Reversible immobilization of glutaryl acylase on sepabeads coated with polyethyleneimine.
The immobilizaton of the enzyme glutaryl-7-aminocephalosporanic acid acylase (GA) was performed via ionic adsorption onto several supports: a new anionic exchange resin, based on the coating of Sepabeads internal surfaces with polyethyleneimine (PEI) of different molecular weights, and conventional EC-Q1A-Sepabeads and DEAE-agarose. Immobilization occurred very rapidly in all cases, but the adsorption strength was much higher in the case of PEI-Sepabeads than in the other supports at pH 7 (e.g., at 150 mM NaCl, 90% of the enzyme was eluted from the DEAE agarose and 15% was eluted from the EC-Q1A-Sepabeads, whereas no desorption was detected with the best PEI-Sepabeads). Interestingly, the adsorption strength of the GA was increased when it was immobilized on PEI-Sepabeads with higher molecular weights. For instance, enzyme desorption was detected from 75 mM NaCl for the derivative prepared onto Sepabeads coated with PEI 700 Da, whereas in the derivative prepared with the highest molecular weight PEI (600 000 Da) no enzyme desorption was detected below 150 mM NaCl. Optimal PEI-Sepabeads (prepared with PEI of 600 000 Da) was even much more thermostable than the covalent derivative prepared onto cyanogen bromide agarose. Moreover, this derivative presented a half-life 26-fold higher than that of the soluble enzyme at 45 degrees C, and the support could be reused 10 times after the full desorption of the enzyme without decreasing loading capacity.
Alonso-Morales
Noelia
N
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain.
López-Gallego
Fernando
F
Betancor
Lorena
L
Hidalgo
Aurelio
A
Mateo
Cesar
C
Fernández-Lafuente
Roberto
R
Guisán
Jose M
JM
D000327:Adsorption; D000837:Anion Exchange Resins / Q000737:chemistry; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000302:isolation & purification; D008422:Materials Testing; D008863:Microspheres; D010399:Penicillin Amidase / Q000737:chemistry* / Q000302:isolation & purification*; D011094:Polyethyleneimine / Q000737:chemistry*
D000837:Anion Exchange Resins; D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D011094:Polyethyleneimine; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase