Fundamental Food Microbiology, Third Edition - Fuad Fathir

252 FUNDAMENTAL FOOD MICROBIOLOGY
Microbial cells can also be immobilized by the methods listed previously, and
the techniques have been studied in the production of some food ingredients and
beverages. Examples include Asp. niger (for citric acid and gluconic acid), Saccharomyces
cerevisiae (for alcoholic beverages), and Lactobacillus species (for lactic
acid).
D. Thermostable Enzymes
The term thermostable enzymes is generally used for those enzymes that can catalyze
reactions above 60�C. 10 There are several advantages of using thermostable enzymes
in a process. The rate of an enzyme reaction doubles for every 10�C increase in
temperature; thus, production rate can be increased or the amount of enzyme used
can be reduced. At high temperatures, when an enzyme is used for a long time (as
in the case of immobilized enzymes), the problems of microbial growth and contamination
can be reduced.
At high temperature, enzymes denature because of unfolding of their threedimensional
structures. The stability of the three-dimensional structure of an enzyme
is influenced by the ionic charges, hydrogen bonding, and hydrophobic interaction
among the amino acids. Thus, the linear sequences of amino acids in an enzyme
greatly influence its three-dimensional structure and stability. Studies have revealed
that increase in both ion pairing and hydrogen bonding on the surface of an enzyme
(on three-dimensional structure) and increases in internal hydrophobicity increase
the thermostability of an enzyme. For example, the enzyme tyrosinase from a
thermolabile strain of Neurospora species denatures in 4 min at 60�C, but from a
thermostable strain of the same species it denatures in 70 min at 60�C. An analysis
of the amino acid sequences revealed that at position 96, tyrosinase has an aspargine
(uncharged) in the thermolabile strain, but aspartic acid (charged) in the thermostable
strain. Thus, an extra ionic charge (on the surface) increases the thermostability of
this enzyme.
Several methods, such as chemical and recombinant DNA techniques, can be
used to increase thermostability of an enzyme. Recombinant DNA technology can
be used in two ways. If the enzyme is present in a thermostable form in a microorganism
that is not in the GRAS list, the gene can be cloned in a suitable vector,
which can then be introduced in a GRAS-listed microorganism and examined for
expression and economical production. The other method is more complicated and
involves determining the amino acid sequence of the enzyme and its three-dimensional
structure (by computer modeling) to recognize the amino acids on the surface
(or inside). The next step involves changing one or more amino acids on the surface
to increase ionic or hydrogen bonding. This can be achieved by site-specific mutagenesis
of base sequences of cDNA for the specific amino acid. The synthesized DNA
can be incorporated in a vector and introduced in a desired microbial strain for
expression of the enzyme and testing for its thermostability.
Several thermostable enzymes obtained from microorganisms on the GRAS list
are currently being used. It is expected that in the future their production by different
methods and use in food will increase.