1 research outputs found
Improvement of enzyme properties with a two-step immobilization process on novel heterofunctional supports
Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of
groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing
proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable
to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was
developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are
complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were
incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the
glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared
with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number
of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on
heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For
example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were
20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from
Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times
more enantioselective than the corresponding monofunctional glyoxyl support derivativeThis work has been sponsored by the
Spanish Ministry of Science and Innovation (project AGL-2009-
07625) and Intramural projects (2008801058); and the Comunidad
AutĂłnoma de Madrid (project S0505/PPQ/03449). We
gratefully recognize to Spanish Ministry of Science and Innovation
for “Ramón y Cajal” contract to Dr. Fernandez-
LorentePeer reviewe