Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports.
In this work, we have used supports activated with m-amino-phenylboronic groups to "reversibly" immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.
Torres
Rodrigo
R
Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Pessela
Benevides
B
Fuentes
Manuel
M
Munilla
Roberto
R
Mateo
Cesar
C
Fernández-Lafuente
Roberto
R
Guisán
José M
JM
D001426:Bacterial Proteins / Q000737:chemistry*; D001897:Boronic Acids / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D008353:Mannitol / Q000737:chemistry; D010399:Penicillin Amidase / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D001426:Bacterial Proteins; D001897:Boronic Acids; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011108:Polymers; D012967:Sodium Dodecyl Sulfate; D008353:Mannitol; C065843:Eupergit C; D010399:Penicillin Amidase; C010686:benzeneboronic acid