Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports.
The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.
Grazú
Valeria
V
Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM Cantoblanco, Madrid, Spain.
Abian
Olga
O
Mateo
Cesar
C
Batista-Viera
Franciso
F
Fernández-Lafuente
Roberto
R
Guisán
José Manuel
JM
D001665:Binding Sites; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004852:Epoxy Compounds / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D008049:Lipase / Q000737:chemistry*; D008422:Materials Testing; D010399:Penicillin Amidase / Q000737:chemistry*; D011485:Protein Binding; D020103:Rhizomucor / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D013438:Sulfhydryl Compounds; D008049:Lipase; D010399:Penicillin Amidase