich.jaeger@fz-juelich.de
Current Opinion in Biotechnology 2002, 13:390397
0958-1669/02/$ see front matter
2002 Elsevier Science Ltd. All rights reserved.

Introduction
Nearly 100 years ago the microbiologist C Eijkmann
reported that several bacteria could produce and secrete
lipases. When it became generally accepted that lipases
remain enzymatically active in organic solvents [1], studies
began to develop these enzymes into ideal tools for the
organic chemist. What is it that makes lipases so attractive?
Firstly, they usually display exquisite chemoselectivity,
regioselectivity and stereoselectivity. Secondly, they are
readily available in large quantities because many of them
can be produced in high yields from microbial organisms,
namely fungi and bacteria. Thirdly, the crystal structures of
many lipases have been solved, facilitating considerably
the design of rational engineering strategies. Finally, they
do not usually require cofactors nor do they catalyse side
reactions. These properties make lipases the most widely
used group of biocatalysts in organic chemistry. This is
reflected not only by more than 1000 original articles on
lipases that appear each year, but also by an impressive
number of excellent reviews covering this topic [27].
Here, we describe recent progress in understanding the
mechanisms of lipase production and secretion, additionally,
we present some interesting novel aspects of using lipases
for biotechnological applications.

Fortunately, two excellent review articles have recently

appeared that summarise many different methods for the
detection of lipase activity [8] and discuss several highthroughput screening methods to determine enantioselectivity
and regioselectivity [9].

Novel lipases
Novel microbial lipases have been isolated, including
extremozymes from both thermophilic and psychrophilic
species [10]. All these enzymes were overexpressed in the
heterologous host Escherichia coli, however, the overexpression
efficiency in terms of yield of enzymatically active protein
was not always reported. Several thousand microbial
samples isolated from soil were tested by screening on solid
and liquid media for the production of lipases, revealing that
about 20% were lipase-producers. Taxonomic identification
resulted in the description of so far unknown lipase-producing
species, including filamentous fungi, yeast and bacteria.
These lipases were extensively characterised with respect
to their hydrolytic and synthetic capacities and also with
respect to their enantioselectivities towards artificial
substrates, such as carboxylic acids, alcohols and amines
including the kinetic resolution of racemic (R,S)-ibuprofen
and ()-menthol as high-value substrates [11,12].
Recently, a framework to classify bacterial lipases was
provided by grouping them into eight families on the basis
of conserved sequence motifs and biological properties
[13]. Meanwhile, several extensions of the original
classification scheme have been proposed [14,15]: the
lipolytic enzymes LipB from Bacillus subtilis and a Bacillus
licheniformis esterase are added to subfamily I.4. The former
subfamily I.5, which contained lipases from the genera
Bacillus and Staphylococcus exclusively contains lipases from
thermophilic Bacilli now renamed Geobacillus, namely
Geobacillus stearothermophilus, Geobacillus thermocatenulatus,
Geobacillus thermoleovorans. The lipases from Staphylococcus
aureus, Staphylococcus hyicus and Staphylococcus epidermidis
constitute subfamily I.6, which additionally contains lipases
from Staphylococcus haemolyticus, Staphylococcus xylosus and
Staphylococcus warneri. Accordingly, the lipases from
Propionibacterium acnes and Streptomyces cinnamoneus were
moved to subfamily I.7 (Table 1).

Production of lipases
Lipase assays

Overexpression

A true lipase (EC 3.1.1.3) is defined as a carboxylesterase,

which catalyses the hydrolysis and synthesis of long-chain
acylglycerols with trioleoylglycerol being the standard
substrate. Biotechnological applications of lipases often
prompt a demand for techniques to determine not only
their activity, but also their substrate- and stereoselectivity;
however, no single universal method exists that allows
the simultaneous determination of these properties.

The first step needed to isolate a lipase for biotechnological

applications is usually overexpression of the corresponding
gene of interest. Frequently, this step is considered to
be trivial, because several proteins can easily be overexpressed and sometimes even secreted using commercially
available systems [16]. Bacterial lipases from various
Bacillus species can be overexpressed in E. coli using
conventional overexpression systems [14,1719]; however,

Lipases for biotechnology Jaeger and Eggert

391

Table 1
Updated classification of bacterial lipolytic enzymes constituting family I [13

].
Subfamily

Enzyme-producing strain

Accession No.

Similarity (%)
Family

Subfamily

Pseudomonas aeruginosa (LipA)*

Pseudomonas fluorescens C9
Vibrio cholerae
Pseudomonas aeruginosa (LipC)
Acinetobacter calcoaceticus
Pseudomonas fragi
Pseudomonas wisconsinensis
Proteus vulgaris

D50587
AF031226
X16945
U75975
X80800
X14033
U88907
U33845

100
95
57
51
43
40
39
38

Burkholderia glumae*
Chromobacterium viscosum*
Burkholderia cepacia*
Pseudomonas luteola

X70354
Q05489
M58494
AF050153

35
35
33
33

100
100
78
77

Pseudomonas fluorescens SIKW1

Serratia marcescens

D11455
D13253

14
15

100
51

Bacillus subtilis (LipA)*

Bacillus pumilus
Bacillus licheniformis
Bacillus subtilis (LipB)

M74010
A34992
U35855
C69652

16
13
13
17

100
80
80
74

Geobacillus stearothermophilus L1
Geobacillus stearothermophilus P1
Geobacillus thermocatenulatus
Geobacillus thermoleovorans

U78785
AF237623
X95309
AF134840

15
15
14
14

100
94
94
92

Staphylococcus aureus
Staphylococcus haemolyticus
Staphylococcus epidermidis
Staphylococcus hyicus
Staphylococcus xylosus
Staphylococcus warneri

M12715
AF096928
AF090142
X02844
AF208229
AF208033

14
15
13
15
14
12

100
45
44
36
36
36

X99255
U80063

14
14

100
50

Propionibacterium acnes
Streptomyces cinnamoneus
*Lipolytic enzymes with known three-dimensional-structure.

Similarities of amino acid sequences were determined with the program Megalign (DNAStar) with the first member of each subfamily arbitrarily
set at 100%.

many enzymes (e.g. different Pseudomonas and Burkholderia

lipases which are used for a variety of biotransformations)
are not amenable to these systems [3]. Lipases from
Pseudomonas species require the functional assistance of
about 30 different cellular proteins before they can be
recovered from the culture supernatant in an enzymatically
active state, indicating that folding and secretion are highly
specific processes that normally do not function properly in
heterologous hosts [20].
Folding and secretion

Lipases are extracellular enzymes and must therefore be

translocated through the bacterial membrane to reach their
final destination. Figure 1 summarises the major secretion
pathways for bacterial lipases. In Gram-positive bacteria,
secreted enzymes have to cross just a single cytoplasmic
membrane. Usually, these proteins contain a signal
sequence, which directs their translocation via the Sec
machinery [21]. More recently, a second translocation
pathway has been described to operate in both Gram-negative and Gram-positive bacteria, named the Tat pathway
because proteins using this pathway contain a unique Twin

arginine translocation motif in their signal sequence. In the

B. subtilis genome, 188 proteins have been identified as
being potentially secreted. These include two lipases of
which LipA contains a Tat signal sequence, whereas the
highly homologous enzyme LipB contains a Sec signal
sequence [22].
Several Gram-negative bacteria are known to efficiently
secrete extracellular lipases, among them Pseudomonas and
Burkholderia species. In Pseudomonas aeruginosa, at least
four main secretion pathways have been identified of
which extracellular lipases use the type II pathway: after
being secreted through the inner membrane via the Sec
machinery they fold in the periplasm into an enzymatically
active conformation. Periplasmic folding catalysts are
needed to ensure the correct folding and proper secretion
of lipases, these include specific intermolecular chaperones
called Lif proteins (lipase-specific foldases) [23]. Recently,
a lipase variant from Pseudomonas species KFCC 10818
carrying just the single amino acid exchange Pro112Gln
folded into its active conformation and displayed 63% of
the wild-type enzymatic activity even in the absence

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Protein technologies and commercial enzymes

Figure 1

Outer membrane (Gram-negative)

Type II secreton
Autotransporter

ABC-transporter
Outer membrane (Gram-positive)

Sec
Tat

ATP

ADP + Pi
Inner membrane (Gram-negative)

ATP

ADP + Pi
Current Opinion in Biotechnology

Pathways used by bacteria to secrete lipolytic enzymes. Gram-positive

bacteria contain an inner or cytoplasmic membrane, Gram-negative
bacteria additionally possess a second so-called outer membrane. The
Sec and Tat secretion pathways mediate translocation of proteins
through the inner membrane and are found in both Gram-positive and

Gram-negative bacteria; the type I (ABC transporter-) and type II

(secreton-) mediated pathways and the self-secreting autotransporter
enzymes are found in Gram-negative bacteria. Relevant original
publications on enzyme secretion are cited in references [3], [20]
and [21].

of its cognate Lif protein [24]. If confirmed with other

Lif-dependent lipases these findings may have important
consequences for the construction of novel high-yield
production host strains.

pharmaceutical used as a coronary vasodilator. A thorough

analysis of the lipase secretion process in S. marcescens
revealed that a C-terminal Val-Ala-Leu motif and its location
relative to the C terminus of the lipase greatly affect the
secretion efficiency [25]. The motif identified here is
different from a previously described secretion motif, a
glycine-rich repeat consisting of the nine-residue sequence
Gly-Gly-X-Gly-X-Asp-X-U-X (where X is any amino acid
and U is a large hydrophobic amino acid). Studies with the
lipase from Pseudomonas species MIS38, which is similar to
the S. marcescens and P. fluorescens lipases, clearly showed
that this latter motif is needed for the binding of 12 Ca2+
ions, thereby inducing the folding of this lipase [26].
Overexpression of additional copies of the ABC exporter

Lipases from Pseudomonas fluorescens and Serratia marcescens

lack a typical N-terminal signal peptide. They are secreted
by the type I secretion pathway (also named the ABC
exporter) consisting of three different proteins. The lipase
from S. marcescens is a biotechnologically important
enzyme because it catalyses with high enantioselectivity
(E = 135) the asymmetric hydrolysis of (rac) trans-3(4-methoxyphenyl)glycidic acid methyl ester yielding a
key intermediate in the synthesis of diltiazem, a major

Lipases for biotechnology Jaeger and Eggert

393

Figure 2

Reaction

Product
S

(a)

Lipase

R = H, epothilone A 4
R = Me, epothilone B 5

O
OH

OAc

OH

OH

O
90% ee

3
OH

(b)
Me

Me

Me

Lipase 'OF-360'
RO

Me
OMe

OMe

OMe
RO

COOMe

RO

COOMe

R = Ac rac-7

R = H (S)-6
R = Ac (S)-7

COOMe

R = H (R)-6
R = Ac (R)-7

OH

Me

Me

Me

(R)-()-8
(c)

Cl

Cl

Lipase (1)
COOMe

Cl
COOMe

Me

Me
OAc

rac-9

COOMe

Me

OAc

OH

(2S,3R)-9

(2S,3R)-10

COOH
C
A

Me

Me
COOMe

N
H

rac-11

Me
B

Me
COOMe

Lipase (2)
N
H

R = OAc (2R,3S)-11

COOMe

+
N
H

N
H

13

R = OAc (2S,3R)-11
R = OH (2S,3R)-12

Examples for lipase-catalysed reactions to produce enantiopure key

intermediates in the synthesis of pharmaceuticals. The reaction is
shown on the left-hand side and the final product on the right.
(a) A lipase from Pseudomonas AK is used to catalyse the reaction
for the production of epothilone A (R = H) or epothilone B
(R = Me) [47]. In the reaction, 2 is a key intermediate of epothilone
A synthesis. (b) In this reaction, 6 and 7 are intermediates in the

synthesis of compounds with antibacterial activities, such as

(R)-()-curcuphenol 8. The reaction is catalysed by lipase OF-360
from Candida rugosa [48]. (c) In this reaction, 9 and 11 are
intermediates in the synthesis of the antibacterial compound
chuangxinmycin [49]. Lipase (1) is from a Pseudomonas sp.
and is termed Amano P. Lipase (2) is lipase OF-360 from
Candida rugosa.

provides a considerable increase in secretion of the lipase

and therefore an increased yield of extracellular lipase
protein, as demonstrated for S. marcescens [27] and
P. fluorescens [28] lipases.

directed evolution. The state-of-the-art technology for

directed evolution of biocatalysts including lipases has
recently been summarised in excellent review articles
[29,30,31]. From the biotechnological point of view, the
most important approach is the evolution of highly enantioselective lipases, pushed forward by a rapidly increasing
demand for enantiomerically pure compounds to be
produced by biocatalytic processes [32]. Bacterial lipases
from P. aeruginosa and B. subtilis served as model enzymes
to demonstrate the potential of directed evolution. Firstly,
P. aeruginosa lipase has been used in the creation of variants
with high enantioselectivity towards both (S)- and (R)-2methyldecanoic acid p-nitrophenylester starting from a

Optimisation of lipases by directed evolution

The commercial use of lipases is a billion dollar business,
which comprises a wide variety of different applications in
the area of detergents and in the production of food ingredients and enantiopure pharmaceuticals [3]. Therefore, a
strong pressure exists to identify and isolate novel lipase
genes and to optimise existing lipases with respect to
desired properties, which is nowadays performed by

394

Protein technologies and commercial enzymes

Figure 3

O
H3C

OH

CH3

C O

B. cepacia
lipase

O C C CH2
CH3

H2C C C O N C

CH3

rac-menthol 14

H3C
C O
HO N C
CH3

CH3

CH3

15

16

17
Current Opinion in Biotechnology

Enantioselective synthesis of menthyl methacrylate 16 catalysed by B. cepacia lipase [52].

non-selective wild-type enzyme [3335]. Secondly, using

an ultrahigh-throughput screening system based on
electrospray ionisation mass spectrometry (ESI-MS), an
enantioselective B. subtilis lipase was evolved that is able
to hydrolyze a meso-compound [9,36]. Thirdly, the
solved crystal structures of both P. aeruginosa [37] and
B. subtilis [38] lipases were used to rationalise amino acid
exchanges leading to increased enantioselectivity.
High-throughput screening to identify enantioselective
lipases is usually an arduous and sometimes expensive task
(see methods reviewed in [9]). Therefore, alternative
methods are being developed that are based on selection
such as phage display, which in principle allows for the
identification of a better enzyme variant in a very
large library consisting of 108 to 1012 members [39]. The
commercially available lipase Lipolase from Thermomyces
lanuginosa was successfully displayed on the surface of
E. coli phage M13 after being fused to coat protein 3 [40]
The selection method employed was based on the covalent
attachment of a biotinylated p-nitrophenylphosphonate to
the active site. Using triolein-inhibitor-coated microtiter
plates, it was possible to enrich lipase variants that retained
activity in the presence of a commercial household detergent.
However, variants performing better than the wild-type
enzyme could not be identified. At present, it seems
questionable as to whether this method may also prove
useful to select for enantioselective lipase variants.

using commercially available lipases from different

sources. High diversity of lipase-catalysed polymer
libraries was achieved by the free combination of diester
and diol monomers, different reaction conditions and the
use of lipases from various sources. In this way, polyester
libraries in 96 deep-well plates were generated in a rapid
and systematic manner. Additionally, the possibility of
ring-opening polymerisation of lactones and cyclic carbonates as well as the transesterification or transacylation of
macromolecules was demonstrated [42].
Biodiesel

An alternative source of energy for public transport is the

so-called biodiesel, which has been produced chemically
using oil from various plants (e.g. rapeseed). Biodiesel fuel
originates from renewable natural resources and concomitantly reduces sulfur oxide production. The conversion of
vegetable oil to methyl- or other short-chain alcohol esters
can be catalysed in a single transesterification reaction using
lipases in organic solvents. However, the production at an
industrial scale failed so far because of the high cost of the
appropriate biocatalyst. Two strategies were presented
recently to solve this problem: immobilisation of P. fluorescens
lipase increased its stability even upon repeated use [43]; and
cytoplasmic overexpression of Rhizopus oryzae lipase in
Saccharomyces cerevisiae with subsequent freeze-thawing and
air drying resulted in a whole-cell biocatalyst that catalysed
methanolysis in a solvent-free reaction system [44].

Biotechnological applications of lipases

Synthesis of fine chemicals

New biopolymeric materials

Key intermediates in the synthesis of therapeutics,

agrochemicals and flavour compounds are usually complex
and/or chiral compounds, which are difficult to synthesise
with chemical methods. Furthermore, just one out of two
drug enantiomers is pharmaceutically functional, making
the synthesis of enantiopure building blocks an important
task for the pharma industry [45]. This is a major reason for
biocatalysis to expand dramatically [46], with lipases
being at the forefront of this development.

Biopolymers like polyphenols, polysaccharides and polyesters show a considerable degree of diversity and
complexity. Furthermore, these compounds are receiving
increasing attention because they are biodegradable and
produced from renewable natural resources. Lipases and
esterases are used as catalysts for polymeric synthesis [41]
with the major advantages being their high selectivity
(e.g. stereoselectivity, regioselectivity and chemoselectivity)
under mild reaction conditions. A combinatorial strategy
was employed to isolate novel polyesters [42]. Structurally
complex monomers with multifunctional reactive groups
were polymerised in a high-throughput enzymatic catalysis

Therapeutics

Several new examples of lipase-catalysed enantioselective

reactions for the synthesis of pharmaceuticals are given in

Lipases for biotechnology Jaeger and Eggert

Figure 2. Pseudomonas AK lipase was used to synthesise

the chiral intermediate 2 in the total synthesis of the
potent antitumour agent epothilone A 4 [47]. Candida
rugosa lipase catalysed the enzymatic resolution of the
antimicrobial compounds (S)- and (R)-elvirol and their
derivatives (S)-(+)- and (R)-()-curcuphenol. (R)-()-curcuphenol 8 exhibits antibacterial activity against
Staphylococcus aureus and Vibrio anguillarum, whereas the
(S)-(+)-enantiomer inhibits the gastric H/ K-ATPase [48].
Biocatalysis by lipases from Pseudomonas species and
C. rugosa led to the chiral intermediates 9 and 11 in the synthesis of the antimicrobial compound chuangxinmycin 13 [49].
Agrochemicals

Lipases are also used in the efficient production of enantiopure (S)-indanofan, a novel herbicide used against grass
weeds in paddy fields [50]. Only the (S)-enantiomer shows
herbicidal activity, which is now synthesised by combined
lipase-catalysed enzymatic resolution and chemical inversion
techniques. The diastereomers of 4-hydroxyproline represent important building blocks for several agrochemicals
and pharmaceuticals. Candida antarctica lipase B was
identified among 43 different commercial lipases and esterases
as an efficient biocatalyst for the enantioselective hydrolysis
of racemic 4-oxo-1,2-pyrrolidinedicarboxylic acid dimethyl
ester [51]. The final compounds cis-4-hydroxy-D-proline or
trans-4-hydroxy-D-proline were produced with 93 to > 99.5%
diastereomeric excess.
Cosmetics and flavours

Several examples of the lipase-assisted synthesis of flavour and

fragrance compounds were reported, with ()-menthol being
the most prominent one. A new way to isolate enantiomerically
pure ()-menthol esters contains a transesterification step with
()-menthol using Burkholderia cepacia lipase (Figure 3) [52].
The final product menthyl methacrylate 16 was subsequently
polymerised to be used as a sustained release perfume. The
plant growth factor ()-methyl jasmonate is another important
perfumery constituent, which can be synthesised with a lipasecatalysed reaction using the commercially available Lipase P
(Amano) to yield the chiral key intermediate (+)-(6S)-methyl
7-epicucurbate [53].
Optimisation of lipase-catalysed reactions

An intelligent combination of improved biocatalysts and

optimised reaction conditions will pave the way to even more
efficient biocatalytic processes. Enzymes are optimised by
directed evolution techniques and several novel approaches
have been described recently to further improve the yield and
purity of the reaction products.

trans-esterification of either -hydroxy esters or -hydroxy

nitriles in combination with ruthenium-catalysed alcohol
racemisation. These reactions proceeded with 92% and 85%
conversions and yielded in high purity products showing
enantiomeric excess (ee) values of up to 99%, thereby providing important building blocks for many pharmaceuticals
and agrochemicals.
Novel solvents for lipase-catalysed reactions

Ionic liquids turned out to be ideal solvents for enzymecatalysed transformations carried out with highly polar
substrates. However, the reproducible preparation of the
solvent seems to be a major challenge, because minor
changes in the structure of the ionic liquid can result in
dramatic changes of the enzymes kinetic properties. A
reliable and reproducible preparation technique for different
ionic liquids was described to overcome this dilemma.
During the preparation, a wash with aqueous sodium
carbonate turned out to be important to yield ionic liquids
suitable for enzymatic reactions [56]. However, much
more work must be invested to understand the key
structural features of ionic liquids that control enzymecatalysed reactions.
Supercritical carbon dioxide (scCO2) with its liquid-like
quality proved to be another promising solvent for lipasecatalysed reactions. The easy and complete removal of this
solvent offers significant advantages for downstream processing,
including product purification. Lipases from Rhizomucor
miehei (Lipozyme) [57] and C. antarctica (Novozym 435) [58]
showed an ideal catalysis performance with respect to activity and stability when tested in scCO2 as the reaction solvent.

Conclusions
The use of lipases for a variety of biotechnological applications
is rapidly and steadily increasing. Many novel lipase genes
are still to be identified and enzymes with new and exciting
properties will be discovered. In parallel, the combination
of optimised lipases with improved reaction conditions will
lead to novel synthetic routes, allowing the production of
high-value chemicals and pharmaceuticals. The new era of
biocatalysis that has just started will undoubtedly see lipases
as the biocatalysts of the future.

Acknowledgements
Our work on lipases is supported by the European Commission in the framework
of the programme Biotechnology (project-no. QLK3-CT-2001-00519).

References and recommended reading

Papers of particular interest, published within the annual period of review,
have been highlighted as:

Dynamic kinetic resolution

The dynamic kinetic resolution approach theoretically

allows the 100% conversion of chiral reaction educts as
compared with a maximum yield of 50% enantiopure
product obtainable from an asymmetric kinetic resolution
reaction. An impressive example is the synthesis of chiral
-lactones [54] and -amino alcohols [55] by lipase-catalysed

395

of special interest
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Jaeger K-E, Dijkstra BW, Reetz MT: Bacterial biocatalysts:

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15. Eggert T, van Pouderoyen G, Pencreach G, Douchet I, Verger R,

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