Enzymes - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 358

TABLE 8.5 Effects of Immobilization Methods on the Retention of Enzymatic Activity of Aminoacylase

Observed
activity, units

Immobilized
enzyme activity, % Support Method

Polyacrylamide Entrapment 526 52.6

Nylon Encapsulation 360 36.0

DEAE-cellulose Ionic binding 668 55.2

DEAE-Spephadex A-50 Ionic binding 680 56.2

CM-Sephadex C-50 Ionic binding 0 0

Iodoacetyl cellulose Covalent binding 472 39.0

CNBr-activated Sephadex Covalent binding 12 1.0

AE-cellulose Cross-linked with glutaraldehyde 8 0.6

Source: Chibata I., Tosa T., Sato T., Mori T. and Matuo Y., Proc. of the 4th Int. Fermentation Symp.: Fermentation Technology Today, 1972,
p. 383 e 389.

material are (1) the binding capacity of the support material, which is a function of charge
density, functional groups, porosity, and hydrophobicity of the support surface, and (2)
stability and retention of enzymatic activity, which is a function of functional groups on
support material and microenvironmental conditions. If immobilization causes some confor-
mational changes on the enzyme, or if reactive groups on the active site of the enzyme are
involved in binding, a loss in enzyme activity can take place upon immobilization.
Usually, immobilization results in a loss in enzyme activity and stability. However, in some
cases, immobilizationmay cause an increase in enzyme activity and stability due tomore favor-
ablemicroenvironmental conditions. Because enzymes often havemore than one functional site
that can bind the surface, an immobilized enzyme preparation may be very heterogeneous.
Evenwhen binding does not alter enzyme structure, some enzyme can be boundwith the active
site oriented away from the substrate solution and toward the support surface, decreasing the
access of the substrate to the enzyme. Retention of activity varies with the method used. Table
8.5 summarizes the retention of activity of arninoacylase immobilized by different methods.

8.3.2. Electrostatic and Steric Effects in Immobilized Enzyme Systems
When enzymes are immobilized in a charged matrix as a result of a change in the micro-
environment of the enzyme, the apparent bulk pH optimum of the immobilized enzyme will
shift from that of soluble enzyme. The charged matrix will repel or attract substrates,
product, cofactors, and H þ depending on the type and quantity of surface charge. For an
enzyme immobilized onto a charged support, the shift in the pH-activity profile is given by

zN F J
RT

D ¼

pH i pH e ¼

(8.100)

pH

where pH i and pH e are internal and external pH values, respectively; z is the charge
(valence) on the substrate; N F is the Faraday constant (96,500 C/eq. g); is the electrostatic

potential; and R is the gas constant. Expressions similar to Eqn (8.100) apply to other

J