Food Biochemistry and Food Processing (2 edition)

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BLBS102-c07 BLBS102-Simpson March 21, 2012 11:12 Trim: 276mm X 219mm Printer Name: Yet to Come


148 Part 2: Biotechnology and Enzymology

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


  1. 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).
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