The industrial applications or therapeutic uses of enzymes/proteins can be appropriately brought into use by increasing their half-lives or thermostability. Proteins with enhanced stability can be obtained by the following methods:
Addition of disulfide bonds,
Changing asparagine to other amino acids,
Reducing the free sulfhydryl groups,
Single amino acid changes, and
Improving kinetic properties of enzymes.
Addition of disulfide bonds
Introduction of disulfide bonds significantly increases the thermostability of enzymes. The disulfide bonds added should not disturb the normal functioning of enzymes. The new protein obtained after the addition of disulfide bonds does not unfold at high temperatures and also does not denatures at non-physiological conditions (i.e., high pH and presence of organic solvents). These features of the new protein facilitate the use of certain enzymes for industrial applications.
T4 Lysozyme : This is an enzyme of bacteriophage T4. Disulfide bonds in T4 lysozyme were introduced by changing two, four or six amino acids (in close proximity) to cysteine residues to form one, two or three disulfide bonds, respectively. In the native T4 lysozyme, two cysteine residues (not held together by a disulfide bond) are present. By oligonucleotide-directed mutagenesis, cysteine residues created disulfide bonds between positions 3 and 97,9 and 164, and 21 and 142 (numbered from N-terminal end) of the enzyme.
On adding disulfide bonds, the folded structure and thermostability of the enzyme increases. Thus, it can be said that T4 lysozyme with three disulfide bonds has high stability and good biological activity.
Xylanase : This is an enzyme used for manufacturing paper from wood pulp. Xylanase should be catalytically active at high temperature. When added with disulfide bonds (one, two or three), it becomes thermostable and more functionally efficient.
Changing Asparagine to Other Amino Acids
The amino acids asparagine and glutamine undergo deamidation (i.e., release ammonia) to form aspartic acid and glutamic acid, respectively at high temperature. These alterations are associated with changes in the protein folding and loss of biological activity.
Triose Phosphate Isomerase : This is a dimeric enzyme with identical subunits, each having two thermosensitive asparagine residues which undergo deamidation. Oligonucleotide-directed mutagenesis was used for introducing threonine or isoleucine instead of asparagine to obtain a new thermostable enzyme. While on replacing the asparagine residues with aspartic acid, an enzyme unstable even at low temperature and having reduced activity is obtained.
Reducing the Free Sulfhydryl Groups
The presence of a large number of free sulfhydryl groups (contributed by cysteine residues) may lower the activity of proteins. In this case, the stability and activity of the protein or enzyme can be improved by reducing the number of sulfhydryl groups.
Human β-Interferon (IFN- β) : IFN- β is produced in E. coli by genetic engineering. Its antiviral activity was found to be only 10% of the original glycosylated form. It was found to exist as inactive dimers and oligomers. Cysteine residues were involved in intermolecular disulfide bonding, thus forming dimers and oligomers.
However, this is the case only in E. coli cells and not in human cells; and this problem was also overcome by replacing the cysteine residues with serine. The structures of these two amino acids (cysteine and serine) are also similar, with the only difference that the sulphur of cysteine has been replaced with oxygen in serine (and this consequently reduces free sulfhydryl groups). This process helps in the production of IFN- β with increased stability and good biological activity.
Single Amino Acid Changes
The stability and biological activity of recombinant proteins can be improved by a second generation variant. This is achieved by a single amino acid change.
α1 Antitrypsin : This amino acid binds to and inhibits the action of neutrophil elastase (an enzyme that damages the lung tissues, and causes emphysema, abnormal distension of lungs by air).
This amino acid binds to and inhibits the action of neutrophil elastase (an enzyme that damages the lung tissues, and causes emphysema, abnormal distension of lungs by air). In this process, α1-antitrypsin is cleaved into serine and methionine (figure a); and the free methionine is oxidised to methionine sulfoxide, thus making α1-antitrypsin a poor inhibitor of elastase.
Methionine is replaced with valine (an oxidative-resistance variant of α1 antitrypsin) (figure a) and the new enzyme obtained is used for treating patients having genetic deficiency of α1-antitrypsin.
Insulin : In neutral solution, therapeutic insulin is present as zinc-containing hexamer. On introducing single amino acid substitutions, insulin exists in monomeric state with good stability and biological activity.
Tissue Plasminogen Activator (tPA) : This is therapeutically used for the lysis of blood clots causing myocardial infarction. The tPA has a shorter half-life of around 5 minutes, thus has to be administered recurrently. The half-life of tPA can be increased by replacing asparagine residue with glutamine as it is less glycosylated than asparagine, thus making a difference in the half-life of tPA.
Improving Kinetic Properties of Enzymes
The functional activities of enzymes can be improved by improving their kinetic properties (Km, specificity, etc.) through oligonucleotide-directed mutagenesis. This is required for enzymes having industrial and therapeutic benefits.
Subtilisin : This is a serine protease enzyme secreted by gram-positive bacteria of Bacillus species. It is extensively used in industries as an enzyme detergent (cleaning agent in laundries). However, its large scale industrial use is restricted as it becomes inactive when the methionine lying close to the active site gets oxidised. This problem is overcome by replacing methionine with other amino acids.
Subtilisin enzyme has also been used for genetic manipulations over the past 15 years. As a result of which about 50% of the native amino acids of this enzyme. have been changed by in vitro mutagenesis, and most of the features of subtilisin (including its stability, substrate specificity, thermal and alkaline inactivation) has been altered.
Asparaginase : This enzyme is used for controlling leukaemia (uncontrolled growth of WBCs). On intravenous administration, asparaginase cleaves asparagine to aspartate (the reduced availability of asparagine restricts cell proliferation).
Asparaginase obtained from different sources shows different effectiveness in controlling leukaemia due to their different kinetic properties. Asparaginase having a low Km (Michaelis constant) value has a high affinity for asparagine (hence more breakdown), thus should be selected to be used therapeutically for controlling leukaemia.
Tyrosyl t-RNA Synthetase : This enzyme is obtained from Bacillus stearothermophilus, and its Km value has been modified with regard to substrate binding. Tyrosyl t-RNA synthetase enzyme catalyses the following two reactions, to yield tyrosine t-RNA:
Tyrosine + ATP → Tyrosyladenylate + PPi
Tyrơsyladenylate + t-RNA → Tyrosine t-RNA + AMP
Replacement of threonine with either alanine or proline has yielded variants of tyrosyl t-RNA synthetase. Alanine variant has a low Km value, thus possesses twofold binding affinity for ATP; while proline variant has a very low Km value, and thus ATP binds about 100-fold more tightly.
Restriction Endonucleases : This enzyme has been modified by oligonucleotide-directed mutagenesis. Most of the restriction endonucleases enzymes recognise the same DNA sequence for their action. There are only 200 different recognition sites, and thus an overlap in the recognition sites of several restriction enzymes has been observed.
There are two types of restriction endonucleases, i.e., the frequent cutters which recognise a sequence of 4−6bp and rare cutters which recognise a sequence above 8bp. The latter ones are more useful for producing large DNA fragments, and therefore protein engineering techniques have been utilised to modify the existing restriction endonucleases and produce rare cutters.
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