Food Biochemistry and Food Processing (2 edition)

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148 Part 2: Biotechnology and Enzymology

Figure 7.17.A generalised schematic for the prediction of protein three-dimensional structure.

5. Genetic manipulation of the wild-type nucleotide
   sequence. A combination of previously published
   experimental literature and sequence/structure analysis
   information is usually necessary for the identification
   of functionally important sites in the protein. Once an
   adequate three-dimensional structural model of the protein
   of interest has been constructed, manipulation of the gene
   of interest is necessary for the construction of mutants.
   Polymerase chain reaction (PCR) mutagenesis is the
   basic tool for the genetic manipulation of the nucleotide
   sequences. The genetically redesigned proteins are
   engineered by the following:
   a. Site-directed mutagenesis: alteration of specific amino
   acid residues. There are a number of experimental
   approaches designed for this purpose. The basic
   principle involves the use of synthetic oligonucleotides
   (oligonucleotide-directed mutagenesis) that are com-
   plementary to the cloned gene of interest but contain
   a single (or sometimes multiple) mismatched base(s)
   (Balland et al. 1985, Garvey and Matthews 1990,
   Wagner and Benkovic 1990). The cloned gene is
   either carried by a single-stranded vector (M13
   oligonucleotide-directed mutagenesis) or a plasmid
   that is later denatured by alkali (plasmid DNA
   oligonucleotide-directed mutagenesis) or heat
   (PCR-amplified oligonucleotide-directed mutagenesis)
   in order for the mismatched oligonucleotide to anneal.

The latter then serves as a primer for DNA synthesis

catalysed by externally added DNA polymerase for the

creation of a copy of the entire vector, carrying, how-

ever, a mutated base. PCR mutagenesis is the most fre-

quently used mutagenesis method (Fig. 7.18). For ex-

ample, substitution of specific amino acid positions by

site-directed mutagenesis (S67D/H68D) successfully

converted the coenzyme specificity of the short-chain

carbonyl reductase from NADP(H) to NAD(H) as well

as the product enantioselectivity without disturbing

enzyme stability (Zhang et al. 2009). In another exam-

ple, engineering of the maize GSTF1–1 by mutating

selected G-site residues resulted in substantial changes

in the pH-dependence of kinetic parameters of the

enzyme (Labrou et al. 2004a). Mutation of a key

residue in the H-site of the same enzyme (Ile118Phe)

led to a fourfold improved specificity of the en-

zyme towards the herbicide alachlor (Labrou et al.

2005).

So far, substitution of a specific amino acid by an-

other has been limited by the availability of only 20

naturally occurring amino acids. However, it is chemi-

cally possible to construct hundreds of designer-made

amino acids. Incorporation of these novel protein

building blocks could help shed new light into the

cellular and protein functions (Wang and Schultz 2002,

Chin et al. 2003, Deiters et al. 2003, Arnold 2009).