Penicillin Acylase Biocatalysts

Enzymes are labile catalysts that require to be stabilized under operating conditions. Penicillin acylase is a moderately expensive enzyme so that its efficient 

5 Synthesis of -Lactam Antibiotics in Homogeneous and Heterogeneous Aqueous and Organic Media 

KCS is the best technological option for the enzymatic synthesis of SS pLA conversion yield is not limited by the equilibrium of the reaction and high productivities 

Ampicillin Eupergit IPA SoUd-state 70 300 Youshko and Svedas (2002) 

5 (g/g) respectively for penicillin acylase CLEA (Illanes et al. 2007a). These results are encouraging and there is still room for further improvement to make biocatalysis of SS 3LA competitive. Actually, better results have been recently obtained with PGA-450 in a fiilly aqueous medium under a green chemistry concept specific productivity was 7.6 mmol/h/g at almost stoichiometric conversion yield (Illanes et al. 2007b). 

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Ospina S, Lopez-Munguia A, Gonzalez R et til. (1992) Characterization tmd use of a penicillin acylase biocatalyst. J Chem Technol Biotechnol 53 205-214 Pickering TJ, Garfoith S, Sayers JR et til. (1999) Variation in the steady state kinetic paiameters of wild type and mutant T5 5 -3 -exonuclease with pH. J Biol Chem 274 17711-17717 Rooney JJ (1995) Eyring transition-state theory and kinetics in catalysis. J Mol Catal A Chemical 96(1) 1-3... 

Novella IS, Fargues C, GreviUot G (1994) Improvement of extraction of penicillin acylase by a combined use of chemical methods. Biotechnol Bioeng 44 379-382 Ospina S, Lopez-Mungufa A, GonztQez R et al. (1992) Characterization and use of a penicillin acylase biocatalyst. J. Chem Technol Biotechnol 53 205-214 Ospina SS, Merino E, Ramirez OT et til. (1995) Recombinant whole cell penicillin acylase biocatalyst production, characterization and use in the synthesis and hydrolysis of antibiotics. Biotechnol Lett 17 615-620... 

Chong, A.S.M. and Zhao, X.S. (2004) Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst. Catalysis Today, 93-95, 293-299. 

Ivanov, A.E., Edink, E., Kumar, A., Galaev I.Y., Arendsen, A.F., Bruggink, A. and Mattiasson, B. (2003) Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide a step toward a thermosensitive industrial biocatalyst. Biotechnol. Prog., 19, 1167. 

In the present enzymatic process, 5 ton 6-APA is obtained using immobilized penicillin acylase (5-10 kg) as the biocatalyst in 100 ton of an aqueous solution containing 10 ton Penicillin G at ambient conditions. During the enzymatic hydrolysis the pH is kept constant by the continuous addition of ammonia (500 kg). In addition, the phenylacetic acid formed upon the hydrolysis of 6-APA can be recycled for use in the Penicillin G fermentation process. 

An example of a commercial process that uses a hydrolase in the hydrolytic mode is the production of the antibiotic intermediate 6-APA by Gist-brocades [13], discussed above in Section 7.2.1 (Fig. 7.1). The biocatalyst is a penicillin acylase, which generates 6-APA from the starting material penicillin G in one step at a moderate temperature in water. The atom utilization of the enzymic process is much higher than that of the corresponding chemical process [14]. 

In spite of usefulness of the simplification obtained by decreasing the experimental substrate concentration, many studies are aimed at the investigation of kinetic properties of immobilized biocatalysts within broader concentration ranges. In a previous paper [29], cells of Escherichia coli with penicillin acylase activity were immobilized by entrapment in calcium pectate gel and tested on the transformation of penicillin G to 6-amino penicillanic acid. Figure 9 shows experimental data from a microcalorimetric investigation of the penicillin G transformation in steady state. As appreciable particle-mass transfer was expected, the mathematical model that includes particle-mass balance was used. 

Another type of stability of immobilized biocatalysts is the retention of activity after periodic use in batch processes, as has been reported previously for penicillin acylase entrapped in polyacrylamide gel [40]. This option can be used to advantage for rapid monitoring of biocatalyst activity under conditions of industrial application. Apart from the measurement of activity as an indication of the necessity to replace the biocatalyst, the periodic analysis of the variation of kinetic properties permits greater insight into deviation from the optimal parameters. 

Illanes A, Altamirano C, Zuniga ME (1996) Thermal inactivation of penicillin acylase in the presence of substrate and products. Biotechnol Bioeng 50 609-616 Illanes A, Altamirano C, AUlapan A et al. (1998a) Packed-bed reactor performance with immobilised lactase under thermal inactivation. Enzyme Microb Technol 23 3-9 Illanes A, Wilson L, Altamirano C et al. (1998b) Reactor performance under thermal inactivation and temperature optimization with chitm-immobilized lactase. In Ballesteros A, Plou F, Iborra JL et al. (eds). Stability and stabilization of biocatalysts. Elsevier, Amsterdam, pp 27-34 Illanes A (1999) Stability of biocatalysts. Elect J Biotechnol 2 1-9... 

Cao L, van Rantwijk F, Sheldon RA (2000) Cross-linked enzymes aggregates a simple and effective method for the immobilization of penicillin acylase. Org Lett 2 1361-1364 Cao L, van Langen LM, van Rantwijk F et al. (2001) Cross-linked aggregates of penicillin acylase robust biocatalysts for the synthesis of P-lactam antibiotics. 1 Mol Catal B Enzym 11 665-670 Cao L, van Langen LM, Sheldon RA (2003) Immobilised enzymes carrier-bound or carrier-free Curr Opin Biotechnol 14 1-8... 

Kurochkina VB, Nys PS (2002) Kinetic and thermodynamic approach to design of processes for enzymatic synthesis of betalactams. Biocatal Biotransform 20(1) 35-41 Lee SB, Ryu DDY (1982) Reaction kinetics and mechanism of penicillin amidase a comparative study of computer simulation. Enzyme Microb Technol 4 35-38 Lin WJ, Kuo BY, Chou CP (2001) A biochemical engineering approach for enhancing production of recombinant penicillin acylase in Escherichia coli. Bioproc Biosys Eng 24 239-247 Lindsay JP, Clark DS, Dordick JS (2004) Combinatorial formulation of biocatalyst preparation for increased activity in organic solvents salt activation of penidllin amidase. Biotechnol Bioeng 85(5) 553-560... 

Biocatalysts that are reversibly soluble as a function of pH have been obtained by the covalent coupling of lysozyme to alginate (113) of trypsin to poly(acrolein-co-acrylic acid) (114) and of cellulase (115), amylase (115) a-chymotrypsin, and papain (116) to poly(methyl methacrylate-co-methacrylic acid). A reversibly soluble cofactor has been produced by the covalent binding of NAD to alginate (117). Reversibly soluble a-chymotrypsin, penicillin acylase, and alcohol dehydrogenase were produced by coupling to the polycation component of polyelectrolyte complexes formed by poly(methacrylic acid) and poly(iY-ethyl-4-vinyl-pyridinium bromide) (118). 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

The microbial sources of penicillin amidases/acylases required for side-chain removal were found and were quickly commercialised as whole-cell biocatalysts. 

Enantioselective enzymatic amide hydrolyses can also be applied for the preparation of optically active organosilicon compounds. The first example of this is the kinetic resolution of the racemic [l-(phenylacetamido)ethyl] silane rac-84 using immobilized penicillin G acylase (PGA E.C. 3.5.1.11) from Escherichia coli as the biocatalyst (Scheme 18)69. (R)-selective hydrolysis of rac-84 yielded the corresponding (l-aminoethyl)silane (R)-85 which was obtained on a preparative scale in 40% yield (relative to rac-84). The enantiomeric purity of the biotransformation product was 92% ee. This method has not yet been used for the synthesis of optically active silicon compounds with the silicon atom as the center of chirality. 

Immobilized forms of penicillin amidases and acylases have replaced whole-cell biocatalysts for the production of 6-APA and 7-ACA as they can be reused many times, in some cases for over 1000 cycles. Another major advantage is the purity of the enzyme, lacking the /3-lactamase contaminants often present in whole cells. The productivity of these biocatalysts exceeds 2000 kg prod-uct/kg catalyst. A typical process for the production of 6-APA employs immobilized penicillin G acylase covalently attached to a macroporous resin. The process can be run in either batch or continuous modes. The pH of the reaction must be maintained at a value between 7.5 and 8 and requires continuous adjustment to compensate for the drop caused by the phenylacetic acid generated during the course of the reaction. Recycle reactors have been used, as they allow both pH control and the use of packed bed reactors containing the immobilized catalyst. The enzymatic process is cheaper, although not... 

Wilson L, Illanes A, Pessela CB et al. (2004a) Encapsulation of crossUnked penicillin G acylase aggregates in Lentikats evaluation of a novel biocatalyst in organic media. Biotechnol Bioeng 86(5) 558-562... 

Wilson L, Illanes A, Abian O et al. (2004a) Co-aggregation of penicillin G acylase and polyiomc polymers an easy methodology to prepare enzyme biocatalysts stable in organic media. Biomacromolecules 5 852-857... 

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