Food Ingredients and Enzymes of Microbial Origin ◾ 239
enzymes. The method may not be applicable if the substrate molecules are large. α-Amylase may
not be a good candidate for immobilization because starch molecules, its substrate, are fairly large.
However, glucose isomerase can be immobilized as its substrate is small glucose molecules. The
supporting materials can be contaminated with microorganisms that are difficult to remove and
can be a source of contamination in food. The materials to be used as support should not be made
of substances that are unsafe and should be approved by regulatory agencies. Some of the immobilized enzymes currently used are glucose isomerase, β-galactosidase, and aminoacylase.
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).
Thermostable Enzymes
The term “thermostable enzymes” is generally used for those enzymes that can catalyze reactions
above 60°C. 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, the 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 temperatures, enzymes denature because of the unfolding of their three-dimensional
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 increases in both ion pairing and hydrogen bonding on the
surface of an enzyme (on a 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 extraionic charge (on the surface) increases the
thermostability of this enzyme.
Several methods, such as chemical and recombinant DNA techniques, can be used to increase
the thermostability of an enzyme. 17 Recombinant DNA technology can be used in two ways. If
the enzyme is present in a thermostable form in a microorganism that is not on 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.
enzymes. The method may not be applicable if the substrate molecules are large. α-Amylase may
not be a good candidate for immobilization because starch molecules, its substrate, are fairly large.
However, glucose isomerase can be immobilized as its substrate is small glucose molecules. The
supporting materials can be contaminated with microorganisms that are difficult to remove and
can be a source of contamination in food. The materials to be used as support should not be made
of substances that are unsafe and should be approved by regulatory agencies. Some of the immobilized enzymes currently used are glucose isomerase, β-galactosidase, and aminoacylase.
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).
Thermostable Enzymes
The term “thermostable enzymes” is generally used for those enzymes that can catalyze reactions
above 60°C. 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, the 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 temperatures, enzymes denature because of the unfolding of their three-dimensional
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 increases in both ion pairing and hydrogen bonding on the
surface of an enzyme (on a 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 extraionic charge (on the surface) increases the
thermostability of this enzyme.
Several methods, such as chemical and recombinant DNA techniques, can be used to increase
the thermostability of an enzyme. 17 Recombinant DNA technology can be used in two ways. If
the enzyme is present in a thermostable form in a microorganism that is not on 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.