Chemistry Reference
In-Depth Information
2.3.3 Selection and screening
In order to sort out the mutant protein with properties of interest, a strategic selection or
screening method is crucial. Selection involves applying one or more procedures in the assay
system so that only desired mutants are left over. Screening puts all the expressed mutants
under an assay system for the desired property. The results of these assays are compared and
mutants with the best performance are chosen. Very often the selected mutants are not good
enough, therefore mutagenesis needs to be repeated. During each round of mutagenesis, small
changes are accumulated until eventually a mutant stands out as a satisfactory candidate.
There can be rules applicable to selection methods for certain desired properties, for
example thermostability. Mutants can be treated at a required temperature for a certain period
of time so that, after the treatment, only surviving mutants have the desired thermostability.
In most cases, however, selection or screening methods have to be tailored for individual
enzymes for specific requirements of their properties to be improved.
2.3.3.1
High-throughput screening
Because most mutations are negative and only small portions of subtle changes lead to
real improvement of an enzyme, a library with a sufficient number of mutants should be
constructed and screened in order to find the desired mutants. If an easy detection system
is available, such as determining positive mutants with a colour reagent, an automatic ap-
paratus can be applied for efficient screening. Nowadays, high-throughput screening has
become a routine practice in research facilities involved in developing industrially feasible
enzymes.
2.3.4
Applications of protein engineering - a powerful
tool for the development of enzymes as
applied biocatalysts
2.3.4.1
Examples for improving thermostability and altering pH optimum
Industrial enzymes need to be stable in their working environments. To improve an enzyme's
stability at elevated temperatures is one of the earliest attempts of protein engineering. A
review by Goodenough
26
has summarized the factors related to thermostability. By under-
standing the chemistry of folding and unfolding of proteins, hydrophobic forces are found
to be the major driving force for folding, thus having a major role in thermostability. Pro-
tein engineering, by increasing the hydrophobic forces of secondary helical structures, has
proven a feasible strategy for increasing the thermostability of a known protein structure.
Liu and Wang
27
have reported on engineering a glucoamylase from
Aspergillus awamori
for increased thermostability. Glucoamylase (EC3.2.1.3) is one of the most important en-
zymes in the food industry. It catalyzes the hydrolysis reaction of starch molecules from
non-reducing ends. The condition for using the enzyme requires a high temperature, which
improves the reaction rate, reduces viscosity of the syrup for easy processing and reduces the
risk of bacterial contamination. The tertiary and the secondary structure of the enzyme have
been determined, but their structure-function relationship remained unclear. Liu and Wang
27
have performed a so-called 'molecular dynamics simulation' and found out that the 11th
α
-helix out of the 13
α
-helixes, which is located on the surface of the catalytic domain, is
