212 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
the interstitial spaces of lattice. Many materials, for
example, alginate, agarose, gelatin, polystyrene, and
polyacrylamide, have been used. As an example of
this latter method, the enzyme’s surface lysine re-
sidues may be derivatized by reaction with acryloyl
chloride (CH 2 uCH⎯CO⎯Cl) to give the acryloyl
amides. This product may then be copolymerized
and cross-linked with acrylamide (CH 2 uCH⎯
CO⎯NH 2 ) and bisacrylamide (H 2 N⎯CO⎯CHu
CH⎯CHuCH⎯CO⎯NH 2 ) to form a gel.
Encapsulation of enzymes can be achieved by en-
veloping the biological components within various
forms of semipermeable membranes as shown in
Figure 8.20E. Encapsulation is most frequently car-
ried out using nylon and cellulose nitrate to con-
struct microcapsules varying from 10 to 100 m. In
general, entrapment methods have found more ap-
plication in the immobilization of cells.
NEWAPPROACHES FORORIENTEDENZYME
IMMOBILIZATION: THEDEVELOPMENT OF
ENZYMEARRAYS
With the completion of several genome projects,
attention has turned to the elucidation of the func-
tional activities of the encoded proteins. Due to the
enormous number of newly discovered open reading
frames (ORF), progress in the analysis of the corre-
sponding proteins depends on the ability to perform
characterization in a parallel and high throughput
(HTS) format (Cahill and Nordhoff 2003). This typ-
ically involves construction of protein arrays based
on recombinant proteins. Such arrays are then ana-
lyzed for their enzymatic activities, for the ability to
interact with other proteins or small molecules, and
so forth. The development of enzyme array technol-
ogy is hindered by the complexity of protein mole-
cules. The tremendous variability in the nature of
enzymes, and consequently in the requirements for
their detection and identification, makes the devel-
opment of protein chips a particularly challenging
task. Additionally, enzyme molecules must be im-
mobilized on a matrix in a way that preserves their
native structures and makes them accessible to their
targets (Cutler 2003). The immobilization chemistry
must be compatible with preserving enzyme mole-
cules in native states. This requires good control of
local molecular environments for the immobilized
enzyme molecule (Yeo et al. 2004). There is one
major barrier in enzyme microarray development:
the immobilization chemistry has to be such that it
preserves the enzyme in a native state and allows
optimal orientation for substrate interaction. This
problem may be solved by the recently developed in
vitro protein ligation (IPL) methodology. Central to
this method is the ability of certain protein domains
(inteins) to excise themselves from a precursor pro-
tein (Lue et al. 2004). In a simplified intein expres-
sion system, a thiol reagent induces cleavage of the
intein-extein bond, leaving a reactive thioester group
on the C-terminus of the protein of interest. This
group can then be used to couple essentially any poly-
peptide with an N-terminal cysteine to the thioester-
tagged protein by restoring the peptide bond. In
another methodology, optimal orientation is based on
the unique ability of protein prenyl transferases to
recognize short but highly specific C-terminal pro-
tein sequences (Cys-A-A-X-, where A is any aliphat-
ic amino acid), as shown in Figure 8.22. The enzyme
accepts a spectrum of phosphoisoprenoid analogs
while displaying a very strict specificity for the pro-
tein substrate. This feature is explored for protein
derivatization. Several types of pyrophosphates
(biotin analogs, photoreactive analogs, and benzo-
phenone analogs; Figure 8.22) can be covalently
attached to the protein tagged with the Cys-A-A-X
motif. After modification the protein can be immobi-
lized directly, either reversibly through biotin-avidin
interaction on avidin-modified support or covalently
through the photoreactive group on several supports.ENZYME UTILIZATION IN
INDUSTRYEnzymes offer the potential for many exciting appli-
cations in industry. Some important industrial en-
zymes and their sources are listed in Table 8.9. In
addition to the industrial enzymes listed below, a
number of enzyme products have been approved for
therapeutic use. Examples include tissue plasmino-
gen activator and streptokinase for cardiovascular
disease; adenosine deaminase for the rare severe
combined immunodeficiency disease; -glucocere-
brosidase for Type 1 Gaucher disease; L-asparaginase
for the treatment of acute lymphoblastic leukemia;
DNAse for the treatment of cystic fibrosis; and neu-
raminidase, which is being targeted for the treatment
of influenza (Cutler 2003).
There are also thousands of enzyme products
used in small amounts for research and development