Karl-Erich Jaeger and Thorsten Eggert- Lipases for biotechnology

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Lipases for biotechnology Jaeger and Eggert 391

many enzymes (e.g. differentPseudomonas andBurkholderia

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-neg-

ative 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 themPseudomonas andBurkholderia 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

Table 1

Updated classification of bacterial lipolytic enzymes constituting family I [13

].

Subfamily Enzyme-producing strain Accession No. Similarity(%)

Family Subfamily

1 Pseudomonas aeruginosa(LipA)* D50587 100Pseudomonas fluorescensC9 AF031226 95Vibrio cholerae X16945 57Pseudomonas aeruginosa(LipC) U75975 51Acinetobacter calcoaceticus X80800 43Pseudomonas fragi X14033 40Pseudomonas wisconsinensis U88907 39Proteus vulgaris U33845 38

2 Burkholderia glumae* X70354 35 100Chromobacterium viscosum* Q05489 35 100Burkholderia cepacia* M58494 33 78Pseudomonas luteola AF050153 33 77

3 Pseudomonas fluorescensSIKW1 D11455 14 100Serratia marcescens D13253 15 51

4 Bacillus subtilis(LipA)* M74010 16 100

Bacillus pumilus A34992 13 80Bacillus licheniformis U35855 13 80Bacillus subtilis(LipB) C69652 17 74

5 Geobacillus stearothermophilusL1 U78785 15 100Geobacillus stearothermophilusP1 AF237623 15 94Geobacillus thermocatenulatus X95309 14 94Geobacillus thermoleovorans AF134840 14 92

6 Staphylococcus aureus M12715 14 100Staphylococcus haemolyticus AF096928 15 45Staphylococcus epidermidis AF090142 13 44Staphylococcus hyicus X02844 15 36Staphylococcus xylosus AF208229 14 36Staphylococcus warneri AF208033 12 36

7 Propionibacterium acnes X99255 14 100Streptomyces cinnamoneus U80063 14 50

*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%.


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392 Protein technologies and commercial enzymes

of its cognate Lif protein [24

]. If confirmed with otherLif-dependent lipases these findings may have important

consequences for the construction of novel high-yield

production host strains.

Lipases fromPseudomonas fluorescens andSerratia 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

pharmaceutical used as a coronary vasodilator. A thoroughanalysis 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 fromPseudomonas 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 Ca 2+

ions, thereby inducing the folding of this lipase [26].

Overexpression of additional copies of the ABC exporter

Figure 1

Inner membrane (Gram-negative)

Outer membrane (Gram-negative)

Sec

Outer membrane (Gram-positive)

Autotransporter

ABC-transporter

Type II secreton

Tat

ATP ADP + Pi

ATP ADP + Pi

Current Opinion in Biotechnology

Pathways used by bacteria to secrete lipolytic enzymes. Gram-positivebacteria contain an inner or cytoplasmic membrane, Gram-negativebacteria additionally possess a second so-called outer membrane. TheSec and Tat secretion pathways mediate translocation of proteinsthrough 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-secretingautotransporterenzymes are found in Gram-negative bacteria. Relevant originalpublications on enzyme secretion are cited in references [3], [20]and [21].


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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.

Optimisation of lipases by directed evolutionThe 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 ingre-

dients 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

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 enan-

tioselective lipases, pushed forward by a rapidly increasing

demand for enantiomerically pure compounds to be

produced by biocatalytic processes [32]. Bacterial lipases

fromP. aeruginosa andB. 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)-2-

methyldecanoic acid p-nitrophenylester starting from a

Figure 2

S

N

OH

S

N

OH

S

N

OAc

Me

OMe

RO

COOMe

Me

OMe

RO

COOMe

Me

OMe

RO

COOMe

+

+

MeCOOMe

Cl

OAc

MeCOOMe

Cl

OH

+

NH

COOMe

Me

R

+

NH

COOMe

Me

R

S

N

O

O O

OH

O

OH

R

Me

H

OH

Me Me

Me

N

H

S

COOH

Me

(a)

(b)

(c)

90% ee

1 2 3

A B

C

13

rac-9

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

R = Ac (S)-7

R = H (R)-6

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

R = Ac (R)-7

(R)-()-8

rac-11

Lipase (2)

Lipase (1)

Lipase 'OF-360'

(2S,3R)-9

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

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

(2S,3R)-10

Lipase

Cl

Me

COOMe

OAc

NH

COOMe

Me

R

Reaction Product

Examples for lipase-catalysed reactions to produce enantiopure keyintermediates in the synthesis of pharmaceuticals. The reaction isshown on the left-hand side and the final product on the right.(a) A lipase from Pseudomonas AK is used to catalyse the reactionfor the production of epothilone A (R = H) or epothilone B(R = Me) [47]. In the reaction, 2 is a key intermediate of epothiloneA 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-360from Candida rugosa [48]. (c) In this reaction, 9 and 11 areintermediates in the synthesis of the antibacterial compoundchuangxinmycin [49]. Lipase (1) is from a Pseudomonas sp.and is termed Amano P. Lipase (2) is lipase OF-360 fromCandida rugosa.


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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 ableto 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 Thermomyceslanuginosa was successfully displayed on the surface of

E. coliphage 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.

Biotechnological applications of lipasesNew biopolymeric materialsBiopolymers like polyphenols, polysaccharides and poly-

esters 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

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 theuse 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 carbon-

ates 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 concomi-

tantly reduces sulfur oxide production. The conversion ofvegetable 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 ofP. fluorescens

lipase increased its stability even upon repeated use [43]; and

cytoplasmic overexpression of Rhizopus oryzae lipase in

Saccharomyces cerevisiaewith subsequent freeze-thawing and

air drying resulted in a whole-cell biocatalyst that catalysed

methanolysis in a solvent-free reaction system [44].

Synthesis of fine chemicals

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.

Therapeutics

Several new examples of lipase-catalysed enantioselective

reactions for the synthesis of pharmaceuticals are given in

Figure 3

CH3

OH

+ H2C C C

CH3

O

O

N C

CH3

C

H3C

OB. cepacia

lipase

CH3

O C C

O

CH2

CH3

+HO N C

CH3

C

H3C

O

rac-menthol 14 15 16 17

Current Opinion in Biotechnology

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


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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)-()-cur-cuphenol 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 synthe-

sis of the antimicrobial compound chuangxinmycin 13 [49].

Agrochemicals

Lipases are also used in the efficient production of enan-

tiopure (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 inversiontechniques. The diastereomers of 4-hydroxyproline repre-

sent 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 beingthe 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 lipase-

catalysed 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 andoptimised 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.

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

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 pro-

viding important building blocks for many pharmaceuticalsand agrochemicals.

Novel solvents for lipase-catalysed reactions

Ionic liquids turned out to be ideal solvents for enzyme-

catalysed 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 liquidssuitable for enzymatic reactions [56]. However, much

more work must be invested to understand the key

structural features of ionic liquids that control enzyme-

catalysed reactions.

Supercritical carbon dioxide (scCO2) with its liquid-like

quality proved to be another promising solvent for lipase-

catalysed 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 activi-

ty and stability when tested in scCO2 as the reaction solvent.

ConclusionsThe 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.

AcknowledgementsOur work on lipases is supported by the European Commission in the frameworkof the programme Biotechnology (project-no. QLK3-CT-2001-00519).

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Karl-Erich Jaeger and Thorsten Eggert- Lipases for biotechnology

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