Properties of Immobilised Penicillin G
Acylase in
β
-Lactam Antibiotic Synthesis
Picture at the cover (© M.H.A. Janssen):
Properties of Immobilised Penicillin G
Acylase in
β
-Lactam Antibiotic Synthesis
Proefschrift
ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,
op gezag van de Rector Magnificus prof. dr. ir. J.T. Fokkema, voorzitter van het College voor Promoties,
in het openbaar te verdedigen op maandag 22 mei 2006 om 12:30 uur
door
Dit proefscrift is goedgekeurd door de promotor: Prof. dr. R.A. Sheldon
Toegevoegd promotor: Dr. ir. F. van Rantwijk
Samenstelling promotiecommissie:
Rector Magnificus, Technische Universiteit Delft, voorzitter Prof. dr. R.A. Sheldon, Technische Universiteit Delft, promotor
Dr. ir. F. van Rantwijk, Technische Universiteit Delft, toegevoegd promotor Prof. dr. A. Bruggink, Radboud Universiteit Nijmegen, DSM
Prof. dr. L. Fischer, Universität Hohenheim
Prof. dr. V.K. Švedas, Lomonosov Moscow State University Prof. dr. ir. L.A.M. van der Wielen Technische Universiteit Delft
Dr. M.C.R. Franssen Wageningen Universiteit
em. Prof. dr. ir. H. van Bekkum Technische Universiteit Delft, reservelid
The research described in this thesis was financially supported by DSM and the Netherlands Ministry of Economic Affairs.
ISBN 90-9020754-6
© 2006 by M.H.A. Janssen
Contents
Abbreviations and symbols xi
1. Introduction to β-lactam antibiotics 1
1.1 β-Lactam antibiotcs 2
1.2 Developments in β-lactam antibiotic synthesis 4
1.3 Enzymatic developments 11
Appendix 1.1: ACV synthase 13
References and notes 14
2. Penicillin G acylase: Structure and enzymatic β-lactam antibiotic synthesis 17
2.1 Penicillin G acylase 18
2.1.1 Discovery and occurrence 18
2.1.2 Classification 18
2.1.3 Structure and catalytic machinery 19
2.2 Enzymatic synthesis of β-lactam antibiotics 22
2.2.1 Thermodynamic and kinetic controll 22
2.2.2 Coupling procedures 24
2.2.3 Penicillin acylase kinetics 27
2.2.4 Immobilisation of penicillin acylase 31
2.2.5 Increasing the efficiency through additives 33
2.3 Motivation, objective, and outline of the thesis 35
References and notes 36
3. Evaluation of the performance of immobilised penicillin G acylase using active-site titration 43
3.1 Introduction 44
3.2.1 Immobilisation of penicillin acylase on Eupergit C 46
3.2.2 Active-site titration 47
3.2.3 Diffusion limitation effects 48
3.2.4 Calculating the minimum substrate bulk concentration 49
3.2.5 Calculation of the zero substrate concentration radius 52
3.2.6 Diffusion limitations and product inhibition 54
3.2.7 Cephalexin synthesis 55
3.3 Conclusions 61
3.4 Materials and methods 61
3.4.1 Materials 61 3.4.2 Immobilisation 62 3.4.3 Hydrolysis of penicillin G 62 3.4.4 Synthesis of cephalexin 63 3.4.5 Active-site titration 63 Acknowledgements 63 References 64
4. Properties and immobilisation of penicillin G acylase on macroporous acrylic supports 67
4.1 Introduction 68
4.2 Results and discussion 69
4.2.1 Immobilisation on Eupergit C 250 L 70
4.2.2 Immobilisation on Sepabeads FP-EP 71
4.2.3 Immobilisation on Sepabeads FP-EA and FP-HA 72
4.2.4 Active-site titration 73
4.2.5 Cephalexin synthesis at low substrate concentrations 75
4.2.6 Cephalexin synthesis at high substrate concentrations 77
4.3 Conclusions 79
4.4 Materials and methods 80
4.4.2 Immobilisation of penicillin acylase 81
4.4.3 Crushing of immobilised penicillin acylase catalysts 82
4.4.4 Hydrolysis of penicillin G 82
4.4.5 Active-site titration 82
4.4.6 Synthesis of cephalexin 83
Acknowledgements 83
References 84
5. Fluoride burst active-site titration of penicillin G acylase using a fluoride ion selective electrode 85
5.1 Introduction 86
5.2 Results and discussions 88
5.2.1 Fluoride burst active-site titration 88
5.2.2 Indirect active-site titration and comparison 94
5.3 Conclusions 96
5.4 Materials and methods 98
5.4.1 Materials 98
5.4.2 Hydrolysis of penicillin G 99
5.4.3 Fluoride burst active-site titration 99
5.4.4 Indirect active-site titration 100
Acknowledgements 100
References and notes 100
6. Dendrimer-activated supports for the immobilisation of penicillin G acylase 103
6.1 Introduction 104
6.2 Results and discussion 107
6.2.1 Immobilisation of dendrimer and enzyme 107
6.2.2 Synthesis of ampicillin and cephalexin 108
6.4 Materials and methods 111
6.4.1 Materials 111
6.4.2 Hydrolysis of penicillin G 111
6.4.3 Polyacrylamide gel dispersion polymerisation 112
6.4.4 Astramol-polyacrylamide gel beads immobilisation 112
6.4.5 Starburst/Astramol-Assemblase immobilisation 113
6.4.6 Astramol-polyketone immobilisation 113
6.4.7 Synthesis of ampicillin and cephalexin 114
Abbreviations and Symbols
6-APA 6-aminopenicillanic acid 7-ACA 7-aminocephalosporanic acid
7-ADCA 7-aminodeacetoxycephalosporanic acid ASU amoxicillin trihydrate synthesis unit [g kg-1dw h-1] BPU penicillin G hydrolysis unit [µmol min-1]
Cs substrate concentration [mol m-3]
Csi Cs at particle medium interface [mol m-3]
CSU cephalexin synthesis unit [µmol min-1]
Dsp effective diffusion coefficient of substrate in the biocatalyst particle [m2 s-1]
η dynamic viscosity [N s m-2] HPG 4-hydroxyphenylglycine
HPGM 4-hydroxyphenylglycine methyl ester
Ki inhibition constant [mol l-1]
Km Michaëlis-Menten constant [mol l-1]
NIPAB 2-nitro-5-[(phenylacetyl)amino]benzoic acid NIPGB D-2-nitro-5-[(phenylglycyl)amino]benzoicacid pAA polyacrylamide
PAA phenylacetic acid
PenG penicillin G (benzylpenicillin)
PenV penicillin V (phenoxymethylpenicillin) PG phenylglycine
PGA phenylglycine amide PGCN phenylglycine nitrile PGM phenylglycine methyl ester PMSF phenylmethanesulfonyl fluoride
r spherical coordinate in the biocatalyst [m]
R0 radius in the biocatalyst particle at which Cs is just zero [m] Rp biocatalyst particle radius [m]
S/H molar ratio of synthesis over hydrolysis product in the reaction mixture [-]
g
Introduction to
β
-Lactam Antibiotics
+ +
L-α-AAA L-Cys L-Val
ACVS 3 ATP 3 AMP+ 3 PPi
1.1
β
-Lactam Antibiotics
Antibiotics are secondary metabolites produced by (micro)organisms that specifically kill or inhibit the growth of other microorganisms. The β-lactam family of antibiotics includes many of the most heavily used antibacterials in clinical medicine. These are antibiotics bearing a β-lactam structure (a four-membered cyclic amide), which is the basis of the antimicrobial activity because it interferes in the cell wall synthesis of growing bacteria. Well-known members of this family are the penicillins and cephalosporins in which the β-lactam ring is fused to a thiazolidine or dihydrothiazine ring, respectively. Other members of the family are the carbapenems and monobactams. The β-lactamase inhibitor clavulanic acid is structurally related, though it is not an antibiotic. Some examples are presented in Figure 1.1. In this chapter, only the penicillin and cephalosporin antibiotics will be discussed. Most penicillins are 6-aminopenicillanic acid (6-APA) derivatives and
Imipenem (a carbapenem) N O SO3H H CH3 H N H O N S N H2N O CH3 COOH H3C · H2O Aztreonam (a monobactam) S COOH N H O H H3C H HO N NH H N O O H OH COOH H Clavulanic acid O N H O N S CH3 CH3 H H COOH H O N H O N H H S COOH O O CH3 NH2 HOOC H Penicillin G Cephalosporin C
most cephalosporins are 7-aminodeacetoxycephalosporanic acid derivatives, see Figure 1.2. By changing the so-called side-chain moiety R, and/or R', numerous penicillins and cephalosporins have been obtained with various antibiotic properties and activities. As the β-lactam nucleus is a fermentation product and the side-chain is of chemical origin, these antibiotics are also known as semi-synthetic penicillin (SSP) or semi-synthetic cephalosporin (SSC) antibiotics.
In 2002, the antibiotics market was valued at 23×109 US$ [1]. It was dominated, and N S O CH3 CH3 H H H N O R H COOH N O H H N O R H S COOH R' Penicillins Cephalosporins N S O CH3 CH3 H H H H2N COOH 6-APA N O H H H2N S CH3 COOH 7-ADCA
Figure 1.2 General structures of the penicillin and cephalosporin antibiotics and the
corresponding β-lactam nuclei 6-APA and 7-ADCA.
Table 1.1 The major β-lactam antibiotics.
Antibiotic product Year of introduction [2]
Estimated market volume in 2002 (tons)a Penicillin G Penicillin V 1941 1953 22,000 3,000 Ampicillin Amoxicillin 1961 1972 20,000 Cephalexin Cefadroxil Cefaclor 1970 1977 1979 3,000 350 300
still is, by the SSP and SSC antibiotics (estimated market volume in 2000: 45,000 tons) [2]. Until recently they were among the top 10 drug sales worldwide. Table 1.1 lists the estimated market volumes of today’s largest SSP and SSC antibiotics and the chemical structures are presented in Figure 1.3.
1.2 Developments in
β
-Lactam Antibiotic Synthesis
The fine chemical and pharmaceutical industries are gradually replacing existing chemical transformations by cleaner and more environmentally benign (bio)catalytic steps. The principal driving forces for cleaner technologies are increasingly stringent governmental policies, economic competitiveness, and scientific and technological developments, triggered by issues such as quality, productivity, impact on the environment, energy consumption, and resource depletion [3]. The need for so-called “green” transformations is easily exemplified by regarding the amount of waste produced per kg product by the different chemical industry branches [4] also known as the E factor [5], see Table 1.2.
Ampicillin Amoxicillin Cephalexin, R = CH3 Cefaclor, R = Cl Cefadroxil N S N H H CH3 CH3 O H O NH2 H H COOH N S N H H CH3 CH3 O H O NH2 H H HO COOH N N H H O O NH2 H H S COOH R N N H H O O NH2 H H S COOH CH3 HO
Arguably, recent trends are more promising [6]. The developments in the β-lactam antibiotics manufacture exemplify the above as will be shown in the following sections. In 1928, Alexander Fleming discovered a compound with antibacterial action and gave it the name penicillin [7], after its fungal producer Penicillium notatum. Fleming devoted a considerable amount of time to study its effectiveness against bacterial infections but his attempts to isolate the active compound failed due to its instability. This was eventually achieved by Chain and Florey and co-workers in 1940 [8] in a follow up on Fleming’s work. It is worth mentioning that in the same year, Abraham and Chain reported on “An enzyme from bacteria able to destroy penicillin”, which they named penicillinase [9] and that would be the omen of widespread penicillin resistance some 15 years later. In recognition of their efforts in the discovery of this “wonder drug” that has saved millions of lives, Fleming, Chain, and Florey together were awarded the Nobel Prize in Physiology or Medicine in 1945.
Penicillin and cephalosporin antibiotics are produced by several microorganisms, most importantly the fungi Penicillium notatum, P. chrysogenum, and Acremonium
chrysogenum (Cephalosporium acremonium). The biosynthesis of isopenicillin N and
Table 1.2 Production of waste in various branches of the chemical industry [4].
Industry branch Product scale (t/a) E factora
cephalosporin C has been studied extensively, see Scheme 1.1 [10]. It starts from the condensation of three amino acids L-α-aminoadipic acid, L-cysteine and L-valine catalysed by the enzyme ACV synthetase, see Appendix 1.1 page 13. The next step is an oxidative cyclisation catalysed by the enzyme isopenicillin N synthase to yield isopenicillin N, the first compound in the biosynthesis with antibiotic activity. Here, the biosynthetic pathways diverge to give different final products, e.g. penicillin G (benzylpenicillin) and cephalosporin C.
In 1959, the rise of antibiotic resistance triggered the development of semi-synthetic
+ +
L-α-AAA L-Cys L-Val
ACVS 3 ATP 3 AMP+ 3 PPi
Mg2+ H2N CO2H CO2H H CO2H SH H2N H CO2H H2N H ACV tripeptide IPN IPNS ascorbate Fe2+ O2 cephalosporins PA-CoA L-α-AAA + CoA AT DTT N O S H CO2H H H N O H penicillin G CO2H H2N O H H CO2H H S H N O N H H H2N CO2H H H H H N CO2H O N SH O H
Scheme 1.1 Biosynthetic pathway of penicillin G. AAA, aminoadipic acid; ACVS, ACV
penicillin (SSP) antibiotics based on numerous variations in the side-chain structure by means of chemical modification of the free amino group of the at that time recently discovered penicillin nucleus, 6-APA, mostly via the acid chloride derivative of a suitable side-chain [11]. Thus, attention was focused on the production of 6-APA. This resulted in the development of an enzymatic process for the deacylation of penicillin G and, to a lesser extent, penicillin V, based on the newly discovered enzymes penicillin G acylase and penicillin V acylase [12,13]. However, the shortcomings of this premature biocatalytic process (low productivity, large volume, no enzyme reuse) triggered the development of a chemical alternative. Ultimately, an inexpensive, highly efficient one-pot chemical deacylation (Scheme 1.2) was developed by the Koninklijke Nederlandsche Gist- en Spiritusfabriek (now a subsidiary of DSM) [14].
In the early 1950s Newton and Abraham discovered a new class of β-lactam antibiotics, produced by Acremonium chrysogenum. This resulted in the isolation of cephalosporin C in 1955 [15] and its structure elucidation in 1961 [16]. Initially, research was focused on the production of this new β-lactam antibiotic nucleus, 7-aminocephalosporanic acid (7-ACA), but this proved difficult because of the aminoadipoyl side-chain (extraction from the water phase was difficult). Moreover,
Ph O N H N O S H H H CO2- K+
1. Me3SiCl, tert. amine 2. PCl5, CH2Cl2, -40 °C 3. n-BuOH, -40 °C 4. H2O H2N N O S H H H CO2H +
penicillin G (potassium salt) 6-APA
PhCH2CO2Bu
Scheme 1.2 Chemical deacylation of penicillin G into the penicillin nucleus 6-APA and butyl
fermentation of cephalosporin C gave relatively low yields and the product contained considerable amounts of the closely related penicillin N and deacetoxycephalosporin C. Besides, the enzymatic deacylation analogous to 6-APA production was not feasible with cephalosporin C, although the 40% yield of a chemical deacylation [17] permitted the synthesis of a large number of 7-acylamidocephalosporanic acid derivatives with as good as or better antibiotic properties than the corresponding penicillins [18].
Thus, research on cephalosporin chemistry was continued and a breakthrough was achieved when it was found that the penicillin nucleus could be chemically converted into deacetoxycephalosporin G via sulfoxidation, silylation, ring-expansion, and hydrolysis [19]. Consequently, the obtained 7-ADCA nucleus became readily available making use of the already established chemical deacylation (Delft Cleavage).
Eventually, an enzymatic process was also developed for the production of 7-ACA in which first the δ-amino group was removed by an oxidative deamination catalysed by a D-amino acid oxidase. Baeyer-Villiger oxidation of the resulting α-keto acid by H2O2 gave, after spontaneous decarboxylation, glutaryl-7-ACA. This was finally hydrolysed by a glutaryl acylase, see Scheme 1.3 [20].
With the β-lactam nuclei readily available, developments in the field of semi-synthetic β-lactam antibiotics synthesis boomed. These have been excellently reviewed from several points of view [2,14,23]. As an example, three present day synthesis concepts will be shortly discussed. Ampicillin, introduced in 1961, is nowadays prepared either via the D-phenylglycine acid chloride hydrochloride salt method under Schotten-Baumann conditions (Scheme 1.4) or via the Dane salt/mixed anhydride process (Scheme 1.5). Amoxicillin (1972) is almost exclusively produced via the Dane salt/mixed anhydride
N S O COOH OAc N HO O O NH2 H N S O COOH OAc N HO O O H O O2, H2O NH3, H2O2 DAAO N S O COOH OAc N O O H O O HO H2O2 H2O N S O COOH OAc N HO O H O CO2 N S O COOH OAc H2N GA cephalosporin C glutaryl-7-ACA 7-ACA α-ketoadipoyl-7-ACA H2O HOOC COOH
Scheme 1.3 Two-enzyme system for the production of 7-ACA from cephalosporin C. First,
cephalosporin C is converted into the corresponding α-keto acid via an oxidative deamination catalysed by a D-amino acid oxidase (DAAO). The liberated H2O2 is used in the subsequent
process because the acid chloride hydrochloride salt gives difficulties in the preparation in terms of purity and stability, thereby becoming too expensive [2]. Cephalexin (1970) is prepared similarly to ampicillin with one costly difference: dissolution of 7-ADCA is only efficient using a strong organic base (not Et3N) or in situ silylation. Note that the preferred solvent for all coupling reactions is dichloromethane, which is on the black list of many (Western) countries.
1.3 Enzymatic Developments
For many years the thus developed chemistries for the manufacture of 6-APA and 7-ADCA, as well as for the semi-synthetic β-lactam antibiotics were universally applied. But meanwhile, developments in the field of biotechnology became more and more advanced, e.g. microbial screening methods, DNA technology, fermentation, enzyme production and purification, enzyme immobilisation (recycling), and (bio)chemical engineering. Eventually, the chemical deacylation using stoichiometric amounts of activation and protection groups, halogenated solvent, and low temperature,
energy-N S H2N H H O H CO2H Me3SiCl, Et3N CH2Cl2, RT N S H2N H H O H CO2SiMe3 2. H2O, +5 °C 3. OH -O Cl NH2·HCl , -5 °C 1. N S N H H O H CO2H H O NH2
intensive reaction conditions (Scheme 1.2) was replaced by an enzymatic step, catalysed by immobilised penicillin acylase, performed in aqueous environment at ambient temperature, see Scheme 1.6 [24]. This enzymatic process generates 5 times less waste than the chemical one [24] and it is estimated that at present almost 90% of the worldwide production of 6-APA is produced via enzymatic deacylation.
While developing the enzymatic hydrolysis process of penicillin G, researchers in academia and industry further explored the potential of the same enzyme for enzymatic (kinetically controlled) β-lactam antibiotic synthesis, first demonstrated by Cole in 1969 for ampicillin [25]. At that time however, the application of an enzymatic process was not commercially viable because biocatalysis, or biotechnology in general, was still in its infancy (high enzyme production costs, low enzyme stability), whereas the chemical
CH2Cl2, base, < -15 to -70 °C O OH NH2 R O O O H + -K+ O Cl O O NH R O O O mixed anhydride ampicillin amoxicillin(R=H)(R=OH) 1. 6-APA/Et3N, -30 to -55°C 2. H+, H2O, 5 °C CH3OH, RT O O- K+ NH R O O Dane salt
routes were very effective. The situation changed in the 1980’s as a result of far-reaching advances in biotechnology and growing environmental awareness [23a].
During the last decade biocatalytic processes for the synthesis of several β-lactam antibiotics have also been developed, but due to complications in the market and process development they have not yet become economically viable alternatives for the existing chemical processes, with three notable exceptions. In 1997, DSM completed the development of a biocatalytic process for the synthesis of cephalexin [26] and in December 2004 DSM announced the production of enzymatically produced cephalexin, cefadroxil, and amoxicillin [27] (see Chapter 2). This can be considered as a real highlight of the industrial application of biocatalysis in β-lactam antibiotic synthesis as a part of sustainable development. Ph O N H N O S H H H CO2 K+ H2O, penicillin G acylase H2N N O S H H H CO2H + 1. sulfoxidation
2. silyl protection, ring expansion 3. hydrolysis H2O, penicillin G acylase + Ph O N H N O H H S CO2H H2N N O H H S CO2H cephalosporin G 7-ADCA PhCH2CO2- K+ PhCH2CO2- K+
Scheme 1.6 Penicillin G acylase catalysed deacylation of penicillin G and cephalosporin G into
Appendix 1.1: ACV Synthetase
The enzyme δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase or ACV synthetase (ACVS, E.C. 6.3.3.36) is a member of the class of non-ribosomal peptide synthetases (NRPS), which are extraordinary multienzyme complexes. Contrary to ribosomal peptide synthesis that can only contain coded amino acids, peptides synthesised by NRPS often contain non-coded amino acids, e.g. D-amino acids and non-proteogenic amino acids. Many peptides synthesised by NRPS are biologically active molecules that have antimicrobial activity, e.g. cyclosporin, penicillin G, gliotoxin, gramicidin, vancomycin, and fengycin.
One NRPS consists of several modules (subunits), one for each amino acid that is incorporated in the peptide chain. Each module contains a minimum of 3 domains, each having one specific task: an adenylation domain (A), a peptidyl carrier domain (P), and a condensation domain (C). The largest NPRS identified so far is the peptaibol synthetase from Trichoderma virens consisting of 20,925 amino acids forming 18 modules with a total molecular mass of 2.3 MDa [28]. The much smaller ACVS of Penicillium
chrysogenum is a trimodular NRPS of 470 kDa producing the ACV tripeptide [29]. The
forming a peptide bond. Next, δ-(L-α-aminoadipyl)-L-cysteinyl moiety is transferred by the C domain of the second module to L-valine bound to the P-domain of the third module, forming the second peptide bond. The third module also contains an epimerisation domain (E) that catalyses the inversion of L-valine into D-valine. Last, the final tripeptide δ-(L-α -aminoadipyl-L-cysteinyl-D-valine) is released from the P domain by the integrated thioesterase domain (TE).
References and Notes
1. IMS Health 2002, see http://www.ims-global.com.
2. Bruggink, A. and Roy, P.D. Industrial synthesis of semisynthetic antibiotics. In: Bruggink, A. (ed.), Synthesis of β-Lactam Antibiotics: Chemistry, biocatalysis, and process integration, Dordrecht: Kluwer, 2001, 12-55.
3. Curzons, A.D., Constable, D.J.C., Mortimer, D.N., and Cunningham, V.L. Green Chem.
2001, 3, 1-6.
4. Sheldon, R.A. Chemtech. 1994, 38-47.
5. Sheldon, R.A. Chem. Ind. 1997, 12-15.
6. Bruggink, A. Chimica Oggi 1998, 16, 44-47.
7. Fleming, A. Brit. J. Exp. Pathol. 1929, 10, 226-236.
8. Chain, E., Florey, H.W., Gardner, A.D., Heatley, N.G., Jennings, M.A., Orr-Ewing, J., and Sanders, A.G. Lancet 1940, 2, 226-228.
9. Abraham, E.P. and Chain, E. Nature 1940, 146, 837.
10. For a review, see: Luengo, J.M. J. Antibiot. 1995, 48, 1195-1212.
11. Barchelor, F.R., Doyle, F.P., Nayler, J.H., and Rolinson, G.N. Nature 1959, 183, 257-258. 12. Sakaguchi, K. and Murao, S. J. Agr. Chem. Soc. Japan 1950, 23, 411.
Gourevitch, A., and Lein, J. Nature 1960, 187, 237-238. (d) Huang, H.T., English, A.R., Seto, T.A., Shull, G.M., and Sobin, B.A. J. Am. Chem. Soc. 1960, 82, 3790-3791.
14. Verweij, J. and De Vroom, E. Recl. Trav.Chim. Pays-Bas 1993, 112, 66-81. 15. Newton, G.G.F. and Abraham, E.P. Nature 1955, 175, 548.
16. (a) Abraham, E.P. and Newton, G.G.F. Biochem. J. 1961, 79, 377-393. (b) Hodgkin, D.C. and Maslem. Biochem. J. 1961, 79, 393-402.
17. Morin, R.B., Jackson, B.G., Flynn, E.H., and Roeske, R.W. J. Am. Chem. Soc. 1962, 84, 3400-3401.
18. Chauvette, R.R., Flynn, E.H., Jackson, B.J., Lavagnino, E.R., Morin, R.B., Mueller, R.A., Pioch, R.P., Roeske, R.W., Ryan, C.W., Spencer, J.L., and Van Heyningen, E. J. Am. Chem.
Soc. 1962, 84, 3401-3402.
19. Morin, R.B., Jackson, B.G., Mueller, R.A., Lavagnino, E.R., Scanlon, W.B., and Andrews, S.L. J. Am. Chem. Soc. 1963, 85, 1896-1897.
20. Tischer, W., Giesecke, U., Lang, G., Röder, A., and Wedekind, F. Ann. N. Y. Acad. Sci. 1992,
672, 502-503.
21. Wegman, M.A., Janssen, M.H.A., Van Rantwijk, F., and Sheldon, R.A. Adv. Synth. Catal.
2001, 343, 559-576.
22. Tramper, J., Beeftink, H.H., Janssen, A.E.M., Ooijkaas, L.P., Van Roon, J.L., Stubel, M., and Schroën, C.G.P.H. Biocatalytic production of semi-synthetic cephalosporins: process technology and integration. In: Bruggink, A. (ed.), Synthesis of β-Lactam Antibiotics, Dordrecht: Kluwer, 2001, 206-249.
23. Rolinson, G.N. J. Antimicrob. Chemother. 1998, 41, 589-603.
24. For further reading see e.g., Van de Sandt, E.J.A.X. and De Vroom, E. Chimica Oggi 2000,
18, 72-75.
25. Cole, M. Biochem. J. 1969, 115, 757.
26. Bruggink, A. Roos, E.C., and De Vroom, E. Org. Process. Res. Dev. 1998, 2, 128-133. 27. DSM Anti-Infectives. DSMPureActives™ A new standard in antibiotics. DSM Press Release,
Delft, 7 December 2004.
28. Wiest, A., Grzegorski, D., Xu, B.-W., Goulard, C., Rebuffat, S., Ebbole, D.J., Bodo, B., and Kenerly, C. J. Biol. Chem. 2002, 277, 20862-20868.
CHAPTER
Penicillin G Acylase: Structure and
Enzymatic
β
-Lactam Antibiotic Synthesis
2.1 Penicillin G Acylase
2.1.1 Discovery and Occurrence
The first report on the enzyme penicillin acylase was in 1950 by Sakaguchi and Murao [1] when they found the enzyme in mycelium of a Penicillium sp. capable of hydrolysing penicillin G into phenylacetic acid and the then unknown 6-APA (named “penicin”). In the early years it was thought that penicillin G acylases were mainly produced by bacteria and penicillin V acylases mainly by moulds. With increasing exceptions to the general belief, it is now well established that they are ubiquitous in bacteria, actinomycetes, fungi, and yeasts [2]. It is interesting to note that still today the biological function of the enzyme remains unknown. It is not for protection against natural penicillins in the environment as the well-know β-lactamases notoriously do. In Escherichia coli the gene encoding for penicillin acylase is located in a cluster of genes involving the metabolism of 4-hydroxyphenylacetic acid, where it is thought to have a function in the degradation of aromatics [3].
2.1.2 Classification
Despite the almost complete absence of an amino acid sequence homology, the Ntn-hydrolases share eight totally conserved secondary structural elements. The catalytic machinery is located within these conserved elements and is probably the same for this enzyme superfamily. Still, several structure-activity relationships remain to be answered [5]. Another characteristic of this enzyme superfamily is the autocatalytic posttranslational modification of the precursor protein [6] consisting of a signal peptide followed by the α-subunit, a spacer sequence, and the β-subunit in which the N-terminal Serβ1 plays a vital role in the case of penicillin acylase [7].
2.1.3 Structure and Catalytic Machinery
The crystal structure of E. coli penicillin G acylase was resolved by Duggleby et al. in 1995 [8]. The enzyme appeared to be a periplasmatic heterodimeric N-terminal serine hydrolase with a molecular mass of 86,183 Da, with a 23,817 Da (209 amino acids) α-subunit and a 62,366 Da (566 amino acids) β-subunit. The enzyme is kidney-shaped (approximate dimensions are 70×50×55 Å) with a deep cup-shaped depression leading to the active site. It has a single-amino-acid catalytic centre, the β-chain N-terminal serine γ-hydroxyl. Common for other serine hydrolases having an Asp-His-Ser catalytic triad is the activation of the Serβ1Oγ by an adjacent histidine base. Contrary, penicillin acylase is believed to be activated by its own Serβ1 free α-amino group using a bridging water molecule [8], though more recently it was found that the reaction proceeds by a direct nucleophilic attack by the Serβ1Oγ without the help of a bridging water molecule [9].
enzyme-substrate complexes, e.g. with phenylacetic acid, phenylmethanesulfonyl fluoride, penicillin G sulfoxide, penicillin G, and D-α-methylphenylacetic acid [8,9,10].
The catalytically important amino acids can be divided into several classes. First is the already mentioned catalytically active nucleophile Serβ1. Second are the oxyanion hole formers Alaβ69 and Asnβ241 (and indirectly Argβ263 that interacts with Asnβ241) that stabilise the oxyanion transition-state intermediate. Third are the residues that play a role in enhancing the nucleophilicity of the Serβ1. These are Glnβ23 (at 2.9 Å and 3.2 Å from Serβ1) and Asnβ241 (3.0 Å from Serβ1). Fourth are the residues that are important for substrate binding. From a structural point of view, penicillin acylase has two substrate binding pockets. The most specific one, S1 (better known as the acyl donor binding pocket), is made up by mainly hydrophobic residues. The enclosed structure and largely hydrophobic character of the S1 pocket makes the enzyme very selective to the benzyl structure with some room for substitutions on the bridging Cα and aromatic ring, e.g., OH, NH2, CH3, OCH3, CN, F, Cl, Br. Ring structures other than phenyl are also accepted, e.g., pyridyl, thiophene, thiazole, 1H-tetrazole, and furanyl. The principle residues that enclose the S1 pocket are Metα142, Pheα146, Pheβ24, Pheβ57, Trpβ154, Ileβ177 with Serβ67 at the closed end [5,8]. The residues that complete the enclosed structure are Proβ22, Glnβ23, Valβ56, Thrβ68, Pheβ71, Leuβ253, and Pheβ256.
The catalytic mechanism of E. coli penicillin G acylase catalysed amide hydrolysis is shown in Scheme 2.1. The first step in penicillin acylase catalysis is the nucleophilic attack of the active Serβ1:Oγ hydroxyl on the electrophilic carbonyl carbon of the amide substrate. The tetrahedral oxyanion transition-state intermediate is stabilised by hydrogen bonding with two amino acids in the oxyanion hole (Alaβ69:N and Asnβ241:Nδ2). Next, the covalent acyl-enzyme intermediate is formed when the carbonyl group is restored under release of the product P (e.g., 6-APA, NH3, CH3OH in the case of penicillin G, D-phenylglycine amide (D-PGA) or methyl ester (D-PGM), resp.). In the following step the acyl moiety is transferred to either a water molecule (hydrolysis) or to an amine nucleophile (e.g., a β-lactam nucleus or any other amine) in which case an amide bond is
SerB1 C O NHR' OH H NH2 H O H N H H O AsnB241 AlaB69 SerB1 AsnB241 AlaB69 SerB1 C O O N H H O N H AsnB241 AlaB69 N H H O NHR' O C H N O H O H NH3 H O H2NR' H O H NH2 H O SerB1 C O OH OH H NH2 H O H N H H O N H AsnB241 AlaB69 SerB1 AsnB241 AlaB69 SerB1 C O O N H H O N H AsnB241 AlaB69 N H H O OH O C H N O H O H NH3 H O O H H O H NH2 H O H H N SerB1 SerB1 SerB1 SerB1 SerB1 SerB1 SerB1 SerB1 SerB1 SerB1 SerB1
SerB1 SerB1 SerB1
formed (synthesis).
2.2 Enzymatic Synthesis of
β
-Lactam Antibiotics
2.2.1 Thermodynamic and Kinetic Control
The enzymatic synthesis of a β-lactam antibiotic or any amide in general can be done either under thermodynamic control (reversed hydrolysis) or kinetic control. This was first demonstrated by Kaufmann and Bauer in 1960 [12] who reported on the resynthesis (reversed hydrolysis) of penicillin G from 6-APA and phenylacetic acid (thermodynamically controlled) or phenylacetylglycine (kinetically controlled). In the latter case they noted in particular a very fast reaction and a considerably higher yield of penicillin G, compared to the thermodynamically controlled synthesis. Indeed, these are the advantages of a kinetically controlled synthesis.
Under thermodynamic control the β-lactam nucleus and the side-chain acid are mixed and the enzyme catalyses the establishment of the thermodynamic equilibrium of the reactants and the product [13]. For penicillin G [12,14], cephalosporin G and cephalotin (see Scheme 2.2) yields of 95% or more have been reported (in the presence of cosolvents) [15]. However, the thermodynamically controlled enzymatic synthesis of more
N O S H2N COOH OAc + N O S COOH OAc N S O H + H2O S CO2H
Scheme 2.2 Penicillin acylase-catalysed thermodynamically controlled synthesis of cephalotin
useful antibiotics e.g. ampicillin, amoxicillin [16] and cephalexin [17] proved impossible due to the zwitterionic nature of the side-chain amino acids phenylglycine (PG) and D-4-hydroxyphenylglycine (D-HPG) over a very broad pH range. For example, it can be calculated that at pH 4, still 99% of the carboxyl group of D-PG is in the carboxylate form [18]. At this pH or lower, the enzyme is no longer active [19].
Following a kinetically controlled penicillin acylase-catalysed synthesis strategy of a β-lactam antibiotic means using an activated acyl donor, typically an ester, amide or anhydride derivative of the side-chain acid, instead of the free side-chain acid itself, to acylate the β-lactam nucleus. A simplified kinetic scheme is presented in Scheme 2.3. Here, the formation of the covalent enzyme-acyl intermediate (EA) is much faster and practically irreversible, which can lead to non-equilibrium concentrations of EA and P. As [AD] becomes lower, the rate of formation of P decreases. In addition, P itself is accepted
Thermodynamic E + A - H2O EA N E + P Kinetic H2O E + AD EA N E + P Q E + A Thermodynamic Kinetic 0 0 [P]equilibrium [P]max Time [P]
as a substrate for enzyme acylation. In time a maximum in [P] is observed, which can be higher than the thermodynamic equilibrium concentration, when the formation rate equals the consumption rate. Subsequently, any excess of P is hydrolysed and in the end equilibrium is restored. When using amino acid side-chains, the hydrolysis of EA to E + A is irreversible so that in the end all P will be hydrolysed. The competing hydrolysis reactions of acyl-donor (primary hydrolysis) and antibiotic product (secondary hydrolysis) are the main disadvantages of the penicillin acylase-catalysed kinetically controlled synthesis of β-lactam antibiotics because it complicates the downstream processing (separation, recycling) and necessitates the use of excess of activated acyl donor.
2.2.2 Coupling Procedures
The reaction conditions vary with the physicochemical properties of the reactants and products, e.g. solubility [24] and stability, both depending on pH and temperature (typically 5-15 °C). Heterogeneous reactant conditions are favourable over homogeneous conditions because they give better yields. Synthesis of semi-synthetic penicillins is performed at pH 6-7, depending on the side-chain donor used, and is focussed on nearly quantitative conversion of the expensive 6-APA nucleus because recycling is inefficient. The acyl donor D-4-hydroxyphenylglycine methyl ester (D-HPGM) is more base sensitive than D-PGA, therefore amoxicillin synthesis (from 6-APA and D-HPGM) is performed at pH 6.5. Ampicillin synthesis from 6-APA and D-PGA is generally performed at pH 7 although recently Youshko et al. [25] demonstrated a significant increase in yield by lowering the pH during the reaction, starting at pH 7 and ending at pH 6.3. The effect becomes understandable when regarding the substrates and antibiotic product solubilities and the corresponding nucleophile reactivity as a function of pH. The solubility of ampicillin decreases with decreasing pH whereas the nucleophile reactivity of 6-APA increases with decreasing pH (from 7 to 6.3).
the downstream processing and recycled [27].
The synthesis of cefadroxil is perhaps the most difficult of the four β-lactam antibiotics discussed here. Again, the solubility of cefadroxil in water is relatively high whereas the stability and solubility of the reactants 7-ADCA and D-HPGM show little or no overlap. 7-ADCA is only sufficiently soluble at pH 7.5 and higher but the ester functionality of D-HPGM is insufficiently stable at pH 7.5-8. A very elegant solution was developed by DSM researchers in which a concentrated basic (pH 8.1) 7-ADCA solution is prepared. Also, a concentrated acidic (pH 2.3) solution of D-HPGM is prepared, which is then added over a period of time to the reactor already containing the immobilised enzyme and the 7-ADCA solution. During the addition the reaction medium is allowed to acidify to pH 6.8 and maintained at that pH. In this way, 7-ADCA is kept at its original
H2O H2O N N KP E + P E···P EA···N Q k-4 k5 k4 k3 k2 KN KAD A + E EA E···AD E + AD
Scheme 2.4. “Minimal” kinetic scheme for penicillin acylase (E) catalysed kinetic β-lactam antibiotic synthesis in which the formation of the covalent acyl-enzyme complex (EA) plays a central role. When attacked by a β-lactam nucleophile N (e.g., 6-APA, 7-ADCA) the synthesis product P may be obtained (aminolysis). When attacked by a water molecule the hydrolysis product A is obtained, which is unreactive in the case of D-PG and D-HPG. AD, activated acyl
donor (e.g., D-PGA, D-PGM); E···AD, non-covalent enzyme-acyl donor complex; Q, leaving
group of AD (e.g., NH3, CH3OH); EA···N, acyl-enzyme with non-covalently bound nucleophile;
concentration by means of supersaturation. Cefadroxil was obtained in 87% yield and S/H = 4.0 [28].
2.2.3 Penicillin Acylase Kinetics
Kinetic studies on the penicillin acylase-catalysed kinetic synthesis of β-lactam antibiotics resulted in a “minimal” kinetic scheme with intermediate formation of an acylated enzyme-nucleophile complex (EA···N), as shown in Scheme 2.4 [29]. In this scheme formation of the covalent acyl-enzyme intermediate (EA) plays a central role and is the rate-limiting step [30]. The scheme can be used for accurate modelling of a β-lactam antibiotic synthesis reaction [29c], though a more simplified scheme has also been used [31]. Clearly, the result of every catalytic cycle is either the formation of the β-lactam antibiotic product P (synthesis or aminolysis) or formation of A (hydrolysis). The ratio between [P] and [A] or their rates of formation (vs and vh, resp.) determines the efficiency of the reaction. This is quantified by the efficiency parameter “synthesis/hydrolysis ratio” or S/H, which is the molar ratio of synthesis product P over hydrolysis product A. In practice, the efficiency of a β-lactam antibiotic synthesis reaction is determined by the rate of formation of P (vs) versus the rate of formation of A (vh). At the time when vs = vh, the maximum yield of P has been reached and the reaction should be stopped as from that time onwards the net result of the reaction becomes hydrolysis of P.
determined directly from simple (initial) rate experiments [29c,32]. Each one relates to one part of Scheme 2.4, i.e. formation of EA, transformation of EA, and transformation of EA···N, resp. Combined with the initial concentrations of reactants AD and N, the maximum conversion of N into P can be calculated [32]. Instead of using the efficiency parameter S/H, the ratio of rates s
h v v ⎛ ⎞⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎜⎝ ⎠is used when dealing with kinetics.
The parameter α relates to the formation of EA, which depends on the relative specificity ratio of the enzyme for both acylation substrates, i.e. the activated acyl donor
AD and the antibiotic product P, thus depending on apparent second-order rate constants of the hydrolysis reaction [29c,30,32]:
cat m cat m k K k K k K k K P 4 P AD 2 AD α − ⎛ ⎞⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜⎝ ⎠ = = ⎛ ⎞⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜⎝ ⎠ (2.1)
concentration reached values > 300 mM, far above its saturation concentration (supersaturation).
Parameter β0 relates to the transformation of the covalent acyl-enzyme complex EA, which is a competition between the β-lactam nucleophile N and water, the solvent of choice. From Scheme 2.4 it follows that [29c]
· · · s h k v v k K k ⎛ ⎞⎟ ⎜ ⎟ = ⎜ ⎟ ⎜ ⎟ ⎜ + ⎝ ⎠ 4 3 N 5 0 [N] [N] (2.2)
were subscript 0 indicates initial rates. Eq. (2.2) describes a hyperbolic dependence of s h v v 0 ⎛ ⎞⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎜⎝ ⎠ on [N], similar to a Michaelis-Menten plot [33] and is independent of [AD]. Eq. (2.1) can also be written as
Table 2.1. Reported values for the steady-state parameters of the penicillin G acylase catalysed
max max · · · · · · s s h h s h v v v v v k v K K k 3 m,N 0 N 0 5 1 [N] [N] [N] [N] 1 [N] [N] [N] γ β β γ ⎛ ⎞⎟ ⎛ ⎞⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎜ ⎛ ⎞⎟ ⎝ ⎠ ⎝ ⎠ ⎜ ⎟ = = = = ⎜ ⎟ ⎜ ⎟ ⎜ + ⎝ ⎠ + + (2.3)
with (vs/vh)max = k4/k5, k3·KN/k5 = [N] at which (vs/vh)0 = 0.5 × (vs/vh)max, and β0 = k4/k3KN (relative reactivity of N at low [N]) and γ = k5/k4 (ratio of conversion of EA···N into P over A) and β indicating the efficiency of acyl-transfer to N [29c]. Comparing some values for ampicillin and cephalexin synthesis reported in the literature [29c,30,32,33,36,37] shows remarkable differences, see Table 2.1. For cephalexin synthesis both a nucleophile saturation effect (γ > 0) [32,36,38] and no nucleophile saturation effect (γ = 0) [30,37] have been reported. This was possibly due to a narrow nucleophile concentration range that was studied and the possibility of a pH and temperature dependence of γ [30]. From the high Km,N values it is clear that the enzyme is much more selective for the 7-ADCA species than for the 6-APA one.
successful [38,39].
2.2.4 Immobilisation of Penicillin Acylase
The immobilisation of enzymes is common practise, mainly in order to minimise the share of the enzyme costs on the process economics by making it possible to reuse the enzyme many times. This means that the enzyme is physically confined, often in a polymer matrix in the form of beads, in such a manner that it cannot go into solution. The use of an immobilised enzyme also generally facilitates the downstream processing because it can simply be removed by sieving, whereas a considerable effort and money would have to be invested in removing a soluble enzyme from a reactor stream. In addition, immobilised enzymes also tend to be more stable than the dissolved enzyme. Unfortunately, there are also disadvantages, e.g. a (partial) loss of activity, changed kinetics, and diffusion or mass
transfer limitations [40].
There are five commonly applied methods for enzyme immobilisation (Figure 2.1): 1) physical adsorption via electrostatic or hydrophobic interactions to a surface; 2)
covalent attachment via chemical bonding to a surface; 3) physical entrapment in a
gel-type matrix that allows for diffusion of smaller sized substrate and product molecules but not the enzyme; 4) physical confinement in a semi-permeable membrane; 5) chemical cross-linking of dissolved enzyme molecules, or precipitated enzyme aggregates (CLEA) or enzyme crystals (CLEC), often using glutaraldehyde as the cross-linker.
literature has been used for the immobilisation of penicillin acylase [41,42]. These include: activated agarose derivatives [43], polyacrylamide and other acrylic copolymers [44] such as commercial Eupergit C and Eupergit C 250 L [45], kieselguhr and celite (porous siliceous or diatomaceous earth) [46], (nanoporous) silica materials [47,48], grafted nylon membranes or particles [49], cross-linked enzyme aggregates (CLEAs), coagulates, and crystals (CLECs) [50], cross-linked gelled gelatine copolymer derivatives (Assemblase) [51,52], polyvinyl alcohol [53], and a thermoreactive “smart” polymer which precipitates at temperatures above 32 °C [54]. The majority of these immobilised preparations have only been studied in the hydrolysis of penicillin G to determine the stability of the immobilised enzyme. However, some have also been studied in the synthesis of β-lactam antibiotics but due to the various reaction conditions (e.g. temperature, pH, concentrations
of reactants, activity of the immobilised enzyme, and acyl donor reactant species) that
A B C D E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E F
Figure 2.1 Common methods of immobilisation: adsorption (A), covalent attachment (B),
have been used, the results cannot be compared. In general, immobilised penicillin acylase never performs as good as the dissolved enzyme. Thus, the (initial) S/H and maximum yield are always lower. For example: in the synthesis of ampicillin from 300 mM 6-APA and 500 mM D-PGA at pH 7.0 and 21 °C the initial S/H = 5.3 for the dissolved enzyme, 4.2 for CLEA, and 2.0 for CLEC (SynthaCLEC-PA) [55]. Diffusion limitations play a significant role as will be demonstrated in chapters 3 and 4. Obviously, diffusion of reactants and products inside an inflexible, microporous, densely packed CLEC particle [56] is very slow, despite the small size of such a particle. However, it also cannot be ruled out that the kinetics of a CLEC enzyme or other immobilised enzyme have been altered due to the immobilisation. Thus, making an efficient immobilised penicillin acylase catalyst for antibiotic synthesis is a compromise between activity and efficiency and this varies with the carrier.
2.2.5 Increasing the efficiency through additives
yield increased from 1.1 to 3.1. Unfortunately, β-naphthol had a severe inhibition effect on the enzyme (98% loss of activity after 1 day). However, no inactivation was observed by the less effective complexant 1,5-dihydroxynaphthalene [58]. In another study [59], complexation of cephalexin with 1,5-dihydroxynaphthalene was studied in an integrated two-step, one-pot synthesis of cephalexin from D-PGA (formed in-situ from
D-phenylglycine nitrile (D-PGCN) using a nitrile hydratase) and 7-ADCA. In the presence of 1,5-dihydroxynaphthalene, the cephalexin yield increased from 48% (without) to 79% and the S/H at maximum yield increased from 1.9 to 7.7 when using Assemblase as the catalyst. The same methodology was also successfully applied for the synthesis of cefaclor [60]. Despite the improvements by adding these complexants, their use on an industrial scale is not likely because of the accumulation of residual amounts in the final product that cannot be avoided.
using 100 mM 7-ADCA and 150 mM D-PGM at pH 6.5 and 15 °C.
By lowering the water activity aw, claimed as such by adding cosolvents such as methanol, glycerol, ethylene glycol, 1,2-propanediol or even sucrose and sorbitol, to the reaction mixture, product hydrolysis can also be reduced though the effects are not always universal and sometimes difficult to explain [63]. For instance, the rate of cephalexin hydrolysis by a Xanthomonas citri acylase was actually increased in the presence of 20%
sorbitol, though cephalexin synthesis was still much more efficient in the presence of 20% sorbitol. Strangely, a sharp optimum in cephalexin yield was observed for aw = 0.96-0.98 using glycerol, sucrose or sorbitol [63a], suggesting that the claimed reduction in aw was not responsible for the altered yields, but probably a more complicated (kinetic) effect. In a similar study with the standard penicillin acylase from E. coli [63b] the presence of 20%
methanol also increased the yield in the synthesis of α-hydroxypenicillin and ampicillin. Again, the extent of the effect depended on the reactants (and products) and reaction conditions, clearly not depending on one parameter. Probably, the answer lies in a good kinetic analysis, starting with the dissolved enzyme, because when dealing with immobilised enzymes mass-transport limitations add to the complexity of the reaction system (see chapter 3). From structural studies [9] it is known that multiple cosolvent molecules such as ethylene glycol can enter the active site (up to four molecules), which are replaced upon substrate binding.
2.3 Motivation, Objective, and Outline of the Thesis
high immobilisation yield, and a long-term operational stability are the main requirements. For application in β-lactam antibiotic synthesis an immobilised penicillin acylase biocatalyst must also have a high S/H ratio because the hydrolysis product has no value. Instead of a trial and error approach, the objective of this thesis was to study the immobilisation of penicillin acylase by a more rational approach in order to understand the effects that often result in lower activities and S/H ratio. In Chapter 3, the immobilisation and catalytic performance of penicillin acylase on Eupergit® C is studied as a function of the enzyme concentration in the immobilised catalysts. In Chapter 4 a similar study is performed using macroporous Eupergit® C 250 L and Sepabeads® as the immobilisation support. In Chapter 5 a new active-site titration method using a fluoride ion-selective electrode is compared with the established method of residual activity titration. In Chapter 6 the immobilisation of penicillin acylase on several dendrimer-activated supports is studied.
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CHAPTER
Evaluation of the Performance of
Immobilised Penicillin G Acylase
Using Active-Site Titration
3.1 Introduction
Penicillin G acylase (penicillin amidase, penicillin amidohydrolase; E.C. 3.5.1.11) is an N-terminal serine hydrolase [1] that catalyses the hydrolysis of penicillin G as well as the synthesis of many semi-synthetic penicillin and cephalosporin antibiotics [2]. The penicillin acylase from Escherichia coli is by far the best-studied enzyme in β-lactam antibiotic research. Much research has been focused on the immobilisation of the enzyme, to enable its recycling in industrial applications with the objective of lowering its share in the costs of the final products. This has resulted in numerous publications on the immobilisation of penicillin acylase, with use of almost every possible immobilisation technique in the field.
The immobilisation of enzymes on Eupergit® C (macroporous oxirane-activated acrylic polymer beads) is well described and its industrial potential was recently reviewed [3]. Moreover, the carrier has a good performance and is easy to use and handle. Penicillin acylase on Eupergit C is well known and is used commercially in the manufacture of 6-aminopenicillanic acid (6-APA) via hydrolysis of penicillin G. Herein we describe the application of penicillin acylase on Eupergit C in the synthesis of cephalexin (CEX). In particular we have investigated the effects of enzyme loading on the activity in penicillin G hydrolysis and the synthetic performance of the immobilised catalysts.
enzyme, and to investigate whether a decrease in activity is caused by a decreased turnover rate or by the complete inactivation of a fraction of enzyme molecules [4]. Wangikar et al. [5] demonstrated the usefulness of active-site titration in comparing the availability of active-sites of subtilisin BPN′, subtilisin Carlsberg and α-chymotrypsin in organic media with that in aqueous media.
Several methods have been reported for the active-site titration of penicillin acylase and other serine hydrolases [6,7,8]. All are based on the very effective and irreversible inhibition by a protease inhibitor and assaying the residual enzyme activity, or by detecting the stoichiometrically released leaving group of the active serine reagent. Because of the enzyme’s selectivity for phenylacetyl species, active-site titration of penicillin acylase formulations is practically restricted to phenylmethanesulfonyl fluoride (PMSF). The sulfonylated N-terminal serine remains as a stable “intermediate” [1] although recent studies using LC-MS have shown a post-inactivation processing of this intermediate into a tentative aziridine intermediate [9] as well as back into the native enzyme (reactivation) upon nucleophilic attack by 6-APA [6,10].
3.2 Results and Discussion
3.2.1 Immobilisation of Penicillin Acylase on Eupergit C
An amount of penicillin acylase corresponding with approximately 2000-8000 BPU was dissolved and allowed to react with 1 g (dry weight) of Eupergit C. The uptake of penicillin acylase by Eupergit C was rapid within the first 24 h and proceeded to completion in the following hours. The activities of the immobilised catalysts are presented in Table 3.1. Up to approx. 1300 BPU g-1 wet weight was immobilised without any residual enzyme activity being detected in the supernatant (in this study 1.0 g dry weight of Eupergit C corresponded to 3.9 g wet weight and all numbers listed below with the unity g -1 refer to wet weight). The activity of the immobilised catalysts increased with the amount that had been presented to the carrier and reached a maximum of 580-590 BPU g-1 wet weight. Attempts to load more enzyme on the carrier resulted in saturation, as there was hardly any further increase in carrier-bound activity. Simultaneously, there was a large increase in residual activity in the supernatant. The immobilisation yield or binding yield,
i.e., the expressed activity over presented activity ratio, declined strongly with increasing
3.2.2 Active-Site Titration
Further insight was gained by titrating the number of immobilised active sites with PMSF, using the method described in the literature [7,4], see Table 3.2. For the first three entries there is a clear linear correlation between the presented amount of active sites and the number of catalytically competent active-sites that was bound to the carrier. A maximum loading was found of approx. 1.80×1017 active-sites g-1 or 0.30 µmol g-1. This is equivalent to 100 mg enzyme g-1 dry Eupergit C, which is the tentative maximum protein loading for Eupergit C [3], though the partial purity of the used enzyme solution suggests that this maximum should be even higher. The corresponding binding yields of 85-89%, are normal values for the immobilisation of penicillin acylase on Eupergit C. The active-site titration results confirm the presence of the expected number of active sites, but apparently they are
Table 3.1 Immobilisation of E. coli PAC on Eupergit C with different loading. Catalyst Presented activity
(BPU g-1) Immobilised activity (BPU g-1) Yieldc (%) Eup-EC-1 428 322 75 Eup-EC-2 856 465 54 Eup-EC-3 1285 556 43 Eup-EC-4 1713 581 34 Eup-EC-5 2141 587 27 Native enzyme - 1670b -
a Native and immobilised enzyme activity were measured by hydrolysis of 2% (w/v) of
