Bioprocess Engineering
39
2
Enzyme Engineering
Enzymes are an alternative to viable
cells for synthesis of biological prod-
ucts. The applications vary from alter-
native fuel synthesis to environmental
remediation to biosensors to food pro-
cessing to textiles. Enzymes themselves
are also bioprocessing products. With
the increased knowledge of recombinant
DNA technology, rare enzymes are now
made easily in large quantities and are
obtained from organisms grown in un-
usual environments like hot springs or
the ocean. One increasing area of en-
zyme use is in the separation of chi-
rally
pure
compounds.
Typically, one
enantiomer in a mixture is often not
useful while the other has therapeu-
tic value. This unique enzyme function
leads to the development of processes
that use both chemical and enzymatic
synthesis
for
the
production
of
new
pharmaceuticals.
The most common reactor conFgura-
tion for enzyme synthesis of a product is a
packed or fluidized immobilized enzyme
reactor. This system has the advantage of
being a continuous process able to han-
dle high throughputs of process streams.
Enzymes are reasonably protected in this
environment. Cellulase is an example of
an enzyme that is used in an immo-
bilized conFguration for the conversion
of biomass feedstock in ethanol produc-
tion for alternative fuels. In addition, these
systems show promise for environmental
remediation to selectively remove heavy
metals or transform hazardous organic
substances into nontoxic compounds.
Enzyme kinetics for simple enzyme-
catalyzed reactions are often referred to as
saturation or Michaelis–Menten kinetics.
The reaction mechanism describing this
process is
E
+
S
k
1
−−−
*
)
−−−
k
1
ES
k
2
−−−→
E
+
P
where E is the enzyme, S is the substrate, P
is the product, ES is the enzyme–substrate
complex, and the
k
values are the reac-
tion rate constants. The scheme involves
a reversible step for formation and disso-
ciation of the enzyme–substrate complex.
It assumes that the second reaction, prod-
uct generation, is irreversible, which is a
good assumption when product accumu-
lation is negligible at the beginning of
the reaction. To develop the rate expres-
sion for enzyme-catalyzed reaction, either
a rapid equilibrium approach or a quasi-
steady state approach is used. Either way,
the resulting rate expression is
v
=
d
P
d
t
=
V
m
S
K
m
+
S
(
1
)
where
v
is the reaction rate,
t
is time,
V
m
is the maximum forward reaction rate,
and
K
m
is the Michaelis–Menten constant.
This rate expression is commonly used
to describe enzyme-catalyzed reactions.
Many enzymatic reactions are described
by Michaelis–Menten kinetics. The rate
constants are usually determined from
initial rate experiments.
Enzymes are effected by pH and tem-
perature. Typically, an enzyme is active
only over a certain pH (1 to 2 pH units)
and temperature range (5 to 10
). The
pH and temperature effects are incorpo-
rated into the enzyme kinetic expression
when needed. Enzymes are also inhibited
by many heavy metals or organic materi-
als. The inhibitors bind to the active site
of the enzyme directly (competitive inhibi-
tion), to another site on the enzyme, thus
reducing the enzyme afFnity for the sub-
strate (noncompetitive inhibition), or to
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