Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions.
The importance of the stabilization of the quaternary structure of multimeric enzymes has been illustrated using a model reaction with great industrial relevance: the enzymatic synthesis of ampicillin from 6-amino penicillanic acid (6APA) and phenylglycine methyl ester (PGM) catalyzed by the tetrameric enzyme alpha-amino acid ester hydrolase from Acetobacter turbidans. The stabilization of the multimeric structure of the enzyme was achieved by multi-subunit immobilization of the enzyme followed by its further solid-phase chemical intersubunit cross-linking with polyfunctional macromolecules (dextran-aldehyde). This stabilized derivative has permitted the study of the reaction under conditions where nonstabilized enzyme molecules tended to dissociate (e.g., absence of phosphate ions). Synthetic yields improved from around 65%, under conditions where the nonstabilized derivative was stable, to around 85% in conditions where only the stabilized derivative could be utilized (40% methanol and absence of phosphate ions). When using high concentrations of PGM, a significant worsening of the reaction performance was detected with a significant decrease in the yields (below 55%, using 50 mM 6APA and PGM). This problem has been sorted out by using a fed-batch reaction system. By addition of PGM continuously to the reaction mixture (to maintain the concentration between 0.5 and 3 mM), 95% of 6-APA could be transformed to antibiotic (47.5 mM) by only using a 20% excess of acylating ester.
Fernández-Lafuente
R
R
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autonoma, 28049 Madrid, Spain.
Hernández-Jústiz
O
O
Mateo
C
C
Terreni
M
M
Fernández-Lorente
G
G
Moreno
M A
MA
Alonso
J
J
García-López
J L
JL
Guisan
J M
JM
D000091:Acetobacter / Q000201:enzymology; D000667:Ampicillin / Q000378:metabolism*; D019149:Bioreactors; D002384:Catalysis; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000432:Methanol; D015394:Molecular Structure; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D010399:Penicillin Amidase / Q000737:chemistry* / Q000378:metabolism*; D010710:Phosphates; D020836:Protein Structure, Quaternary; D013696:Temperature
D004800:Enzymes, Immobilized; D010710:Phosphates; C033393:methyl phenylglycine; D000667:Ampicillin; D010397:Penicillanic Acid; C049057:ampicillin acylase; D010399:Penicillin Amidase; C004408:aminopenicillanic acid; D005998:Glycine; D000432:Methanol