157
amino acid sequence for the desired improvement in function/structure of the
enzyme.
3. Site-directed mutagenesis: The next step consists of constructing a gene that will
encode the specific amino acid sequence. This is best achieved by isolation and
cloning of the natural gene encoding the concerned enzyme and using this gene
for site-directed mutagenesis. In the case of site-directed mutagenesis, specified
changes in the base sequence are introduced at specified sites of genes. The
genes for the specific amino acid are first isolated, modified appropriately by
site-directed mutagenesis, and cloned into an expression vector to obtain a gene
construct. Once the appropriate gene is constructed, it is introduced and expressed
in a suitable host, e.g., Escherichia coli. The recombinant or mutant enzyme so
produced is isolated, purified, and used for the determination of its structure and
properties. The alterations made in the amino acid sequence will be reflected in
the characteristics of enzymes produced by recombinant DNA, which can be
compared with that of the native enzymes. The information so obtained is added
to the database. If the enzyme structure and function are not altered as desired,
the next cycle of experimentation is undertaken.
6.4.1 Examples of Enzyme Engineering
Even though enzyme engineering strategies have been widely employed for studying the relationship between amino acid sequences and the structures and functions
of various enzymes, commercial examples are few. Some of the enzymes studied
are tyrosyl-tRNA synthetase, β-lactamase, dihydrofolate reductase, subtilisin, lysozyme, alcohol dehydrogenase, and lactate dehydrogenase. Various studies have also
focused on alterations of industrial enzymes to augment their efficiency. The results
from these studies have helped to exhibit the strength of the technique, to generate
highly beneficial data on sequence–structure–function relationships, and also to
reveal the limitations of our knowledge.
Subtilisin Subtilisin (EC 3.4.21.62) is a non-specific protease initially obtained
from Bacillus subtilis. The advantage of enzyme engineering can be illustrated by
utilizing subtilisin (produced from B. amyloliquefaciens), the principal enzyme in
the detergent enzyme preparation, Alcalase (Ottesen and Svendsen 1970). In order
to enhance the enzyme activity in detergents, it is important to improve the enzyme
stability at higher temperatures, pH, and oxidant (bleach) strengths. The P 1 cleft of
this enzyme possesses the amino acid residue on C-terminal side of the targeted
peptide bond. Substitutions of amino acid in the P 1 cleft help in enhancing the specificity of the enzyme for specific peptide bonds, while specificity for others is diminished. This alteration can be predicted reasonably precise. For some applications,
the increase in relative specificities may be beneficial. Subtilisin is exceptional in
that it has a large hydrophobic site, which is fairly non-specific in activity that can
be made more specific quite easily (e.g. by reducing its size). The effect of a substitution in the P 1 cleft on the relative specific activity between substrates may be
6 Enzyme Engineering
amino acid sequence for the desired improvement in function/structure of the
enzyme.
3. Site-directed mutagenesis: The next step consists of constructing a gene that will
encode the specific amino acid sequence. This is best achieved by isolation and
cloning of the natural gene encoding the concerned enzyme and using this gene
for site-directed mutagenesis. In the case of site-directed mutagenesis, specified
changes in the base sequence are introduced at specified sites of genes. The
genes for the specific amino acid are first isolated, modified appropriately by
site-directed mutagenesis, and cloned into an expression vector to obtain a gene
construct. Once the appropriate gene is constructed, it is introduced and expressed
in a suitable host, e.g., Escherichia coli. The recombinant or mutant enzyme so
produced is isolated, purified, and used for the determination of its structure and
properties. The alterations made in the amino acid sequence will be reflected in
the characteristics of enzymes produced by recombinant DNA, which can be
compared with that of the native enzymes. The information so obtained is added
to the database. If the enzyme structure and function are not altered as desired,
the next cycle of experimentation is undertaken.
6.4.1 Examples of Enzyme Engineering
Even though enzyme engineering strategies have been widely employed for studying the relationship between amino acid sequences and the structures and functions
of various enzymes, commercial examples are few. Some of the enzymes studied
are tyrosyl-tRNA synthetase, β-lactamase, dihydrofolate reductase, subtilisin, lysozyme, alcohol dehydrogenase, and lactate dehydrogenase. Various studies have also
focused on alterations of industrial enzymes to augment their efficiency. The results
from these studies have helped to exhibit the strength of the technique, to generate
highly beneficial data on sequence–structure–function relationships, and also to
reveal the limitations of our knowledge.
Subtilisin Subtilisin (EC 3.4.21.62) is a non-specific protease initially obtained
from Bacillus subtilis. The advantage of enzyme engineering can be illustrated by
utilizing subtilisin (produced from B. amyloliquefaciens), the principal enzyme in
the detergent enzyme preparation, Alcalase (Ottesen and Svendsen 1970). In order
to enhance the enzyme activity in detergents, it is important to improve the enzyme
stability at higher temperatures, pH, and oxidant (bleach) strengths. The P 1 cleft of
this enzyme possesses the amino acid residue on C-terminal side of the targeted
peptide bond. Substitutions of amino acid in the P 1 cleft help in enhancing the specificity of the enzyme for specific peptide bonds, while specificity for others is diminished. This alteration can be predicted reasonably precise. For some applications,
the increase in relative specificities may be beneficial. Subtilisin is exceptional in
that it has a large hydrophobic site, which is fairly non-specific in activity that can
be made more specific quite easily (e.g. by reducing its size). The effect of a substitution in the P 1 cleft on the relative specific activity between substrates may be
6 Enzyme Engineering