12th Class Biology Biotechnology : Principles And Processes Biotechnology

Study of use of living organisms and the substances produced due to their activities is called biotechnology.

Mostly micro-organisms are used in many industries as alcohol, enzymes, vaccines, vitamins, antibiotics, organic acids, etc. So biotechnology is controlled use of microbes for benefit of human beings. In this science, principles of biochemistry, molecular biology and microbiology are mostly used. Some important examples of biotechnology products are :

Alcohol : It is the result of yeast fermentation, which is the incomplete oxidation of complex organic compounds with the help of enzymes produced by yeast (Invertase and Zymase)

\[\underset{(\text{Sucrose})}{\mathop{{{C}_{12}}{{H}_{22}}{{O}_{11}}}}\,+{{H}_{2}}O\underset{\text{Invertase}}{\mathop{\xrightarrow{\text{Yeast}}}}\,\underset{(\text{Glucose})}{\mathop{{{C}_{6}}{{H}_{12}}{{O}_{6}}}}\,+\underset{(\text{Fructose})}{\mathop{{{C}_{6}}{{H}_{12}}{{O}_{6}}}}\,\]

\[{{C}_{6}}{{H}_{12}}{{O}_{6}}\underset{\text{Zymase}}{\mathop{\xrightarrow{\text{Yeast}}}}\,\underset{(\text{Ethyl}\,\text{alcohol})}{\mathop{2{{C}_{2}}{{H}_{5}}OH}}\,+2C{{O}_{2}}\]

In the middle of the 19^th century, Louis Pasteur reported that alcohol, beer and butter milk are the result of yeast fermentation. Yeast fermentation is the basis of baking as well as brewing industries, besides preparation of fermented foods like idli, dosa etc.

Types of yeasts

(1) Baker’s yeast : These include the selected strains of Saccharomyces cerevisiae and Torulopsis utilis grown on molasses. They are available in the form of powder or cakes. These yeasts have ability to ferment the sugar in the dough. The carbon dioxide produced during fermentation is responsible for the leavening (i.e., raising the dough). Both \[C{{O}_{2}}\] and alcohol escape during baking so that the bread and other baked products become porous and soft. The baker's yeast is manufactured by growing the 'stock' strain on molasses and then collected after centrifugation and washing. These are used to flavour the food, as nutrient ingredients, to ferment and raise dough in bread making (leavening agent).

(2) Brewer’s yeast : Brewing industry produces alcoholic beverages of several types depending upon the fermenting agent and the medium. Fermenting agents are Saccharomyces cerevisiae, S. sake, S. ellipsoidens (wine yeast) and S. pireformis (ginger yeast) is a saprophytic unicellular ascomycetes fungus. Molasses is dark coloured syrup left after extraction of sugar. It still contains 30% sucrose and about 32% invert sugar (mixture of glucose and fructose).

Some other common products of yeast fermentation are :

Beer : It is produced from Hordeum vulgare (barley) malt and alcohol content is 4-8%.

Wine : Produced from grapes by fermentation and alcohol content is 10-20%.

Brandy : Produced by distillation of wine and alcohol content is 43-57%.

Gin : Produced from fermented European rye, i.e., Secale cereale.

Rum : Produced from molasses of sugarcane and alcohol content is 40%.

Industrial production of alcohol : Although industrial fermentations to produce alcohol are performed almost exclusively with S. cerevisiae, the bacterium Zymomonas mobilis is a potentially useful organism for the commercial production of alcohol. Zymomonas can permit glucose, fructose and sucrose, thus producing relatively high yields of alcohol. 

Organic acids : Microbes are useful in the manufacture of a number of organic acids as follows :

(1) Acetic acid (Vinegar) : It is most important acid being produced by two step fermentation of sugarcane juice by yeast and Acetobacter aceti bacteria.

\[{{C}_{6}}{{H}_{12}}{{O}_{6}}\underset{\text{Anaerobic}}{\mathop{\xrightarrow{\text{Yeast}}}}\,\underset{\text{(Ethyl}\,\text{alcohol)}}{\mathop{2{{C}_{2}}{{H}_{5}}OH+2C{{O}_{2}}}}\,\]

\[{{C}_{2}}{{H}_{5}}OH+{{O}_{2}}\underset{Aerobic}{\mathop{\xrightarrow{Acetobacter\,aceti}}}\,\underset{\text{(Acetic}\,\text{acid)}}{\mathop{C{{H}_{3}}COOH+{{H}_{2}}O}}\,\]

(2) Lactic acid : It was the first organic acid to be produced from microbial fermentation of lactose (milk sugar). Fermenting agents are bacteria, e.g., Streptococcus lactis and Lactobacillus and Fungi, e.g., Rhizopus. Lactic acid is used in confectionery, fruit juices, essence, pickles, canned vegetables and fish products.

\[\underset{\text{Lactose}}{\mathop{{{C}_{12}}{{H}_{22}}{{O}_{11}}}}\,+{{H}_{2}}O\xrightarrow{S.\,lactis}\underset{\text{Lactic}\,\text{acid}}{\mathop{4C{{H}_{3}}CHOHCOOH}}\,\]

(3) Citric acid : It is obtained by the fermentation of sugar syrup by Aspergillus niger and Mucor species. It is used in dyeing, engraving, medicines, flavouring and preservation of food and candies.

(4) Gluconic acid : This acid is produced, during glucose oxidation by most Aspergillus sps. and many species of other fungal genera like Penicillium, Mucor, Fusarium, Pullutaria, etc.

It is used in pharmaceuticals, food, infant feed, detergent textile, leather, photographic and concrete industries.

(5) Fumaric acid : This is obtained by the activity of Rhizopus nigricans on sugar.

Enzymes : The term 'enzyme' was coined by William Kuhne in 1876. Buchner (1897) found that living cells of yeast are not required for alcoholic fermentation, but their extract also causes fermentation. In dialysis process, which is used to remove small molecules from enzymes. Enzymes are the organic substances which enhance the rate of biochemical reactions (without themseleves undergoing any change). Enzymes are also called biological catalysts or biocatalysts. Enzymes can also be used outside the cell to catalyse some specific biochemical reactions to manufacture many industrial products. Out of total over 2200 enzymes known today, only a few are used in industries, medicines and food.

Enzymes obtained by microbial activity

(1) Proteases or proteolytic enzymes : Obtained from Aspergillus oryzae and Bacillus subtilis and are used in detergents to remove some proteinaceous stains on clothes.

(2) Amylases : Obtained from Bacillus subtilis, Aspergillus niger, A. oryzae etc., and are used in beer and bread making. Also used for softening starched clothes.

(3) Lactase : Obtained from Saccharomyces fragilis and Torula cremoris. Lactases convert lactose (milk sugar) into lactic acid which is responsible for coagulation of milk protein (casein).

(4) Rennin (Rennet) : Danish chemist Christian Hensen (1874) first obtained it from calf stomach and used for cheese formation.

(5) Invertase : Obtained from Saccharomyces cerevisiae and is used to prevent sandiness in dairy products.

(6) Pectinases : Obtained from Aspergillus lucherisis and used for destruction of penicillin action.

(7) Cellulase : Obtained from Myrothecium verrucaria and used in production of dextrin and fructose.

Role of enzyme in medicine

(1) Thrombin : Obtained from beef plasma and is used for blood clotting in surgical operations.

(2) TPA (Tissue Plasminogen Activator) : This enzyme is specifically used in heart patients to dissolve blood clots. Some enzymes are also used to modify protein molecules. Pig insulin has been converted to human insulin (or humulin) by the action of enzymes.

(3) Papain : This protein digesting enzyme obtained from latex of papaya, is used for making meat tender.

(4) Protein modifying enzymes : Some enzymes are known to modify proteins for human used e.g., conversion of pig insulin into human insulin and called Pseumcelin.

Vitamins : Vitamins are the organic compounds provided to the body with diet and are required for some biochemical processes in very small amounts. First vitamin was discovered by Casimir Funk in (1911) when he succeeded in isolating vitamin B₁ (thiamine), the deficiency of which causes beri beri. This discovery was followed by the discovery of vitamin A by Mc Collum, vitamin D by Mellanby and vitamin C by Albert Van Szent Gyorgy. The first vitamin which was produced during fermentation was vitamin C. This fermentation was carried out by using a wild bacterium. Some important examples of microbes-produced vitamins are :

(1) Riboflavin (Vit.\[{{B}_{2}}\]) : It is synthesized for industrial purposes by fermentation with a filamentous yeast, Ashbya gossypii. It was produced for the first time in 1938 using the microbial fermentation. The main sources of Vit. B₂ are cereals, yeast, leafy vegetables, milk etc. It is precursor of FMN and FAD. Which act as coenzymes for dehydrogenases enzymes. It is a bitter, crystalline, odourless, yellowish brown substance essential for the growth and reproduction in animals.

(2) Cobalamine/Cyanocobalamin (Vit.\[{{B}_{12}}\]) : This vitamin is of animal protein origin like liver, meat, fish. It is not present in vegetarian diet and its deficiency causes pernicious anemia. This vitamin was isolated for the first time in 1948 from liver extract and as a by product during production of antibiotics by fermentation. The first commercial fermentation production of vitamin B₁₂ was as a by products of fermentation for streptomycin and acetone-butanol fermentation. Nowadays this vitamin is being produced by fermentation using Pseudomonas denitrificans, Bacillus coagulans, B.megatherium and propionic acid bacteria, i.e., Propionibacteria.

(3) Ascorbic acid (Vitamin C) : It is produced by a complicated modification of glucose by different species of Acetobacter. Acetobacter is helpful in dehydrogenation of
D-Sorbital and its conversion to L-Sorbose. The lattes is precursor of vitamin (or L-ascorbic acid).

Dextrins : These are soluble polysaccharides formed by simple sugars through the agency of micro-organisms like Leuconostoc mesenteroides bacteria or its enzyme called dextran sucrose. Fermentation is allowed to proceed till dextrins are obtained. Dextrans represent the 6-10% solution of dextrins. It is used in plasma transfusions.

Tissue culture : This is also a latest method of crop improvement. Besides also used in manufacture of antibiotics, alkaloids and dyes (like Shikonin, a red dye used in silk-industry and in treatment of burns is obtained from culture of cells of Lithospermum root).

Antibiotics : These are the substances of microbial origin and having antimicrobial activity or these are the chemical substances secreted by one micro-organism, which inhibit or check the growth of other micro-organisms. Antibiotics are secondary metabolites and therefore, medium is so selected that the organism is diverted to its secondary metabolism.

First antibiotic discovered was Penicillin (wonder drug), from Penicillium notatum by Sir Alexander Fleming (1928). However the term "antibiotic" was given by Waksman (1942). Waksman himself discovered two antibiotics i.e., Actinomycin (1941) and Streptomycin (1942). Main sources of antibiotics are fungi, bacteria and actinomycetes and about 7,000 antibiotics are known at this time. There are two main groups of antibiotics :

(1) Broad spectrum antibiotics are those which inhibit the growth of many pathogenic species, differing from one another in their structure.

(2) Narrow spectrum antibiotics are those which inhibit the growth of a specific pathogenic species. Different antibiotics have different modes of action, e.g., by destruction of cell membrane, inhibition of cell wall synthesis or checking protein synthesis or inhibition of nucleic acid synthesis.

Antibiotic produced by micro-organisms

Steroids : Steroids are high molecular weight complex crystallisable fatty compounds. Which are having one 5-carbon ring and three 6-carbon rings. Steroids are of wide distribution in both plants and animals. Cholesterol is one of the most important steroids of animals, which is a precursor of animal hormones (steroid hormones) like progesterone and oestrogen (female sex hormones) and testosterone (male sex hormone).

Steroids are nowadays used as valuable drugs, e.g., in treatment of hormonal disturbances, for suppressing immune response in auto immune diseases, used in formation of birth control pills or contraceptive pills (oestrogens and progesterones) and anit-inflammatories.

Rhizopus stolonifer is capable of hydroxylation, necessary for steroid synthesis. (Murray and Peterson, 1950).

Progesterone is precursor of 4 different useful steroids and the micro-organisms used in this conversion are Rhizopus arrhizus, R. stolonifer (R. nigricans), Curvularia lunata (all fungi) and a bacterium Streptomyces argenteolus.

Vaccines : The vaccines are preparations of infectious or toxic agents that have been altered so as not to cause disease. It is injected into an antibody producing organism to produce immunity in the body against diseases. The process of inoculation of vaccine is known as Vaccination and Louis Pasteur (1850) is responsible for understanding the basis of vaccination and immunization. (Edward Jenner 1790, however made earlier studies in relation to smallpox). A vaccine contains either weakened (polio, smallpox and measles vaccines) or even killed pathogens (typhoid vaccine) which have still antigens to induce antibody production. In some cases (like botulism and tetanus), toxins produced by pathogens serve as vaccines. A large number of vaccines (called first generation vaccines) against different viral and bacterial diseases have been produced.

In recent years, some new vaccines called second generation vaccines and third generation vaccines have been developed. Second generation vaccines produced by recombinant DNA technology or genetic engineering, e.g., vaccines are for Herpes virus and Hepatitis B. Third generation vaccines are produced synthetically or are synthesized vaccines, e.g., for feline leukemia virus and foot and mouth disease virus.

Insulin : It is a hormone secreted by \[\beta -\]cells of islets of langerhans of pancreas in human body. Insulin is a hormone of protein nature (51 amino acids), made of 2 polypeptide chains having 21 and 30 amino acids respectively and joined by disulphide bonds. Deficiency of this insulin leads to a disease called diabetes mellitus. Insulin supplied from outside can cure this disease.

Sharpy-Shafer (1916) proposed that diabetes is due to failure pancreas to secrete a chemical named insulin. Banting and Best (1921) first of all isolated insulin from dog's pancreas and used it for curing diabetes patients. Insulin is now obtained from pancreas of slaughtered pigs and cattle. This insulin slightly differs from man's insulin and effectively controls diabetes.

By using genetic engineering or recombinant DNA technology, insulin producing genes from human beings have been transferred into E.coli bacteria, which produce insulin called 'Humulin' for clinical use. This type of synthetic insulin was produced by an American pharmaceutical firm Eli-Lily on July 5, 1983.

Monoclonal antibodies : Monoclonal antibodies (Mabs) are the specialized antibodies, which are specific to only one type of antigen. Antibodies are produced in lymph nodes, spleen and liver. Antibodies are often required for research in medicine. The concept of monoclonal antibodies was put forward by Georges Kohler and Cesal Milstein in 1974 and these antibodies are produced outside the body by hybrid cell culture technique, known as Hybridoma technology. For this technology Kohler and Milstein were awarded Nobel Prize in 1984. Lymphocytes and myeloma cells fuse together to form hybridoma cells (by somatogamous fusion).

These hybridoma cells have capacity of antibody production (of lymphocytes) and rapid cell division (of cancer cells). These hybridoma cells in cultural conditions produce large quantities of specific and pure (monoclonal) antibodies, which are separated and used in cure of different diseases.

Yoghurt : Yoghurt is a form of sour milk. It is a special diet, which is prepared by fermentation of concentrated milk first with Lactobacillus bulgaricus and Streptococcus thermophilus at 40–46°C for 4 hrs and then fermentation with yeast. America produces 75 Lac kg of yoghurt yearly.

Interferons : Interferons (antiviral proteins) were produced by Charles Weismann of Zurich University through recombinant DNA technology in E. coli in 1980.

Amino acids : An important basic amino acid, lysine is produced from diaminopimelic acid (constituent of cell wall of bacteria, e.g., E.coli) by activity of bacterium Enterobacter aerogens.

Cheese : Danish chemist Christian Hensen (1874) first obtained Rennet from calf stomach and used for cheese formation.

It is made by separating casein and fat of milk form the liquid. About 400 kinds of cheese are known which can be made from many types of milk (goats, cows, buffaloes or camels) by employing microbes under favourable conditions.   

Cheese are following types

(1) Ripened Cheese : Blocks of cottage cheese are salted and placed in brine solution for a few days. Pressing helps in removing whey. Salting removes moisture and checks growth of microbes. The salt is now wiped and the cheese allowed to ripen for \[1-16\] months. Ripening is carried out with the help of different strains of bacteria and fungi. Roquefort Cheese, Camembert Cheese, Limburger Cheese, Swiss Cheese. Temperature is kept low during ripening. It provides a particular consistency and flavour to the cheese.

(2) Unripened/Cottage Cheese : Pasteurised skimmed milk is inoculated with cheese culture. Within 1-2 hours, rennet is added. Formerly, stomach of sheep and goat (lining has rennin) or sap of fig trees (enzyme ficin) was used. Fruit extract of Withania coagulans is also effective. Curd is placed in cloth lined porous containers for draining the whey. The left out solidified material is called cottage cheese. It can be consumed as such. Whey contains lactic acid, lactose and minerals.

(3) Soft cheese has moisture content of more than 40%.

(4) Hard cheese has lower moisture content (39% or less)

(5) Processed cheese is formed by melting, blending and emulsifying one or more types of cheese.

(6) Raw Cheese : Milk is curdled with the help of cheese culture Streptococcus lactis, S. cremoris, Leuconostoc citrovorum, etc. Curd is heated gently to separate cheese from liquid called whey. Any liquid left in cheese is allowed to drain by hanging it in cloth. It is salted and mixed with cream for marketing. Raw cheese has short shelf life.

Butter : Butter contains 80% fat, 2-6% lactose and proteins and often 2% salt. Butter is made by churning cream until the fatty globules of butter separate from the liquid buttermilk. The cream is inoculated with mixed cultures of Streptococcus lactis, S. diacetylactic and species of Leuconostoc citrovorum and L. dextranicum.

Biosynthesis of somatostatin : This hormone is secreted from front lobe of pitutary gland. It is made up of 191 amino acid units. Ross isolated first this hormone. The individual suffers dwarfness due to its deficiency. The gene or DNA of this hormone is introduced in E.coli, near the gene which codes for
\[\beta -\]galactosidase.