Immobilization-stabilization of Penicillin G acylase fromEscherichia coli

Abstract

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.

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports

“…Immobilized-stabilized glyoxyl agarose/ penicillin G acylase derivatives were prepared as previously described (Alvaro et al, 1991), using a penicillin G acylase extract from E. coli donated by Antibioticos S.A. (Leon, Spain) with a 25% purity. We immobilized 210 IU (measured in hydrolysis of penicillin G at 37°C and pH 8), completely preserving the enzymatic activity and increasing the enzyme stability by 1,000-fold (Alvaro et al, 1991).…”

“…Immobilized-stabilized glyoxyl agarose/ penicillin G acylase derivatives were prepared as previously described (Alvaro et al, 1991), using a penicillin G acylase extract from E. coli donated by Antibioticos S.A. (Leon, Spain) with a 25% purity. We immobilized 210 IU (measured in hydrolysis of penicillin G at 37°C and pH 8), completely preserving the enzymatic activity and increasing the enzyme stability by 1,000-fold (Alvaro et al, 1991). Cephalexin, 7-aminodesacetoxicephalosporanic acid (7-ADCA), and phenylglycine methyl ester were also kindly donated by Antibioticos S.A. Polyethylene glycols and dextrans were from Sigma Chemical Co. (St. Louis, MO), and polyethylene glycol 10,000 was from Pharmacia Biotech (Uppsala, Sweden).…”

Improvement of Catalytic Properties of Escherichia coli Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag

“…Wild-type and modified PGA were immobilized on aldehyde agarose (glyoxyl agarose) by loading 0.7 to 1 mg of protein per gram of support and subsequently compared with a commercial acylase from E. coli immobilized by the same procedure (5,12). 3G3KPGA was completely immobilized under standard conditions (pH 10 and 25°C), but the resulting derivative turned out to be inactive due to low stability (Fig.…”

Improved Stabilization of Genetically Modified Penicillin G Acylase in the Presence of Organic Cosolvents by Co‐ Immobilization of the Enzyme with Polyethyleneimine

“…This support has been chosen because it has allowed one to stabilize many enzymes via multipoint covalent attachment [33] and has been specifically used to stabilize PGA. [34][35][36] The immobilization of proteins on this support is based on its reaction with primary amino groups of the enzyme. [37] Therefore, the modifications of Asn and Gln on the protein surface may not reduce the reactivity of PGA with glyoxyl-agarose.…”