Kinetics of beta-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization.

Competition with well-established, fine-tuned chemical processes is a major challenge for the industrial implementation of the enzymatic synthesis of beta-lactam antibiotics. Enzyme-based routes are acknowledged as an environmental-friendly approach, avoiding organochloride solvents and working at room temperatures. Among different alternatives, the kinetically controlled synthesis, using immobilized penicillin G acylase (PGA) in aqueous environment, with the simultaneous crystallization of the product, is the most promising one. However, PGA may act either as a transferase or as a hydrolase, catalyzing two undesired side reactions: the hydrolysis of the acyl side-chain precursor (an ester or amide, a parallel reaction) and the hydrolysis of the antibiotic itself (a consecutive reaction). This review focuses specially on aspects of the reactions' kinetics that may affect the performance of the enzymatic reactor.

[1]  A. Illanes,et al.  Synthesis of cephalexin in ethylene glycol with glyoxyl-agarose immobilised penicillin acylase: temperature and pH optimisation , 2003 .

[2]  C G P H Schroën,et al.  Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin. , 2002, Biotechnology and bioengineering.

[3]  V. Kasche.  Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of β-lactam antibiotics, peptides and other condensation products , 1986 .

[4]  R. Fernández-Lafuente,et al.  Influence of the enzyme derivative preparation and substrate structure on the enantioselectivity of penicillin G acylase , 2002 .

[5]  H. Schoemaker,et al.  Application of Enzymes in Industrial Organic Synthesis , 1997, CHIMIA.

[6]  D. Janssen,et al.  Identification of the Catalytic Residues of α-Amino Acid Ester Hydrolase from Acetobacter turbidans by Labeling and Site-directed Mutagenesis* , 2002, The Journal of Biological Chemistry.

[7]  A. Böck,et al.  The penicillin acylase from Escherichia coli ATCC11105 consists of two dissimilar subunits , 1983 .

[8]  Anja E.M. Janssen,et al.  Cephalexin synthesis by immobilised penicillin G acylase under non-isothermal conditions: reduction of diffusion limitation , 2001 .

[9]  J Tramper,et al.  Modeling of the enzymatic kinetic synthesis of cephalexin--influence of substrate concentration and temperature. , 2001, Biotechnology and bioengineering.

[10]  V. Svedas,et al.  Kinetics of Ampicillin Synthesis Catalyzed by Penicillin Acylase from E. coli in Homogeneous and Heterogeneous Systems. Quantitative Characterization of Nucleophile Reactivity and Mathematical Modeling of the Process , 2000, Biochemistry (Moscow).

[11]  Anja E.M. Janssen,et al.  Thermodynamically controlled synthesis of β-lactam antibiotics. Equilibrium concentrations and side-chain properties , 1999 .

[12]  R. Fernández-Lafuente,et al.  Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions. , 2001, Biomacromolecules.

[13]  R. Sheldon,et al.  Cross-linked aggregates of penicillin acylase: robust catalysts for the synthesis of β-lactam antibiotics , 2001 .

[14]  M. V. Savchenko,et al.  Enantioselective Penicillin Acylase—catalyzed Reactions , 1996 .

[15]  R. Fernández-Lafuente,et al.  Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports. , 1998, Biotechnology and bioengineering.

[16]  D. Janssen,et al.  Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase. , 2004, Protein engineering, design & selection : PEDS.

[17]  J. J. Heijnen,et al.  Feasibility of the thermodynamically controlled synthesis of amoxicillin , 1998 .

[18]  R. Fernández-Lafuente,et al.  The presence of methanol exerts a strong and complex modulation of the synthesis of different antibiotics by immobilized penicillin G acylase , 1998 .

[19]  O. Abián,et al.  Co-aggregation of penicillin g acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media. , 2004, Biomacromolecules.

[20]  D. Janssen,et al.  Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding an α-Amino Acid Ester Hydrolase from Acetobacter turbidans , 2002, Applied and Environmental Microbiology.

[21]  L. Riechmann,et al.  Equilibrium and kinetically controlled synthesis with enzymes: semisynthesis of penicillins and peptides. , 1987, Methods in enzymology.

[22]  A. Nurk,et al.  Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli. , 1999, Biochimica et biophysica acta.

[23]  R E Hubbard,et al.  Ligand-induced conformational change in penicillin acylase. , 1998, Journal of molecular biology.

[24]  Johannes Tramper,et al.  Process Design for Enzymatic Adipyl‐7‐ADCA Hydrolysis , 2002, Biotechnology progress (Print).

[25]  D. Janssen,et al.  Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies. , 2000, Protein engineering.

[26]  E. Bárzana,et al.  Effect of pH in the synthesis of ampicillin by penicillin acylase , 1996 .

[27]  R. Elander.  Industrial production of β-lactam antibiotics , 2003, Applied Microbiology and Biotechnology.

[28]  A G Murzin,et al.  SCOP: a structural classification of proteins database for the investigation of sequences and structures. , 1995, Journal of molecular biology.

[29]  F. Bolivar,et al.  The role of penicillin amidases in nature and in industry. , 1991, Trends in biochemical sciences.

[30]  S. Rossi,et al.  Enzyme Reaction Engineering: Effect of Methanol on the Synthesis of Antibiotics Catalyzed by Immobilized Penicillin G Acylase under Isothermal and Non‐Isothermal Conditions , 2002, Biotechnology progress.

[31]  R. Fernández-Lafuente,et al.  Synthesis of antibiotics (cephaloglycin) catalyzed by penicillin G acylase: Evaluation and optimization of different synthetic approaches , 1996 .

[32]  M. Cole.  Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli. , 1969, The Biochemical journal.

[33]  V. Kasche,et al.  Enzyme catalyzed biotransformations in aqueous two‐phase systems with precipitated substrate and/or product , 1995, Biotechnology and bioengineering.

[34]  K. Wilson,et al.  Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism. , 2001, Journal of molecular biology.

[35]  C G P H Schroën,et al.  Modelling of the enzymatic kinetically controlled synthesis of cephalexin: influence of diffusion limitation. , 2002, Biotechnology and bioengineering.

[36]  R. Fernández-Lafuente,et al.  Enzyme reaction engineering: synthesis of antibiotics catalysed by stabilized penicillin G acylase in the presence of organic cosolvents. , 1991, Enzyme and microbial technology.

[37]  R. Sheldon,et al.  Penicillin acylase-catalyzed ampicillin synthesis using a pH gradient: a new approach to optimization. , 2002, Biotechnology and bioengineering.

[38]  Eleanor J. Dodson,et al.  Penicillin acylase has a single-amino-acid catalytic centre , 1996, Nature.

[39]  A. Murzin,et al.  A protein catalytic framework with an N-terminal nucleophile is capable of self-activation , 1995, Nature.

[40]  O. Abián,et al.  Thermodynamically Controlled Synthesis of Amide Bonds Catalyzed by Highly Organic Solvent‐Resistant Penicillin Acylase Derivatives , 2008, Biotechnology progress.

[41]  R. Sheldon,et al.  Enzymatic synthesis of β‐lactam antibiotics using penicillin‐G acylase in frozen media , 1999, FEBS letters.

[42]  R. Giordano,et al.  Improving selectivity and productivity of the enzymatic synthesis of ampicillin with immobilized penicillin G acylase , 2004 .

[43]  Ruy Sousa,et al.  Enzymatic synthesis of amoxicillin: avoiding limitations of the mechanistic approach for reaction kinetics. , 2002, Biotechnology and bioengineering.

[44]  M. Sastry,et al.  Penicillin G Acylase‐Fatty Lipid Biocomposite Films Show Excellent Catalytic Activity and Long Term Stability/Reusability , 2002, Biotechnology progress.

[45]  A. Bruggink.  Synthesis of β-Lactam Antibiotics , 2001 .

[46]  A. Straathof,et al.  MODELLING THE ENZYMATIC DEACYLATION OF PENICILLIN G: EQUILIBRIUM AND KINETIC CONSIDERATIONS , 1997 .

[47]  D. Janssen,et al.  Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of beta-lactam antibiotics. , 2003, European journal of biochemistry.

[48]  T. Earnest,et al.  Precursor Structure of Cephalosporin Acylase: Insights into Auto-Proteolytic Activation in a New N-Terminal Hydrolase Family , 2002, The Journal of Biological Chemistry.

[49]  O. Abián,et al.  Preparation of artificial hyper-hydrophilic micro-environments (polymeric salts) surrounding enzyme molecules: New enzyme derivatives to be used in any reaction medium , 2002 .

[50]  Min-Gon Kim,et al.  Effect of organic solvents on penicillin acylase-catalyzed reactions: interaction of organic solvents with enzymes , 1996 .

[51]  R. Sheldon,et al.  Biocatalysts and Biocatalysis in the Synthesis of ß-Lactam Antibiotics , 2001 .

[52]  J. García,et al.  Stabilization of Penicillin G Acylase from Escherichia coli: Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment , 2004, Applied and Environmental Microbiology.

[53]  R. Sheldon,et al.  Penicillin acylase-catalyzed synthesis of ampicillin in "aqueous solution-precipitate" systems. High substrate concentration and supersaturation effect , 2000 .

[54]  H. Ertan,et al.  STABILIZATION OF PENICILLIN G ACYLASE AGAINST PH BY CHEMICAL CROSS-LINKING , 1996 .

[55]  R. Giordano,et al.  Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates , 2005 .

[56]  A. Margolin,et al.  Substrate specificity of penicillin amidase from E. coli. , 1980, Biochimica et biophysica acta.

[57]  D. Janssen,et al.  The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase. , 2002, European journal of biochemistry.

[58]  D. G. Mita,et al.  Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model. , 2002, Biotechnology and bioengineering.

[59]  D. Janssen,et al.  The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases* , 2003, Journal of Biological Chemistry.

[60]  Heijnen,et al.  Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction. , 2000, Enzyme and microbial technology.

[61]  P. Gunasekaran,et al.  Recent biotechnological interventions for developing improved penicillin G acylases. , 2004, Journal of bioscience and bioengineering.

[62]  Dohyun Han,et al.  A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase* , 2004, Journal of Biological Chemistry.

[63]  R. Fernández-Lafuente,et al.  Dynamic reaction design of enzymic biotransformations in organic media: equilibrium‐controlled synthesis of antibiotics by penicillin G acylase , 1996, Biotechnology and applied biochemistry.

[64]  A. Blinkovsky,et al.  Synthesis of β-lactam antibiotics containing α-aminophenylacetyl group in the acyl moiety catalyzed by d-(—)-Phenylglycyl-β-lactamide amidohydrolase , 1993 .

[65]  O. Abián,et al.  Stabilization of Immobilized Enzymes Against Water-Soluble Organic Cosolvents and Generation of Hyper-Hydrophilic Micro-Environments Surrounding Enzyme Molecules , 2001 .

[66]  M. Arroyo,et al.  Biotechnological applications of penicillin acylases: state-of-the-art , 2002, Applied Microbiology and Biotechnology.

[67]  J. Guisán.  Aldehyde-agarose gels as activated supports for immobilization-stabilization of enzymes , 1988 .

[68]  Dongzhi Wei,et al.  Enzymatic synthesis of cephalexin in aqueous two-phase systems , 2002 .

[69]  R. Fernández-Lafuente,et al.  Stabilization of a tetrameric enzyme (α-amino acid ester hydrolase from Acetobacter turbidans) enables a very improved performance of ampicillin synthesis , 2001 .

[70]  O. Abián,et al.  Encapsulation of crosslinked penicillin G acylase aggregates in lentikats: Evaluation of a novel biocatalyst in organic media , 2004, Biotechnology and bioengineering.

[71]  W. Quax,et al.  Improved beta-lactam acylases and their use as industrial biocatalysts. , 2004, Current opinion in biotechnology.

[72]  A. Illanes,et al.  Optimization of cephalexin synthesis with immobilized penicillin acylase in ethylene glycol medium at low temperatures , 2004 .

[73]  K. Wilson,et al.  Structural studies of penicillin acylase , 2000 .

[74]  R. Sheldon,et al.  Highly efficient synthesis of ampicillin in an "aqueous solution-precipitate" system: repetitive addition of substrates in a semicontinuous process. , 2001, Biotechnology and bioengineering.

[75]  M. A. Prieto,et al.  The PaaX Repressor, a Link between Penicillin G Acylase and the Phenylacetyl-Coenzyme A Catabolon of Escherichia coli W , 2004, Journal of bacteriology.

[76]  J Hinrichs,et al.  pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis. , 1999, Biotechnology and bioengineering.

[77]  J. Kennedy,et al.  Advances in enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA). , 2000, Biotechnology advances.

[78]  D. Kazan,et al.  Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose , 2002 .

[79]  R. Sheldon,et al.  Towards Biocatalytic Synthesis of β-Lactam Antibiotics , 2001 .