General Nature and Basic Structure of Enzyme

 A living cell is capable of performing a multitude of  biochemical reactions in order to survive grow and multiply. This involves

  • Degradation of complex nutrients into simpler forms so that they can be absorbed by the cell.
  • Uptake of these simple nutrients.
  • Chemical transformation of these simple nutrient molecules into various precursor metabolites, so that they are available for biosynthesis.
  • Generation of ATP and other bio reactive molecules, which co-operate in cellular biochemical reactions.
  • Biosynthesis of cellular molecules and structural components of the cell etc.

All these diverse types of chemical reactions can occur with a high degree of specificity and rate, under normal growth conditions for the organism. These chemical changes are brought about by the living cell through the role and participation of a special class of molecules known as enzymes . Enzymes function in the cell by acting as a biocatalyst.

Discovery of Enzymes

The term enzyme was coined by Kuhne in 1878, suggesting in yeast (en = in, zyme = yeast). It was examined that cell free extract from yeasts is capable of causing conversion of sugar into alcohol. This lead to the understanding that the molecules present inside the cells of yeasts are responsible for these chemical transformations. Kuhne called them as enzymes.

Later on, it was established that all biochemical activities of a living cell are attributed to these magic molecules called enzymes. The first enzyme to be discovered was 'amylase'. Its presence in malt extract was detected in 1833 by two French chemists Pain and Persuses.

General Nature of Enzymes

Enzymes are regarded as organic biocatalysts which are capable of functioning both extra cellular and intra cellular. Following are the major characteristics of enzymes. 

1] With exception of a few catalytic RNA molecules or ribozymes, all enzymes are protein in nature. Over 90 % enzymes are globular proteins. Many of them are conjugated proteins, having non protein component. 

Enzymes may be monomeric or multimeric proteins. As they are proteins, they share all major characteristic of proteins .

  • They are macromolecules with high MW.
  • They are non dialyzable and are unable to pass through semi permeable membrane.
  • They are amphoteric in nature. i.e. they possess both types of lonizable groups : -NH₂ and -COOH. which on ionization . yield positively charged ammonium (NH4+) ion and negatively charged carboxyl (COO-) ion.
  • As they are amphoteric, they possess specific isoelectric pH. At this pH, both -NH₂ and -COOH groups get equally ionized such that their net electrical charge becomes minimum.
  • They show electrophoretic mobility. If the enzyme solution is at pH, above isoelectric value, they acquire negative charge and hence move towards anode and vice versa.
  • They are colloidal in nature.
  • They can be salted out by salts like ammonium sulfate
  • They can be precipitated out by protein denaturing solvents like acetone and alcohol.
  • They can absorb maximum UV light at 280 um wavelength.

2]. Enzymes are mostly thermo labile and get denatured at high temperature, usually above 60°C. However, some enzymes are found thermo stable and can withstand high temperature up to 70°C - 80°C or even more.

3]. Enzymes are highly specific in action. They can act on a specific substrate molecule to bring about specific biochemical reaction .

Catalytic RNA - Ribozyme

  Apart from proteins, a few RNA have also been recognized to have catalytic activity. They are called ribozymes or catalytic RNA. They were first recognized by Thomas Cech in 1982. The ribozymes have two types of common roles :
  1. RNA processing, where the RNA is involved in RNA splicing, RNA ligation and RNA replication.  
  2. Peptide bond formation during protein synthesis. In ribosomes, they function as a part of rRNA and participate in peptide bond formation.

Basic structure of Enzymes

In general, enzyme molecule consists of two components.
  1. Apo enzyme and
  2. Prosthetic group or cofactor

Apo enzyme is protein part of enzyme.
Prosthetic group or cofactors are non protein part of enzymes. They consist of vitamin or their derivatives or metal lons. (If these non protein components are bound loosely to protein, they are called cofactors) and if they are bound firmly (covalently), are called prosthetic groups. Apo enzyme and prosthetic group form complex to form active form of enzyme, known as Holoenzyme.

Classification of Enzymes

What is GMP ?

 Good Manufacturing Practices (GMP) is the minimum standard that a pharmaceutical manufacturer must meet in their production processes. Process must:

  • Be of consistent high quality.
  • Be appropriate to their intended use.
  • Meet the requirements of the marketing authorization (MA) and product specification.

  Pharmaceutical organizations which comply with GMP must have manufacturer license. Regulatory agencies carries out in section on these pharmaceutical organizations to check if manufacturing sites comply with GMP. Sites are inspected when applied for a manufacturer license and then periodically based on risk assessments.

CGMP Legal Principles

Quality built into product

  • By "taking care" in making medicine.
  • Quality system is based on the principle of Quality by Design instead of quality by Inspection.
Without/Inadequate cGMP
  • Product(s) adulterated(defects need not be shown).
  • Firm and its management are responsible.
Current = Dynamic
  • Standards evolve over time.
Good Practices
  • Minimal standards.
  • Not "best practices". (Unless "best" is, in fact, current minimal.)


  • Is a legal requirement enforced and mandated through law, regulations and directives by each country government.
  • To protect public health of each and every individual.
  • Prevent contamination and mix-ups.
  • Prevents mislabeling and adulteration.
  • Consistent maintenance of Quality product supply throughout the product life cycle.
  • Ensure high standard quality product supplied into the market is safe and effective.
  • Satisfy stakeholders, customers and consumers.
  • To meet the requirement of the marketing authorization (MA) and product specification.
  • Enhance the organization image and reputation.

Responsibility of GMP

  • Quality and GMP compliance are independent of job title and have no boundaries.
  • Everyone involves in the process regulatory compliance, manufacturing, packing, Quality Control, distribution and supply of pharmaceuticals product has the responsibility to make sure it reaches to the patient with registered quality standards.
  • Building quality into the entire process of an operation makes sustainable compliance more achievable. However, it requires commitment by Senior management and the allocation of adequate resources (personnel, facilities, training, etc.).

Applications of Biotechnology in Various Fields

 Biotechnology is a branch of biology involving the use of living organisms and bioprocesses in engineering, technology, medicine, and other fields using bioproducts. The term biotechnology indicates the use of living organisms or their products for modifying the human health and environment.

Applications of Biotechnology in various fields

1] Medicine:

Modern biotechnology finds promising applications in medicine as in:

i) Drug Production :

  • Most of the traditional pharmaceutical drugs used for treating the symptoms of a disease are simpler molecules found through trials and errors. Small molecules are manufactured chemically, but the larger ones are created by human cells, bacterial cells, yeast cells, and animal or plant cells.
  • Modern biotechnology involves the use of genetically altered microorganisms (e.g., E.coli or yeast) for producing insulin or antibiotics via synthetic means. Modern biotechnology can also be used for producing plant-made pharmaceuticals. Biotechnology is also used in the development of molecular diagnostic devices used to define the target patient population for a given biopharmaceutical. For example, herceptin was the first drug to be used with a matching diagnostic test for treating breast cancer in women whose cancer cells expressed HER2 protein.

ii) Pharmacogenomics :

  • It is the study of how the genetic inheritance of an individual affects his/her body's response to drugs. The term pharmacogenomics was derived from the words pharmacology and genomics, thus it involves studying the relationship between pharmaceuticals and genetics. 
  • Pharmacogenomics aims to design and produce drugs adapted to each individual's genetic makeup.

iii) Gene Therapy :

  • It is used for the treatment of genetic and acquired diseases like cancer and AIDS. Gene therapy utilises normal genes for supplementing or replacing the defective genes or for strengthening immunity. This therapy targets either the somatic cells (i.e., body) or the gametes (ie., egg and sperm). 
  • In somatic gene therapy, the recipient's genome is altered; however, this alteration is not passed on to the next generation. On the other hand, in germline gene therapy, the egg and sperm cells of the parents are altered to be passed on to their offspring.

iv) Genetic Testing : 

This involves direct examination of the DNA, and is used for:
  • Carrier screening, or identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest,
  • Confirming the diagnosis of symptomatic individuals,
  • Determining sex,
  • Forensic/identity testing.
  • New-born screening.
  • Prenatal diagnostic screening.
  • Pre-symptomatic testing for determining the risk of developing adult-onset cancers, and
  • Pre-symptomatic testing for predicting adult-onset disorders.

2] Cloning :

  In this method, the nucleus from one cell is removed and is transferred to an unfertilised egg cell whose nucleus has either been deactivated or removed. Cloning can be done in the following two ways:

i) Reproductive Cloning :

  •  In this method, the egg cell after a few divisions is transferred to a uterus for its development into a foetus that is genetically identical to the donor of the original nucleus.

ii) Therapeutic Cloning :

  • In this method, the egg is placed in a petridish for its development into embryonic stem cells that are potential for treating several ailments.

3] Agriculture :

Biotechnology in the agricultural field is used for the following purposes:

i) Crop Yield :

  • For increasing the crop yield, one or two genes are transferred to a highly developed crop variety for imparting a new character. The current techniques of genetic engineering are best for the effects controlled by a single gene. Some genetic characteristics related to yield (e.g., enhanced growth) can be controlled by various genes, each posing a nominal effect on the yield.

ii) Reduced Vulnerability of Crops to Environmental Stresses :

  • Such crops which can be made resistant to biotic and abiotic stresses can be developed with the help of genes; for example, drought and salty soil are the two limiting factors in crop productivity.

iii) Increased Nutritional Qualities :

  • The nutritional value of proteins contained in foods can be enhanced; for example, proteins in legumes and cereals can be transformed such that they also provide amino acids required in the balanced diet of humans.

iv) Reduced Dependence on Fertilisers :

  • Modern biotechnology can also be used to reduce the dependence of farmers on agrochemicals; for example, Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein having insecticidal properties. Conventionally, these bacteria were used to produce an insecticidal spray by a fermentation process. 
  • In this form, the Bt toxin occurs as an inactive protoxin, which becomes effective when digested by an insect. There are several Bt toxins and each has a specificity for some target insects.

v) Production of Novel Substances in Crop Plants :

  • Biotechnology is also applied for novel uses apart from food; for example, oilseed is genetically modified to produce fatty acids for detergents, substitute fuels, and petrochemicals. Potatoes, tomatoes, rice tobacco, lettuce, safflowers, and other plants are genetically engineered to produce insulin and certain vaccines.

4] Biological Engineering :

 It is a branch of engineering that involves biotechnologies and biological science. It includes different disciplines such as biochemical engineering, biomedical engineering, bio-process engineering, biosystem engineering, etc.

Classification of Bacteria Based on Temperature Requirement


 The temperature at which the growth of organisms is maximum and most rapid is called the optimum growth temperature. The range of temperature between which the organisms can grow is called the temperature range for growth. Shift in temperature on either side of optimum value retards growth. Usually the maximum temperature up to which the organisms can grow, is close to the optimum growth temperature. Whereas the minimum temperature required for growth can be much lower than the optimum value. Minimum, optimum and maximum growth temperatures are called cardinal temperatures.

   Cardinal temperatures vary greatly between microorganisms. Optima normally range from 0°C to 75°C; whereas growth can take place between -20°C to 100°C. Organisms having a narrow range of growth temperature are called stenothermal while the organisms having wide range of growth temperature are called eurythermal.

Based on the temperature requirements for growth, bacteria can be divided into three groups:

  1. Psychrophiles
  2. Mesophiles
  3. Thermophiles


The organisms able to grow at low temperature i.e. below 10°C, are called psychrophiles. They can grow even at 0°C or even lower, if the medium is not frozen due to high salt concentration. The optimum temperature for growth is 15°C or lower. The maximum temperature of growth is 20°C. There are two types of psychrophiles.

  1. Obligate or true psychrophiles :  These are the organisms which grow at 0°C or lower with optimum growth temperature 15°C or below. e.g. Vibrio marinus, Vibrio psychroerythreous.
  2. Facultative psychrophiles or psychrotroph : These are the organisms which can grow at 0°C. But optimum temperature for growth is between 20°C to 30°C. e.g. Pseudoinonas flourescens.

The physiological factors for the psychrophilic nature of organisms are not much clear. But it is observed that their ribosomes and other cellular enzymes are unstable at high temperature. Hence, they cannot grow at higher temperature. Even their membrane permeability is altered at temperature above optimum value, leading to leakage of cell material, impairment of membrane permeability and hence results in loss of viability.


  These are the organisms that can grow at moderate temperatures of incubation, i.e. within range of 25°C to 40°C. Their optimum growth temperature falls within this range. All pathogenic bacteria for humans and warm blooded animals grow best at body temperature, i.e. at 37°C.


The organisms capable of growing best at temperature above 45°C are called thermophiles. They can be grouped into two categories.

1) Facultative thermophiles

These organisms can grow even in the mesophilic range of temperature.

2) Stereothermophiles or hyperthermophiles

  These bacteria cannot grow in the mesophilic range of temperature. They grow well between temperatures 45°C to 70°C. There are some organisms which can grow even at 110°C. These bacteria are also referred to as strict or obligate thermophiles. These bacteria are normally found from hot water springs, salt lakes etc.
e.g. Bacillus coaggulans, B. stereothermophilus. Thermus aquaticus can grow even at 100°C.

The basis of thermal resistance of these bacteria is the thermal stability of most of their cellular proteins.

Fermentation : Defination, Principle and Batch Fermentation Method

 The process of fermentation involves biochemical activity of organisms during their growth, development, reproduction, senescence, and death. Fermentation technology employs organisms to produce food, pharmaceuticals, and alcoholic beverages in industries on a large scale.

Principle of Fermentation

The principle involved in industrial fermentation technology is that organisms are grown under optimum conditions and are provided with raw materials and other necessary requirements like carbon, nitrogen, salts, trace elements, and vitamins. The end products formed due to their metabolism during their life span are released into the media. These end products are extracted by human beings as they are commercially valuable.

Some major end products of fermentation produced on a large scale industrial basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete proteins, enzymes, and other beneficial products.

Batch Fermentation

In batch fermentation process, the microorganisms are inoculated in a fixed volume of batch culture medium. The organisms during their growth consume the nutrients, and the growth products (i.e., biomass and metabolites) start accumulating. Since the nutrient environment within the Fermenter is continuously changed, the rate of cell metabolism also changes, and ultimately, cell multiplication stops due to limitation of nutrients and accumulation of toxic excreted waste products.

Growth Characteristics in a Batch Culture of a Microorganism. 1) Lag Phase, 2) Transient Acceleration, 3) Exponential Phase, 4) Deceleration Phase, 5) Stationary Phase, 6) Death Phase

There is the complex nature of batch growth of microorganisms. In the initial lag phase, no apparent growth is observed; however, biochemical analyses show metabolic turnover signifying that the cells are acclimatising to the environmental conditions and will start growing. Then comes the transient acceleration phase when the inoculum begins to grow. This phase is quickly followed by the exponential phase where the organisms are growing at fastest rate as the nutrients are in excess, environmental conditions are optimum and growth inhibitors are absent.

In the batch fermentation process, the exponential growth occurs for a limited period. With the change in nutrient conditions, the growth rate decreases and begins deceleration phase followed by the stationary phase at which the growth sto completely because of nutrient exhaustion. The death phase when the grow rate has come to an end is the final phase of the cycle. Mostly t biotechnological batch processes are stopped before this stage because decreasing metabolism and cell lysis.

Advantages of Batch Fermentation

  1. It Requires less space.
  2. It Can be easily handled, and
  3. There is Less chances of contamination.

Disadvantages of Batch Fermentation

  1. It is time consuming method.
  2. It requires more time for cleaning, sterilisation, and cooling.
  3. Product yield is low.

Applications of rDNA Technology and Genetic Engineering in Medicine

 Genetic engineering has an important role to play in the production of medicines. Microorganisms and plant-based substances are used for producing various drugs, vaccines, enzymes, and hormones at low costs. Genetic engineering involves the study of inheritance pattern of diseases in man and collection of human genes that provide a complete map for inheritance of healthy individuals.


  Recombinant DNA technology is used for producing vaccines against diseases by isolating antigen or protein present on the surface of viral particles. Vaccines contain a form of an infectious organism that does not cause disease but allow the body immune system to form antibodies against the infective organism.

   When an individual receives vaccination against any viral disease, the antigens produce antibodies to act against and inactivate the viral profeins. The scientists with the help of recombinant DNA technology have transferred the genes for some viral sheath proteins to vecinia virus which was used against small pox. Vaccines produced by gene cloning are non-contaminated and safe as they contain only coat proteins against which antibodies are produced. Vaccines against viral hepatitis influenza, herpes simplex virus, virus-induced foot and mouth disease in animals are being produced by gene cloning.


  Insulin was commercially produced in 1982 through biogenetic or recombinant DNA technology. Its medicinal use was approved by Food and Drug Administration (FDA) of the USA in the same year. The human insulin gene has been cloned in large quantities in E. coli bacterium that can be used for synthesising insulin. Humilin is the commercially available genetically engineered insulin.


Lymphokines are proteins regulating the immune system in the human body. u. Interferon is an example of lymphokines that are used to fight viral diseases (such as hepatitis, herpes, and common cold) and cancer. Such drugs can be manufactured in large quantities in bacterial cells. Lymphokines are also helpful in AIDS. Interleukin-II is a commercially available genetically engineered substance that stimulates the multiplication of lymphocytes.


  Somatostatin is used in some of the growth related abnormalities. This drug appears to be species specific and the polypeptide obtained from other mammals has no effect on humans, hence it is extracted from the hypothalamus of cadavers. Genetic engineering has helped in the chemical synthesis of gene which is joined to the PBR 322 plasmid DNA and cloned into a bacterium. This transformed bacterium is converted into a somatostatin synthesising factory.

Production of Blood Clotting Factors

  Blockage of coronary arteries by cholesterol or blood clots causes heart attack. Plasminogen is a substance found in blood clots. Genetically engineered tissue Plasminogen Activator (tPA) enzyme is used to dissolve these clots in individuals who have suffered heart attacks.


  Antibodies cloned from a single source and targeted for a specific antigen (monoclonal antibodies) have proved useful in the treatment of cancer. Monoclonal antibodies have been targeted with radioactive elements of cytotoxins (e.g., Ricin from castor seed) to make them more deadly. Such antibodies seek cancer cells and kill them with their radioactivity or toxin.