BLBS102-c07 BLBS102-Simpson March 21, 2012 11:12 Trim: 276mm X 219mm Printer Name: Yet to Come
7 Biocatalysis, Enzyme Engineering and Biotechnology 145(A)(B)(C)Figure 7.15.Structure of several representative triazine dyes: (A) Cibacron Blue 3GA, (B) Procion Red HE-3B and (C) Procion Rubine MX-B.The rapid development of recombinant DNA technology since
the early 1980s has changed the emphasis of a classical enzyme
purification work. For example, epitope tagging is a recombinant
DNA method for inserting a specific protein sequence (affinity
tag) into the protein of interest (Terpe 2003). This allows the
expressed tagged protein to be purified by affinity interactions
with a ligand that selectively binds to the affinity tag. Examples
of affinity tags and their respective ligands used for protein and
enzyme purification are shown in Table 7.7.ENZYME ENGINEERING
Another extremely promising area of enzyme technology is en-
zyme engineering. New enzyme structures may be designed and
produced in order to improve existing ones or create new ac-
tivities. Over the past two decades, with the advent of protein
engineering, molecular biotechnology has permitted not only
the improvement of the properties of these isolated proteins, but
also the construction of ‘altered versions’ of these ‘naturally
occurring’ proteins with novel or ‘tailor-made’ properties (RyuTable 7.7.Adsorbents and Elution Conditions of Affinity TagsAffinity Tag Matrix Elution ConditionPoly-His Ni^2 +-NTA Imidazole 20–250 mM or low pH
FLAG Anti-FLAG monoclonal antibody pH 3.0 or 2–5 mM EDTA
Strep-tag II Strep-Tactin (modified streptavidin) 2.5 mM desthiobiotin
c-myc Monoclonal antibody Low pH
S S-fragment of RNaseA 3 M guanidine thiocyanate, 0.2 M citrate pH 2, 3 M
magnesium chloride
Calmodulin-binding peptide Calmodulin EGTA or EGTA with 1 M NaCl
Cellulose-binding domain Cellulose Guanidine HCl or urea>4M
GlutathioneS-transferase Glutathione 5–10 mM reduced glutathione