Esterases as stereoselective biocatalysts

Biotechnology Advances 33 (2015) 547–565

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Esterases as stereoselective biocatalysts Diego Romano a, Francesco Bonomi a, Marcos Carlos de Mattos b, Thiago de Sousa Fonseca b, Maria da Conceição Ferreira de Oliveira b, Francesco Molinari a a b

Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, Via Mangiagalli 25, 20133 Milan, Italy Department of Organic and Inorganic Chemistry, Federal University of Ceará, Campus do Pici, Postal Box 6044, 60455-970 Fortaleza, Ceará, Brazil

a r t i c l e

i n f o

Available online 10 February 2015 Keywords: Esterases Enantioselectivity Carboxylester hydrolases Biocatalysis Protein engineering Medium engineering Kinetic resolution

a b s t r a c t Non-lypolitic esterases are carboxylester hydrolases with preference for the hydrolysis of water-soluble esters bearing short-chain acyl residues. The potential of esterases as enantioselective biocatalysts has enlarged in the last few years due to the progresses achieved in different areas, such as screening methodologies, overproduction of recombinant esterases, structural information useful for understanding the rational behind enantioselectivity, and efficient methods in protein engineering. Contributions of these complementary know-hows to the development of new robust enantioselective esterases are critically discussed in this review. © 2015 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction, definitions and classifications . . . . . . . . . . . . . . . . . . . . . . . . . . Structural determinants of activity and enantioselectivity . . . . . . . . . . . . . . . . . . . . 2.1. Structural determinants of activity . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Enantioselectivity and the active site structure . . . . . . . . . . . . . . . . . . . . . 2.3. Enantioselectivity and substrate access . . . . . . . . . . . . . . . . . . . . . . . . . 3. Assays for esterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Identification of new esterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Methods for improvement of enantioselectivity . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Improvement by protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Improvement by medium engineering . . . . . . . . . . . . . . . . . . . . . . . . . 6. Application of non-lypolitic esterases as enantioselective biocatalysts . . . . . . . . . . . . . . 6.1. Enantioselective hydrolysis of chiral and prochiral esters of primary and secondary alcohols. 6.2. Kinetic resolution of racemic esters of tertiary alcohols. . . . . . . . . . . . . . . . . . 6.3. Enantioselective extremophilic esterases . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Immobilization of esterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Use of esterases on a preparative scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Summary and outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction, definitions and classifications Carboxylester hydrolases (EC 3.1.1.1) encompass a large and sundry group of enzymes able to catalyse the cleavage and formation of carboxyl ester bonds. These enzymes have been used as biocatalysts due to their good stability, high chemo-, regio- or stereoselectivity, while working

http://dx.doi.org/10.1016/j.biotechadv.2015.01.006 0734-9750/© 2015 Elsevier Inc. All rights reserved.

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547 548 548 548 549 549 552 553 553 558 559 559 561 561 562 562 562 563 563

without organic cofactors. Traditionally, they have been classified as (carboxyl)esterases and lipases, based on experimental data and theoretical hypothesis, often quite uncertain. Lipases are generally considered as lipolytic carboxylester hydrolases capable of hydrolysing water-insoluble esters, releasing long-chain fatty acids (N 8 carbon atoms), whereas esterases have been mostly recognized as enzymes

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D. Romano et al. / Biotechnology Advances 33 (2015) 547–565

acting on water-soluble esters bearing short-chain acyl residues (b 8 carbon atoms). Various benchmarks (primary sequence, structural features, kinetics, and use of specific inhibitors) have been proposed to clearly differentiate lipases from esterases, but all the proposed criteria for differentiation have been applied with little success. All the suggested criteria for discriminating lipases from esterases were recently reconsidered and it was concluded that none of them was suitable, as lipases are just a kind of esterases (Ben Ali et al., 2012). Therefore, they pragmatically suggested organizing the world of carboxylester hydrolases in lipolytic esterases (lipases or LEst, proposed EC: L3.1.1.1) and non-lipolytic esterases (NLEst, proposed EC: NL3.1.1.1), although the official nomenclature is still referred to (carboxyl)esterase as EC 3.1.1.1 and lipases (triacylglycerol) as EC 3.1.1.3. This review deals with the use of non-lypolitic esterases (from here forward simply called esterases) as biocatalysts and is organized according to important concepts related to stereoselective hydrolysis of chiral and prochiral esters developed in the last few years, addressing specific aspects of the interactions underlying the stereoselective action of these proteins and methods for their improvement. Examples where the use of esterases allows the development of chemoenzymatic routes to industrially relevant molecules by obtaining products with high optical purity and high space-time yields are discussed. This review is also written as an update to previous reviews concerning the use of nonlypolitic esterases as biocatalysts (Bornscheuer, 2002a; Bornscheuer and Kazlauskas, 1999; Panda and Gowrishankar, 2005). 2. Structural determinants of activity and enantioselectivity

molecule (or alcohol molecule) to the acyl–enzyme complex. Next step involves proton transfer to the Ser–OH group followed by product release, as in the “consensus” mechanism. It has been suggested that different conformations of the oxyanion loop in esterases (and acyltransferases) may also control which nucleophile (water or alcohol) is favoured in the attack at the C_O of the acyl–enzyme complex, activating or deactivating the attacking water via a second water molecule (Jiang et al., 2011). The hypothesis is based upon the comparison of X-ray structures of a number of esterases, where a carbonyl oxygen points towards the active site (conformation A of Fig. 1), whereas in acyltransferases a \NH\ of polypeptide chain points towards the active site (conformations B and C of Fig. 1). Databases of elucidated 3D-structures of α/β hydrolase fold proteins are available, being quite helpful for structure analysis and predictions. The α/β-Hydrolase Fold Enzyme Family 3DM Database (ABHDB or 3DM) is a structure-based classification of most of the available sequences of α/β-hydrolase fold enzymes, thought as a tool for the analysis of structure–function relationships and the mechanistic determinants of substrate specificity (Kourist et al., 2010). Another helpful tool is the ESTHER database (http://bioweb.ensam.inra.fr/ esther), which collects information related to this superfamily (from genes to protein sequences, including structural and applicative data (Hotelier et al., 2004)). Esterases can be classified using the so-called superfamily-based approach, which generates a superfamily based on structural and sequence similarity; furthermore, 3DM has also been proven to be suitable for the recruitment of esterases based on similarity in sequence–structure alignments of known esterases, and for understanding/predicting rational modification of proteins.

2.1. Structural determinants of activity 2.2. Enantioselectivity and the active site structure Many studies have attempted to elucidate – on a structural basis – the determinants of stereoselectivity in the various classes of proteins from the hydrolase family. This family is possibly the largest in enzymology, and includes lipases, esterases, amidases, epoxide hydrolases, dehalogenases and hydroxynitrile lyases. From a structural standpoint, all of them are sharing the so-called α/β hydrolase fold, where eight strands in a central β-sheet are connected by α-helixes that surround the protein core. Many α/β-hydrolase fold enzymes also contain cap domains of highly variable structure, typically sitting on top of the active site in the hydrolase domain. From a merely mechanistic standpoint, all proteins in this vast family seem to share a common fundamental mechanism, having at their active site a catalytic triad consisting of a nucleophile (serine, aspartate or cysteine), a histidine and a carboxylic acid (aspartate or glutamate). These residues occur on conserved locations in loops, and the protein fold brings them together to form the active site. The catalytic mechanism for carboxylesterases (representing the most investigate members of the family) starts with nucleophilic attack by the serine hydroxyl on the substrate carbonyl. The serine hydroxyl group is activated by the catalytic histidine/aspartate, which takes up the proton from the Ser– OH group. A transient tetrahedral intermediate is formed, which is stabilized by two peptide nitrogen atoms that form the so-called “oxyanion hole”. The proton is then transferred from the histidine to the leaving group, and the acid group of the substrate becomes covalently bound to the serine. The histidine then activates a water molecule, which hydrolyses the covalent intermediate via nucleophilic attack on the carbonyl carbon of the intermediate. After the hydrolysis, the histidine donates a proton to the serine, releasing the acyl component of the substrate. This “consensus” two-step mechanism has been challenged very recently (Aranda et al., 2014) on the basis of computational studies. A four-step mechanism was suggested, which includes the formation of two tetrahedral intermediates: the first one involves a Ser residue bounded to the previous carbonyl group of the substrate as in the classical mechanism. The second tetrahedral intermediate of the mechanism is formed upon attack of a histidine-activated water

Given the structure of the active site and of the surrounding substrate binding pocket, two major hypotheses have been put forward to provide some basis for explaining enantioselectivity. One relates to specific geometric features of the active site itself, as specific distances between some substrate atoms and groups in the active site sidechain have a different impact on kcat and Km for different substrates, thus providing a simple rationale for enantioselectivity as determined by the reaction kinetics (Ema, 2004; L. Zhang et al., 2014). Most relevant to the case of the esters of tertiary alcohols, a role has been suggested also for the “oxyanion hole” pocket (as relevant to substrate orientation), and for the so called “nucleophilic elbow” around the catalytic serine residue (as relevant to catalysis proper) (Bassegoda et al., 2010). This “elbow” surrounds the active site serine residue in a conserved pentapeptide sequence motif Gly-X1-Ser-X2-Gly, and results in the formation of a very sharp turn between strand β5 and the following α-helix. Enzymes from the hormone-sensitive lipase-like family share the GGG(A)X motif in the oxyanion hole and a highly conserved GDSAGG motif close to the catalytic serine, whereas acetylcholine esterases and mammalian liver esterases contain a GESAGA consensus motif in their “nucleophilic elbow”. The results of mutagenesis studies indicated that the consensus motif is of considerable plasticity and can be varied to a certain extent without compromising conversion of tertiary alcohol (Bassegoda, 2010). However, the second glycine seems to be a key position for the enantioselectivity of these esterases. As a matter of fact, hydrolases bearing the GGG(A)X-motif (e.g., Candida rugosa lipase, Candida antarctica lipase A, pig liver esterase (PLE), an acetyl choline esterase from banded krait and a recombinant esterase from Bacillus subtilis) proved to be active against tertiary alcohol esters (Henke et al., 2003). Enzymes having the common GX-motif are not able to accommodate these sterically demanding compounds. Due to the GGG(A)X-motif, the space in the oxyanion hole pocket seems to be enlarged enough to allow a quaternary carbon to enter the active site (Kourist and Bornscheuer, 2011).


549

Fig. 1. Favoured and disfavoured water–water interactions are determined by different conformations of the oxyanion loop (Jiang et al., 2011).

2.3. Enantioselectivity and substrate access Substrate access to the active site has been indicated by many authors as one of the main factors governing the specificity of catalysis and enantioselectivity (Hasenpusch et al., 2011). The selective access of a given substrate and/or of one of its possible enantiomeric forms may be affected by the position and nature of the “lid” or “cap” that in many esterases closes the buried active site to molecules coming from the protein exterior. In most cases, the “cap” or “lid” domain shields the catalytic triad, thus contributing directly to substrate binding (Carr and Ollis, 2009). This domain is usually inserted at the C-terminal ends of strands β4, β6, β7, or β8, and may differ considerably in size in the various enzymes. Even in “capless” proteins in this class, the nature of the channels allowing entrance of the substrate and release of the products (not necessarily coincident in some of the enzymes studied so far) seems to exert a relevant effect on both affinity and stereoselectivity towards the substrate binding. Although the active site itself is the spot most frequently mutated to increase the activities or enantioselectivities of α/β-hydrolase fold enzymes, studies aiming at focused directed evolution with the entrance to the active site as a target have led to interesting results. For instance, mutants at the entrance tunnel of Rhodococcus rhodochrous haloalkane dehalogenase gave a 32-fold increase in activity (Pavlova et al., 2009), whereas those at the entrance into Aspergillus niger M200 epoxide hydrolase gave increased enantioselectivity in the hydrolysis of various epoxides (Kotik et al., 2007). It is worth noting that both the position of the “cap” or the proper alignment of amino acidic side chains along the “entrance” and “exit” channels may be affected by interaction with other chemical species, sometimes resulting in significant changes of the specific activity and/ or of stereoselectivity. In this frame, it could be interesting to investigate further the reported effects of specific co-solutes on the stereospecificity of some hydrolases. Effects of co-solutes have been reported — for example, in the case of simple cations or of detergents, but seldom these studies have addressed the structural effects of the co-solutes in terms of possible alterations of the access/exit channels, and in terms of the possible effects of the co-solutes on the stability of the enzyme during turnover. For example, the optimum cation might serve as an inherent additive, stabilizing the enzyme against substrate/product deactivation. Generally, by simply switching the substrate counterion from Na+ to K+ satisfactory results at high substrate concentration

were obtained. Both these effects were reported to have a significant impact on the practical uses of various sub-classes of hydrolases as for producing chiral intermediates under conditions of practical relevance (Ma et al., 2014). In the same general frame, the possibility that impaired local flexibility may be one of the reasons for the observed increase in stereoselectivity at low temperatures awaits direct experimental demonstration. On the other hand, there is no clear-cut evidence that the multimeric structures in several enzymes in this class seem may play a significant role with respect to other structural features (including the immediate surroundings of the catalytic triad and the structure of the oxyanion hole, as discussed above) in determining affinity and chiral specificity. Authors suggested the oligomerization as a stabilizing factor for hyperthermophilic proteins compared to their mesophilic homologous (Palm et al., 2011). However, the exchange route for substrate and product between the active site and the solvent is not obvious from the structures. Also, the flexibility of the cap domain has been suggested to facilitate such exchange (kinetic data of Est2 indicate that the first 35 residues of the protein play a role in the conformational stability of the protein and have a regulatory role) (Rozeboom et al., 2014). Other studies have concluded that setting up a pattern that relates esterase activity and the amino acid sequence of the oxyanion hole motif appears difficult so that, in conclusion, simple prediction of esterase mutant performance through information derived from other enzymes (or their mutants) seems to be hardly reliable (no operational problems with modified enzymes were reported in the experimental section) (Fillat et al., 2014). 3. Assays for esterases Discovery of new esterases has been facilitated by the occurrence of a number of solid and liquid assays for their detection. A reliable, fast and informative assay method is a prerequisite for screening new natural esterases or libraries obtained from genomes/metagenomes, or by protein engineering, especially when directed evolution is involved (Mateos-Díaz et al., 2012; Schmidt and Bornscheuer, 2005). Medium- or high throughput screenings (HTS) can be based on the alcohol or on the acid released upon ester hydrolysis. The fatty acids released by enzymatic hydrolysis can be determined qualitatively by gel-diffusion techniques or quantitatively using titrimetry, colorimetric and fluorometric assays, chromatographic procedures (TLC, GC, HPLC), turbidimetry, and immunological methods. The most common assays

Fig. 2. Hydrolysis of phenol esters for generating chromophores and/or fluorophores.

550


Table 1 Most common colourimetric and fluorometric for the detection of esterase activity. Reaction

Pros

Cons

Common equipment; easily available substrates

Spontaneous hydrolysis

Common equipment; easily available substrates; suitable for zymograms and solid-phase assays

Spontaneous hydrolysis; use of surfactants for solubilizing long-chain fatty esters

Monofunctionalized fluorescein esters are water soluble esters; lipophilic fluorescein diacetate (FDA) can cross cell membranes and be activated by (intra)cellular esterases.

Spontaneous hydrolysis

Liberation of free resorufin can be monitored spectrophotometrically or fluorometrically

The resorufin moiety may not be readily hydrolysed since its polycyclic structure

Highly specific; acyloxymethyl ethers of umbelliferone are more stable against non-specific degradation; fluorescence of the product is pH-independent at pH higher than 2.2

Esters serving as substrates are usually not stable at high or low pH and/or elevated temperatures and only work in aqueous solutions.

Useful for screening stereoselective hydrolysis; the substrates are highly stable at elevated temperatures and over a broad range of pH-values.

Reduced reactivity towards non-catalytic proteins such as bovine serum albumin (BSA) compared to its ester analogues. The major advantage of this approach is, that only applicable to

Coloured p-nitrophenol is measured at 410 nm

Coloured products of azo-coupling are measured at 560 nm

Fluorescence of fluorescein (λex 485 nm, λem 538 nm)

Fluorescence of resorufin (λex 544 nm, λem 590 nm) Resorufin can be also detected spectrophotometrically at 572 nm

Fluorescence of umbelliferone (7-hydroxycoumarin) (λex 360 nm, λem 460 nm)


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Table 1 (continued) Reaction

Pros

Cons (chiral) carboxylic acids as substrates.

Chiral umbelliferone derivatives Stable under strongly alkaline conditions (pH 11)

Synthesis of the substrate is requested

Common equipment; suitable for zymograms and solid-phase assays

Substrate not recognized by many esterases

Fluorescence of pyrenebutyric acid (λex 300 nm, λem 370 nm) 3-((2,4-Dinitrophenyl)(methyl)amino)propane-1,2-diol can be detected by HPLC

Blue indigo is generated by oxidation of indoxyl released after hydrolysis

for preliminary screening are pH-stat methods (based on the continuous titration of fatty acids released upon hydrolysis), spectrophotometric and fluorometric methods. Various phenolics (i.e., p-nitrophenol, umbelliferone, fluorescein, resorufin) are chromophores and/or fluorophores and their esters have been widely used for assaying esterases activity. The colour/fluorescence of these phenols is due to the spectral properties of the phenolate anion. Acylation of the phenolic hydroxyl group conceals the chromogenic/ fluorogenic nature of the free phenol, which can be re-established upon hydrolysis. Hydrolysis is favoured by the low pKa of the leaving group (pKas of phenols are around 7), which makes phenolic esters generally quite unstable in water and esterase assays sometimes unreliable. Insertion of an acetoxymethyl group into the fluorophore ester bond endows molecules with higher chemical stability (Fig. 2) (Reymond, 2008). Table 1 summarizes the most common assays based on colourimetry and fluorimetry. Enantioselectivity of esterases is not easily measured by colorimetric or fluorometric assays, whereas conventional chiral GC and HPLC are still the most consistent methods. This appears to be the main bottleneck for screening thousands of mutants obtainable by protein engineering (Böttcher et al., 2010). Moreover, determination of the real enantioselectivity of an esterase towards racemic mixtures can be inaccurate if the assessment of the enzymatic activity is performed with single enantiomers, since competitive binding of the two enantiomers is ignored.

“Quick E” was one of the first HTS assay protocol for detecting the activity of esterases towards chiral substrates (Janes et al., 1997). It is based on the comparison of the independent enzymatic hydrolysis of two enantiomers of chiral chromogenic substrates (p-nitrophenolate); the hydrolysis of the single enantiomers are carried out in the presence of an achiral substrate (a resorufin ester) used as a reference for mimicking the competition observed when a racemic mixture is hydrolysed (Fig. 3). The initial rates are monitored at 404 nm for the hydrolysis of the p-nitrophenolate esters and at 572 nm for the hydrolysis of the resorufin ester. Acetic acid released by the esterase-catalysed hydrolysis of acetate esters can be detected by an enzyme-coupled assay, as shown in Fig. 4. Determination is based on the formation of NADH measured by the increase in absorbance at 340 nm. Enantiopure (R)- and (S)-acetate esters can be independently assayed for determining the apparent enantioselectivity of different esterases (Baumann et al., 2001). Isotopically labelled (e.g., deuterated) esters as pseudo-enantiomers (Reetz, 2001) can be used for assaying enzymatic hydrolysis using mass spectrometry (Reetz et al., 1999), NMR (Reetz et al., 2002), or FTIR spectroscopy (Tielmann et al., 2003). Isotopically labelled pseudoenantiomers differ not only in the configuration of the stereocenter, but also because of the presence of an isotopic labelling. When two pseudo-enantiomers are mixed in equimolar amounts, a pseudoracemate is created, consequently mimicking the real case of a racemic mixture. Enantiopreference of esterases can be, therefore, determined

552


Fig. 3. Quick E: an esterase assay protocol for detecting the activity of enantioselective esterases (Janes et al., 1997).

by the comparison of the unlabelled and labelled acyl group released upon hydrolysis from the two pseudo-enantiomers (Fig. 5). 4. Identification of new esterases New enantioselective esterases can be identified by conventional microbial screening, sequence-based analysis of genomes and metagenomes, and function-based analysis of metagenomes; moreover, protein engineering can evolve known esterases into variants with improved enantioselectivity together with good activity and stability (Panda and Gowrishankar, 2005). Programmes of conventional microbial screening imply culturing microbiologically pure strains, followed by the addition of the substrate at different growth times. Bioconversion activity is then evaluated after extraction of the substrate/product and their analysis; consequently, microbial screenings are often vastly time consuming. Automated liquid handling systems facilitate culturing and extraction, and screening restricted to strains already known for enantioselective activity can

Fig. 4. Acetic-acid assay for the identification of enantioselective esterases (Baumann et al., 2001).

accelerate the process (Gandolfi et al., 2000; Ramírez et al., 2008). The main advantages of this approach are that real chiral or prochiral substrates can be used and stereobias evaluated already during the screening phase. A brilliant screening for probing enantioselectivity towards racemic esters of primary alcohols was developed using an aspartate auxotroph Escherichia coli as host strain; the mutant library is plated on minimal media supplemented with the aspartate ester of the desired enantiomer of the alcohol (Boersma et al., 2008). The growth of less enantioselective variants is inhibited by adding a phosphonate ester of the undesired enantiomer, which covalently inhibits the less enantioselective enzyme variants; as a result, only clones able to hydrolyse the desired enantiomer can grow by releasing aspartate, whereas the growth of colonies with preference for the opposite enantiomer is inhibited (Fig. 6). (Micro)organisms adapted to extreme environments have developed a number of structural and physiological strategies of molecular evolution. Esterases from microorganisms or metagenomes found in extreme environments have a powerful potential as robust biocatalysts to be used under harsh and/or non-conventional conditions. Some researchers have been focused on the recruitment of esterases with thermophilic (Panda and Gowrishankar, 2005), psychrophilic (Tutino et al., 2010), thermoacidophilic (i.e., activity at low pH and high temperatures), and halophilic (Trincone, 2011) properties. To overcome the difficulties associated with cultivation techniques of microorganisms from unconventional environments, metagenomes are often exploited for identifying genes of extremophilic esterases for cloning in suitable hosts. This strategy has proven effective for accessing to a number of new enzymes, but often the stereoselectivity of these new enzymes can be very poor, since their screening has been performed only by homology of sequences not necessarily involved in stereorecognition. Screening from metagenomics is used to find enzymes without culturing the (micro)organisms which primarily produced the protein (Ferrer et al., 2005). This approach relies on sequence analysis to provide the basis for predictions about function. A disadvantage of sequence-based (meta)genomic for the identification of new esterases with new or improved stereopreferences, is the tendency to find enzymes related to previously reported families, whereas esterases with significantly new sequences might be ignored. Function-based


553

Fig. 5. Detection of enantioselective esterase activity based on the pseudo-enantiomers concept.

metagenomics does not require that genes have homology to genes of known similar function, allowing for the access to definitely new proteins. Metagenomic libraries can be subjected to functional screening to detect clones showing peculiar esterase activity (i.e., new stereoselectivity, new substrate specificity) (Martínez-Martínez et al., 2013; Uchiyama and Miyazaki, 2009). The success of these screening techniques strongly depends on transcription, translation and correct folding in the heterologous host, since typically only a subset of proteins from a metagenomic library are recovered by functional metagenomic screening using E. coli as host (Lorenz et al., 2002). Functional metagenomic screenings for identifying new esterases are generally carried out using achiral substrates (e.g., p-nitrophenyl acetate or butyrate, tributyrin) suitable for high throughput solid plate or liquid assays under different conditions (e.g., different temperatures, presence of solvents). As a consequence, enzymes with positive properties in terms of generic activity, stability and robustness are found, but not necessarily with valuable stereoselectivity. Interestingly, also the opposite case is observed, since enzymes found after metagenomic screenings may display remarkable characteristics in terms of activity/stereoselectivity, but often these features cannot be related to the original environment from which they were derived. This is the case of EstCE1, an esterase gene identified from the metagenome of drinking water, which is able to enantioselectively hydrolyse (+)-menthylacetate, while it is not active on (−)-menthylacetate (entry 1, Table 2) (Elend et al., 2006). A brilliant example of identification of new esterases from unconventional metagenomes for the resolution of chiral aromatic and glycidol esters was reported by the group of Ferrer (entry 3, Table 2) (Martínez-Martínez et al., 2013). The metagenome of a sub-saline lake

located at an altitude of 655 m in Spain encodes esterases/lipases with broad substrate specificity and characteristics reflecting the original habitat. Maximal activity was reached at alkaline pH (8.0 to 8.5), temperature ranging from 16 to 40 °C, and the activity of these enzymes was often stimulated (from 1.5 to 2.2 times) by chloride ions in different concentrations (0.1 to 1.2 M). Functional metagenomic screening using E. coli as expression system can be furthermore affected by the presence of endogenous esterases; eight esterases active towards p-nitrophenyl butyrate were found in a strain of E. coli, showing the typical sequence motif G-x-S-x-G, conserved in all known α/β-hydrolase fold hydrolases (Kuznetsova et al., 2005). Godinho et al. found that crude extracts prepared from E. coli cells contained an esterase (YbfF) with enantioselective activity towards racemic esters of isopropylidene glycerol (IPG) (entry 14, Table 2) (Godinho et al., 2011). 5. Methods for improvement of enantioselectivity 5.1. Improvement by protein engineering Improvement of the performances of esterases by protein engineering using directed evolution and rational design has been a main goal in the development of esterases as enantioselective biocatalysts (Bornscheuer, 2002b; Schmidt et al., 2009). The dramatic development of complementary techniques (bioinformatics, protein crystallography, spectroscopic methods) for deeply analysing the mechanisms of enzymatic activity (including stereoselectivity) makes possible today an extensive use of rational, semirational and random protein

Fig. 6. Detection of enantioselective esterases based on hydrolysis of aspartate esters and its analogue.

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Table 2 Application of esterases as enantioselective biocatalysts. Entry Esterase

Notes

Reference

1

EstCE1 from the metagenome of drinking water

Wild-type

Elend et al. (2006)

2

Est34 from metagenome mining

GGG(A)X family

Kourist et al. (2007)

3

Esterases LAE3 (from the metagenome of Lake Arreo)

Wild-type

Martínez-Martínez et al. (2013)

4

Arthrobacter sp. ZJB-09277 (Est arth) Bacillus sp. BP-7 (EstBP7)

Wild type Stable up to 80% (v/v) DMSO

Zheng et al. (2014)

It is difficult to set up a pattern relating esterase activity towards pNPA and the exact amino acid sequence of the oxyanion hole motif. Predictions of esterase mutant performance through information derived from previously assayed enzymes or mutants are hardly reliable. Semirational protein engineering

Fillat et al. (2014)

5

Stereoselective application

6

Bacillus amyloliquefaciens (BAE)

7

Bacillus coagulans (BCE)

Wild-type

Molinari et al. (1996)

8

Bacillus stearothermophilus (BsteE)

Wild-type thermophilic

Henke and Bornscheuer (2002)

9

Bacillus subtilis DSM 402 (BsubE) differs from BS2 for 11 aa

10

B. subtilis DSM402 (BS2)

Improved by protein engineering

Schmidt et al. (2007) Heinze et al. (2007)

11

Bacillus subtilis Thai I-8 carboxylesterase NP

The different enantioselectivities of NP and CesB results from a few amino acid substitutions in the cap domain. In addition, Ala156 may be a determinant of enantioselectivity, as its side chain interferes with binding of some R-enantiomers in the active site of NP.

Smeets and Kieboom (1992) Quax and Broekhuizen (1994) Steenkamp and Brady (2008)

12

Bacillus subtilis 168 CesA

13

Bacillus subtilis 168 CesB

Liu et al. (2013, 2014, in press)

Henke and Bornscheuer (2002)


555

Table 2 (continued) Entry Esterase

Notes

Reference

14

Escherichia coli YbfF W235I

Improved by rational protein engineering

Godinho et al. (2011, 2012b).

15

Halomonas aquamarina 9B (Hmest)

Whole cells

De Vitis et al. (in press)

16

Kluyveromyces marxianus (KME)

Wild-type

Monti et al. (2008)

17

Nocardia farcinica IFM 10152 (Est01)

Wild-type; available from Enzymicals

Nguyen et al. (2011)

18

Paenibacillus barcinonensis (EstA)

Improved by rational protein engineering; available from Enzymicals

Bassegoda et al. (2010)

19

PLEs

Crude mixture

Namiki et al., 2014 For a review: Domínguez de María et al. (2007)

20

Pig liver esterase (PLE) PLE06 is the recombinant isoenzyme

21

PLE V263D

Süss et al. (2014) Kietzmann et al. (2012) Hasenpusch et al. (2011) Hermann et al. (2008) Hasenpusch et al. (2011)

22

Alternative pig liver esterase (APLE) Novel isoform of PLE expressed in P. pastoris PLE (microsome extracts)

The enzyme model has a rigid carapace made up from β-strands and a soft body of α-helices determining the experimental substrate specificity. The access of the substrate to the active site passes through two α-helices. Their structure and even the folding state of one of them are specifically affected by mutations, which modify the substrate specificity. Thus, the entrance channel determines the substrate specificity of the enzyme, rather than the structure of the active site. The single recombinant isoforms are available from Enzymicals.

Immobilized pig liver microsomal fraction

Shesterenko et al. (2014)

23


24

Pseudomonas fluorescens esterase I (PFE I)

Mutants by rational engineering; available from Enzymicals

Schliessmann et al. (2009)

25

Pseudomonas fluorescens Double mutant M2 (V121I, F198C) Pseudomonas fluorescens Double mutant I76V/V175A Pseudomonas putida

Double mutant by semi-rational protein engineering

Jochens and Bornscheuer (2010)

Double mutant by directed evolution

Schmidt et al. (2006)

The protein sequence of PPE revealed two motifs: besides the catalytic triad (Ser151, Asp246, His276),

Rehdorf et al. (2012)

26

27

(continued on next page)

556


Table 2 (continued) Entry Esterase PPEst


Notes

Reference

PPE also possesses the classical pentapeptide G149-D150-S151-A152-G153, a consensus sequence around the active site serine, and a GGGX-motif (G77–G79) associated with activity towards TAs. Due to the GGG(A)X-motif, the space in the oxyanion hole pocket is enlarged allowing sterically more demanding substrates such as a quaternary carbon to enter the active site. Ma et al. (2013) Two designed mutants (D287A and W187H) were constructed. W187H retained excellent enantioselectivity (E N 200), whereas enantioselectivity of D287A variant decreased significantly (from E N 200 to 8.7); this might be explained by excessive enlargement of the binding pocket. The W187H variant exhibited significantly increased catalytic efficiency and retained excellent enantioselectivity. Wild type thermophilic Ozaki and Sakimae (1997) Shaw et al. (2006)

28

Pseudomonas putida ECU1011 (rPPE, E. coli)

29

Pseudomonas putida IFO12996

30

Pyrobaculum calidifontis VA1 (PestE)

31

Serratia marcescens (SmarE)

32

Rhodococcus sp. ECU1013 (RhEst1)

Wild-type

Liu et al. (2013)

33

Sulfolobus solfataricus (Sso-Est1)

Wild type thermophilic

Sehgal and Kelly (2003)

34

Sulfolobus tokodaii (Est0071)


Wada et al. (2013)

35

Thermomyces lanuginosus DSM 10635


Li et al. (2014)

36

Thermotoga maritima genome

Recombinant protein

Tao et al. (2013)

37

Trichosporon beigelli (TBE)

Whole cells

Kapoor et al. (2003)

38

Trichosporon beigelli (TBE)

Lyophilised whole cells

Koul et al. (2005)

39

Trichosporon brassicae TbraE

Whole cells

Shen et al. (2002)

Low enantioselectivity on esters of tertiary alcohols: in Palm et al. (2011) this case, orientations of the two enantiomers in the binding pocket were characterised by no significant energy differences, corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the unhindered carboxylic acid esters, two different orientations with significant energy differences for each enantiomer were found matching the high E values. Available from Enzymicals Available from Tanabe Seiyaku, Japan Fang et al. (2001)


Fig. 7. Different enantioselectivity of recombinant PLE isoform towards acetates of aromatic secondary alcohols. Adapted from http://www.enzymicals.com/downloads/enzymicals_portfolio_2014.pdf.

engineering of esterases (Jochens et al., 2011). Protein engineering has been used not only for improving useful properties of esterases as biocatalysts, but also for better understanding the mechanism of substrate–protein interactions, including phenomena of stereorecognition. Directed evolution improves protein activities taking into account the whole protein structure, regardless the position of the mutation inserted, hence generating a high number of variants, which are often difficult to screen. Resolution of racemic but-3-in-2-yl acetate has been investigated with a number of wild-type esterases, and the best result was obtained with an esterase from Pseudomonas fluorescens giving an E of 63 (Baumann et al., 2000). Directed evolution of this enzyme was carried out using the HTS assay based on the acetic acid released, leading to the creation of a double mutant (PFE Ile76Val/ Val175Ala), which showed improved enantioselectivity (E = 96) and high reaction rate (entry 26, Table 2) (Schmidt et al., 2006). In all these cases, the prediction of mutagenesis sites affecting the overall stability of the protein is difficult. To overcome these challenges, computational tools have been developed to estimate the consequence of mutations on protein stability or binding affinity (Potapov et al., 2009). Rational protein design suggests a number of limited mutations restricted to residues of the protein known for their potential functionality, thus limiting mutations with negative impact on the activity and/ or stability. Semi-rational approaches require that directed evolution is only focused to specific residues critical for activity, stability and enantioselectivity; these residues have been identified on the basis of experimental data and structural alignment of thousands of sequences

557

of α/β-hydrolases now available in databases, such as the aforementioned ESTHER or ABHDB. A compromise between directed evolution and rational protein engineering is the preparation of small libraries, while maintaining a smart and rational approach in determining the possible mutations; this can be achieved by analysing the potential structural determinants of activity and stereoselectivity (Nobili et al., 2013; Schmidt et al., 2009). Recognition of amino acidic residues playing a crucial role for the access and interaction with the binding site may lead to mutation of these “hotspots”. Mutations are either introduced by saturation mutagenesis (Reetz and Carballeira, 2007) or by introducing only the most frequently occurring amino acids (also denoted as allowed amino acids) based on the structural alignment of thousands of amino acid sequences of α/β-hydrolase fold enzymes with bioinformatic tools, such ABHDB (Kourist et al., 2010). A so-called small and smart approach has been often developed in the last few years, involving focused evolution based on the mutation of residues recognized for their potential effect on activity/enantioselectivity (Jochens and Bornscheuer, 2010). Preparation of small libraries, while maintaining a smart approach in determining the possible mutations, was achieved by introducing only the most frequently occurring amino acids (also denoted as allowed amino acids) based on the structural alignment of thousands of amino acid sequences of α/β-hydrolase fold enzymes with the bioinformatic tool 3DM. The rational of this choice is founded upon the observation that among α/β-hydrolase fold enzymes, it was statistically observed that only certain “allowed” amino acids in given positions lead to functional proteins, whereas activity was negatively affected by mutations with amino acids rarely occurring in those positions (Jochens and Bornscheuer, 2010). Four active-site positions of the well-characterised esterase from P. fluorescens (Cheeseman et al., 2004) were recognized using the 3DM database and subjected to sitesaturation mutagenesis for improving the enantioselectivity on 3phenylbutyric acid esters (Jochens and Bornscheuer, 2010). Significant improvements of the reaction rates and enantioselectivity (E = 80 vs 3.2 of the wild-type; entry 25, Table 2) were achieved with the double mutant M2 (V121I, F198C), found after screening a considerable reduced library size compared with the ones obtainable by unbiased directed evolution. Another structural determinant often employed for semi-rational protein engineering approaches is the GGG(A)X string, a conserved motif in esterases accepting bulky substrates, such as esters of tertiary alcohols, albeit often with low enantioselectivity. In fact, it has been found that the third glycine can be replaced by other amino acid, such as serine (Bassegoda et al., 2010) or threonine (Fillat et al., 2014). Hence, these amino acids have been recognized as hotspots for beneficial mutations aimed at achieving higher activity/enantioselectivity. A brilliant semi-rational approach for enhancing the enantioselective hydrolysis of tetrahydrofuran-3-ol acetate using Bacillus stearothermophilus esterase (BsteE) was recently proposed

Fig. 8. Stereopreferences of esterases towards racemic 1,2-O-isopropylideneglycerol.

558


Fig. 9. Domino reactions for the preparation of (S)-naproxen using the thermostable Est0071 from Sulfolobus tokodaii.

(Nobili et al., 2013). The thermophilic esterase BsteE has been overexpressed in different hosts and successfully applied for the kinetic resolution of (±)-menthyl acetate (entry 8, Table 2) (Henke and Bornscheuer, 2002). Resolution of THF-3-ol esters appears to be somehow troublesome, since substituents at the stereocenter do not show big differences in terms of bulkiness; wild-type BsteE gave a modest (E = 4.3) preference for the (S)-enantiomer. Docking studies with the wild-type enzyme showed that also in this case, a hydrophobic pocket determines the steric interactions of binding; four residues (Asp31, Leu93, Met195 and Val224) were identified as the hotspots for achieving the most significant effects on the stereorecognition of the protein, since their side chains are located b3.5 Å away from the substrate. Two double mutants libraries were created by concurrently mutating the amino acids placed at the closest positions (Asp31 and Leu93 for library I, and Met195 and Val224 for library II). The best mutant (D31T/L93F) was found among the double mutants from library I, showing a marked increase in the enantioselectivity (E = 10.4 vs 4.3 of the wild-type). Although this improvement may seem quite limited, it should be noted that analogous improvement in the enantioselectivity was obtained for other esterases after screening of thousands of clones from notfocused directed evolution (Reetz et al., 1997). In all these examples, the thoughtful choice of the amino acid to be mutated resulted in an improvement of the enantioselectivity without negative effect on thermostability of the protein.

5.2. Improvement by medium engineering Stereoselectivity of reactions catalysed by esterases can be influenced by different parameters, especially medium composition. No general predictions for improving activity and/or enantioselectivity can be deduced by literature and the effects of the addition of cosolvents or other additives are poorly understood; nevertheless, the critical parameters to be studied when optimising an esterase-catalysed biotransformation are related to the presence of organic cosolvents (either water-miscible or water-immiscible), inorganic salts, surfactants, substrate/enzyme concentration, and temperature. Medium engineering can be dependent upon biocatalyst purity, since the occurrence of other competing enzymes or isoforms can affect the overall stereoselectivity of the biotransformation. One of the most dramatic effects of medium engineering was observed in the case of PLEs, where the addition of water-miscible cosolvents remarkably increased the enantioselectivity towards different meso-cyclic diesters. Interestingly, improvement of enantioselectivity was accompanied by a decrease of relative rates, likely suggesting that the cosolvents may cause a differential inactivation of one or more PLEs composing the crude mixture (Bjorkling et al., 1985); consequently, only the more enantioselective isoform is active, but a general decrease of activity is detected. Another common phenomenon in ester hydrolysis is spontaneous hydrolysis, which can be reduced by adding suitable amounts of organic cosolvents (Godinho et al., 2011; Heinze et al., 2007).

Fig. 10. Enzymatic hydrolysis involved in the chemoenzymatic synthesis of Cardura®.


559

A

B

Fig. 11. A. Chemoenzymatic synthesis of Pregabalin. B. Use of immobilized cholesterol esterase for the enantioselective hydrolysis involved in the preparation of the antiviral drug emtricitabine (Emtriva).

Tolerance towards organic solvents is an important requisite for esterases to be developed as biocatalyst on a preparative scale. A novel kinetic resolution for the obtainment of (S)-3-cyano-5-methyl hexanoic acid (entry 4, Table 2) has been developed using an esterase from Arthrobacter sp. ZJB-09277 (Zheng et al., 2014). This biocatalyst shows remarkable activity in the presence of high concentrations of water-miscible solvents (80% DMSO), allowing the enzymatic resolution of the substrate at a concentration of 100 mM. Organic cosolvents, salts and surfactants can change enzyme conformation with consequence on activity/enantioselectivity. A new esterase APE1547 from the hyperthermophilic archaeon Aeropyrum pernix K1 had a 5.7-fold activity increase and a 9-fold enantioselective increase towards 2-octyl acetate when treated with acetone. A similar effect was observed in the case of the esterase from Trichosporon brassicae (TbraE) when treated with 2-propanol prior its use for the resolution of racemic ethyl ester of ketoprofen (entry 39, Table 2) (Shen et al., 2002). Although mechanisms underlying this activity/enantioselectivity improvement were not further investigated, the authors suggest a possible conformational change involving the hydrophobic cavity through which substrate accesses the catalytic triad (Cong et al., 2011). Enhancement of the enantioselectivity of kinetic resolutions can be accomplished by performing reaction at relatively low temperatures. Psychrophilic esterases can be used for this purpose and, more generally, for the production of unstable compounds (Tutino et al., 2010). Coldadapted Bacillus amyloliquefaciens esterase (BAE) hydrolyses 1-(3′,4′methylenedioxyphenyl)ethyl acetate with limited enantioselectivity (E = 35); the stereopreference was raised from 35 up to 140 by combining the use of low temperature (0 °C) and addition of Tween 80 (Liu et al., 2014). Inorganic cations can also influence esterase-catalysed biotransformations by different means; aggregation and active conformation of proteins can be determined by salt type and concentrations, consequently influencing esterase activity and enantioselectivity. Enantioselective hydrolysis of different salts of rac-2-acetoxy-2-(2chlorophenyl) acetate (rac-AcO-CPA) was carried out using a mutant of Pseudomonas putida esterase (rPPE01-W187H) (entry 28, Table 2) (Dou et al., 2014). For improving the activity, besides sodium salt, potassium and ammonium salts were used as counterions on the basis of the Hofmeister series (Ma et al., 2013) Better results were obtained with K+ (50% conversion after 16 h, N 99% ee) than with Na+ (40% conversion

after 20 h, N 99% ee), while the use of NH+ 4 as counterion, strongly limited activity, since only 9.4% conversion could be reached after 20 h. This salt effect can be due the different steric recognition within the oxyanion hole of the different salts used as substrates, more than an influence of the cation on protein conformation. 6. Application of non-lypolitic esterases as enantioselective biocatalysts 6.1. Enantioselective hydrolysis of chiral and prochiral esters of primary and secondary alcohols Pig liver esterases (PLEs) have been widely used for a number of stereoselective hydrolysis of racemic and prochiral esters, since the pioneering work on desymmetrization of prochiral dimethyl 3-hydroxy3-methylglutarate in 1975 (Huang et al., 1975), being quite stable and accepting a wide range of substrates. Isoenzymes with different stereoselectivities compose the preparation extracted from animal tissues, leading to problems in reproducibility, potential lack of selectivity and regulatory issues. In spite of these drawbacks, PLEs have still been used in a number of different enantioselective hydrolysis of racemic and prochiral esters. The preparation of chiral building blocks using PLEs has been thoroughly reviewed (Domínguez de María et al., 2007). The potential of PLEs was recently confirmed in the asymmetric hydrolysis of prochiral dimethyl 3,3-dimethyl-2-methylenecyclohexane-1,1dicarboxylate, affording the corresponding half-ester in 96% yield and 99% ee (entry 19, Table 2); the optically pure product is employable for the total synthesis of kauranoids and ent-kauranoids (Namiki et al., 2014). Isolation of microsomal fraction from raw pig liver can be easily performed by calcium sedimentation and low speed centrifugation, and directly used for biotransformations without further purification; immobilized (Ca-alginate) pig liver microsomal fraction was used for the enantioselective hydrolysis of 3-hydroxy-1,4-benzodiazepin-2-one acetates (entry 23, Table 2) (Shesterenko et al., 2014). Six isoenzymes have been identified in the original formulations of pig liver esterases and their use as single pure recombinant proteins guarantees a reproducible enzyme activity especially concerning stereoselectivity. The single isoenzymes composing the crude extract have been produced as recombinant proteins in conventional microbial hosts and are now commercially available (http://www.enzymicals. com/Enzymicals_Enzymes_v011.pdf). It is noteworthy to observe that

560


the six recombinant enzymes (named ECS-PLE01-06) showed very different stereobias towards secondary alcohols (Fig. 7), highlighting the importance of the availability of individual proteins with welldefined properties as biocatalysts (Hummel et al., 2007). PLEs have been also functionally expressed in Pichia pastoris, allowing the detection of a novel isoform (called alternative pig liver esterase, APLE) capable to enantioselectively hydrolysing methyl (2R,4E)-5-chloro2-isopropyl-4-pentenoate (entry 21, Table 2) (Hermann et al., 2008). A mutant (V263D) (entry 22, Table 2) was further evolved for increasing activity and productivity of the biotransformation (Kietzmann et al., 2012). Various enantioselective esterases have been found in strains belonging to the genus Bacillus. Naproxen esterase (NP) from B. subtilis Thai I-8 is a well-known carboxylesterase that catalyses the enantioselective hydrolysis of naproxen methyl ester to produce S-naproxen (Quax and Broekhuizen, 1994). Hydrolysis of several 2arylpropionates, 2-(aryloxy)propionates and N-arylalanine esters is also efficiently catalysed by NP esterase (entry 11, Table 2) (Smeets and Kieboom, 1992). The analysis of the genome sequence of B. subtilis 168 (one of the first bacterial genome annotated) allowed for the identification of two proteins (CesA, 98% sequence identity; CesB, 64% identity), showing high homology towards carboxylesterase NP of B. subtilis Thai I-8 (Droge et al., 2005). These enzymes have different potentials as enantioselective biocatalysts, which are shown to result from only a few amino acid substitutions in the cap domain. Modelling of a substrate in the active site of NP allowed explaining the different enantioselectivities. Activity and enantioselectivity of esterases NP, CesA and CesB were compared using racemic esters of arylpropionic acids (ibuprofen methyl ester and naproxen methyl ester); NP and CesA esterases showed a similar behaviour, being enantioselective towards S-naproxen methyl ester (entries 11 and 12, Table 2), whereas CesB was poorly active on this substrate. Optically pure (S)-1,2-O-isopropylideneglycerol (IPG or solketal) is an important building block for the preparation of β-blockers, prostaglandins, phospholipids and leukotrienes; thus, its obtainment by enantioselective hydrolysis of racemic IPG esters has been extensively studied using non-lipolytic esterases, since commercial lipases are not enantioselective. Short chain IPG esters are not enantioselectively hydrolysed by esterase NP or CesA. Esterase CesB showed high enantioselectivity towards IPG caprylate producing S-IPG, while showing modest enantioselectivity with IPG acetate and butyrate (entry 13, Table 2) (Droge et al., 2001). The main structural determinants of the different activity towards IPG esters observed with CesA and CesB were identified by sequence and structure comparison (Godinho et al., 2012a). Crystal structures and molecular modelling of esterases NP and CesB were used for explaining the different enantiopreferences. Crystal structures show that (also in these enzymes) the catalytic triad is placed at the bottom of a deep hydrophobic cavity where preferential stereoisomeric interactions with specific residues pointing towards the free space inside the cavity favour or limit the access of the two enantiomers (Rozeboom et al., 2014). A moderate thermophile (optimal temperature 65 °C) esterase from Bacillus coagulans (BCE) catalyses the hydrolysis of IPG benzoate with E = 100 (entry 7, Table 2) (Molinari et al., 1996; Romano et al., 2005). Another esterase with high hydrolytic activity towards IPG esters with different alkyl chains is the cytoplasmic esterase YbfF from E. coli (Godinho et al., 2011). Molecular docking of the R-enantiomers of butyrate and caprylate IPG esters into the active site of YbfF enabled the identification of active site residues crucial for enantioselectivity. Random mutagenesis at the four identified positions gave a YbfF variant (W235I) with improved enantioselectivity (E = 38 for IPG butyrate and E = 77 for IPG caprylate; entry 14, Table 2) (Godinho et al., 2012b). Microbial esterases show preference for the hydrolysis of R-IPG esters, thus producing S-IPG, with the exception of an esterase from Streptomyces violaceusniger (Molinari et al., 2005a) and an esterase from the yeast Kluyveromyces marxianus displays opposite stereopreference (Molinari

et al., 2005b; Monti et al., 2008) (entry 16, Table 2). Fig. 8 summarizes the stereopreferences of the esterases with enantioselectivity towards esters of 1,2-O-isopropylideneglycerol. Enzymatic resolution of racemic esters of 2,2-dimethylcyclopropane carboxylic acid (DMCPA), furnishing (S)-DMCPA (the correct enantiomer used as intermediate for Cilastatin synthesis) was recently studied (Liu et al., 2013). A conventional microbial screening programme allowed for the identification of a bacterial strain able to hydrolyse different racemic esters of DMCPA with low conversions and high enantioselectivity. A shot-gun gene library of the genome of Rhodococcus sp. ECU1013 allowed the identification of a nonlypolytic esterase (designated as RhEst1) able to enantioselectively hydrolyse rac-DMCPA; yields were improved by selecting the suitable concentration of enzyme (25 mg/mL), 47.8% yield and 97.5% eep (E = 240) were obtained starting from 500 mM substrate (entry 32, Table 2). An enantioselective esterase from Trichosporon beigelli (TBE) proved to be highly stereoselective on esters of racemic 1-chloro-3(1-naphthyloxy)-2-propanol furnishing the corresponding (S)-alcohol, which is used as optically pure intermediate in the chemoenzymatic synthesis of (S)-propanolol (entry 37, Table 2) (Kapoor et al., 2003). This esterase has been used without purification, by employing preparations of lyophilised whole cells showing excellent enantioselectivities in the resolution of racemic mixtures of different esters of racemic alcohols and carboxylic acids (entry 38, Table 2) (Koul et al., 2005). A rational improvement of esterase activity was recently reported for the hydrolysis of 1-(3′,4′-methylenedioxyphenyl)ethyl acetate by B. amyloliquefaciens esterase (BAE) (Liu et al., 2015). The wild-type enzyme showed limited enantioselectivity (E = 35) and good activity (kcat / KM = 0.88 s− 1 mM− 1) under non-optimised conditions (Liu et al., 2013). Modelling of the protein elucidated the positions of the catalytic triad (Ser190, Glu306, and His395) and two residues (Ala108 and Ala191) involved in the catalytic machinery; eight sites surrounding the nucleophilic Ser190 (distances within 8 Å) were selected for saturation mutagenesis. Notably, three of these amino acids (the ones adjacent to the oxyanion residues Ala108 and close to catalytic Ser190 and His 396) are hotspots found in other esterases for generating variants to enhance enantioselectivity for tertiary alcohol (Kourist et al., 2007). A variant (designed as V10-BAE) with double mutations at positions 358 and 396 exhibited a four-fold enhanced enantioselectivity towards 1-(3′,4′-methylenedioxyphenyl)ethyl acetate than the wildtype enzyme (entry 6, Table 2). Position 396 is the one adjacent to catalytic histidine: the amino acid residue in this position seems to play a crucial role in determining the accessibility to the active site, with marked increases of the selectivity; mutant A396C gave almost a three-fold increase in enantioselectivity and increase activity, due to higher steric hindrance and positive interactions of cysteine with a glutamic acid surrounding the active site, but not directly involved in the catalytic mechanism (Liu et al., in press). Esterases are widely distributed in Pseudomonas, and several have been identified and cloned, such as the ones from P. fluorescens (Cheeseman et al., 2004) and P. putida (Ozaki and Sakimae, 1997). P. fluorescens esterase I (PFE I) is one of the most studied bacterial esterase since it is stable, easy to express and most of the tools for rational understanding of the enzymatic activity (crystal structure, different mutants) are available (Cheeseman et al., 2004). The detailed knowledge of the structure allowed identifying four bulky amino acid residues (F126, F144, F159, and I225) sterically influencing the substrate approach to the active site. These hotspots were used for generating single and double mutants endowed with higher enantioselectivity (and sometimes higher reaction rates) towards different racemic aromatic esters (entry 24, Table 2) (Schliessmann et al., 2009). An esterase (PpEST) from P. putida IFO12996 catalyses the stereoselective hydrolysis of the racemic ethyl ester of 3-(acetylthio)-2-methylpropionic acid, furnishing the corresponding (R)-3-(acetylthio)-2-methylpropanoic acid


(entry 29, Table 2), a key chiral intermediate for the synthesis of angiotensin-converting enzyme inhibitors (Shaw et al., 2006). The crystal structure of PpEST showed that in the oxyanion hole, the NH groups of Thr98 and Trp31 preferentially form hydrogen bonds with the carbonyl oxygen of the ester group, determining both enantioselectivity and chemoselectivity, since the thioester group remains untouched (Elmi et al., 2005). 6.2. Kinetic resolution of racemic esters of tertiary alcohols Hydrolysis of esters of tertiary alcohols under mild conditions is useful in organic chemistry, particularly for kinetic resolution and removal of carboxyl protective groups. Until few years ago, commercially available enzymes do not accept esters of tertiary alcohols as substrates, making difficult the kinetic resolution of these bulky substrates (Pogorevc and Faber, 2000). Several subclasses of the α/βhydrolases have a wider active site with a conserved motif containing three glycines in the oxyanion hole (in a few esterases the third glycine is replaced by an alanine) followed by a bulky residue X (Henke et al., 2002; Henke et al., 2003). Esterases belonging to this GGG(A)X family are able to catalyse the enzymatic hydrolysis of esters of tertiary alcohols (and more generally sterically hindered carboxyl esters) and are now available also as commercial enzymes (Kourist et al., 2008). The α/β-Hydrolase Fold Enzyme Family 3DM Database (ABHDB or 3DM) includes two large groups of esterases able to hydrolyse esters of tertiary alcohols. The first one contains enzymes from the hormone-sensitive lipase-like family, which share the GGG(A)X motif in the oxyanion hole and a highly conserved motif (GDSAGG) adjacent to the serine involved in the catalytic triad. The second family has a high level of similarity to acetylcholine esterases and mammalian liver esterases, sharing the conserved GESAGA consensus motif on the catalytic elbow (the so-called “nucleophilic elbow”) (Kourist et al., 2010). Among the GGG(A)X enzymes, an esterase from B. subtilis (BS2) and lipase A from C. antarctica (CAL-A) have been used for the removal of tert-butyl alcohol from different esters (Schmidt et al., 2005), but most of the wild-type esterases found in nature are not very enantioselective (Bassegoda et al., 2010, 2013; Oh et al., 2012; Rehdorf et al., 2012; Schmidt et al., 2007; Suzuki et al., 2004). Wildtype esterase BS2 is poorly enantioselective towards (R,S)-2phenylbut-3-in-2-yl acetate and 1,1,1-trifluoro-2-phenylbut-3-in-2-yl acetate; the large active site of BS2 allows the access of the bulky alcohol portion of the substrate with a number of possible orientations. Hence, a series of mutants were created by rational and semirational approaches based on computer modelling and manual docking with the aim to obtain stereodivergent esterases able to furnish both the enantiomers of the chiral tertiary alcohols. Two single-mutated variants (Gly105Ala, Ala400Leu) gave the S-enantiomer with high enantioselectivity (entry 10, Table 2) after optimisation of the reaction conditions (Heinze et al., 2007; Schmidt et al., 2007), while a double mutant (E188W/ M193C) furnished the R-enantiomer (E = 64 in the case of 1,1,1trifluoro-2-phenylbut-3-in-2-yl acetate) (Bartsch et al., 2008). Interestingly, also the isoenzyme of pig liver esterase PLE01 contains a GGG(A)X motif and hydrolyses acetates of tertiary alcohols with limited enantioselectivity; attempts to increase the enantioselectivity by rational protein engineering did not succeed (Gall et al., 2010). The knowledge of this structural determinant, which confers activity of these enzymes towards bulky esters, allows screening of genome sequences for the presence of esterase genes containing the GGG(A)X motif. Thirty-five esterases bearing a GGG(A)X motif were derived from an environmental metagenome and one of them (Est34) was highly enantioselective towards racemic 1,1,1-trifluoro-2-phenylbut3-in-2-yl acetate, after optimisation of the reaction conditions (entry 2, Table 2) (Kourist et al., 2007). Two GGG(A)X esterases (EstA1 and EstA2) were identified from the genome of Nocardia farcinica IFM10152 and used for the hydrolysis of different esters with hindering

561

group near the ester function. Albeit no interesting enantioselectivity was found towards esters of tertiary alcohols, EstA1 was highly enantioselective in the hydrolysis of racemic menthyl acetate (entry 17, Table 2) (Nguyen et al., 2011). Another esterase from B. subtilis (BsteE) belonging to this family has been used for enantioselective hydrolysis of racemic esters of secondary aromatic alcohols (entry 9, Table 2) (Henke and Bornscheuer, 2002); BsteE shares high homology with BS2, being different for only 11 aa residues. An esterase from Paenibacillus barcinonensis (EstA) was identified through the use of 3DM approach due to its sequence similarity of 49% to the GGG(A)X-esterase BS2 from B. subtilis; EstA has an unusual serine (GGS) in the third position of the GGG(A)X motif and it is poorly enantioselective towards 1,1,1-trifluoro-2-phenylbut-3-in-2-yl acetate. Variants at the shared motif (GGG, GGA, GAG, AGG, and AGA) were created and checked for the hydrolysis of racemic 1,1,1-trifluoro-2phenylbut-3-in-2-yl acetate, showing that EstA-AGA was highly enantioselective (entry 18, Table 2) (Bassegoda et al., 2010). A similar approach of protein engineering was followed for increasing the enantioselectivity of EstBP7 (formerly EstA1) from Bacillus sp. BP-7, an esterase with a rare GGG(A)X-motif, where threonine is found at the third position of the oxyanion hole (Fillat et al., 2014); one of the mutants (EstBP7-AGA) was highly enantioselective (E N 100) towards 2-(4-pyridyl)but-3-in-2-yl acetate at low reaction temperature (4 °C) (entry 5, Table 2). 6.3. Enantioselective extremophilic esterases Thermophile microorganisms are valuable sources of thermostable esterases with potential qualities connected with industrial applications, such as stability towards organic solvents, detergents and additives (Levisson et al., 2009). A thermostable esterase (Est0071) was identified from genome of the thermophilic archaeon Sulfolobus tokodaii. This esterase possesses the GGG(A)X motif and it hydrolyses several arylaliphatic tertiary alcohols with modest enantioselectivity (Suzuki et al., 2004). Thermophilic Est0071 can enantioselectively catalyse the hydrolysis of sterically hindered malonate diesters at room temperature (entry 34, Table 2) (Wada et al., 2013). Est0071 works also at temperatures higher than 70 °C, where spontaneous decarboxylation occurs, affording the racemic arylpropionic ester, which undergoes further enantioselective hydrolysis. This domino reactions promoted by wild-type Est0071 lead to high conversion (N99%), but low enantioselectivity ee 21% in the case of naproxen preparation (Fig. 9). A novel thermophilic esterase (TLE) from Thermomyces lanuginosus DSM 10635 was recently overexpressed in E. coli and used for the enantioselective hydrolysis of diethyl 2-(1-cyano-3-methylbutyl) malonate for the preparation of (3S)-3-cyano-2-(ethoxycarbonyl)-5methylhexanoic acid (entry 35, Table 2), a chiral intermediate in the synthesis of Pregabalin, a drug used for treatment of several central nervous system disorders (Li et al., 2014). PestE, a highly thermostable esterase, was isolated from the thermophilic archaeon Pyrobaculum calidifontis VA1. This enzyme efficiently catalyse the kinetic resolution of esters of racemic carboxylic acids (entry 30, Table 2), but shows poor enantioselectivity towards esters of tertiary alcohols. The different selectivity was explained by in silico experiments of docking, showing that each enantiomer of the chiral esters of carboxylic acids accesses the active site with different orientations having significant energy differences, whereas the two enantiomers of the chiral esters of tertiary alcohols showed no significant differences in steric hindrance in approaching the active site (Palm et al., 2011). Attempts to find homologues to esterase NP among hyperthermophilic organisms led to the discovery of the Sso-EST1 esterase from Sulfolobus solfataricus P1, which catalysed hydrolysis of methyl ester of racemic naproxen with limited enantioselectivity at 50 °C (entry 33, Table 2) (Sehgal and Kelly, 2003). A putative esterase gene was identified from the genome of the hyperthermophilic bacterium Thermotoga maritima and expressed in E. coli; the recombinant protein (Tm1160)

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displayed optimal activity at 70 °C and enantioselectively hydrolysed the racemic ketoprofen ethyl ester furnishing (S)-ketoprofen with an E = 91.4 (entry 37, Table 2) (Tao et al., 2013). Another interesting class of potentially robust biocatalysts are enzymes produced by marine microorganisms (Trincone, 2011). Marine derived enzymes are generally moderate to extreme halophilic, often displaying peculiar substrate selectivity and enantioselectivity, coupled with stability in conditions of high ionic strength and high concentrations of organic solvents (S. Zhang et al., 2014). Furthermore, marine biocatalysts are often cold-adapted and psychrophilic esterases are particularly suited for the enantioselective transformation of unstable substrate or just (as mentioned before) for exploiting better enantioselectivity achievable at low temperatures (Tutino et al., 2010). Cold-adapted esterases from bacteria (estO from Pseudoalteromonas arctica) (Al Khudary et al., 2010) and yeast (yli180Est from Yarrowia lipolytica CL180) (Kim et al., 2007) have been studied for their activity at low temperatures, but attempts to use them for the kinetic resolution of racemic pharmaceutically valuable chiral esters (i.e., ofloxacin) showed low activity and low enantioselectivity, albeit yli180Est showed preference for the right (levofloxacin) enantiomer. A conventional screening among bacteria isolated from water-brine interface of the deep hypersaline anoxic basins (DHABs) of the Eastern Mediterranean was aimed at selecting strains able to enantioselectively hydrolyse racemic esters of anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid, a key intermediate for the synthesis of prostaglandins (entry 15, Table 2). An esterase from Halomonas aquamarina 9B (Hmest) catalysed the resolution of the racemic substrate with an enantioselectivity (E = 60–70) suitable for the chemoenzymatic synthesis of D-cloprostenol (De Vitis et al., 2015). 6.4. Immobilization of esterases Immobilization is a general method for improving the properties of enzymes, such as easy recycle, operational stability, and even selectivity (Rodrigues et al., 2011). The properties of thermophilic hydrolases can be dramatically improved by immobilization (Cowan and FernandezLafuente, 2011). A thermophilic esterase from B. stearothermophilus was immobilized by multipoint covalent attachment to glyoxyl agarose with a consequent increase in the stability of the esterase and a retention of 65% of the initial activity after one week in 50% DMF or DMS at 30 °C (Fernandez-Lafuente et al., 1995). Immobilization of lypolitic and nonlypolitic esterases is also a requisite for performing continuous biotransformations in flow reactors (Tamborini et al. 2012; Itabaiana et al., 2013). In contrast to the extensive studies devoted to lipase (Adlercreutz, 2013), esterases have been scarcely studied as immobilized enzymes, especially for stereoselective applications. The recombinant esterase from B. subtilis (BSE) was immobilized with the method of cross-linked enzyme aggregates (CLEAs); cross-linked aggregates of BSE (CLA-BSE) were used for the kinetic resolution of DL-menthyl acetate to produce Lmenthol, showing an improved operational stability (Zheng et al., 2011). The recombinant esterase from P. putida IFO12996 covalently bound to magnetic nanoparticles via glutaraldehyde coupling and used for 10 cycles of successive batches in the resolution of racemic methyl β-acetylthioisobutyrate (Shaw et al., 2006). Thermal stability of CLABSE at 30 °C was 360 times higher than that observed with free BSE and could be reused for 10 times with only about 8% reduction in activity. A cell-bound esterase from Pseudomonas sp. (PsE) was used for the kinetic resolution of different 2-acetoxyphenylacetic acids; whole cells entrapped in calcium alginate gave 171% activity recovery with a halflife of 123 h at 30 °C (Ju et al., 2011). Racemic ethyl 3-hydroxy-3phenylpropionate was enantioselectively hydrolysed using an esterase (SNSM-87) from Klebsiella oxytoca immobilized on Sepabeads® (Wang and Tsai, 2009). Cholesterol esterase was successfully immobilized onto hydrophobic polypropylene (Accurel PP) for the chemoenzymatic synthesis of the antiviral drug emtricitabine (Emtriva) (Osborne et al., 2006), as described in the following section dedicated to the use of esterases on a preparative scale. A recombinant esterase from Aspergillus

nidulans (NStcI) was recently adsorbed on a similar support (Accurel MP1000) and used for the enzymatic kinetic resolution of (R,S)-1phenylethanol (Peña-Montes et al., 2013). 7. Use of esterases on a preparative scale The production of (S)-naproxen has been improved by using NP esterase as recombinant protein in Bacillus strain BCL1050 (with activity 800 times higher than that of the wild type strain). The enzyme is poorly stable in the presence of product concentrations above 15 g/L, most likely for the negative interactions of the carboxyl group of (S)naproxen with basic amino groups of the protein. Complementary methods were used for enhancing the stability of NP esterase towards high product concentrations: the recombinant enzyme was stabilized using formaldehyde to block the lysine side-chain amines and sitedirected mutagenesis was applied for replacing Lys34 with Glu (Broekhuizen et al., 1990). Under optimised conditions, 150 g/L of racemic naproxen methyl ester were converted with 45.1% yield and 99% ee of the (S)-naproxen; the overall process can be improved by recycle of the unreacted (R)-naproxen methyl ester after racemization with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (Steenkamp and Brady, 2008). Recombinant pig liver esterase has been employed also for the scaleup of the desymmetrization of dimethyl cyclohex-4-ene-cis-1,2dicarboxylate (entry 20, Table 2). Whereas crude extracts of PLEs gave variable enantioselectivity ranging from 80 to 97% ee, recombinant PLE06 is reproducibly enantioselective, allowing for the set-up of a bioprocess producing 265 g of enantiopure (1S,2R)-1-(methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid with ee N 99.5% (Süss et al., 2014). (S)-1,4-Benzodioxan-2-carboxylic acid is the key optically pure intermediate for the synthesis of (S)-doxazosin (traded as Cardura®); Cardura has proven beneficial in the treatment of hypertension and effective in the treatment of benign prostatic hyperplasia. (S)-1,4Benzodioxan-2-carboxylic acid has been obtained by kinetic resolution of the corresponding ethyl ester using the commercially available esterase from Serratia marcescens (entry 31, Table 2). Enzymatic hydrolysis was carried out in a two-liquid phase system composed with water (pH 9.0) and toluene for favouring the partitioning of the product in water and the substrate in toluene; the unreacted (R)-ethyl ester was racemized and recycled by using a catalytic amount of tert-BuOK (Fig. 10). Enantiomerically pure (S)-1,4-benzodioxan-2-carboxylic acid was obtained by crystallization of the crude reaction mixture on a 1–2 kg scale (Fang et al., 2001). The optimised chemoenzymatic synthesis of Pregabalin is reported in Fig. 11A, and it was originally developed using commercially available Lipolase 100L-EX (a commercially available lypolitic esterase from T. lanuginosus) (Martinez et al., 2008). TLE shows high stability at temperatures up to 60 °C and pH 8.5, being a good candidate for possible preparative applications. A robust bioprocess for the preparation of immobilized cholesterol esterase and its use in the resolution of the racemic butyrate ester of 2,3-dideoxy-5-fluoro-3-thiacytidine (emtricitabine, FTC) was developed at Dowpharma (Osborne et al., 2006). Cholesterol esterase immobilized on porous polypropylene (Accurel EP-100) was stable over 15 successive cycles of use; racemic FTC butyrate (∼ 8 kg, 200 g/L) was resolved with immobilized cholesterol esterase to give 2.17 kg of (−)-FTC with 98% ee. 8. Summary and outlook This review gave an overview of the recent developments about the use of non-lypolitic esterases for stereoselective applications in organic chemistry. The number of stable esterases from natural environments and/or rationally designed for being successfully used in stereocontrolled hydrolysis of racemic or prochiral esters is rapidly growing. New detailed


information about structural determinants involved in activity and stereoselectivity has helped for rational or semi-rational protein engineering with excellent results. In this respect, it is likely that esterases will become more widely used in the near future for preparative purposes, particularly for the production of chiral pharmaceutical intermediates and fine chemicals. However, a better understanding of molecular interactions is still needed for more efficient designing of proteins suitable for industrial application. Another area of study to be developed involves the effects of co-solutes (co-solvents, detergents or salts) on possible structural changes (i.e., alterations of the access/exit channels of the enzyme) affecting the enantioselectivity.

Acknowledgements We would like to thank the Brazilian funding agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing the Special Visiting Researcher fellowship (process 400171/ 2014-7) under the Brazilian Scientific Program “Ciência sem Fronteira”.

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Esterases as stereoselective biocatalysts

Esterases as stereoselective biocatalysts

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