Genetic modification of the penicillin G acylase surface to improve its reversible immobilization on ionic exchangers.
A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.
Montes
Tamara
T
Instituto de Catálisis, CSIC, Campus Universidad Autónoma, 28049 Madrid, Spain.
Grazú
Valeria
V
López-Gallego
Fernando
F
Hermoso
Juan A
JA
García
Jose L
JL
Manso
Isabel
I
Galán
Beatriz
B
González
Ramón
R
Fernández-Lafuente
Roberto
R
Guisán
José M
JM
D001709:Biotechnology / Q000379:methods; D002411:Cation Exchange Resins; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*
D002411:Cation Exchange Resins; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D010399:Penicillin Amidase