Stabilization of a multimeric beta-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking.
This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable beta-galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-epoxy-IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized beta-galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long-term alkaline incubation, the enzyme immobilized on Sepabeads-epoxy-boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS-PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross-linking with aldehyde-dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 degrees C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.
Pessela
Benevides C C
BC
Department of Biocatalysis, Instituto de Catálisis (CSIC), Campus Universidad Autónoma, Cantoblanco 28049 Madrid, Spain.
Mateo
Cesar
C
Fuentes
Manuel
M
Vian
Alejandro
A
García
José L
JL
Carrascosa
Alfonso V
AV
Guisán
José M
JM
Fernández-Lafuente
Roberto
R
D000327:Adsorption; D000447:Aldehydes / Q000737:chemistry*; D003911:Dextrans / Q000737:chemistry*; D019281:Dimerization; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004852:Epoxy Compounds / Q000737:chemistry*; D006868:Hydrolysis; D007785:Lactose / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D013045:Species Specificity; D013824:Thermus / Q000145:classification / Q000201:enzymology*; D001616:beta-Galactosidase / Q000737:chemistry*
D000447:Aldehydes; D003911:Dextrans; D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D011108:Polymers; D001616:beta-Galactosidase; D007785:Lactose