ISSN 1070-4272. Russian Journal of Applied Chemistry, 2006, Vol. 79, No. 5, pp. 827 !832. + Pleiades Publishing, Inc., 2006.
Original Russian Text + I.Yu. Bakunina, O.I. Nedashkovskaya, T.N. Zvyagintseva, Yu.A. Shchipunov, 2006, published in Zhurnal Prikladnoi Khimii,
2006, Vol. 79, No. 5, pp. 839!844.
AND POLYMERIC MATERIALS
Immobilization of =-Galactosidase inside Hybrid Silica
Nanocomposites Containing Polysaccharides
I. Yu. Bakunina, O. I. Nedashkovskaya, T. N. Zvyagintseva, and Yu. A. Shchipunov
Pacific Ocean Institute of Bioorganic Chemistry, Vladivostok, Russia
Institute of Chemistry, Far-East Division, Russian Academy of Sciences, Vladivostok, Russia
Received July 12, 2005
Abstract-a-Galactosidase was encapsulated into sol3gel derived hybrid polysaccharide3silica nanocom-
posites. The main properties of the immobilized enzyme were studied.
Immobilization has long been successfully used for
storage and prolongation of action of enzymes, as well
as for development of artificial biocatalysts [1, 2].
Encapsulation into a matrix or immobilization on a
support allows repeated use of enzymes and facilitates
isolation of products of enzymatic reaction, which is
essential for development of biotechnological proc-
esses. An important advantage of immobilization is
the stabilization effect, i.e., significant prolongation
of the lifetime and enhancement of the stability of en-
zymes. Therefore, new efficient supports and matrices
for encapsulation of enzymes are constantly sought
a-Galactosidase (a-D-galactoside galactohydrolase,
EC 188.8.131.52) is a promising biochemical tool for
studying the antigene structure of various biological
objects, in particular, blood cells. Immobilization of
a-galactosidase was repeatedly attempted. For exam-
ple, enzyme from coffee beans was covalently bound
to dextran activated with cyanogen bromide .
a-Galactosidase from Pycnoporus cinnabarinus
was immobilized on chitin dispersions with glutaric
dialdehyde . Enzyme from Aspergillus oryzae was
immobilized on Eupergit C containing epoxy groups,
which provided covalent bonding of the protein by
amino, SH, and carboxy groups . a-Galactosidase
from the same source was placed into calcium alginate
gel, in which it was additionally immobilized with
glutaric dialdehyde . Also, this enzyme was im-
mobilized with polyacrylamide gel .
It should be noted that, in the cited studies, the en-
zyme was encapsulated via cross-linking. Immobiliza-
tion inside a matrix or on a support by covalent bond-
ing can adversely affect the enzyme through disturb-
ing the conformation of the protein macromolecule.
In some cases this is the main reason for partial or
complete denaturation of not very stable enzymes
[8, 9]. In the case of sol3gel technology, immobiliza-
tion involves no cross-linking agents. The protein
macromolecules are encapsulated into the inorganic
matrix formed in situ by polycondensation reactions.
The macromolecules undergo minimal structural
changes, and the enzymes can preserve their activity
. a-Galactosidase from Pseudoalteromonas sp.
KMM 701 sea bacterium can eliminate a-1,3-bound
galactose residues from the nonreducing end of gluco-
conjugates, blood group substances, and antigene de-
terminants for erythrocytes in B(III) blood group via
suppressing their serologic activity . a-Galactosi-
dase is a thermally labile enzyme. In concentrated
solutions at 20oC a-galactosidase remained active for
1 week, and at 30oC lost 50% activity within 10 min
. Dilution of concentrated solutions of the enzyme
accelerated its inactivation. A low thermal stability
and short lifetime of a-galactosidase in solution
hinder its biotechnological application.
Our preliminary studies showed that encapsulation
of a-galactosidase into polysaccharide3silica matrices
obtained by the method we suggested previously [11,
12] significantly prolonged the lifetime of the enzyme.
In this study we examined in more detail the condi-
tions for a-galactosidase immobilization inside new
hybrid nanocomposites and the properties of immobi-
lized a-galactosidase in order to select candidate nano-
composites for development of biocatalysts suitable
for biotechnological and biosensing applications.