The β-lactam family of antibiotics is the most important class of antibacterial compounds in clinical application. The narrow bactericidal spectrum of naturally occurring β-lactam antibiotics, their low acid stability and increasing resistance problems have triggered the development of semi-synthetic antibiotics (SSA's) such as the semi-synthetic penicillins (SSP's) and semi-synthetic cephalosporins (SSC's). In general, chemical synthesis of semi-synthetic β-lactam antibiotics is performed under harsh conditions using reactive intermediates and organic solvents at low temperatures, causing high downstream processing costs and processes that are environmentally unfriendly. Therefore, there is an ongoing effort to replace the traditional chemical processes by enzymatic conversion, in order to obtain a more sustainable production of semi-synthetic β-lactam antibiotics.
Natural β-lactams typically consist of the β-lactam nucleus (e.g. 6-amino-penicillanic acid (6-APA), 7-amino-desacetoxy-cephalosporanic acid (7-ADCA) and others) and a so-called side chain, which is connected to the nucleus via an amide bond. Penicillin G acylase (EC 3.5.1.11) is a hydrolytic enzyme which is broadly used to remove the side chain of penicillin G (PenG), cephalosporin G (CefG) and related antibiotics to produce the corresponding deacylated nucleus 6-APA and 7-ADCA respectively together with the liberated side chain (phenylacetic acid (PAA) in the case of PenG and CefG). These deacylated β-lactam intermediates are the building blocks of SSA's such as ampicillin, amoxicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, cefalexin, cefadroxil, cefradine, cefaclor, cefprozil, cefatoxime and others. For recent reviews on PenG acylases see Rajendhran J, and Gunasekaran P., J Biosci. Bioeng. (2004), 97, 1-13, Arroyo M et al., Appl Microbiol Biotechnol. (2003) 60, 507-14, Sio C F and Quax W J. Curr Opin Biotechnol. (2004), 15, 349-55, Chandel, A. K. et al. Enzyme and Microbial Technology (2008), 42, pp. 199-207.
Apart from deacylating β-lactam compounds, it has been found that PenG acylase and amino ester hydrolases can also be used to synthesize β-lactam antibiotics. In this process, the PenG acylase catalyses the condensation of an activated side chain with a deacylated β-lactam intermediate (such as 6-APA, 7-ADCA, 7-ACA and others). The enzyme-catalyzed synthesis of β-lactam antibiotics can be carried out in either an equilibrium-controlled or a kinetically controlled conversion process. Under conditions of an equilibrium-controlled conversion, the level of product accumulation that can be reached is governed by the thermodynamic equilibrium of the reaction, which is unfavourable in the case of the synthesis of semi-synthetic antibiotics, in particular when the reaction is carried out in aqueous media. In a kinetically controlled conversion the enzyme catalyses the transfer of the acyl group from the activated side chain, i.e. the acyl donor, to the β-lactam nucleus, i.e. nucleophilic acceptor. For the preparation of semi-synthetic penicillins, the activated side chain may be the amide-derivative or the methylester of an aromatic carboxylic acid. In this case, the level of product accumulation is governed by the catalytic properties of the enzyme and high non-equilibrium concentrations of the acyl-transfer product can transiently be obtained. Examples of side chain used in the synthesis of SSA's are activated phenylglycine, activated hydroxyphenylglycine, activated dihydro-phenylglycine and others.
PenG acylase catalyzes the hydrolysis of amides and esters via an acyl-enzyme intermediate in which the N-terminal serine of the β-subunit is esterified to the acyl group. In the case of hydrolysis, water attacks the acyl-enzyme and drives the hydrolysis to completion. When an amino group of an added external nucleophile (e.g. 6-APA, 7-ADCA) is present, both the nucleophile and the water may attack the acyl enzyme, yielding the desired acyl-transfer product (antibiotic) and the undesired hydrolyzed acyl donor, respectively.
The ability of PenG acylase to act as an acyl transferase, i.e. to synthesize SSA's, is already exploited on an industrial scale in the enzymatic production of various semi-synthetic β-lactam antibiotics. However, in the production of SSA's, the hydrolysis reaction by water reduces the efficiency of the transfer reaction, due to the loss of activated precursor side chains. The ratio between the rate of synthesis (S) and rate of hydrolysis (H) is an important parameter for evaluating the synthetic performance of a PenG acylase. The S/H ratio equals the molar ratio of synthesized product (SSA) compared to the hydrolysis product at defined conditions during the enzymatic acylation reaction. The synthesized product is defined herein as the β-lactam antibiotic formed from the activated side chain and the β-lactam nucleus. The hydrolysis product is defined herein as the corresponding acid of the activated side chain. For an economically attractive process, it is desirable that the S/H ratio is high, while at the same time, the enzymatic activity preferably is also sufficiently high.
The S/H ratio that is observed in a conversion is dependant on the reactants, the reaction conditions and the progress of the conversion. Youshko et al. showed that the initial value of the S/H ratio is dependent both on the kinetic properties of the enzyme and the concentration of the nucleophilic acceptor (e.g. 6-APA)—see Youshko, M. I. and Svedas, V. K., Biochemistry (Moscow) (2000), 65, 1367-1375 and Youshko, M. I. et al. Biochimica et Biophysica Acta—Proteins and Proteomics (2002), 1599, 134-140. At fixed conditions and nucleophile concentration, the initial S/H ratio can be used to compare the performance of different PenG acylases and/or different PenG acylase mutants. In addition, the performance of different PenG acylases can be compared by measuring the synthesis and the hydrolysis during the conversion as function of time, which allows for calculation of the S/H ratio at different stages of the conversion. The synthetic activity (=the rate at which the product of synthesis is formed=rate of synthesis=production rate) of a PenG acylase in an acylation reaction refers to the amount of β-lactam antibiotic formed in the acylation reaction per unit time at defined conditions. Preferably, the initial activity is determined. The initial enzymatic activity can be determined by carrying out the acylation reaction and then constructing a graph of the amount of product synthesized versus the reaction time, a so-called progress curve. In general, at the start of the conversion, the rate of product formation is relatively constant and the activity can be derived directly from the slope of the progress curve. In case the synthetic activity already starts to decline at the beginning of the conversion the initial rate should be obtained by extrapolation of the progress curve and calculation of the slope at t=0. In order to compare the activity of different PenG acylases the synthetic activity should be normalised to the same amount of protein. In the same way as for the initial rate of synthesis the initial rate of hydrolysis can be determined from a graph of the amount of the activated side chain hydrolyzed versus the reaction time.
PenG acylases have been subject of several studies involving PenG acylase mutants. An extensive list of published mutations is given Rajendhran and Gunasekara (2004)—supre vide. More recently, further studies were published by Gabor, E. M. and Janssen, D. B., Protein Engineering, Design and Selection (2004), 17, 571-579; Jager, S. A. W. et al. Journal of Biotechnology (2008), 133, 18-26; Wang, J., et al. Applied Microbiology and Biotechnology (2007), 74, 1023-1030.
International Patent Application WO96/05318 to Gist-brocades teaches how the specificity of PenG acylases can be modified by mutating the substrate binding site at one or more amino acid positions. It was shown that the S/H ratio of PenG acylases can also be tuned in this way.
In addition, International Patent Applications WO98/20120 (to Bristol-Meyers Squibb), WO03/055998 (to Gist-brocades) and Chinese Patent Application CN101177688 (to Shanghai Institute for Biological Sciences) describe a process for the enzymatic preparation of a β-lactam antibiotic from a β-lactam nucleus and an activated side chain with the aid of a PenG acylase mutant. WO98/20120 discloses mutations at amino acid positions 142 and 146 in the α-subunit and at amino acid positions 24, 56 or 177 in the β-subunit of Escherichia coli PenG acylase. Particularly, the PenG acylase variant with a mutation at position β24 (Fβ24A), whereby phenylalanine is replaced by alanine, appears to produce a significantly higher yield in the synthesis of penicillins and cephalosporins. However, in WO03/055998 it was shown that in processes where, instead of an ester precursor, an amide precursor, is used in combination with said mutant Fβ24A, the S/H ratio is still high, but the enzymatic activity is so low that the use of this mutant is economically much less attractive. Instead, it was shown in WO03/055998 that a PenG acylase mutant wherein arginine at position 145 in the α-subunit was replaced by leucine (Rα145L), cystein (Rα145C) or lysine (Rα145K), also showed an improved S/H ratio but, in addition, had retained a higher level of synthetic activity, especially with amide precursors. Nevertheless, the synthetic activity of all these mutants was less the synthetic activity of the wild-type PenG acylase.
CN101177688 disclosed that also of mutants of the Bacillus megaterium PenG acylase, an improvement of the S/H ratio was accompanied by a decrease of the synthetic activity.
EP-1418230 to TUHH-Technologie GmbH, discloses Alcaligenes faecalis PenG acylases for which the post-translational maturation of the α-subunit is incomplete resulting in a higher hydrolytic activity for penicillin G and 6-nitro-3-phenylacetamide benzoic acid (further referred to as NIPAB). Incomplete processing of said α-subunit was invoked by amino-acid substitutions in the so-called linker region between α and β subunit. It was not described whether or not such mutations could also increase the synthetic activity.
The prior art discussed above shows that, although it is possible to increase the S/H ratio of various mutants of PenG acylase, such improvements in the S/H ratio are accompanied by a decrease of the synthetic activity compared to the wild type PenG acylase. Therefore, the disadvantage of these mutants is that long conversion times are needed or very high concentration of mutant PenG acylases in such conversion, which makes industrial applications of such mutants economically unattractive if not impossible.
It is the purpose of the present invention to provide mutant PenG acylases which have increased S/H ratio's while maintaining or more preferably increasing the synthetic activity compared to the wild type enzyme in order to be suitable for industrial processes.