1070-4272/01/7405-0897$25.00C2001 MAIK [Nauka/Interperiodica]
Russian Journal of Applied Chemistry, Vol. 74, No. 5, 2001, pp. 897!899. Translated from Zhurnal Prikladnoi Khimii, Vol. 74, No. 5,
2001, pp. 869!871.
Original Russian Text Copyright + 2001 by Shkutina, Stoyanova, Selemenev.
Sorption Immobilization of Glucoamylase
on Nonionic Sorbent Styrosorb
I. V. Shkutina, O. F. Stoyanova, and V. F. Selemenev
Voronezh State University, Voronezh, Russia
Received January 30, 2001
Abstract-Glucoamylase was immobilized on super-cross-linked nonionic sorbent Styrosorb by the sorption
procedure. The features of native and immobilized glucoamylase are compared.
Glucoamylase catalyzes hydrolytic cleavage of
starch. This enzymatic reaction is widely used in
alcohol industry and in glucose production. Immobi-
lized enzymes are characterized by several advantages
as compared to native enzymes: possibility of repeated
and continuous use, stability to denaturing agents, and
simplicity of preparation of high-purity product .
In this work was studied the possibility of sorption
immobilization of glucoamylase on super-cross-linked
nonionic sorbent Styrosorb used as the efficient sor-
bent of large biomolecules .
In our experiments we used the enzymic prepara-
tion of glucoamylase precipitated with ethanol from
an extract of the surface culture Aspergillus awamori.
Two samples of Styrosorb (Styrosorb-1 with a specific
surface area of 440 m
and Styrosorb-2 with a
specific surface area of 260 m
) were used as the
supports. These sorbents containing no ion-exchange
groups were prepared from super-cross-linked poly-
styrene. Their structure contains certain amount of
hydrophobic pockets capable of binding large organic
molecules . Glucoamylase was immobilized by
sorption from its solution in a 0.1 M acetate buffer
under static conditions at 18320oC for 24 h. The en-
zyme amount associated with the support was deter-
mined as the difference between the protein amount in
the solution before and after sorption. The concentra-
tion of immobilized protein was expressed as mg of
protein per 1 g of the support. The protein concentra-
tion in solution was determined by the Lowry method
, and the glucoamylase activity, by the glucoseoxi-
dase method . The enzyme amount producing
1 mmol of glucose per minute in catalytic hydrolysis
of starch at 30oC was accepted as the unit of the
Glucoamylase molecule consists of the amino acid
units which, depending on pH, form various ionic
species. Therefore, the hydrogen ion concentration
in the enzyme solution can significantly affect the
enzyme sorption. We found that the maximal amount
of the protein is sorbed at pH 4.735.0, i.e., at the iso-
electric point of glucoamylase in the solution (Fig. 1).
Figure 2 shows that the curves of glucoamylase
sorption on Styrosorb-1 and Styrosorb-2 within the
pH range of the maximal sorption rate differ in shape.
In passing from Styrosorb-2 to Styrosorb-1 having a
larger specific surface area the sorption curve becomes
S-shaped, indicating the change in the sorption mech-
anism. In the first stage of sorption the monomolecu-
lar layer of glucoamylase is formed owing to the
sorbent3sorbate interactions. Further sorption of en-
zyme occurs by the mechanism of the sorbate3sorbate
interactions. Due to intermolecular association of the
enzyme molecules, a polymolecular layers on the sor-
Fig. 1. Dependence of glucoamylase sorption q on pH
of the equilibrium solution. (1) Styrosorb-1 and (2) Styro-
sorb-2; the same for Figs. 2 and 3.