Food Biochemistry and Food Processing

8 Enzyme Engineering and Technology 205

cleotide-directed mutagenesis) in order for the

mismatched oligonucleotide to anneal. The latter

then serves as a primer for DNA synthesis cat-

alyzed by externally added DNA polymerase for

the creation of a copy of the entire vector, carry-

ing however, a mutated base. PCR mutagenesis is

the most frequently used mutagenesis method

(Fig. 8.18). For example, substitution of specific

aminoacid positions by site-directed mutagenesis

altered the preference for coenzyme from

NADPto NADfor Chromatium vinosumglu-

tathione reductase (Scrutton et al., 1990).

So far substitution of a specific amino acid by

another has been limited by the availability of

only 20 naturally occurring amino acids. How-

ever, it is chemically possible to construct hun-

dreds of designer-made amino acids. Incorpora-

tion of these novel protein building blocks could

help shed new light onto the cellular and protein

functions (Wang and Schultz 2002, Chin et al.

2003, Deiters et al. 2003).

b. Construction of deletion mutants: deletion

of specified areas within or at the 5/3 ends

(truncation mutants) of the gene.

c. Construction of insertion/fusion mutants:

insertion of a functionally/structurally important

epitope or fusion to another protein fragment.

There are numerous examples of fusion proteins

designed to facilitate protein expression and pur-

ification, display of proteins on surfaces of cells

or phages, cellular localization, and metabolic

engineering as well as protein-protein interaction

studies (Nixon et al. 1998).

d. Domain swapping: exchanging of protein

domains between homologous or heterologous

proteins.For example, exchange of a homologous

region between Agrobacterium tumefaciens-

glucosidase (optimum at pH 7.2–7.4 and 60°C)

and Cellvibrio gilvus-glucosidase (optimum at

pH 6.2–6.4 and 35°C) resulted in a hybrid

enzyme with optimal activity at pH 6.6–7.0 and

45–50°C (Singh et al. 1995).

Figure 8.17.A generalized schematic for the prediction of three-dimensional protein structure.