Topic Covered!
History, scope and importance of Biotechnology; Specializations in
Agricultural Biotechnology: Genomics, Genetic engineering, Tissue Culture,
Bio-fuel, Microbial Biotechnology, Food Biotechnology ete. Basics of
Biotechnology, Primary metabolic pathways, Enzymes and its activities.
History of Biotechnology
Definition
“Biotechnology is any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.” Convention on Biological Diversity (CBD), 1992
- Biotechnology: Integration of natural sciences and engineering to achieve applications of organisms, cells, parts thereof, and molecular analogues for products and services
- Term coined by Karl Ereky (1919) - Hungarian engineer
- Modern definition by EFB (European Federation of Biotechnology): Integration of biochemistry, microbiology, and engineering sciences
Historical Timeline
8000 BC
Fermentation used by Sumerians and Babylonians for brewing beer
6000 BC
Egyptians used yeast for baking bread; cheese and yogurt production
500 BC
Chinese used moldy soybean curds as antibiotics
1665
Robert Hooke discovered cells
1857
Louis Pasteur discovered microbial fermentation
1865
Gregor Mendel established principles of heredity
1919
Karl Ereky coined term "Biotechnology"
1953
Watson & Crick discovered DNA double helix structure
1973
Stanley Cohen & Herbert Boyer created first recombinant DNA organism
1983
Kary Mullis invented PCR (Polymerase Chain Reaction)
1994
First genetically modified food (Flavr Savr tomato) approved by FDA
2003
Human Genome Project completed
2012
CRISPR-Cas9 gene editing technology developed
Scope of Biotechnology
Color-Coded Branches of Biotechnology
| Color Code | Branch | Applications |
|---|---|---|
| 🟢 Green | Agricultural Biotechnology | GM crops, biofertilizers, biopesticides, tissue culture |
| 🔴 Red | Medical Biotechnology | Pharmaceuticals, vaccines, gene therapy, diagnostics |
| 🔵 Blue | Marine/Aquatic Biotechnology | Aquaculture, marine drugs, biofuels from algae |
| ⚪ White | Industrial Biotechnology | Enzymes, biofuels, bioplastics, bioprocessing |
| 🟤 Brown | Environmental Biotechnology | Bioremediation, waste treatment, pollution control |
| 🟡 Yellow | Nutritional Biotechnology | Functional foods, nutraceuticals, food processing |
| ⚫ Grey | Biodefense | Bioterrorism detection, biosecurity |
| 🟣 Purple | Patent & IP Biotechnology | Patent law, intellectual property rights |
Major Application Areas
- Healthcare: Production of insulin, growth hormones, monoclonal antibodies, vaccines
- Agriculture: Development of Bt crops, herbicide-resistant crops, Golden Rice
- Industrial: Enzyme production, biofuel generation, biodegradable plastics
- Environmental: Bioremediation of polluted sites, sewage treatment
- Forensics: DNA fingerprinting, paternity testing
- Food Technology: Probiotics, fermented foods, improved nutritional content
Importance of Biotechnology
- Food Security: Increased crop yield, drought-resistant varieties, pest-resistant crops
- Healthcare Advancement: Personalized medicine, targeted drug delivery, gene therapy
- Environmental Sustainability: Reduction in chemical usage, biodegradable alternatives, clean energy
- Economic Growth: Job creation, export opportunities, industrial development
- Disease Management: Early diagnosis, effective treatments, preventive measures
- Resource Conservation: Efficient use of water, soil, and nutrients
- Climate Change Mitigation: Carbon sequestration, biofuels reducing fossil fuel dependency
Agricultural Biotechnology
Definition & Scope
- Application of scientific tools and techniques (including genetic engineering, molecular markers, molecular diagnostics, vaccines, and tissue culture) to modify living organisms
- Aims to improve plants, animals, and microorganisms for food, fiber, and fuel production
- Encompasses traditional breeding techniques and modern molecular biology methods
🌾 Agricultural Biotechnology Workflow
Problem Identification
(Pest attack, drought, low yield, nutritional deficiency)
(Pest attack, drought, low yield, nutritional deficiency)
Gene Identification
(Find gene responsible for desired trait)
(Find gene responsible for desired trait)
Gene Isolation & Cloning
(Extract and multiply the gene)
(Extract and multiply the gene)
Vector Construction
(Insert gene into plasmid/Ti plasmid)
(Insert gene into plasmid/Ti plasmid)
Transformation
(Introduce into plant cells via Agrobacterium/gene gun)
(Introduce into plant cells via Agrobacterium/gene gun)
Selection & Regeneration
(Select transformed cells, grow into plants)
(Select transformed cells, grow into plants)
Testing & Field Trials
(Laboratory → Greenhouse → Contained field → Open field)
(Laboratory → Greenhouse → Contained field → Open field)
Regulatory Approval & Commercialization
(Safety assessment, release for cultivation)
(Safety assessment, release for cultivation)
Key Techniques in Agricultural Biotechnology
- Genetic Engineering: Insertion of foreign genes to express desired traits
- Tissue Culture: Micropropagation, somaclonal variation, protoplast fusion
- Molecular Markers: RFLP, RAPD, SSR, SNP for marker-assisted selection (MAS)
- Genetic Transformation Methods:
- Agrobacterium-mediated transformation (for dicots)
- Biolistics/Gene gun (for monocots)
- Electroporation
- Microinjection
- CRISPR-Cas9: Precise gene editing for crop improvement
- RNA Interference (RNAi): Gene silencing for pest/disease resistance
Major GM Crops & Their Traits
| Crop | Gene/Trait | Benefit |
|---|---|---|
| Bt Cotton | Cry1Ac, Cry2Ab genes from Bacillus thuringiensis | Resistance to bollworm |
| Bt Brinjal | Cry1Ac gene | Resistance to fruit and shoot borer |
| Golden Rice | psy, crtI genes for β-carotene synthesis | Vitamin A enrichment |
| Flavr Savr Tomato | Antisense PG gene | Delayed ripening, longer shelf life |
| Roundup Ready Soybean | EPSPS gene | Herbicide (glyphosate) tolerance |
| Rainbow Papaya | PRSV coat protein gene | Resistance to papaya ringspot virus |
Applications in Agriculture
- Crop Improvement:
- Increased yield potential
- Enhanced nutritional quality (biofortification)
- Improved shelf life
- Better taste and appearance
- Stress Tolerance:
- Drought tolerance (DREB genes)
- Salinity tolerance (LEA proteins)
- Heat and cold tolerance
- Heavy metal tolerance
- Pest & Disease Resistance:
- Insect resistance (Bt toxins)
- Viral resistance (coat protein, RNAi)
- Fungal resistance (chitinase, glucanase)
- Bacterial resistance (Xa21 gene in rice)
- Herbicide Tolerance:
- Glyphosate tolerance (EPSPS gene)
- Glufosinate tolerance (bar gene)
- Plant as Biofactories:
- Molecular pharming (production of pharmaceuticals)
- Production of industrial enzymes
- Biofuel production
Molecular Breeding Approaches
- Marker-Assisted Selection (MAS): Using DNA markers linked to desired traits for selection
- Marker-Assisted Backcrossing (MABC): Introgression of specific genes while maintaining genetic background
- Genomic Selection (GS): Using genome-wide markers for prediction of breeding values
- QTL Mapping: Identifying chromosomal regions affecting quantitative traits
- Association Mapping/GWAS: Linking phenotypic variation to genetic variation
🎯 Important Points for CSIR NET & ICAR SRF
- CSIR NET Karl Ereky coined "Biotechnology" in 1919
- ICAR SRF First GM food approved by FDA: Flavr Savr Tomato (1994)
- CSIR NET Bt cotton contains Cry1Ac and Cry2Ab genes from Bacillus thuringiensis
- ICAR SRF Golden Rice is enriched with β-carotene (Vitamin A precursor)
- CSIR NET Agrobacterium tumefaciens is used for dicot transformation, Gene gun for monocots
- ICAR SRF CRISPR-Cas9 discovered by Jennifer Doudna and Emmanuelle Charpentier (2012)
- CSIR NET Human Genome Project completed in 2003
- ICAR SRF PCR invented by Kary Mullis (1983)
- CSIR NET India's only approved GM crop for cultivation: Bt Cotton
- ICAR SRF Marker-Assisted Selection (MAS) increases efficiency and speed of breeding programs
Introduction to Genomics
Definition & Scope
- Genomics: Study of the complete set of DNA (genome) of an organism including all genes and non-coding sequences
- Term coined by Thomas Roderick (1986)
- Involves sequencing, mapping, and analyzing genomes
- Differs from genetics (studies individual genes) - genomics studies all genes collectively
Types of Genomics
| Type | Focus | Applications |
|---|---|---|
| Structural Genomics | Physical nature of genomes (DNA sequence, chromosome structure) | Genome mapping, sequencing projects |
| Functional Genomics | Function and expression of genes | Transcriptomics, proteomics, metabolomics |
| Comparative Genomics | Comparing genomes of different species | Evolutionary studies, gene identification |
| Metagenomics | Genomic study of environmental samples | Microbial community analysis |
| Pharmacogenomics | How genes affect drug response | Personalized medicine |
Key Technologies
- Next-Generation Sequencing (NGS):
- Illumina sequencing (most widely used)
- Ion Torrent sequencing
- 454 Pyrosequencing
- SOLiD sequencing
- Third-Generation Sequencing:
- PacBio (Pacific Biosciences) - Single Molecule Real-Time (SMRT)
- Oxford Nanopore - reads very long DNA fragments
- Microarray Technology: Gene expression profiling
- Bioinformatics Tools: BLAST, Clustal, MEGA, etc.
🧬 Human Genome Project (HGP) Timeline
1990 - Project Initiated
Goal: Sequence all 3 billion base pairs of human DNA
Goal: Sequence all 3 billion base pairs of human DNA
2000 - Draft Sequence Released
90% of genome sequenced
90% of genome sequenced
2003 - Project Completed
99.99% accuracy achieved
99.99% accuracy achieved
Key Findings
~20,000-25,000 genes | 98% non-coding DNA | Humans share 99.9% DNA similarity
~20,000-25,000 genes | 98% non-coding DNA | Humans share 99.9% DNA similarity
🎯 Important for Exams
- CSIR NET Human genome has approximately 3.2 billion base pairs
- ICAR SRF Only ~2% of human genome codes for proteins
- CSIR NET Smallest genome: Mycoplasma genitalium (580 kb)
- ICAR SRF Largest plant genome: Paris japonica (150 billion bp)
- CSIR NET First complete genome sequenced: Haemophilus influenzae (1995)
Genetic Engineering
Definition & Principles
- Genetic Engineering: Direct manipulation of an organism's genes using biotechnology
- Also called Recombinant DNA Technology
- Involves isolation, modification, and insertion of genes into host organisms
- Produces Genetically Modified Organisms (GMOs) or Transgenic organisms
Tools of Genetic Engineering
| Tool | Function | Examples |
|---|---|---|
| Restriction Enzymes | Molecular scissors - cut DNA at specific sites | EcoRI, BamHI, PstI, HindIII |
| DNA Ligase | Molecular glue - joins DNA fragments | T4 DNA ligase, E. coli DNA ligase |
| Vectors | Carrier DNA molecules | Plasmids, Bacteriophages, Cosmids, BAC, YAC |
| DNA Polymerase | Synthesizes new DNA strands | Taq polymerase (PCR), Pfu polymerase |
| Host Cells | Recipient organisms | E. coli, Yeast, Mammalian cells |
🔬 Steps in Genetic Engineering
1. Identification of Desired Gene
Select gene with desired trait
Select gene with desired trait
2. Isolation of Gene
Use restriction enzymes to cut gene from donor DNA
Use restriction enzymes to cut gene from donor DNA
3. Gene Cloning
Amplify gene using PCR
Amplify gene using PCR
4. Vector Preparation
Cut vector (plasmid) with same restriction enzyme
Cut vector (plasmid) with same restriction enzyme
5. Ligation
Join gene with vector using DNA ligase → Recombinant DNA
Join gene with vector using DNA ligase → Recombinant DNA
6. Transformation
Insert recombinant DNA into host cell
Insert recombinant DNA into host cell
7. Selection
Identify and select transformed cells (antibiotic resistance)
Identify and select transformed cells (antibiotic resistance)
8. Expression
Desired protein produced by host cell
Desired protein produced by host cell
Types of Restriction Enzymes
- Type I: Cut DNA at random sites away from recognition sequence
- Type II: Cut DNA at specific recognition sites (most used in genetic engineering)
- Type III: Cut DNA about 25 bp away from recognition site
- Type IV: Recognize and cut modified DNA
Sticky vs Blunt Ends
Sticky Ends (Cohesive Ends)
- Staggered cuts with overhanging sequences
- Easy to ligate due to complementary base pairing
- Example: EcoRI, BamHI
- Preferred for cloning
Blunt Ends
- Straight cuts with no overhangs
- More difficult to ligate
- Example: SmaI, EcoRV
- Can join any blunt-ended DNA
Gene Transfer Methods
- Transformation: Uptake of naked DNA by bacterial cells (CaCl₂ method, heat shock)
- Transfection: Introduction of DNA into eukaryotic cells
- Transduction: Transfer via bacteriophages
- Electroporation: Using electric pulses to create pores in cell membrane
- Microinjection: Direct injection into nucleus
- Biolistics/Gene Gun: Bombarding cells with DNA-coated gold/tungsten particles
- Liposome-mediated: DNA packaged in lipid vesicles
Applications
- Medicine: Production of insulin, growth hormone, interferons, vaccines
- Agriculture: Bt crops, herbicide-resistant crops, drought-tolerant varieties
- Industrial: Production of enzymes, biofuels, biodegradable plastics
- Research: Gene function studies, disease models
- Environmental: Bioremediation organisms
Tissue Culture
Definition & Principles
- Tissue Culture: Growth of cells, tissues, or organs in artificial nutrient medium under sterile conditions
- Based on Totipotency - ability of a single cell to divide and develop into complete organism
- First demonstrated by Gottlieb Haberlandt (1902) - Father of Plant Tissue Culture
- First successful plant tissue culture: White (1934) - cultured tomato roots
Types of Plant Tissue Culture
| Type | Explant Used | Purpose |
|---|---|---|
| Callus Culture | Any plant part | Undifferentiated cell mass production |
| Cell Suspension Culture | Callus cells | Secondary metabolite production |
| Organ Culture | Root, shoot, flower | Studying organ development |
| Protoplast Culture | Cell without wall | Somatic hybridization, genetic transformation |
| Anther/Pollen Culture | Anthers/Pollen grains | Haploid plant production |
| Embryo Culture | Embryo | Overcome seed dormancy, hybrid embryo rescue |
| Meristem Culture | Shoot apex (0.1-0.3 mm) | Virus-free plant production |
Components of Culture Medium
- Macronutrients: N, P, K, Ca, Mg, S (in mM concentrations)
- Micronutrients: Fe, Mn, Zn, Cu, B, Mo, Co, I (in μM concentrations)
- Carbon source: Sucrose (2-3%), Glucose
- Vitamins: Thiamine (B₁), Nicotinic acid, Pyridoxine (B₆), Myoinositol
- Plant Growth Regulators:
- Auxins: IAA, NAA, 2,4-D, IBA (promote root formation, callus induction)
- Cytokinins: BAP, Kinetin, Zeatin (promote shoot formation, cell division)
- Gibberellins: GA₃ (promote elongation)
- Gelling agent: Agar (0.8-1%), Gelrite
- pH: 5.5-5.8
Common Culture Media
- MS Medium (Murashige and Skoog, 1962) - Most widely used, high salt concentration
- B5 Medium (Gamborg, 1968) - Lower salt, good for cell suspension
- White's Medium (1943) - Low salt, for root culture
- N6 Medium (Chu, 1975) - For cereal tissue culture
- Woody Plant Medium (WPM) - For woody species
🌱 Micropropagation Process
Stage 0: Mother Plant Selection
Select healthy, disease-free plant
Select healthy, disease-free plant
Stage I: Initiation
Surface sterilization + inoculation on medium
Surface sterilization + inoculation on medium
Stage II: Multiplication
Shoot proliferation using cytokinins
Shoot proliferation using cytokinins
Stage III: Rooting
Root induction using auxins
Root induction using auxins
Stage IV: Acclimatization
Transfer to greenhouse → field conditions
Transfer to greenhouse → field conditions
Applications
- Clonal Propagation: Rapid multiplication of elite plants
- Germplasm Conservation: Long-term storage of plant genetic resources
- Disease-free Plant Production: Meristem culture eliminates viruses
- Secondary Metabolite Production: Pharmaceuticals, dyes, flavors
- Somaclonal Variation: Genetic variation for crop improvement
- Protoplast Fusion: Somatic hybridization (Pomato = Potato + Tomato)
- Haploid Production: Anther/pollen culture for breeding programs
- Synthetic Seed Production: Encapsulated somatic embryos
Biofuels
Definition & Types
- Biofuels: Fuels derived from biological sources (biomass) that can replace or supplement fossil fuels
- Renewable and potentially carbon-neutral energy sources
Generations of Biofuels
| Generation | Source | Examples | Limitations |
|---|---|---|---|
| First Generation | Food crops (sugar, starch, vegetable oil) | Bioethanol from corn/sugarcane, Biodiesel from soybean/palm | Food vs Fuel debate, land use issues |
| Second Generation | Non-food biomass (lignocellulosic materials) | Cellulosic ethanol from agricultural waste, wood chips | Complex pretreatment required, high cost |
| Third Generation | Algae and microorganisms | Algal biodiesel, microbial oils | Scale-up challenges, cultivation costs |
| Fourth Generation | Genetically modified organisms | Engineered algae/bacteria for enhanced fuel production | Regulatory concerns, GMO acceptance |
Major Types of Biofuels
- Bioethanol:
- Produced by fermentation of sugars using yeast (Saccharomyces cerevisiae)
- Sources: Sugarcane, corn, molasses, lignocellulosic biomass
- Used as gasoline substitute or blend (E10, E85)
- Formula: C₂H₅OH
- Biodiesel:
- Produced by transesterification of vegetable oils/animal fats
- Sources: Jatropha, soybean, rapeseed, palm oil, algae
- Used in diesel engines (B20, B100)
- Chemical name: Fatty Acid Methyl Esters (FAME)
- Biogas:
- Mixture of CH₄ (50-70%), CO₂ (30-40%), traces of H₂S, N₂
- Produced by anaerobic digestion of organic waste
- Microorganisms involved: Methanogens (Methanobacterium, Methanococcus)
- Used for cooking, heating, electricity generation
- Biohydrogen:
- Produced by photofermentation or dark fermentation
- Microorganisms: Clostridium, Enterobacter, Cyanobacteria
- Clean fuel - only water as byproduct
- Biobutanol:
- Superior to ethanol (higher energy content, less corrosive)
- Produced by ABE fermentation (Clostridium acetobutylicum)
🔥 Bioethanol Production Process
Feedstock Preparation
Grinding/chopping of biomass
Grinding/chopping of biomass
Pretreatment (for lignocellulose)
Acid/alkali/steam treatment to break lignin
Acid/alkali/steam treatment to break lignin
Hydrolysis/Saccharification
Enzymatic breakdown to simple sugars (cellulase, amylase)
Enzymatic breakdown to simple sugars (cellulase, amylase)
Fermentation
Yeast converts sugars to ethanol (30-35°C, 48-72 hrs)
Yeast converts sugars to ethanol (30-35°C, 48-72 hrs)
Distillation
Separation of ethanol from water (95% purity)
Separation of ethanol from water (95% purity)
Dehydration
Molecular sieves to achieve 99.5% purity
Molecular sieves to achieve 99.5% purity
Algae for Biofuel Production
- Advantages:
- High lipid content (20-50%)
- Rapid growth rate (doubles biomass in 24 hrs)
- No competition with food crops
- Can grow in wastewater/saline water
- CO₂ sequestration during growth
- Important Algae Species:
- Chlorella - high lipid content
- Botryococcus braunii - produces hydrocarbons
- Dunaliella salina - grows in high salinity
- Nannochloropsis - fast-growing, high EPA content
- Cultivation Systems:
- Open ponds/raceways - low cost, contamination risk
- Closed photobioreactors - better control, high cost
Microbial Biotechnology
Definition & Scope
- Microbial Biotechnology: Use of microorganisms and their products for industrial, agricultural, medical, and environmental applications
- Includes bacteria, fungi, algae, protozoa, viruses
Industrial Microbiology Applications
| Product | Microorganism | Application |
|---|---|---|
| Antibiotics | ||
| Penicillin | Penicillium chrysogenum | Antibacterial |
| Streptomycin | Streptomyces griseus | Tuberculosis treatment |
| Tetracycline | Streptomyces aureofaciens | Broad-spectrum antibiotic |
| Organic Acids | ||
| Citric acid | Aspergillus niger | Food acidulant, beverages |
| Lactic acid | Lactobacillus | Food preservative, bioplastics |
| Acetic acid | Acetobacter aceti | Vinegar production |
| Enzymes | ||
| Amylase | Bacillus subtilis | Starch hydrolysis, baking |
| Protease | Bacillus licheniformis | Detergents, cheese making |
| Lipase | Candida rugosa | Fat digestion, biodiesel |
| Vitamins | ||
| Vitamin B₁₂ | Pseudomonas denitrificans | Dietary supplement |
| Riboflavin (B₂) | Ashbya gossypii | Food fortification |
Microbial Fermentation
- Batch Fermentation:
- Closed system with finite amount of nutrients
- Growth phases: Lag → Log → Stationary → Death
- Harvested at end of fermentation
- Fed-Batch Fermentation:
- Nutrients added periodically
- Extends production phase
- Used for penicillin, baker's yeast
- Continuous Fermentation:
- Nutrients continuously added, products removed
- Steady-state maintained
- Used for single-cell protein, ethanol
Bioremediation
- Bioremediation: Use of microorganisms to degrade environmental pollutants
- Types:
- In situ: Treatment at contaminated site (bioventing, biosparging)
- Ex situ: Excavated material treated elsewhere (biopiles, bioreactors)
- Examples:
- Pseudomonas putida - degrades toluene, naphthalene
- Deinococcus radiodurans - radioactive waste treatment
- Alcanivorax borkumensis - oil spill cleanup
- Mycorrhizal fungi - heavy metal phytoremediation
Biofertilizers
- Nitrogen-Fixing:
- Rhizobium - symbiotic with legumes
- Azotobacter, Azospirillum - free-living
- Anabaena, Nostoc - cyanobacteria in rice fields
- Phosphate Solubilizing:
- Bacillus megaterium
- Pseudomonas striata
- Mycorrhizae:
- VAM (Vesicular Arbuscular Mycorrhizae) - Glomus species
- Enhance P, Zn uptake
Biopesticides
- Bacterial:
- Bacillus thuringiensis (Bt) - produces insecticidal crystal proteins
- Bacillus sphaericus - mosquito larvae control
- Fungal:
- Trichoderma - biocontrol of soil-borne pathogens
- Beauveria bassiana - insect pathogen
- Viral:
- NPV (Nuclear Polyhedrosis Virus) - caterpillar control
Food Biotechnology
Definition & Applications
- Food Biotechnology: Application of biological techniques to improve food production, processing, safety, and nutritional quality
Fermented Foods
| Food Product | Microorganism | Process |
|---|---|---|
| Yogurt/Curd | Lactobacillus bulgaricus, Streptococcus thermophilus | Lactic acid fermentation of milk |
| Cheese | Lactococcus, Leuconostoc | Milk coagulation + aging |
| Bread | Saccharomyces cerevisiae | Alcoholic fermentation (CO₂ causes rising) |
| Beer | Saccharomyces cerevisiae | Fermentation of malted barley |
| Wine | Saccharomyces cerevisiae | Fermentation of grape juice |
| Vinegar | Acetobacter aceti | Oxidation of ethanol to acetic acid |
| Soy sauce | Aspergillus oryzae | Fermentation of soybeans |
| Idli/Dosa | Leuconostoc mesenteroides | Fermentation of rice-lentil batter |
| Tempeh | Rhizopus oligosporus | Fermentation of soybeans |
Enzymes in Food Processing
- Amylases: Starch hydrolysis in baking, brewing, syrup production
- Proteases: Meat tenderization, cheese making, beer clarification
- Pectinases: Fruit juice clarification, extraction
- Lactase: Lactose-free milk production
- Glucose oxidase: Removing oxygen from beverages, egg products
- Rennet (Chymosin): Milk coagulation in cheese making
- Transglutaminase: Protein cross-linking (meat restructuring)
Probiotics & Prebiotics
- Probiotics: Live microorganisms that confer health benefits
- Lactobacillus acidophilus
- Bifidobacterium bifidum
- Lactobacillus casei
- Benefits: Improved digestion, immunity boost, vitamin synthesis
- Prebiotics: Non-digestible food components that promote probiotic growth
- Inulin, Fructooligosaccharides (FOS)
- Found in: Chicory, garlic, onions, bananas
- Synbiotics: Combination of probiotics and prebiotics
GM Foods
- Flavr Savr Tomato - Delayed ripening (antisense PG gene)
- Golden Rice - β-carotene enriched (Vitamin A)
- Bt Brinjal - Insect resistant
- AquAdvantage Salmon - Fast-growing (growth hormone gene)
- Arctic Apple - Non-browning (PPO gene silenced)
Single Cell Protein (SCP)
- Protein-rich biomass from microorganisms
- Sources:
- Spirulina (Cyanobacteria) - 60-70% protein
- Chlorella (Green algae)
- Saccharomyces cerevisiae (Yeast)
- Fusarium graminearum (Fungus) - Quorn™
- Advantages: High protein, rapid growth, substrate versatility
- Uses: Animal feed, human food supplement
Primary Metabolic Pathways
Definition
- Primary Metabolism: Essential biochemical pathways directly involved in growth, development, and reproduction
- Products: Amino acids, nucleotides, sugars, lipids, proteins
- Present in all organisms
🔄 Cellular Respiration Overview
Glycolysis
Cytoplasm
Glucose → 2 Pyruvate
Net: 2 ATP + 2 NADH
Cytoplasm
Glucose → 2 Pyruvate
Net: 2 ATP + 2 NADH
Krebs Cycle
Mitochondrial Matrix
Pyruvate → CO₂
2 ATP + 6 NADH + 2 FADH₂
Mitochondrial Matrix
Pyruvate → CO₂
2 ATP + 6 NADH + 2 FADH₂
ETC
Inner Membrane
NADH/FADH₂ → ATP
~34 ATP
Inner Membrane
NADH/FADH₂ → ATP
~34 ATP
1. Glycolysis (EMP Pathway)
- Location: Cytoplasm
- Overall Reaction: Glucose (C₆H₁₂O₆) → 2 Pyruvate (C₃H₄O₃) + 2 ATP + 2 NADH
- Key Enzymes:
- Hexokinase - Phosphorylates glucose
- Phosphofructokinase (PFK) - Rate-limiting enzyme
- Pyruvate kinase - Final step
- Phases:
- Energy investment phase (consumes 2 ATP)
- Energy payoff phase (produces 4 ATP)
- Net Yield: 2 ATP + 2 NADH per glucose
2. Krebs Cycle (TCA Cycle/Citric Acid Cycle)
- Location: Mitochondrial matrix
- Precursor: Pyruvate → Acetyl CoA (by pyruvate dehydrogenase complex)
- Key Intermediates: Citrate, α-ketoglutarate, Succinate, Malate, Oxaloacetate
- Products per Acetyl CoA:
- 3 NADH
- 1 FADH₂
- 1 GTP (= 1 ATP)
- 2 CO₂
- Key Enzymes:
- Citrate synthase
- Isocitrate dehydrogenase (rate-limiting)
- α-ketoglutarate dehydrogenase
- Succinate dehydrogenase (only enzyme in inner membrane)
3. Electron Transport Chain (ETC)
- Location: Inner mitochondrial membrane
- Complexes:
- Complex I: NADH dehydrogenase
- Complex II: Succinate dehydrogenase
- Complex III: Cytochrome bc₁ complex
- Complex IV: Cytochrome c oxidase
- Complex V: ATP synthase (F₀F₁ ATPase)
- ATP Yield:
- 1 NADH = ~2.5 ATP
- 1 FADH₂ = ~1.5 ATP
- Chemiosmotic Theory: Peter Mitchell (1961) - proton gradient drives ATP synthesis
4. Photosynthesis
- Location: Chloroplasts
- Overall Equation: 6CO₂ + 6H₂O + Light → C₆H₁₂O₆ + 6O₂
- Light-Dependent Reactions (Light Reactions):
- Location: Thylakoid membrane
- Photosystem II (P680) - water splitting
- Photosystem I (P700) - NADPH formation
- Products: ATP + NADPH + O₂
- Light-Independent Reactions (Calvin Cycle):
- Location: Stroma
- CO₂ fixation by RuBisCO enzyme
- 3 Phases: Carboxylation, Reduction, Regeneration
- Net product: G3P (glyceraldehyde-3-phosphate)
5. Pentose Phosphate Pathway (HMP Shunt)
- Location: Cytoplasm
- Function: Produces NADPH and pentose sugars
- Two Phases:
- Oxidative phase: Generates NADPH
- Non-oxidative phase: Produces ribose-5-phosphate
- Significance:
- NADPH for biosynthesis and antioxidant defense
- Ribose-5-P for nucleotide synthesis
Primary vs Secondary Metabolism
Primary Metabolism:- Essential for life
- Present in all organisms
- Examples: Glycolysis, Krebs cycle
- Products: Amino acids, nucleotides
- Not essential for survival
- Species-specific
- Examples: Alkaloids, antibiotics
- Products: Pharmaceuticals, pigments
Anabolism vs Catabolism
Anabolism:- Building-up reactions
- Requires energy (endergonic)
- Examples: Protein synthesis, photosynthesis
- Complex molecules from simple ones
- Breaking-down reactions
- Releases energy (exergonic)
- Examples: Glycolysis, protein degradation
- Simple molecules from complex ones
Enzymes and Their Activities
Definition & Properties
- Enzymes: Biological catalysts (mostly proteins) that speed up chemical reactions without being consumed
- First enzyme discovered: Diastase by Anselme Payen (1833)
- Term "enzyme" coined by Wilhelm Kühne (1878)
- First enzyme crystallized: Urease by James Sumner (1926)
- Key Properties:
- Highly specific (substrate specificity)
- Lower activation energy
- Remain unchanged after reaction (reusable)
- Work under mild conditions (pH, temperature)
- Can be regulated
Enzyme Classification (EC Number System)
| Class | Type of Reaction | Examples |
|---|---|---|
| 1. Oxidoreductases | Oxidation-reduction reactions (electron transfer) | Dehydrogenases, Oxidases, Reductases, Peroxidases |
| 2. Transferases | Transfer of functional groups | Kinases, Transaminases, Methyltransferases |
| 3. Hydrolases | Hydrolysis reactions (breaking bonds with water) | Lipases, Proteases, Amylases, Nucleases |
| 4. Lyases | Addition/removal of groups to form double bonds | Decarboxylases, Aldolases, Dehydratases |
| 5. Isomerases | Intramolecular rearrangements (isomerization) | Racemases, Epimerases, Mutases |
| 6. Ligases | Formation of bonds with ATP cleavage | DNA ligase, Synthetases, Carboxylases |
Enzyme Structure
- Apoenzyme: Protein part of enzyme (inactive alone)
- Cofactor: Non-protein component required for activity
- Metal ions: Zn²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Mn²⁺
- Coenzymes: Organic molecules (NAD⁺, FAD, CoA, TPP)
- Prosthetic group: Tightly bound cofactor (heme in hemoglobin)
- Holoenzyme: Apoenzyme + Cofactor = Complete, active enzyme
- Active Site: Region where substrate binds
- Small portion of enzyme (10-20 amino acids)
- 3D cleft or crevice
- Exhibits substrate specificity
Mechanism of Enzyme Action
- Lock and Key Model (Emil Fischer, 1894):
- Rigid complementarity between enzyme and substrate
- Substrate fits exactly into active site like key in lock
- Induced Fit Model (Daniel Koshland, 1958):
- Active site undergoes conformational change upon substrate binding
- More accurate representation
- Explains enzyme specificity better
⚡ Enzyme-Substrate Reaction Pathway
E + S
Free enzyme + Substrate
Free enzyme + Substrate
ES Complex
Enzyme-Substrate Complex formed
Enzyme-Substrate Complex formed
EP Complex
Enzyme-Product Complex
Enzyme-Product Complex
E + P
Enzyme released + Product formed
Enzyme released + Product formed
Factors Affecting Enzyme Activity
- Temperature:
- Optimum temperature: 37°C (human enzymes)
- Low temp: Reduced kinetic energy, slow reaction
- High temp: Denaturation of enzyme protein
- Q₁₀ effect: Rate doubles for every 10°C rise (up to optimum)
- pH:
- Each enzyme has optimal pH
- Pepsin: pH 2 (stomach)
- Trypsin: pH 8 (intestine)
- Arginase: pH 10
- Extreme pH causes denaturation
- Substrate Concentration:
- Rate increases with [S] up to saturation point
- At saturation, all enzyme molecules occupied
- Further increase in [S] has no effect on rate
- Enzyme Concentration:
- Rate directly proportional to [E] when substrate is in excess
Enzyme Kinetics - Michaelis-Menten Equation
- Equation: V = (Vmax × [S]) / (Km + [S])
- Vmax: Maximum velocity when enzyme is saturated
- Km (Michaelis constant):
- Substrate concentration at half Vmax (V = Vmax/2)
- Measure of enzyme-substrate affinity
- Low Km = High affinity (enzyme binds substrate easily)
- High Km = Low affinity
- Lineweaver-Burk Plot:
- Double reciprocal plot: 1/V vs 1/[S]
- Linear transformation of Michaelis-Menten equation
- Y-intercept = 1/Vmax
- X-intercept = -1/Km
- Useful for determining Km and Vmax accurately
Enzyme Inhibition
| Type | Mechanism | Effect on Km/Vmax | Examples |
|---|---|---|---|
| Competitive Inhibition | Inhibitor resembles substrate, competes for active site | Km increases, Vmax unchanged | Malonate inhibits succinate dehydrogenase |
| Non-competitive Inhibition | Inhibitor binds to site other than active site (allosteric site) | Km unchanged, Vmax decreases | Heavy metals (Pb²⁺, Hg²⁺) |
| Uncompetitive Inhibition | Inhibitor binds only to ES complex | Both Km and Vmax decrease | Some toxins and drugs |
| Irreversible Inhibition | Covalent bond formation, permanent inactivation | Enzyme destroyed | Organophosphates (nerve gases), Penicillin |
Allosteric Enzymes
- Regulatory enzymes with multiple binding sites
- Allosteric site: Binding site other than active site
- Positive modulators: Increase enzyme activity
- Negative modulators: Decrease enzyme activity
- Cooperativity: Binding of one substrate molecule affects binding of others
- Sigmoidal curve (S-shaped) instead of hyperbolic
- Example: Phosphofructokinase in glycolysis
- Activated by: AMP, ADP
- Inhibited by: ATP, citrate
Enzyme Regulation
- Feedback Inhibition:
- End product inhibits first enzyme in pathway
- Prevents overproduction
- Example: Isoleucine inhibits threonine deaminase
- Covalent Modification:
- Phosphorylation/dephosphorylation
- Reversible activation/inactivation
- Example: Glycogen phosphorylase
- Zymogen Activation:
- Inactive enzyme precursor (proenzyme)
- Activated by proteolytic cleavage
- Examples: Pepsinogen → Pepsin, Trypsinogen → Trypsin
- Compartmentalization:
- Separation of enzymes in different cellular compartments
- Prevents conflicting pathways
Industrial Applications of Enzymes
- Food Industry:
- Amylases: Baking, brewing, starch processing
- Proteases: Cheese making, meat tenderization
- Pectinases: Fruit juice clarification
- Glucose isomerase: High fructose corn syrup
- Detergent Industry:
- Proteases: Protein stain removal
- Lipases: Fat/oil stain removal
- Amylases: Starch stain removal
- Textile Industry:
- Cellulases: Denim washing, fabric softening
- Amylases: Desizing of fabrics
- Paper Industry:
- Xylanases: Pulp bleaching
- Cellulases: Deinking of recycled paper
- Pharmaceutical Industry:
- Asparaginase: Leukemia treatment
- Streptokinase: Dissolves blood clots
- Restriction enzymes: Genetic engineering
- Diagnostic Applications:
- Glucose oxidase: Blood glucose meters
- Alkaline phosphatase: ELISA tests
Ribozymes
- Ribozymes: RNA molecules with catalytic activity
- Discovered by Thomas Cech and Sidney Altman (1989 Nobel Prize)
- First ribozyme discovered: Self-splicing intron in Tetrahymena
- Examples:
- RNase P: tRNA processing
- Self-splicing introns (Group I and II)
- Ribosome: Peptide bond formation (peptidyl transferase activity)
- Hammerhead ribozyme: RNA cleavage
- Significance: Support RNA world hypothesis (RNA as first genetic material)
🎯 High-Yield Points for CSIR NET & ICAR SRF
- CSIR NET Human genome: ~3.2 billion bp, ~20,000-25,000 genes, 99% non-coding DNA
- ICAR SRF EcoRI recognizes sequence: 5'-GAATTC-3' (palindromic)
- CSIR NET Totipotency concept demonstrated by Gottlieb Haberlandt (1902)
- ICAR SRF MS medium (Murashige & Skoog, 1962) - most widely used plant tissue culture medium
- CSIR NET First generation biofuels: Food crops (corn ethanol, soybean biodiesel)
- ICAR SRF Bt toxin: Cry proteins from Bacillus thuringiensis
- CSIR NET Citric acid produced by Aspergillus niger
- ICAR SRF Penicillin discovered by Alexander Fleming (1928), produced by Penicillium chrysogenum
- CSIR NET Rhizobium: Symbiotic N-fixing bacteria in legume root nodules
- ICAR SRF Probiotics: Lactobacillus, Bifidobacterium
- CSIR NET Glycolysis net yield: 2 ATP + 2 NADH per glucose
- ICAR SRF Krebs cycle: Generates 3 NADH + 1 FADH₂ + 1 GTP per Acetyl-CoA
- CSIR NET RuBisCO: Most abundant protein on Earth, catalyzes CO₂ fixation
- ICAR SRF Km value: Substrate concentration at half Vmax (lower Km = higher affinity)
- CSIR NET Competitive inhibition: Km increases, Vmax unchanged
- ICAR SRF Ribozymes: Catalytic RNA molecules (discovered by Cech & Altman)
- CSIR NET Lock and Key model: Emil Fischer (1894)
- ICAR SRF Induced Fit model: Daniel Koshland (1958) - more accurate
💡 Memory Tricks & Mnemonics
- Enzyme Classes (OTHLIL): Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases
- Glycolysis Steps: "Goodness Gracious, Father Franklin Did Go By Picking Pumpkins (to) Prepare Pies"
- Glucose → G6P → F6P → F1,6BP → DHAP/G3P → 1,3BPG → 3PG → 2PG → PEP → Pyruvate
- Krebs Cycle Intermediates: "Can I Keep Selling Seashells For Money, Officer?"
- Citrate → Isocitrate → α-Ketoglutarate → Succinyl-CoA → Succinate → Fumarate → Malate → Oxaloacetate
- Essential Amino Acids: "PVT TIM HALL" - Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, Lysine
- Restriction Enzymes: Remember they create palindromic sequences (read same on both strands)
- Biofuel Generations: 1st = Food crops, 2nd = Waste biomass, 3rd = Algae, 4th = Genetically modified
📝 Common MCQ Question Patterns
- Enzyme-Organism Matching: Know which organism produces which enzyme/product
- Timeline Questions: Remember key years and scientists
- Numerical Values: ATP yield from glycolysis, Krebs cycle, ETC
- Optimal Conditions: pH and temperature for specific enzymes
- Inhibition Types: Effect on Km and Vmax
- Vector Types: Capacity and applications (Plasmid < Cosmid < BAC < YAC)
- Culture Media: Know MS, B5, White's medium and their uses
- Gene Names in GM Crops: Cry genes, psy, crtI, bar, cp4-epsps
