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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-Universität Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany; e-mail: [email protected] Current Opinion in Biotechnology 2002, 13:390–397 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 [2–7]. 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 [14,17–19]; however,
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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)* 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
2
Burkholderia glumae* Chromobacterium viscosum* Burkholderia cepacia* Pseudomonas luteola
X70354 Q05489 M58494 AF050153
35 35 33 33
100 100 78 77
3
Pseudomonas fluorescens SIKW1 Serratia marcescens
D11455 D13253
14 15
100 51
4
Bacillus subtilis (LipA)* Bacillus pumilus Bacillus licheniformis Bacillus subtilis (LipB)
M74010 A34992 U35855 C69652
16 13 13 17
100 80 80 74
5
Geobacillus stearothermophilus L1 Geobacillus stearothermophilus P1 Geobacillus thermocatenulatus Geobacillus thermoleovorans
U78785 AF237623 X95309 AF134840
15 15 14 14
100 94 94 92
6
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
7
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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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
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Figure 2
Reaction
Product S
(a)
N
Lipase
N
R = H, epothilone A 4 R = Me, epothilone B 5
S
S
S
+
N
N
O
O OH
OAc
OH
OH
O 90% ee
R
2
1
O
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
H
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
R
Me B
Me COOMe
Lipase (2) N H
S
R
R = OAc (2R,3S)-11
COOMe
+ N H
R
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
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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
O
+
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 [33–35]. 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 δ-hydroxy esters or β-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 enzyme’s 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 δ-lactones [54] and γ-amino alcohols [55] by lipase-catalysed
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34. Reetz MT, Wilensek S, Zha D, Jaeger K-E: Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis. Angew Chem Int Ed Engl 2001, 40:3589-3591. 35. Zha D, Wilensek S, Hermes M, Jaeger K-E, Reetz MT: Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution. Chem Commun 2001: 2664-2665. 36. Jaeger K-E, Eggert T, Eipper A, Reetz MT: Directed evolution and the • creation of enantioselective biocatalysts. Appl Microbiol Biotechnol 2001, 55:519-530. This review provides an extensive summary of current directed evolution methods and their use to optimise the enantioselectivity of biocatalysts including several lipases. 37.
Nardini M, Lang DA, Liebeton K, Jaeger K-E, Dijkstra BW: Crystal structure of Pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases. J Biol Chem 2000, 275:31219-31225.
38. van Pouderoyen G, Eggert T, Jaeger K-E, Dijkstra BW: The crystal β hydrolase fold structure of Bacillus subtilis lipase: a minimal α/β enzyme. J Mol Biol 2001, 309:215-226. 39. Soumillion P, Fastrez J: Novel concepts for selection of catalytic activity. Curr Opin Biotechnol 2001, 12:387-394. 40. Danielsen S, Eklund M, Deussen HJ, Graslund T, Nygren PA, • Borchert TV: In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor. Gene 2001, 272:267-274. An impressive phage display approach that attempts to select for optimised lipase variants. 41. Gross RA, Kalra B, Kumar A: Polyester and polycarbonate synthesis by in vitro enzyme catalysis. Appl Microbiol Biotechnol 2001, 55:655-660.
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42. Kim D-Y, Dordick JS: Combinatorial array-based enzymatic • polyester synthesis. Biotechnol Bioeng 2001, 76:200-206. The lipase-catalysed polycondensation of structurally complex monomers demonstrated the feasibility of a combinatorial biocatalytic approach for polymer synthesis. 43. Iso M, Chen B, Eguchi M, Kudo T, Shrestha S: Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase. J Mol Catal B: Enzymatic 2001, 16:53-58. 44. Matsumoto T, Takahashi S, Kaieda M, Ueda M, Tanaka A, Fukuda H, Kondo A: Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production. Appl Microbiol Biotechnol 2001, 57:515-520.
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51. Sigmund AE, Hong W, Shapiro R, DiCosimo R: Chemoenzymatic synthesis of cis-4-hydroxy-D-proline. Adv Synth Catal 2001, 343:587-590. 52. Athawale V, Manjrekar N, Athawale M: Enzymatic synthesis of chiral menthyl methacrylate monomer by Pseudomonas cepacia lipase catalysed resolution of (±)-menthol. J Mol Catal B: Enzymatic 2001, 16:169-173. 53. Kiyota H, Higashi E, Koike T, Oritani T: Lipase-catalyzed preparation of both enantiomers of methyl jasmonate. Tetrahedron Asymmetry 2001, 12:1035-1038.
45. Patel RN: Enzymatic synthesis of chiral intermediates for drug development. Adv Synth Catal 2001, 343:527-546.
54. Pàmies O, Bäckvall J-E: Enzymatic kinetic resolution and chemoenzymatic dynamic kinetic resolution of δ-hydroxy esters. An efficient route to chiral δ-lactones. J Org Chem 2002, 67:1261-1265.
46. Liese A, Seelbach K, Wandrey C: Industrial Biotransformations. •• Weinheim: Wiley-VCH; 2000. An excellent summary of industrially relevant biocatalysts, the corresponding reactions and process essentials.
55. Pàmies O, Bäckvall J-E: Efficient lipase-catalyzed kinetic resolution and dynamic kinetic resolution of β-hydroxy nitriles. A route to useful precursors for γ-amino alcohols. Adv Synth Catal 2001, 343:726-731.
47.
56. Park S, Kazlauskas RJ: Improved preparation and use of room • temperature ionic liquids in lipase-catalyzed enantio- and regioselective acylations. J Org Chem 2001, 66:8395-8401. A thorough experimental analysis of ionic liquids to optimise their use for lipase-catalysed reactions.
Zhu B, Panek JS: Methodology based on chiral silanes in the synthesis of polypropionate-derived natural products — total synthesis of epothilone A. Eur J Org Chem 2001, 001:1701-1714.
48. Ono M, Suzuki K, Tanikawa S, Akita H: First synthesis of (+)- and (–)-elvirol based on an enzymatic function. Tetrahedron Asymmetry 2001, 12:2597-2604. 49. Kato K, Ono M, Akita H: New total synthesis of (±)-, (–)- and (+)chuangxinmycin. Tetrahedron 2001, 57:10055-10062. 50. Tanaka K, Yoshida K, Sasaki C, Osano YT: Practical asymmetric synthesis of the herbicide (S)-indanofan via lipase-catalyzed kinetic resolution of a diol and stereoselective acid-catalyzed hydrolysis of a chiral epoxide. J Org Chem 2002, 67:3131-3133.
57.
Al-Duri B, Goddard R, Bosley J: Characterisation of a novel support for biocatalysis in supercritical carbon dioxide. J Mol Catal B: Enzymatic 2001, 11:825-834.
58. Matsumura S, Ebata H, Kondo R, Toshima K: Organic solvent-free enzymatic transformation of poly(εε-caprolyctone) into repolymerizable oligomers in supercritical carbon dioxide. Macromol Rapid Commun 2001, 22:1325-1329.
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-Universität Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany; e-mail: [email protected] Current Opinion in Biotechnology 2002, 13:390–397 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 [2–7]. 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 [14,17–19]; however,
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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)* 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
2
Burkholderia glumae* Chromobacterium viscosum* Burkholderia cepacia* Pseudomonas luteola
X70354 Q05489 M58494 AF050153
35 35 33 33
100 100 78 77
3
Pseudomonas fluorescens SIKW1 Serratia marcescens
D11455 D13253
14 15
100 51
4
Bacillus subtilis (LipA)* Bacillus pumilus Bacillus licheniformis Bacillus subtilis (LipB)
M74010 A34992 U35855 C69652
16 13 13 17
100 80 80 74
5
Geobacillus stearothermophilus L1 Geobacillus stearothermophilus P1 Geobacillus thermocatenulatus Geobacillus thermoleovorans
U78785 AF237623 X95309 AF134840
15 15 14 14
100 94 94 92
6
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
7
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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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
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Figure 2
Reaction
Product S
(a)
N
Lipase
N
R = H, epothilone A 4 R = Me, epothilone B 5
S
S
S
+
N
N
O
O OH
OAc
OH
OH
O 90% ee
R
2
1
O
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
H
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
R
Me B
Me COOMe
Lipase (2) N H
S
R
R = OAc (2R,3S)-11
COOMe
+ N H
R
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
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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
O
+
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 [33–35]. 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 δ-hydroxy esters or β-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 enzyme’s 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 δ-lactones [54] and γ-amino alcohols [55] by lipase-catalysed
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Reetz MT: Combinatorial and evolution-based methods in the creation of enantioselective catalysts. Angew Chem Int Ed Engl 2001, 40:284-310. A comprehensive review article describing the current status of highthroughput screening technology to determine the enantioselectivity of chemical and biocatalysts. 10. Demirjian DC, Moris-Varas F, Cassidy CS: Enzymes from extremophiles. Curr Opin Chem Biol 2001, 5:144-151. 11. Cardenas F, de Castro MS, Sanchez-Montero JM, Sinisterra JV, Valmaseda M, Elson SW, Alvarez E: Novel microbial lipases: catalytic activity in reactions in organic media. Enzyme Microb Technol 2001, 28:145-154.
24. Kim EK, Jang WH, Ko JH, Kang JS, Noh MJ, Yoo OJ: Lipase and its • modulator from Pseudomonas sp. strain KFCC 10818: proline-toglutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator. J Bacteriol 2001, 183:5937-5941. This is the first report of a Pseudomonas lipase adopting an enzymatically active conformation even in the absence of its cognate intermolecular chaperone. 25. Omori K, Idei A, Akatsuka H: Serratia ATP-binding cassette protein exporter, Lip, recognizes a protein region upstream of the C terminus for specific secretion. J Biol Chem 2001, 276:27111-27119. 26. Amada K, Kwon HJ, Haruki M, Morikawa M, Kanaya S: Ca2+-induced folding of a family I.3 lipase with repetitive Ca2+ binding motifs at the C-terminus. FEBS Lett 2001, 509:17-21. 27. •
Shibatani T, Omori K, Akatsuka H, Kawai E, Matsumae H: Enzymatic resolution of diltiazem intermediate by Serratia marcescens lipase: molecular mechanism of lipase secretion and its industrial application. J Mol Catal B: Enzymatic 2000, 10:141-149. An impressive connection of biological findings concerning expression and secretion of S. marcescens lipase with an important biotechnological application. 28. Ahn JH, Pan JG, Rhee JS: Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp. Appl Environ Microbiol 2001, 67:5506-5511. 29. Powell KA, Ramer SW, del Cardayré SB, Stemmer WPC, Tobin MB, •• Longchamp PF, Huisman GW: Directed evolution and biocatalysis. Angew Chem Int Ed Engl 2001, 40:3948-3959. This review article summarises the state-of-the-art technology of recombinative methods like DNA-shuffling for directed evolution of biocatalysts including lipases. 30. Farinas ET, Bulter T, Arnold FH: Directed enzyme evolution. Curr Opin Biotechnol 2001, 12:545-551.
12. Cardenas F, Alvarez E, de Castro-Alvarez M-S, Sanchez-Montero J-M, Valmaseda M, Elson SW, Sinisterra J-V: Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases. J Mol Catal B: Enzymatic 2001, 14:111-123.
31. Brakmann S: Discovery of superior enzymes by directed molecular evolution. Chembiochem 2001, 2:865-871.
13. Arpigny JL, Jaeger K-E: Bacterial lipolytic enzymes: classification •• and properties. Biochem J 1999, 343:177-183. A frequently cited classification of bacterial lipases based on amino acid homology and biological properties.
32. Reetz MT, Jaeger K-E: Directed evolution as a means to create enantioselective enzymes for use in organic chemistry. In Directed Molecular Evolution of Proteins. Edited by Brakmann S, Johnson K. Weinheim: Wiley-VCH; 2002:245-279.
14. Nthangeni MB, Patterton H, van Tonder A, Vergeer WP, Litthauer D: Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases. Enzyme Microb Technol 2001, 28:705-712.
33. Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW, Reetz MT, Jaeger K-E: Directed evolution of an enantioselective lipase. Chem Biol 2000, 7:709-718.
15. Eggert T, van Pouderoyen G, Pencreac´h G, Douchet I, Verger R, Dijkstra BW, Jaeger K-E: Biochemical properties and threedimensional structures of two extracellular lipolytic enzymes from Bacillus subtilis. Colloids and Surfaces B: Biointerfaces 2002, in press. 16. Baneyx F: Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 1999, 10:411-421. 17.
Eggert T, Pencreac´h G, Douchet I, Verger R, Jaeger K-E: A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase. Eur J Biochem 2000, 267:6459-6469.
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34. Reetz MT, Wilensek S, Zha D, Jaeger K-E: Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis. Angew Chem Int Ed Engl 2001, 40:3589-3591. 35. Zha D, Wilensek S, Hermes M, Jaeger K-E, Reetz MT: Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution. Chem Commun 2001: 2664-2665. 36. Jaeger K-E, Eggert T, Eipper A, Reetz MT: Directed evolution and the • creation of enantioselective biocatalysts. Appl Microbiol Biotechnol 2001, 55:519-530. This review provides an extensive summary of current directed evolution methods and their use to optimise the enantioselectivity of biocatalysts including several lipases. 37.
Nardini M, Lang DA, Liebeton K, Jaeger K-E, Dijkstra BW: Crystal structure of Pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases. J Biol Chem 2000, 275:31219-31225.
38. van Pouderoyen G, Eggert T, Jaeger K-E, Dijkstra BW: The crystal β hydrolase fold structure of Bacillus subtilis lipase: a minimal α/β enzyme. J Mol Biol 2001, 309:215-226. 39. Soumillion P, Fastrez J: Novel concepts for selection of catalytic activity. Curr Opin Biotechnol 2001, 12:387-394. 40. Danielsen S, Eklund M, Deussen HJ, Graslund T, Nygren PA, • Borchert TV: In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor. Gene 2001, 272:267-274. An impressive phage display approach that attempts to select for optimised lipase variants. 41. Gross RA, Kalra B, Kumar A: Polyester and polycarbonate synthesis by in vitro enzyme catalysis. Appl Microbiol Biotechnol 2001, 55:655-660.
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42. Kim D-Y, Dordick JS: Combinatorial array-based enzymatic • polyester synthesis. Biotechnol Bioeng 2001, 76:200-206. The lipase-catalysed polycondensation of structurally complex monomers demonstrated the feasibility of a combinatorial biocatalytic approach for polymer synthesis. 43. Iso M, Chen B, Eguchi M, Kudo T, Shrestha S: Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase. J Mol Catal B: Enzymatic 2001, 16:53-58. 44. Matsumoto T, Takahashi S, Kaieda M, Ueda M, Tanaka A, Fukuda H, Kondo A: Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production. Appl Microbiol Biotechnol 2001, 57:515-520.
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45. Patel RN: Enzymatic synthesis of chiral intermediates for drug development. Adv Synth Catal 2001, 343:527-546.
54. Pàmies O, Bäckvall J-E: Enzymatic kinetic resolution and chemoenzymatic dynamic kinetic resolution of δ-hydroxy esters. An efficient route to chiral δ-lactones. J Org Chem 2002, 67:1261-1265.
46. Liese A, Seelbach K, Wandrey C: Industrial Biotransformations. •• Weinheim: Wiley-VCH; 2000. An excellent summary of industrially relevant biocatalysts, the corresponding reactions and process essentials.
55. Pàmies O, Bäckvall J-E: Efficient lipase-catalyzed kinetic resolution and dynamic kinetic resolution of β-hydroxy nitriles. A route to useful precursors for γ-amino alcohols. Adv Synth Catal 2001, 343:726-731.
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56. Park S, Kazlauskas RJ: Improved preparation and use of room • temperature ionic liquids in lipase-catalyzed enantio- and regioselective acylations. J Org Chem 2001, 66:8395-8401. A thorough experimental analysis of ionic liquids to optimise their use for lipase-catalysed reactions.
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48. Ono M, Suzuki K, Tanikawa S, Akita H: First synthesis of (+)- and (–)-elvirol based on an enzymatic function. Tetrahedron Asymmetry 2001, 12:2597-2604. 49. Kato K, Ono M, Akita H: New total synthesis of (±)-, (–)- and (+)chuangxinmycin. Tetrahedron 2001, 57:10055-10062. 50. Tanaka K, Yoshida K, Sasaki C, Osano YT: Practical asymmetric synthesis of the herbicide (S)-indanofan via lipase-catalyzed kinetic resolution of a diol and stereoselective acid-catalyzed hydrolysis of a chiral epoxide. J Org Chem 2002, 67:3131-3133.
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