Immobilized Enzyme Reactors

Enzymes are proteins that catalyze or enhance the rate of a chemical reaction.
Enzymes are very specific in terms of the substrate (reactant) that they act on.
Enzymes are usually named after the substrate whose reaction is catalyzed.
For example urease acts upon urea, heparinase acts on heparin.

For example collagenase:

E.C. 3.4.24.3
Allosteric interaction
 Vmax S
r
Km  S
 Initial rate of reaction
about equal to 2 mg/ml/min

Out of substrate

1 unit = liberates 1 mol of glucose per minute from starch at

pH = 4.8 and 60 oC
 Our goal now is to develop relationships that can be used to describe the rates or kinetics of
chemical reactions that are catalyzed by enzymes. Description of the enzyme reaction kinetics will

allow us to develop mathematical models for immobilized enzyme reactors that can be used to

analyze experimental data, provide information for scaleup of our devices, and explore the effects

of operating conditions on reactor performance. The heparinase reactor described above will be

used as a model reaction system for these discsussions.

The successful development of an immobilized enzyme reactor requires knowledge of the

enzyme kinetics, an understanding of the effects on the observed reaction rate of reactant or

substrate diffusion, and a design equation for the specific reactor that is used (Fogler, 1992). Each

of these is discussed in greater detail in the following discussion.

So we need to know:
1. Enzyme reaction kinetics
2. The effect of substrate diffusion on the observed reaction rate
3. A design equation for the particular reactor of interest
 Enzyme Kinetics
First we need to define the kinetics or rate of the enzyme reaction and how it depends on the
concentration of the substrate (reactant). This usually entails defining the free enzyme kinetics

(enzyme in solution and not on a solid support) and the kinetics after the enzyme has been

immobilized on the support material. In some cases immobilization has no effect on the intrinsic

activity of the enzyme, whereas sometimes immobilization can significantly alter the kinetics of the

conversion process.

Generally, the kinetics of enzyme reactions are described by the Michaelis-Menten

equation.

Vmax S
r
Km  S
E + S <--> ES ---> E + P
 The Michaelis-Menten equation can be derived by assuming that the conversion of substrate

to product occurs in two steps. In the first step, the substrate (S) combines with the enzyme (E) to
form an enzyme-substrate complex (E*S) as shown below

E  S  E *S (8.49)

In the second step, the enzyme-substrate complex (E*S) is converted into product (P) and free
enzyme (E) which is then available to recombine with substrate.

E*S  E + P (8.50)

The rate controlling step in the above process is assumed to be the conversion of the enzyme-
substrate complex to product. Accordingly, it is therefore assumed that the reaction forming the

enzyme-substrate complex is at equilibrium. The rate of the enzyme reaction is then given by

dS dP
rS     k cat E * S (8.51)
dt dt

where kcat is a first order rate constant that relates the reaction rate (r S) to the concentration of the

enzyme-substrate complex, ie. E*S. S and P are the respective concentrations of the substrate and
the product.
 From Equation 8.49 we can define the enzyme-substrate dissociation constant (reciprocal of
SE
the equilibrium constant) as Km = and then E*S = SE/Km with the result that Equation 8.51
E *S

becomes

dS dP SE
rS    k cat (8.52)
dt dt Km

Letting E0 represent the total concentration of the enzyme, then E0 must equal the sum of the free

enzyme concentration, ie. E, and the amount of enzyme bound to substrate, ie. E*S. Hence we can

write that E0 = E + E*S. Since E*S = SE/Km we then have that E = KmE0/(Km + S). Substituting
this result into Equation 8.52 then gives us an expression for the rate of the enzyme reaction as

shown by the next equation

k cat E 0S V S
rs   max (8.53)
Km  S Km  S
 The reaction rate, rS, has units of moles/(reaction volume)/time. S represents the substrate or

reactant concentration in units of moles/volume. Vmax represents the maximum reaction rate for a

given total enzyme concentration E0 [Units/(reaction volume)], where Vmax = kcat E0 , and kcat is the

reaction rate constant (moles/Units/time). Enzyme activity is commonly expressed in terms of

"Units" (U), and for heparinase, a unit of activity is defined as the amount of enzyme required to

degrade 1 mg of heparin/hr. The amount or mass of enzyme needed is dependent on the enzyme

purity and is usually reported as so many units of activity per mg of enzyme. Km is the Michaelis
constant and may be thought of as that substrate concentration at which the reaction rate is equal to
one-half the maximum rate (Vmax). It is important to note that at high substrate concentrations the

reaction rate saturates at Vmax because in total there are not enough available free enzyme
molecules for the substrate reaction. The reaction rate is then independent of the substrate

concentration and is therefore said to be zero order. At low substrate concentrations (S<<Km), the

reaction rate is linearly proportional to the substrate concentration and is therefore said to be first
order.
Finding Vmax and Km from Experimental Data

1  Km 1 1
   
rS  Vmax  S Vmax
Example 8.5 soluble heparinase kinetics
Note that it is convenient for this type of analysis to
divide the enzyme reaction rate by the corresponding enzyme concentration, thus

expressing the rate on a per unit amount of enzyme that is present in the reactor. One unit of

heparinase (1 U) is defined as the amount of enzyme required to degrade 1 mg heparin/hr.

Hence we can rewrite Equation 8.53 as follows

rS k S
R  cat
E0 Km  S

and this equation can then rearranged to give

1  Km  1 1
   
R  k cat  S k cat
Km = 0.078 mg heparin/ml of solution

Kcat = 0.891 mg heparin/U/hr

1 U = amount of enzyme needed to

degrade 1 mg heparin/hr
Enzyme Activity Strongly Dependent on the pH
Enzyme Activity Depends on Temperature

V = A exp(-E/RT)
Enzymes Also Denature if the Temperature is Too High

Good

Bad

T increasing
Even Fluid Shear Forces Can Deactivate the Enzyme
 Enzyme Immobilization

Enzyme immobiliziation offers several advantages. First, immobilization keeps the enzyme

out of the bulk solution, which in the case of blood returning to the body, could result in an allergic

reaction to the foreign protein of the enzyme. Secondly, immobilization offers the potential to reuse

the enzyme, which may in fact be quite expensive. Finally, in many cases, the enzyme is stabilized

(less labile) when immobilized, retaining its activity for longer periods of time.
Ways to Immobilize An Enzyme
 One example where immobilized enzyme reactors have been proposed is in the treatment of

neonatal jaundice (Lavin et al., 1985; Sung et al., 1986). Newborns tend to have higher levels of the

greenish-yellow pigment bilirubin than those found in adults. Bilirubin is a natural product derived

from red blood cells after they have lived out their lifespan of about 120 days. It is formed from the

heme portion (the four pyrole rings) of the hemoglobin molecule after removal of the iron. The

bilirubin binds to plasma albumin for transport to the liver where it is finally excreted from the

body in the bile fluids.

The fetus's bilirubin readily crosses the placenta and is removed by the mother's liver.

However, in the period after birth, the infant's liver is not fully functional for the first week,

resulting in increased levels of plasma bilirubin. In some cases, the infant's bilirubin levels are

sufficiently high resulting in a jaundiced (yellow) appearance to the skin. High levels of plasma
bilirubin can be toxic to a variety of tissues and in these cases jaundiced infants are commonly

treated by phototherapy or blood transfusions. In phototherapy, the infant is placed under a blue

light that converts the bilirubin to a less toxic byproduct. Phototherapy through the skin is not

capable of controlling cases of severe

jaundice. However, blood transfusions can replace the infant's blood with adult blood effectively

diluting the infant's plasma bilirubin levels. However, blood transfusions pose their own risk,

particularly infectious diseases such as hepatitis and HIV.

 An alternative approach to the treatment of neonatal jaundice is the use of a bilirubin

specific enzyme for removal of bilirubin from the infant's blood (Lavin et al., 1985; Sung et al.,
1986). The enzyme bilirubin oxidase catalyzes the oxidation of bilirubin according to the following
reaction stoichiometry.

bilirubin + 1/2 O2  biliverdin + H2O (8.48)

Calculations indicate that the amount of oxygen needed to convert all of the bilirubin found in the

blood is about 100 times less than the actual oxygen content of blood. Therefore, no external
supply of oxygen is needed within the enzyme reactor to carry out this reaction. Biliverdin itself is

much less toxic than bilirubin and in fact is further oxidized by bilirubin oxidase to other less toxic

substances.
Experiments using a water jacketed reactor (much like that in Figure 8.3a) containing bilirubin
oxidase covalently attached to agarose beads showed that plasma bilirubin levels in rats decreased

by 50 % after 30 minutes of treatment. The rat's blood was recirculated through the 6 ml reactor
volume at a flow rate of 1 ml/min. Clearly these results indicate that an immobilized bilirubin
oxidase reactor could be a new approach for the treatment of neonatal jaundice. It also

shows the feasibility of using immobilized enzyme reactors for the specific removal of a harmful

substance present in the blood.