Immobilization-stabilization of penicillin G acylase from Escherichia coli.
We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.
Alvaro
G
G
Instituto de Catálisis, C.S.I.C., Madrid, Spain.
Fernandez-Lafuente
R
R
Blanco
R M
RM
Guisán
J M
JM
D000447:Aldehydes; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D000432:Methanol; D010399:Penicillin Amidase / Q000378:metabolism*; D012685:Sepharose
D000447:Aldehydes; D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D012685:Sepharose; D010399:Penicillin Amidase; D000432:Methanol