University of Groningen

Selection of novel lipases and esterases for enantioselective biocatalysisDröge, Melloney Joyce

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CChhaapptteerr 11

INTRODUCTION

Based on:

DRÖGE, M. J., PRIES, F. AND QUAX, W.J. (2000). Directed evolution of Bacillus lipase. In: Lipases and Lipids: Structure, Function and Biotechnological Applications. Eds. Kokotos, G., Constantinou-

Kokotou, V.; pp 169-180.

Chapter 1

10

Introduction

11

Chiral Drugs

Concept of chirality In 1815, the French chemist Jean-Baptiste Biot was the first to report that certain naturally occurring organic materials possessed the ability to rotate the plane of polarised light. After this discovery, the concept of optical chirality and the tetrahedral 3D configuration of carbon atoms were further described by Pasteur, Van’t Hoff, and LeBel. Today, chirality is clearly defined and a detailed distinction between stereoisomers, diastereomers and meso-compounds can be made (Challener, 2001).

Many biologically active compounds exist as a mixture of stereoisomers (Roth, 1997). Administration of single enantiomers is associated with improved potency and selectivity of the drug and diminished side events as a result of the activity of the unwanted enantiomer, due to the fact that binding sites of enzymes and receptors preferentially interact with only one of the stereoisomers. As a consequence, the production of single enantiomers instead of racemic mixtures has become an important process in the pharmaceutical and agrochemical industry. Some examples of drugs emphasising the importance of racemate separation into its stereoisomers are the anti-arthritic agent penicillamine, the tuberculostatic drug ethambutol, and the dopamine precursor levodopa (l-dopa) which is used for the treatment of Parkinson’s disease (Hyneck et al., 1990). The latter is marketed enantiopurely because the d-enantiomer is associated with severe leukopenia.

FIGURE 1: The distribution of chiral drugs over the different therapeutic classes (based on Stinson, 2000).

Chapter 1

12

Today, more than 50% of the organic pharmaceuticals are chiral (figure 1). Out of these chiral pharmaceuticals, 36% have a single chiral centre and are mostly, in 80% of all cases, administered as racemates. The remaining 64% of the organic pharmaceuticals contain more than one chiral centre and from these substances only 20% is administered as a racemic mixture (Roth, 1997). Since chiral compounds represent more than 50% of the world-wide most frequently prescribed drugs, the interest in the preparation and isolation of chiral drugs has increased dramatically. More drugs are marketed as single enantiomers instead of a racemic mixture, a process known as ‘chiral switching’ (for a review, see Agranat et al., 2002). However, in spite of the knowledge that the specific effect of a drug is caused by just one enantiomer, racemic mixtures are still frequently applied as it has been virtually impossible to properly separate them until now.

Yet, the increasing knowledge of biotechnology has offered tempting novel techniques for the stereoselective separation of chiral compounds. This thesis will focus on the separation of two enantiomers out of a racemate using bacterial enzymes. The substrates of interest are nonsteroidal anti-inflammatory drugs (NSAIDs) and beta-adrenergic receptor antagonists (beta-blockers).

*

OO

OH

COOH

CH3

H3C

CH3

ibuprofen

O NH CH3

OH

CH3

*

NH

NH CH3

CH3OHSH3C

O

O

*

naproxen

propranolol sotalol

CH3O

CH3

COOH* *

1,2-O-isopropylidene-sn-glycerol FIGURE 2: Chemical structures of racemic naproxen, ibuprofen, propranolol, sotalol, and 1,2-O-isopropylidene-sn-glycerol.

Introduction

13

Nonsteroidal anti-inflammatory drugs (NSAIDs) 2-Aryl propionic acids, such as naproxen and ibuprofen, are examples of chemical compounds with their activity restricted to only one of the isomers (figure 2). These compounds are among the most commonly used analgesics in the treatment of acute and chronic pain and inflammation. Most frequently reported adverse effects are gastric erosion and renal dysfunction. It is generally accepted that the pharmacological and toxicological effects of NSAIDs are caused by a specific inhibition of the binding of arachidonic acid to cyclooxygenase, thereby preventing the production of pro-inflammatory prostaglandins (Kurumbail et al., 1996). Cyclooxygenase is known to exist in three isoforms, COX-1, COX-2 and COX-3, which are similar in size and kinetics, but differ in their expression and distribution in the body. COX-1 is constitutively expressed in most tissues and is involved in a wide variety of physiological processes. Both COX-2 and COX-3 are found to be inducible in inflammatory cells. As such, the anti-inflammatory action of NSAIDs is a rather complex process that is nowadays believed to be associated with the inhibition of all COX isoforms (Schwab et al., 2003). Their gastro-intestinal side effects appear to be associated with inhibition of COX-1. It has been demonstrated that the inhibition of cyclooxygenase by NSAIDs is predominantly due to the enantiomer of the (S)- configuration, while the activity of the (R)-enantiomer is much lower (Carabaza et al., 1996; Warner et al., 1999). Thus, administration of enantiopure NSAIDs appears to be highly desirable. Today, naproxen and ibuprofen are the only isomerically pure NSAIDs on the market, whereas others are still sold as racemic mixtures (Roth, 1997).

Beta-adrenergic receptor antagonists Cardiovascular diseases are the world’s leading cause of death, accounting for 20-50% of the total death rates. Nevertheless, the death rates of cardiovascular diseases vary widely all over the world with a high incidence in the Western World. For this reason, development of effective agents for either treatment or prophylaxis of cardiovascular disease is highly medically and economically attractive. In a total of $185.4 billion expenses for pharmaceutical sales in the world’s twelve largest markets in 1998, cardiovascular drugs account for $36.9 billion. As a consequence, cardiovascular therapeutics are the largest therapeutic category in terms of sales. The sales of cardiovascular drugs contributed almost 20% of the total pharmaceutical market and the sales grows at an annual rate of 8% (Stinson, 2000). Unsurprisingly, among the top 200 world-wide prescriptions of 2002, several beta-blockers are present, such as atenolol, metoprolol, propranolol, bisoprolol and carvedilol (www.rxlist.com). Beta-blockers are drugs that show marked efficacy in angina pectoris, hypertension, cardiac arrhythmias, migraine headaches, and other disorders related to the sympathetic nervous system. Propranolol, approved by the FDA in 1967, is the prototype of the beta-adrenergic receptor antagonists. Propranolol is a competitive, nonselective beta-blocker and is marketed as a racemic mixture (figure 2). Like most beta-blockers, only its l-isomer has an adrenergic blocking activity (Rahn, 1983). This effect is

Chapter 1

14

due to a strong resemblance of the l-isomer with the adrenergic hormone l-noradrenaline. Sotalol is a class III antiarrhythmic drug which also competitively antagonises beta-adrenergic receptors (figure 2). l-Sotalol has beta-blocking effects comparable to a racemic mixture of d,l-sotalol, whereas d-sotalol is practically devoid of beta-blocking properties but remains a potent class III agent (Hoffmeister et al., 1991).

Production of enantiopure drugs Both examples mentioned above illustrate that the isolation and manufacturing of enantiomerically pure compounds is very important: it prevents unwanted side effects caused by the 'wrong' enantiomer and reduces the metabolic task to eliminate the drug from the human body. In recent years, the use of enzymes for the preparation of optically enriched compounds has become an alternative to chemical synthesis. Bacterial enzymes, like lipases and esterases, are capable of enantioselective hydrolysis and esterification in an environmentally friendly and cheap process. Lipases and esterases belong to the group of enzymes generally known as hydrolases. These stable and inexpensive enzymes both catalyse the hydrolysis of ester bonds in aqueous solutions and esterifications and transesterifications in organic solvents. The next paragraph will focus in more detail on both classes of enzymes.

OO

O C

O

CH3

H

N O

Ser

O H NN

His

H-O

Asp

O N

His

O

Asp

-OHN

H

-OH

N O

Ser

O

CH3CO

OO

+

+N

His

O

Asp

-OHN

H

O-H

OH

N O

CH3CHO

Ser

O H

N H-O

Asp

O

His

N

H

N O

C CH3

O

Ser

O

OH

H

FIGURE 3: Hydrolysis of an acetate ester of 1,2-O-isopropylidene-sn-glycerol (IPG) by an esterase/lipase.

Introduction

15

Hydrolases The hydrolase family, amongst others consisting of esterases, (phospho)lipases, proteases, amidases, epoxide hydrolases, nitrilases, and glycosidases, is a group of enzymes that can catalyse bond cleavage by reacting with water. Hydrolases are classified in group 3 of the Enzyme Commission (EC), and are further classified by the type of bond hydrolysed. For instance, esterases are classified as number 3.1.1.1. and lipases as 3.1.1.3.. Currently, the biotechnological potential of hydrolytic enzymes are of special interest as hydrolases have some advantageous characteristics which make them ideally suited for organic chemistry: (i) as a result of a broad substrate specificity, hydrolases often accept various synthetic intermediates as substrates; (ii) high stereoselectivity, also towards unnatural substrates; (iii) catalysis of several related reactions, such as condensations and alcoholysis; (iv) they are often commercially available (Bornscheuer & Kazlauskas, 1999; Patel, 2001). Strikingly, most of the enzymes used in industry are microbial enzymes, originating from bacteria or fungi.

Esterases Esterases (carboxylester hydrolases) are hydrolases catalysing the cleavage and formation of ester bonds. In contrast to lipases their action is generally restricted to water soluble short chain fatty acids. The reaction mechanism of ester hydrolysis by esterases is known and is composed of four steps (figure 3). First of all, the oxygen atom of the hydroxyl group of the active site serine attacks the carbonyl carbon atom of the ester bond, yielding a tetrahedral intermediate stabilised by the catalytic His and Asp. The histidine imidazole ring becomes protonated and positively charged. The positive charge is stabilised by the negatively charged amino acid residue. The tetrahedral intermediate is stabilised by two hydrogen bonds which are formed with the amide bonds of residues belonging to the oxyanion hole. Then, the alcohol moiety is released and the acyl-enzyme complex is formed. Next, the hydroxyl group of a water molecule attacks the carbon of the acyl-enzyme complex and a second tetrahedral intermediate is formed. Finally, the acyl component is released and the active enzyme is regenerated (Derewenda et al., 1994; Egloff et al., 1995; Grochulski et al., 1994; Lang et al., 1998; Longhi et al., 1997; Verschueren et al., 1993). Phosphonates can be used as carboxylester hydrolase inhibitors, since they mimic the first transition state in ester hydrolysis (Deussen et al., 2000a; 2000b; Mannesse et al., 1995; Stadler et al., 1996). This is due to the charge distribution and the geometric configuration of the phosphorus atom, such as the negatively charged oxyanion hole intermediate and sp3 hybridisation of the central atom.

Most esterases, of which the 3D structure has been elucidated, do have a so-called characteristic α/β-hydrolase fold (Ollis, 1992). The ‘canonical’ α/β-hydrolase fold consists of eight mostly parallel β-sheets surrounded on both sides by α-helices (figure 4). This fold provides a typical scaffold for the catalytic triade of the esterase (Ser, Asp and His) and for

Chapter 1

16

the consensus sequence Gly - Xaa - Ser - Xaa - Gly flanking the active site serine. This motif is a consensus for active site serines and is a characteristic pentapeptide for most esterases and lipases. More recently, the existence of a different motif, Gly - Xaa - Xaa - Leu, has been described (Arpigny & Jaeger, 1999).

Esterases are widely distributed in animals, plants and microorganisms, but the majority of their physiological functions still remain more or less unknown. The interesting characteristics of esterases reside in their stability, their activity in organic solvents, the fact that they do not require cofactors, and their high regio- and stereospecificity. These properties make esterases attractive biocatalysts for organic chemistry.

Over the past years, many esterases have been cloned and overexpressed (Bornscheuer, 2002a). Among these, there are a large number of bacterial esterases (e.g. Baigori et al., 1996; Chen et al., 1995; Choi et al., 2003; Jackson et al., 1994; Kim et al., 2003b; Manco et al., 1998; Pogorevc et al., 2000; Prim et al., 2001; Quax & Broekhuizen, 1994; Simoes et al., 1997; Zock et al., 1994). However, only a few of them are useful biocatalyst in kinetic resolution experiments. The most valuable enzymes originate from Bacillus and Pseudomonas species (for a review see Bornscheuer & Kazlauskas, 1999). One of the enzymes of Bacillus, the carboxylesterase NP of B. subtilis Thai I-8, is of particular interest, as it was characterised as a very enantioselective (S)-naproxen esterase (Quax & Broekhuizen, 1994).

Carboxylesterases of Bacillus subtilis In B. subtilis species some intracellular and extracellular esterases have been characterised (Higerd & Spizizen, 1973; Meghi et al., 1990). One of these esterases was identified and isolated from B. subtilis Thai I-8 in 1994 by Quax and co-workers and found to be effective for the resolution of chiral compounds (Mutsaers & Kooreman, 1991; Quax & Broekhuizen, 1994; Smeets & Kieboom, 1992; Van der Laan et al., 1993). This esterase could be used for the kinetic resolution of propionate esters with an aromatic ring containing a 2-substituent, like 2-arylpropionates, 2-(aryloxy)propionates and N-arylalanine esters (Azzolina et al., 1994; 1995; Smeets & Kieboom, 1992).

ß1 ß2 ß4 ß3 ß5 ß6 ß7 ß8aA aB aC aD aE aF

Canonical /ß hydrolase foldα

nucleophile acid histidine

N C

FIGURE 4: Schematic representation of the canonical α/β-hydrolase fold.

Introduction

17

(S)-naproxen (R)-naproxen methyl ester

CH3O

CHCOOCH3

CH3

esterase

CH3O

CH

CH3

H COO-

CH3O

CH

CH3

HCOOCH3

+

FIGURE 5: The kinetic resolution process of naproxen. After reaction the insoluble (R)-naproxen ester is separated from the soluble (S)-naproxen by filtration. The (R)-naproxen ester is racemised and recycled into the process. The gene encoding this esterase was cloned into a productive B. subtilis strain (1-85), resulting in an 800 times higher expression than in the wild strain. The highly selective and efficiently produced esterase, named carboxylesterase NP, offered a competitive method (figure 5) to the classical resolution step in the synthesis of naproxen, which uses a selective diasteromeric crystallisation to separate the (R)- and (S)- enantiomer of naproxen and requires expensive chemicals (Bertola et al., 1989; Mutsaers & Kooreman, 1991; Quax & Broekhuizen, 1994).

Purified carboxylesterase NP can be used for the resolution of (RS)-naproxen methyl ester. Racemic naproxen methyl ester can be incubated with the enzyme in a stirred tank. After conversion, the non-reacted (R)-naproxen methyl ester is filtered off from the product (S)-naproxen. The (R)-naproxen methyl ester is subjected to racemisation and recycled into the process. Overall, this will yield (S)-naproxen with an excellent optical purity (99% enantiomeric excess, ee) at 40% conversion.

In B. subtilis strain 168, eight esterases have been functionally characterised (Bischoff & Ordal, 1991; Chen et al., 1995; Eder et al., 1996; Eggert et al., 2000; Higerd & Spizizen, 1973; Kneusel et al., 1994; Kunst et al., 1997; Moore & Arnold, 1996; Nilsson et al., 1994; Quax & Broekhuizen, 1994; Riefler & Higerd, 1976; Zock et al., 1994) and 9 further genes homologous to esterases have been found (Kunst et al., 1997). Among the characterised esterases, are two carboxylesterases: p-nitrobenzyl esterase (Chen et al., 1995; Moore & Arnold, 1996) and carboxylesterase NA (Quax & Broekhuizen, 1994). p-Nitrobenzyl (PNB) esterase catalyses the deblocking of beta-lactam antibiotic p-nitrobenzyl esters, such as cephalexin-PNB and loracarbef-PNB (Chen et al., 1995; Moore & Arnold, 1996).

Chapter 1

18

1 40 MSNHSSSIPELSDNGIRYYQTYNESLSLWPVRCKSFYIST MSNHSSSIPELSDNGIRYYQTYNESLSLWPVRCKSFYIST 41 80

RFGQTHVIASGPEDAPPLVLLHGALFSSTMWYPNIADWSS RFGQTHVIASGPEDAPPLVLLHGALFSSTMWYPNIADWSS 81 120

KYRTYAVDIIGDKNKSIPENVSGTRTDYANWLLDVFDNLG KYRTYAVDIIGDKNKSIPENLSGTRTDYANWLLDVFDNLG 121 160

IEKSHMIGLSLGGLHTMNFLLRMPERVKSAAILSPAETFL IEKSHMIGLSLGGLHTMNFLLRMPERVKSAAILSPAETFL 161 200

HPFHDFYKYALGLTASNGVETFLNWMMNDQNVLHPIFVKQ PFHHDFYKYALGLTASNGVEKFLNWMMTDQNVLHPIFVKQ 201 240

FKAGVMWQDGSRNPNPNADGFPYVFTDEELRSARVPILLL FQAGVMWQDGSRNPNPKADGFPYVFTDEELRSARVPILLL 241 280

LGEHEVIYDPHSALHRASSFVPDIEAEVIKNAGHVLSMEQ LGEHEVIYDPHSALHRASSFVPDIEAEVIKNAGHVLSMEQ 281 300

PTYVNERVMRFFNAETGISR PAYVNERVMRFFNAETGISR FIGURE 6: Sequence of carboxylesterase NP of B. subtilis Thai I-8 (upper lane) and carboxylesterase NA of B. subtilis 168 (lower lane). The matching amino acids are in black. The Gly - Xaa - Ser - Xaa - Gly motif is underlined. Comparison of the sequence of the carboxylesterase NA in the genome of B. subtilis strain 168 with the carboxylesterase of Quax et al., showed an identity of 98% (figure 6). A Gly – Xaa – Ser – Xaa – Gly motif is present in both carboxylesterases. From this, we expected that the carboxylesterase NA of B. subtilis 168 could also be an valuable catalyst for kinetic resolution purposes as will be shown in this thesis.

Lipases Like esterases, lipases catalyse the hydrolysis of esters. However, lipases preferentially catalyse the hydrolysis of water-insoluble long chain esters such as triglycerides (figure 7). The in vitro enzymatic activity of lipases can be measured by the hydrolysis of a wide variety of carboxylic acids esters (both natural and unnatural substrates), as well as by the highly specific cleavage of long-chain acylglycerols (Beisson et al., 2000; Jaeger et al., 1994). It

Introduction

19

should be mentioned that triacylglycerols are water insoluble. Thus, the conversion to glycerol and organic acids has to take place at the water-lipid interface. This process, known as interfacial activation, is likely to be enhanced by the presence of a lid-like polypeptide, which covers the active site and is present most lipases. After diffusion of the lipase in the interface, the active site cleft of the enzyme is exposed due to a conformational change of the lid (open conformation) (Milled et al., 2001). Due to this interfacial activation, the kinetics of a lipase catalysed hydrolysis reaction can not be described by Michaelis-Menten kinetics since this model is only valid for soluble enzymes and substrates (Verger & de Haas, 1976).

Bacterial lipases vary considerably in size, ranging approximately from 20 to 70 kDa. Surprisingly, all their 3D structures are remarkably similar. Nowadays, several 3D structures of lipases have been elucidated and they all contain an α/β-hydrolase fold (figure 4). In contrast to esterases, most lipases contain an additional helical segment, the so-called lid, covering the active site catalytic triade (Ser, His, Glu or Asp) (Jaeger et al., 1994).

In 1999, bacterial lipolytic enzymes (e.g. lipases and esterases) were classified by Arpigny & Jaeger based on a comparison of their amino acid sequences and some fundamental biological properties. As a result, bacterial lipolytic enzymes were classified in 8 families. Family I, which is subdivided in 6 subfamilies, contains the so called ‘true’ lipases: Pseudomonas lipases; lipases from Gram positive bacteria, such as Bacillus and Staphylococcus; and other lipases, such as lipases from Propionibacterium and Streptomyces. The enzymes of family II lack the classical pentapeptide Gly - Xaa - Ser - Xaa – Gly, but have a Gly – Asp – Ser - Leu motif instead. Within this family, esterases of Strep. scabies, Ps. aeruginosa, Salmonella typhimurium, Photorhabdus luminescens and Aeromonas hydrophila are found. In family III, the extracellular lipases of Streptomyces and Moraxella are included, while family IV contains the enzymes similar to the mammalian hormone-sensitive lipase. Enzymes originating from mesophilic bacteria (e.g. Ps. oleovorans, Haemophilus influenza, Acetobacter pasteurianus), from cold-adapted organisms (e.g. Moraxella sp., Psychrobacter immobilis) and heat-adapted organisms (Sulfolobus acidocaldarius) are grouped in family V. Family VI contains the smallest esterases known, having a molecular mass of 23 – 26 kDa, while family VII contains the large bacterial esterases (55 kDa). In family VIII, enzymes similar to class C β-lactamases are found.

O

HO+ 3

O H

O H

O Hlipase+ 3 H 2O

O

O

O

O

O

O

FIGURE 7: Lipase catalysed hydrolysis of a triglyceride (Jaeger et al., 1994).

Chapter 1

20

TABLE 1: Organisms from which lipases have already been used for enantiomer resolution according to a search in the Biological Abstracts 1994 –July 2003. Organism Number of articles

Candida antarctica

Candida rugosa

Candida cylindracea

Pseudomonas cepacia/Burkholderia

Pseudomonas fluorescens

Pseudomonas aeruginosa

Mucor miehei

Porcine pancreatic lipase

Tripterygium wilfordii

Rhizopus

Rabbit gastric lipase

Pseudomonas KW51

Aspergillus niger

Human lipoprotein lipase

Chromobacterium viscosum

Streptomyces virginiae

Bacillus subtilis

numerous unknown/unclassified

38

36

25

20

12

10

8

2

1

4

1

1

4

1

1

1

1

Compared to esterases, lipases are of particular interest for the enantiopure production of several compounds as a result of a broader substrate specificity, a higher stability in organic solvents, and often a higher enantioselectivity. In general, lipases have become of commercial importance as constituents of washing detergents and catalysts in the synthesis of compounds which serve as a precursor to pharmaceuticals, agrochemicals and other synthetic targets (table 1). Bacterial lipases with interesting industrial application possibilities have thus far been found in Pseudomonas, Thermomyces and Staphylococcus species (Jaeger & Eggert, 2002; Reetz & Jaeger, 1998; Schmidt-Dannert, 1999; Soberón-Chávez & Palmeros, 1994). However, these lipases require specific chaperones for activation, which limits their expression in heterologous hosts. The use of the homologous host for production is usually not desirable since these strains are not 'generally recognised as safe'

Introduction

21

(GRAS). An important exception is the lipase A of B. subtilis (LipA), as this lipase can be overproduced in large quantities in a GRAS host: Bacillus. Moreover, fungal lipases show micro-heterogeneity as a consequence of glycosylation and/or phosphorylation which excludes Escherichia coli and phage fd as a reliable expression host.

Lipases of Bacillus subtilis In 1992, Colson and co-workers have cloned and sequenced the gene responsible for the extracellular lipolytic activity in B. subtilis 168 (lipA gene). After purification of the enzyme from the wild type culture supernatant, an enzyme of 181 amino acids with a molecular mass of 19.348 kDa was obtained. Strikingly, LipA lacked the characteristic Gly - Xaa - Ser - Xaa - Gly pentapeptide. The first Gly residue in the consensus is altered into Ala (Dartois et al., 1992). Biochemical characterisation revealed that LipA showed maximum stability at alkaline pH, ranging up to pH 12, while maximum activity was observed at pH 10. LipA was active towards p-nitrophenylesters and triacylglycerides. A marked preference towards a C8 alkyl chain was observed (Dartois et al., 1992; Eggert et al., 2000; Lesuisse et al., 1993).

Surprisingly, a homologue of lipA was found when the genome sequence of B. subtilis 168 was elucidated in 1997 (Kunst et al., 1997). This second extracellular lipase, called LipB, revealed a 74% identity on protein level to LipA. The gene was cloned and overexpressed and the corresponding protein was purified. The LipB gene encoded a protein of 182 amino acids with a calculated molecular mass of 19.490 kDa. Enzymatic characterisation revealed that LipA and LipB had virtually similar specific activities towards p-nitrophenylesters and triacylglycerides. However, in contrast to LipA, LipB was unable to hydrolyse the typical lipase substrate triolein (Eggert et al., 2000; 2001).

FIGURE 8: 3D structure of the B. subtilis LipA. The structure shows a single compact domain consisting of six ß-strands in a parallel ß-sheet, surrounded by five α-helices. Two α-helices are located at one side of the ß-sheet, whereas the other three are found at the opposite side (including a very small α-helix consisting of only 4 amino acids residues). The active site residues Ser77, His156, and Asp133 are shown in black ball-and-stick representation and are labeled with S, H and D, respectively (Van Pouderoyen et al., 2001).

Chapter 1

22

Like other lipases, the fold of B. subtilis LipA resembles the core of α/ß hydrolase folded enzymes. The structure shows a single compact domain that consists of six ß-strands in a parallel ß-sheet, surrounded by five α-helices (figure 8). Two α-helices are located at one side of the ß-sheet, whereas the other three are found at the opposite side (including a very small α-helix consisting of only 4 amino acids residues). Due to its small size and the absence of a separate lid domain, which is present in the larger lipases, B. subtilis LipA can be regarded as a minimal α/ß hydrolase fold enzyme. The active site is located at the bottom of a small cleft between two loops and is solvent exposed due to the lack of the lid domain. As such, B. subtilis LipA does not show interfacial activation as compared to other bacterial lipases. Within the active site, the catalytic Ser77 is positioned at a very sharp turn between a β-strand and an α-helix, the so-called nucleophilic elbow (Kawasaki et al., 2002; Van Pouderoyen et al., 2001).

In this thesis, we have chosen to engineer the enantioselective characteristics of LipA of B. subtilis for the enantioselective production of beta-blocker compounds. LipA of B. subtilis 168 is of particular interest for the production of enantiopure compounds as it shows some advantageous potentials for industrial application: (i) it has a small size (181 amino acids, 19.348 kDa); (ii) it has a remarkable alkaline pI (9.73) which makes it very tolerant to basic pH (optimal activity is observed at pH 10); (iii) it can be well expressed and engineered in E. coli and B. subtilis; (iv) it can be produced at high yields in a GRAS organism; (v) it does not show interfacial activation in the presence of oil-water interfaces (due to the absence of the so-called lid), allowing characterisation by Michaelis-Menten kinetics; (vi) its crystal structure has recently been elucidated; (vii) expression of the cloned gene in E. coli revealed that it exhibits a broad substrate range, which included various-chain-length p-nitrophenyl esters and triglycerides (Dartois et al., 1992; 1994; Eggert et al., 2000; Lesuisse et al., 1993; Van Pouderoyen et al., 2001).

Protein engineering In the past decade the technique of protein engineering has allowed investigators to create new enzymes and proteins. This new technology has not only increased our knowledge on structure-function relationships of proteins, but it has also led to practical applications (Bornscheuer et al., 2002b; 2002c; Bornscheuer & Pohl, 2001). Interestingly, some of the most stunning commercial successes have not been the result of rational design based on a 3D structure, but are merely the payoff of smart combinations of random mutagenesis and screening or selection. Rational design usually requires the availability of the 3D structure of the enzyme and knowledge about the structure-relationship of the enzyme (Chen, 2001; Kazlauskas, 2000). In contrast, directed evolution involves mutagenesis of the gene encoding the enzyme (Farinas et al., 2001; Powell et al., 2001; Tao & Cornish, 2002). Existing approaches to improve single proteins or parts of proteins include random point

Introduction

23

mutations (i.e. error-prone PCR (Caldwell & Joyce, 1994), mutator strains (for a review, see Miller, 1998) and UV or chemical mutagenesis (Tange et al., 1994), cassette library mutagenesis (i.e. random, biased, saturation and codon-based cassette mutagenesis (Arkin & Youvan, 1992; Reidhaar-Olson et al., 1991)) or recombination of gene fragments (i.e. DNA shuffling (Stemmer, 1994), the staggered extension process, StEP, (Zhao, 1998), random priming recombination (Shao et al., 1998), and synthetic shuffling (Ness et al., 2002; Zha et al., 2003)). Using these mutagenesis strategies, large numbers of mutants can be created in relatively short amounts of time (Ness et al., 2001).

High-throughput screening methods are commonly used to identify proteins with new properties. In such screening strategies, each separate mutant of a library has to be analysed using a high-throughput assay. Unfortunately, for many enzymes, such as lipases and esterases, no high-throughput methods are available. It would therefore be highly advantageous if the screening process could be complemented with a selection method to quickly obtain potentially successful mutants from the library. Existing approaches of selection comprise both in vivo and in vitro techniques. Such techniques can be divided into three subclasses depending on how genotype and phenotype are physically linked and selection is performed (for reviews see: Cohen et al., 2001; Griffiths & Tawfik, 2000; Jaeger et al., 2001; Lin & Cornish, 2002; Pelletier & Sidhu, 2001; Soumillion & Fastrez, 2001):

The genotype-phenotype linkage and the selection can both take place in vivo in compartmentalised cells. The most widely used in vivo method to study protein-protein interactions is the yeast two-hybrid system. Basically, interaction between two proteins of interest results in a functional assembly of the two components of a signalling system: a DNA binding domain and a transcriptional activation domain. As a result, transcriptional activation of a reporter gene leads to a detectable phenotype (for a review, see Pelletier & Sidhu, 2001). Recently, Pabo and co-workers adapted a bacterial two-hybrid assay to evolve zinc finger variants with defined DNA-binding specificities in one round of selection (Joung et al., 2000). Another example for in vivo selection is based on metabolic selection (e.g. growth). For instance, the growth of a bacterial host can be made dependent on the production of a lipase that cleaves a certain lipid or ester provided in the growing medium as the sole carbon source. Only the cells encoding a variant lipase, that can hydrolyse the supplied substrate will be able to survive. After some consecutive rounds of mutagenesis and selection a new variant can be evolved in a stepwise manner.

In a fully in vitro system, the genotype-phenotype linkage and the selection both occur in vitro (Griffiths & Tawfik, 2000). Compared to fully in vivo methods, in vitro techniques are not limited by the number of molecules that can be handled. Large libraries, up to 1014 variants, can be built since the method is not limited by cellular transformation efficacies (Amstutz et al., 2001). Two main groups of in vitro selection technologies can be distinguished. The first group imitates the compartmentalisation of living cells by

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performing translation and selection within a water oil emulsion (Griffiths & Tawfik, 2000). The second group makes use of a physical link between messenger RNA and a nascent polypeptide during translation to couple genotype and phenotype. The most popular in vitro display technologies are ribosome display, based on a non-covalent ternary complex of mRNA and the ribosome and the nascent polypeptide, and mRNA display, based on a covalent coupling of mRNA to the nascent protein (Amstutz et al., 2001).

Partially in vivo techniques, such as cell surface display and phage display, comprise an in vivo step linking the genotype and phenotype. However, the actual selection step occurs in vitro. Cell surface display is based on the principle that proteins are fused to a membrane protein, which can serve as an anchor to present proteins on the cell surface (Wittrup, 2001). Phage-display is one of the major developments leading to a breakthrough in selection methodology. This technique combines random or site-directed mutagenesis with a highly specific selection method, thus allowing rapid screening of many biocatalysts with improved enantioselective characteristics.

Phage display The M13 phage particle consists of a single stranded DNA molecule (the phage genome) which is encapsulated by the phage coat proteins (figure 9). The phage protein coat consists of thousands of copies of the pVIII major coat protein (depending on the length of the phage genome), four to five copies of pIII and pIV (located at the top of the phage), and four to five copies of pVII and pIX (located at the bottom of the phage) (Azzazy & Highsmith, 2002; Smith, 1985; Wilson & Finlay, 1998). Small peptides can be expressed on filamentous phages by inserting the gene of interest into one of the phage coat protein genes, such as the gene III sequence (Smith, 1985). The power of this selection method is elegantly demonstrated in the field of antibody fragments, where new variants have been obtained by affinity maturation using phage display. Selections of 1 out of 109 have been reported (Arkin & Wells, 1998; Atwell & Wells, 1999; Griffith & Duncan, 1998).

FIGURE 9: Illustration of a phage. The single stranded circular genome is surrounded by approximately 2700 copies of the major coat protein pVIII and 4 or 5 copies of each of four species of minor coat proteins, including pIII, which binds to a host cell F-pilus (Atwell & Well, 1999).

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Today, also enzymes, such as amylases (Verhaert et al., 2002), ß-lactamases (Avalle et al., 1997; Ponsard et al., 2001; Soumillion et al., 1994; Vanwetswinkel et al., 1996), endopeptidases (Heinis et al., 2001), DNA-polymerases (Jestin et al., 1999), kinases (Ting et al., 2001), lipases (Danielsen et al., 2001; Deussen et al., 2002b; Dröge et al., 2003a), nucleases (Light & Lerner, 1995; Pedersen et al., 1998), subtiligases (Atwell & Wells, 1999), and transferases (Demartis et al., 1999; Hansson et al., 1997; Widersten & Mannervik, 1995), have successfully been displayed on bacteriophages.

While phage display had proven to be extremely suitable for selections based on binding, it appeared to be a more difficult challenge to adapt this technique for enzyme catalysis. Nowadays, several strategies have been developed for selection on catalytic activity including selection on suicide substrates, transition state analogues, and substrates anchored to the phage (Atwell & Wells, 1999; Avalle et al., 1997; Danielsen et al., 2001; Demartis et al., 1999; Jestin et al., 1999; Soumillion et al., 1994; Vanwetswinkel et al., 1995; 1996). Selection on suicide substrates and transition state analogues have the main limitation that (a) it requires a detailed knowledge of the reaction mechanism, (b) appropriate suicide substrates of transition state analogues have to be designed and synthesised, and (c) selection might be directed to a single step in the catalytic cycle or to a reaction intermediate (Jestin et al., 1999). Anchoring of the substrate to the phage in proximity of the displayed enzyme not always overcomes these problems. Since only a single turnover has to occur per phage particle in order to be selectable, it is difficult to discriminate between two enzymes with different kinetic properties. Furthermore, the substrate has to be covalently attached to the phage particle, which might hamper proper catalysis (Demartis et al., 1999; Forrer et al., 1999).

In respect of this thesis, the gene of interest (e.g. lipase or esterase gene) can be cloned in a phagemid vector (figure 10). This phagemid carries a copy of gene III. When bacterial cells transformed with these phagemids are infected with helper phages, which carry the full complement of capsid-encoding genes but are defective in replication, the secreted phage particles carry the phagemid genome and a mixture of wild-type and fusion pIIIs (figure 9).

FIGURE 10: Schematic representation of the phagemid system. The lipase or esterase gene is inserted into gIII coat protein gene at the pIII N-terminal signal peptidase cleavage site, resulting in a display of the lipase or esterase as a N-terminal fusion protein to the pIII molecule coat protein.

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A library of mutant lipase genes, produced with localised PCR mutagenesis directed to domains identified as being involved in substrate binding (figure 8), can easily be inserted into the pIII coat protein. As a result, pools of enzyme variants will be expressed on the surface of bacteriophages. The expression of mutant enzymes as a fusion to the pIII coat protein then allows selection of enzyme variants with an improved enantioselectivity.

In this thesis, we will describe a novel, immobilised suicide inhibitor for the selection of improved lipase variants on the basis of the universal reaction mechanism of ester hydrolysis by lipases (Jaeger et al., 1994). These inhibitors consist of the target chiral drug linked to an insoluble matrix via a phosphonate moiety. After recognition of the chiral drug, the phosphonate group prevents enzyme leaving due to the formation of a covalently coupled enzyme-inhibitor complex. In this way, mutated enzymes can be selected using the insoluble matrix. Although several lipase/esterase inhibitors do exist, we propose phosphonates as superior inhibitors since: (i) they resemble the substrate hydrolysis by mimicking the first transition state in ester hydrolysis, (ii) they form a covalent bond with the enzyme and (iii) the linkage can be measured spectrophotometrically when a compound like p-nitrophenyl is used as leaving group. In this way, variants with an improved binding capacity can be selected and enriched after several rounds of “panning”. The power of this technique is obvious: the enzymes are linked to the phages which contain the mutated genes, thus speedig up mutant identification tremendously.

Aim of the thesis This thesis describes the application of lipolytic enzymes of B. subtilis for the enantiopure production of the chiral intermediate 1,2-O-isopropylidene-sn-glycerol (IPG) out of its racemic ester (figure 2). IPG is an interesting chiral intermediate for pharmaceutical industries, since it is a starting compound in the synthesis of beta-adrenoceptor antagonists, such as propranolol. To improve the yield of enantiopure IPG as compared to wild type lipase/carboxylestererase production, we have evaluated two different strategies to evolve novel highly enantioselective biocatalysts.

First, we employed paralogous gene analysis for the identification of carboxylesterase NP paralogues in B. subtilis 168. Ever since the sequencing of B. subtilis 168 was completed in 1997 (Kunst et al., 1997), it became clear that this species contains many paralogous genes. As such, it was tempting to determine whether such highly homologous carboxylesterases could have altered or enhanced enantioselective properties.

As an alternative approach, we developed a novel phage display strategy, consisting of a fast and reproducible dual selection method using enantiomeric phosphonate suicide inhibitors for the selection of B. subtilis LipA mutants with altered enantioselective properties towards IPG.