Optimization of an industrial biocatalyst of glutaryl acylase: stabilization of the enzyme by multipoint covalent attachment onto new amino-epoxy Sepabeads.
Glutaryl-7-aminocephalosporanic acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 degrees C and 200 IU/g at 37 degrees C which makes it a biocatalyst of possible industrial application.
López-Gallego
Fernándo
F
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain.
Betancor
Lorena
L
Hidalgo
Aurelio
A
Mateo
Cesar
C
Guisán
Jose M
JM
Fernández-Lafuente
Roberto
R
D000327:Adsorption; D002384:Catalysis; D002617:Chemical Industry / Q000295:instrumentation*; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004853:Epoxy Resins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008422:Materials Testing; D008863:Microspheres; D010399:Penicillin Amidase / Q000737:chemistry*; D011485:Protein Binding; D013696:Temperature
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D004853:Epoxy Resins; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase