enzyme biotechnology

Enzyme Biotechnology- Methods and applications

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Enzyme Biotechnology

Definition of enzyme: Enzymes are high molecular weight proteins made up of a long chain of amino acids that are linked to each other by peptide bonds. Enzymes accelerate/ catalyze the biochemical reactions without itself getting consumed. Thus, enzymes are also known as biocatalyst1,2.

Classification of enzymes: Enzymes are classified based on the type of reaction they catalyze. There is a total of 7 categories/classes of enzymes which are listed below in table 11,2.  

Table 1: Name of the enzyme class, the function of the enzyme, the general type of reaction enzyme-catalyzed, and examples. (X, Y, and Z represents chemical groups)  

Sr noEnzyme class nameFunction/ RoleGeneral type of reaction catalyzedExamples of enzyme
1TransferasesCatalyze the transfer/ exchange of certain chemical groups amongst the substrateX-Y + Z ↔ X + Y-ZMethyltransferase, acyltransferase, sulfotransferase. Glucotransferase.  
2HydrolasesPromotes the hydrolysis of reactant/ substrateX-Y + H2O → X-H + Y-OHEsterase, lipase, phosphatase, peptidase, nucleosidase 
3OxidoreductasesCatalyze the redox type of reactions.Xred + Yoxd ↔ Xoxd + YredOxidase, peroxidases, hydrolases, oxygenase.
4LyasesPromotes the removal of certain chemical groups from the substrate molecules OR promotes the reverse reaction.X-Y ↔ X + YDecarboxylase, aldehyde lyase
5IsomeraseCatalyzes the conversion reaction for isomersX-Y-Z ↔ Y-Z-XGlucose isomerase, maleate isomerase
6LigasePromotes the joining of two molecular substrates to get a single compound. Such type of reaction is characterized by the release of energy.X + Y + ATP → X-Y + ADP + PO4DNA ligase
7TranslocasePromotes the movement of ions/ molecules across the biological membrane. Ornithine translocase.
  • Recombinant enzyme technology:

Recombinant DNA technology produces recombinant DNA also known as rDNA using a specific set of enzymes called recombinant enzymes.

Some of the recombinant enzymes used in rDNA technology are mentioned below:

  1. Restriction enzymes.  Restriction enzymes are a set of enzymes that cleaves the DNA at specific sites. Nucleases belong to the restriction enzymes class that breaks the DNA molecule by breaking the phosphodiester linkage in DNA. Examples of nucleases: RNAse (cleaves the RNA) and DNAse (cleaves the DNA).

Nucleases are of two types: Exonuclease (cleaves the DNA molecule at the end) and endonucleases (cleaves the DNA molecule in the middle of the molecule).

  • Exonucleases: removes the terminal nucleotide of the DNA molecule
  • Endonucleases: cleaves the internal phosphodiester bond. There are 3 types of endonucleases types I, II, and III. Endonucleases scan the entire DNA chain and cut the DNA molecule which leads to the formation of 3’, 5’, blunt ends, or sticky ends29. 
  • Ligases: are a class of enzymes that join the DNA and RNA fragments together. DNA is joined by DNA ligase whereas RNA is joined by RNA ligase. DNA ligase helps in repairing the single-strand breaks in DNA.
  • Polymerases: are a set of enzymes that synthesize the long chain of polymers. DNA and RNA polymerase belong to this class.

The function of DNA polymerase: DNA polymerase helps in synthesizing the DNA molecule from deoxyribonucleotides8.

The function of RNA polymerase: RNA polymerase copies the DNA sequence into an RNA sequence during transcription process9.

  • Enzyme topography

The rate of a biological reaction depends upon the topography or the shape of an enzyme. Each enzyme with a specific shape can attach to only a specific substrate. Each enzyme will fit into only one substate molecule because each enzyme has a specific shape of the active site. For a biological reaction to occur the active site of the enzyme binds to the substrate.

Example: Acetylcholine (Ach) binds to the extracellular domain of the acetylcholine receptor30.

  • PEGylation

PEGylation is a biochemical modification process of the fusion or attachment of polyethylene glycol (PEG) polymer to macromolecules such as drugs, protein, vesicles, antibodies, and enzymes. The macromolecule is covalently or non-covalently conjugated to the PEG polymer. Due to PEGylation desired therapeutic properties are obtained in the macromolecule.

The initial research of PEGylation was done by Frank Davis Sir in the late 1970s. He demonstrated that PEGylated proteins had longer half-lives in the bloodstream and decreased immunogenicity31.

  • Advantages/Significance of PEGylation: The significance of PEGylation is listed below:
  • The in-vivo half-life of the drug, enzyme or protein is increased
  • Improved drug stability
  • Reduced dosage frequency
  • Enhanced protection from proteolytic degradation
  • The immunogenicity and antigenicity of the macromolecule are reduced.
  • Minimal loss of the pharmacological activity
  • The solubility of the macromolecule (drug, antibody, protein, etc..) is increased.
  • Helps in achieving target-specific drug delivery
  • Improves the pharmacokinetic properties of the therapeutic agent31.

The first FDA approved drug Adagen that used PEGylation was launched in the year 1990. Since then various pegylated drug products are approved by FDA which are in use.

Some of the pegylated compounds which are currently available in the market for use are listed in table 2:

Table 2: Examples of few FDA approved PEGylated products31. 

DrugUse
KrystexxaReduces the uric acid levels and aids in removing gout crystals
Adagen (pegademase)The modified enzyme used for Enzyme Replacement Therapy (ERT).
Somavert (pegvisomant)Prescription medicine for acromegaly, a disease caused by the surplus of growth hormones in the body. The goal is to have a normal IGF-1 level in the blood.
Cimzia (certolizumab)An injected prescription medication that works to prevent inflammation that may result from an overactive immune system.

Why PEG is used in therapeutic formulations?

Some of the reasons for use of PEG in the systemic and non-systemic formulations are listed below:

  • Higher solubility in both organic and inorganic solvents
  • Minimum toxicity levels
  • Lower immunogenicity
  • Non-biodegradability
  • Hydrophilic nature
  • Fast renal clearance31

Due to the reasons mentioned above PEG polymer is the most commonly used in comparison to the other polymers of similar molecular weight and size.

There are three types of PEGylation: a) first-generation and b) second generation and c) third generation PEGylation.

  1. First-generation PEGylation: First-generation PEGylation is also known as non-specific PEGylation. Amine conjugation is the most commonly used method for non-specific PEGylation. The PEG conjugates obtained from non-specific PEGylation were not uniform and resulted in the formation of positional isomers. However, there are multiple first-generation PEGylated drug products in the market which are still in use. E.g. Pegasys used in the treatment of Hepatitis C, utilizes non-specific PEGylation.
  2. Second generation PEGylation: also known as site-specific PEGylation. In this method specific site of the PEG molecule interacted with the chemical groups on the protein or therapeutic drug to obtain a PEGylated conjugated product. The various strategies for site-specific PEGylation include N-terminal PEGylation, thiol and bridging PEGylation, histidine tags, and enzymatic PEGylation.
  3. Third generation PEGylation: The main aim of third-generation PEGylation is to improve the potency and half-life, site-specificity, and lower dosage of the drug. The main strategy for third-generation PEGylation is based on electrostatic linkages between PEG and drug.

The main pathway of site-specific PEGylation is reversible conjugation or release of prodrugs. Reversible conjugation is even less inhibiting on drug activity than irreversible conjugation, used in the first-generation PEGylation. Second generation PEGylation looks to temporarily attach PEG molecules via cleavable linkages. This way, drugs can be released according to a specified schedule, in vivo via hydrolytic cleavage31.

  • Limitations of PEGylation:
  • Possibility of the formation of side products.
  • PEG has limited conjugation ability
  • Costly process
  • Steric interference of protein with the target receptor
  • Interference with protein binding site31.
  • Drug cloning

Cloning is a process through which identical copies of the cell, tissues, or organism are produced. There are three types of cloning:

  • Gene cloning: creates replicas of DNA segments.
  • Reproductive cloning: creates copies of whole animals.
  • Therapeutic/drug cloning: creates embryonic stem cells. These embryonic stem cells are used to replace the injured tissues or cells in the body by healthy tissues or cells.

Application of therapeutic cloning:

  1. Cloning is most applicable in the preclinical phase of drug discovery. Cloning helps in the discovery of various receptor types and subtypes. Muscarinic, melanocortin, and dopamine receptors are some of the receptors identified through therapeutic cloning. Examples of successful drug cloning that are now used as therapeutics are insulin, growth hormone, erythropoietin, and tissue factors.
  2. Vaccines are another area of therapeutic cloning application.
  3. Stem cell therapy for treating disorders like cancer, diabetes6.

Advantages of therapeutic cloning: Some of the advantages of therapeutic cloning are mentioned below:

  • It helps in understanding the growth and development of stem cells. Thus, it is helpful to discover new treatments, medicines for various diseases.
  • The use of pluripotent stem cells in stem cell therapy helps in treating diseases in any body organ and tissues by replacing the damaged or mutated tissues7.

Disadvantages of therapeutic cloning:

  • Costly and time-consuming process.
  • Initial research and development require experienced and skilled personnel7.
  • Gene therapy

What is gene therapy?

Gene therapy is a medical field that uses genes to treat diseases. The various strategies for gene therapy are as follows:

  1. Inserting a healthy gene into the target cell to replace the defective/faulty/mutated gene.
  2. Inactivating a defective/faulty/mutated gene
  3. Inserting a new healthy gene in the target cell to prevent diseases3.

How does gene therapy work?

In gene therapy, new genetic material is introduced inside the target cells to replace the mutated/ abnormal genes. The new gene inserted makes a useful protein or restores the function of the mutated gene. When a gene is directly inserted into the target cell it fails to show therapeutic activity. Thus, a carrier also known as a vector is used to insert the gene into the target cell. Most commonly genetically modified viruses that are non-virulent are used as vectors to introduce the new genetic material inside the target cell. The most common viruses used as vectors are adenoviruses, retroviruses3.

There are two approaches to gene therapy:

  1. Introducing the vector by the parenteral route (intravenous route) directly into the specific tissue.
  2. A sample of the patient’s tissue or cell is removed and then exposed to the vector for a defined period in the controlled conditions. The cells containing the vector are again introduced back into the patient’s body.

If the new gene introduced inside the patient’s body starts making functional proteins then it indicates that the gene therapy is successful3.

Advantages of gene therapy

  • Used to prevent and cure hereditary disorders. E.g. Cystic fibrosis
  • It helps to achieve the required pharmacological action4.

Disadvantages of gene therapy:

  • Inside the body, cells undergo constant division due to which long-lasting therapeutic effect is not obtained from gene therapy.
  • Due to the insertion of new genes, there is a possible immune response to be stimulated in the body which can be life-threatening.
  • The use of a virus as a vector in gene therapy may induce toxicity, immune response in the patient’s body.
  • Diseases which occur due to defect in a single gene can only be treated.
  • The entire therapy is costly4.

Applications of gene therapy:

Gene therapy is used to treat various disorders which are mentioned below:

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