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Principles of Biotechnology and Genetic Engineering

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Biotechnology is a fast developing technology as well as a science that has already made an impact to different aspects of day-to-day human life such as Public Health, Pharmaceuticals, Food and Agriculture, Bioenergetics and Information technology. It is very clear that biotechnology will be a key technology for the 21st century or the science of future. It has the potential to ensure food security, dramatically reduce hunger and malnutrition, and reduce rural poverty particularly in the developing countries like India. Considering its commercial potential and its possible impact on the economy, Government of India has taken a number of measures to build up trained human resource in biotechnology and promote research and development and its commercial aspects in the country.

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CONTENTS

2.16

2.17

2.18

2.19

2.20

2.21

2.22

2.23

2.24

2.25

2.26

Chapter 3

Goodness of FIT (Chi-Square Distribution)

Use of Graph Paper with Logarithmic Coordinates

Process Flow Diagram

Material and Energy Balances

Fluid Flow and Mixing

Mass Transfer

Heat Transfer

Bioreactor Designing

Unit Operations

Homogeneous Reactions

Reactor Engineering

Review Questions

54

54

55

58

60

60

61

61

62

63

67

69

Biotechnology and Society

70

3.1

3.2

3.3

3.4

3.5

3.6

3.7

70

73

73

76

77

79

79

81

Public Perception of Biotechnology

Patenting (Intellectual Property Rights—IPR)

Patents

International Patent Laws

Patenting in Biotechnology

Varietal Protection

Ethical Issues in Biotechnology—Agriculture and Health Care

Review Questions

Part 2

BIOMOLECULES

83

Chapter 4

Building Blocks of Biomolecules—Structure and Dynamics

85

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

4.10

4.11

4.12

4.13

Introduction

Functional Groups of Biomolecules

Building Blocks of Carbohydrates

Building Blocks of Proteins

Building Blocks of Nucleic Acids: Nucleotides

Building Blocks of Lipids: Fatty Acids, Glycerol

Optical Activity and Stereochemistry of Biomolecules

 

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Chapter

OVERVIEW

In This Chapter

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

1.10

1.11

1.12

1.1

Introduction and Definition

Historical Perspectives

Scope and Importance of Biotechnology

Commercial Potential

An Interdisciplinary Challenge

A Quantitative Approach

Classical vs Modern Concepts

Quality Control in Manufacturing

Product Safety

Good Manufacturing Practices (GMP)

Good Laboratory Practices (GLP)

Marketing

INTRODUCTION AND DEFINITION

T

he term ‘biotechnology’ was used before the twentieth century for traditional activities such as making dairy products such as cheese and curd, as well as bread, wine, beer, etc. But none of these could be considered biotechnology in the modern sense. Genetic alteration of organisms through selective breeding, plant cloning by grafting, etc. do not fall under biotechnology.

The process of fermentation for the preparation and manufacturing of products such as alcohol, beer, wine, dairy products, various types of organic acids such as vinegar, citric acid, amino acids, and vitamins can be called classical biotechnology or traditional biotechnology. Fermentation is the process by which living organisms such as yeast or bacteria are employed to produce useful compounds or products.

 

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FUNDAMENTALS

2.1

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BIOCHEMICAL ENGINEERING

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INTRODUCTION

W

e are all aware of opportunities created by advances in molecular biology. Living cells and their components can be used to produce a large number of useful compounds such as therapeutics and other products. But to obtain significant benefits as a commercial operation, molecular biology needs the support of biochemical engineering. The vital area of biotechnology that is concerned with practical application of biological agents (whole cell systems and biocatalysts) and the methodologies and processes associated with it on an industrial scale is biochemical engineering. Biochemical engineering is applicable in different areas of biotechnology such as biochemical reactions, enzyme technology, environmental biotechnology, microbial manipulations, bioseparation technology, plant and animal cell cultures, and food technology. It consists of the development of new process technology, designing bioreactors, developing efficient, and economically feasible extraction and purification procedures (downstream processing).

 

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FUNDAMENTALS

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BIOCHEMICAL ENGINEERING

!'

For example, measurements such as area, volume, velocity, etc. are derived from base quantities or fundamental quantities.

Area

= length × length

Volume

= length × length × length

Velocity

= distance/time

Units

Physical variables are measured against certain standards known as units. Base units are those used to express the dimensions or the fundamental quantities, and the derived units are those derived from the fundamental or base units. There are different systems of units such as MKS,

CGS, SI, and FPS units. Units of one system can be converted into units of another system. SI units is the officially accepted system and is widely in use. There are two clusterings of metric units in science and engineering. One cluster, based on the centimeter, the gram, and the second, is called the CGS system. The other, based on the meter, kilogram, and second, is called the MKS system.

Similarly, FPS system is the old British system that uses foot, pound, and second as the basic units.

 

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PRINCIPLES

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GENETIC ENGINEERING

supports the natural process of cells by trying to maintain their environment to provide optimum growth conditions by providing appropriate temperature, pH, substrates, salts, vitamins, and oxygen. In most of the bioreaction processes the substrate of the biotransformation and the carbon source of the organisms will be the same. Table 2.6 gives some of the carbohydrates commonly used in the various fermentation processes as the carbon source and substrate for the reaction.

Bioreactors can be classified according to the type of biocatalysts and the type of bioreaction. The first classification is based on the type of biological agent used:

• microbial fermentors or

• enzyme (cell-free) reactors.

Further classification is possible based on biochemical reactions and process requirements.

Downstream processing: The recovery and purification of the required product from the growth medium through a set of separation and purification techniques is called downstream processing. Each stage in the overall separation procedure is strongly dependent on the history and quality of the biological production process.

 

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BIOTECHNOLOGY

AND SOCIETY

In This Chapter

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.1

Public Perception of Biotechnology

Patenting (Intellectual Property Rights�IPR)

Patents

International Patent Laws

Patenting in Biotechnology

Varietal Protection

Ethical Issues in Biotechnology�Agriculture and Health Care

PUBLIC PERCEPTION OF BIOTECHNOLOGY

Science and Society

O

ur perceptions or attitudes toward things are not always rational and are often culturally influenced. They are a combination of thoughts or the cognitive dimension, feelings, or the affective dimension, and the way we react—the behavioral dimension. The cognitive dimension consists of things we know, the affective dimension comprises of things we feel, and the behavioral dimension is how we will act on the attitudes we build. Attitudes help us to become socially acceptable; belonging to a group is very important, and it gives meaning to things we experience.

Advancements in science and technology have made our life very simple and fast. At the same time some of this advancement has caused great concern regarding the long-term impacts on environment and life. In 1985, the World Commission on Environment and Development (WCED), also known as Brundtland Commission appointed by United Nations (UN), recommended sustainable development preserving the environment without any degradation. The Commission defined sustainable development as ‘the development that meets the needs of the present without

 

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Part

BIOMOLECULES

B

iomolecules are those compounds synthesized by living organisms. These groups of compounds have different sizes, shapes, chemical and physical properties, and biological functions. These biomolecules include different classes of compounds, which are broadly divided into two categories, depending on size and nature. Those molecules, which are polymers and bigger in size, are known as macromolecules and other molecules, which are simple and small in size, are biomolecules. There are four types of macromolecules in biological systems; namely, carbohydrates, proteins, lipids, and nucleic acids. Out of these four types three are polymers composed of monomers, or building blocks. Lipids are not polymers.

This part is divided into three chapters. In the first chapter we study the small molecules including the building blocks of macromolecules. This includes monosaccharides or sugars, amino acids, nucleotides, vitamins, coenzymes, and fatty acids. Some of these molecules form the building blocks of macromolecules. For example, amino acids are the building blocks of proteins. In biological systems, all these molecules, both macro and micro, are in a state of flux or in a dynamic state. That is, they are always subjected to chemical transformations in order to maintain the state of life.

 

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PRINCIPLES

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The Peptide Bond

Peptide-bond formation is a condensation reaction leading to the polymerization of amino acids into peptides and proteins. Peptides are small consisting of few amino acids. A number of hormones and neurotransmitters are peptides. Additionally, several antibiotics and antitumor agents are peptides. Proteins are polypeptides of greatly divergent length. The simplest peptide, a dipeptide, contains a single peptide bond formed by the condensation of the carboxyl group of one amino acid with the amino group of the second with the concomitant elimination of water. The presence of the carbonyl group in a peptide bond allows electron resonance stabilization to occur such that the peptide bond exhibits rigidity not unlike the typical —C = C— double bond. The peptide bond is, therefore, said to have partial double-bond character.

O

O

C

C +

N

N

H

H

FIGURE 4.14 Resonance stabilization forms of the peptide bond.

TABLE 4.5

Amino Acid

The 20 protein amino acids classified according to the nature of their R groups.

 

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PRINCIPLES

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BIOTECHNOLOGY

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GENETIC ENGINEERING

Oxidations-Reductions-Redox Reactions

Redox reactions are among a cell’s most important enzyme-catalyzed reactions. Oxidation and reduction refer to the transfer of one or more electrons from a donor to an acceptor, generally of another chemical species. The donor is oxidized, the acceptor reduced.

A substance that donates electrons is called a reductant or reducing agent, while the electron acceptor is called oxidant or oxidizing agent. Both, together, represent a redox couple:

Electron donor → e– + electron acceptor

Oxidation-reduction reactions are accompanied by a change in free energy. The free energy is a measure for the tendency to donate or to accept electrons. The flow of electrons can be measured and is called redox potential or electromotive force. An element is found to be in its highest degree of oxidation when it is in the compound that is poorest in energy. To depict redox reactions consistently, a common standard is needed, whose potential has arbitrarily been defined as zero.

 

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BUILDING BLOCKS

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BIOMOLECULES�STRUCTURE

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DYNAMICS

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the cycle continues. The details of the reactions leading to the regeneration of RUBP are represented in Figure 4.42 in detail. Many of the steps in the regeneration reaction are similar to that of the pentose phosphate pathway except that they are operating in the reverse direction. That is why the

Calvin cycle is also called the reductive pentose phosphate pathway. The sugar interconversions are mediated fundamentally by transketolase and transaldolase. All the enzymes required for the

Calvin cycle are located in the stroma of chloroplast.

For every molecule of triose synthesized from CO2, 6 NADPH2 and 9 ATP are required.

That is, each molecule of CO2 reduced to a sugar [CH2O]n requires two molecules of NADPH and three molecules of ATP.

CAM and C4 Plants

The enzyme RBISCO is a dual enzyme having both carboxylase and oxygenase activity. The probability with which RuBisCO reacts with oxygen versus with CO 2 depends on the relative

12 ATP

12 NADPH

 

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STRUCTURE AND

FUNCTION OF

MACROMOLECULES

In This Chapter

5.1 Introduction

5.2 Carbohydrates

5.3 Proteins

5.4 Enzymes

5.5 Nucleic Acids

5.6 Lipids and Biological Membranes

5.1

INTRODUCTION

W

e have discussed the structure and dynamics of smaller biomolecules, which form the building blocks of the important cellular macromolecules, in earlier chapters. These biomolecules can undergo polymerization or condensation to form specific polymers of high molecular weight known as macromolecules. These macromolecules are of four distinct groups—carbohydrates, proteins, nucleic acids, and lipids. All these macromolecules are specialized for carrying out specific cellular functions, which are very closely related to their functions. So a clear understanding of their structure is required for the proper understanding of their functions in the cell metabolism.

5.2

CARBOHYDRATES

Carbohydrates, as we have discussed in the previous chapter, consist of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Monosaccharides, or the simple sugars, are the building blocks or the monomers by which other forms are constructed. Disaccharides consist of two monosaccharide residues linked together by glycosidic bonds. This bond forms between the OH group of anomeric carbon (carbon No.1) of one sugar and with the OH group of any other carbon atom, preferably of 4th or 6th position of another sugar. The number of monomers varies

 

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STRUCTURE

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FUNCTION

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Enzymes function as catalysts, which are substances that facilitate (speed up) reactions without actually entering into the reaction. They are used over and over, and a single enzyme molecule may mediate thousands of reactions in a single second. Even simple reactions like dissolution of carbon dioxide in water will not take place to an appreciable extent by itself. But we can make it dissolve in water in higher concentrations under high pressure. Carbonated drinks have CO2 under high pressure. On releasing the pressure by removing the cap, lots of CO 2 bubbles will release. But in biological systems the dissolution of CO2 takes place under normal conditions at a rate more than 10.6 times that of uncatalyzed reactions. This is possible because of an enzyme known as carbonic anhydrase, which mediates the reaction. Similarly, all reactions in biological systems are mediated by one or more enzymes and so reactions take place at higher speeds.

Enzymes operate on reactants, which are known as substrates, and convert them into products. The reaction may require energy or it may release energy. The enzyme is unaffected by the reaction.

 

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PRINCIPLES

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Techniques based on spectroscopy include colorimetry, UV-visible spectrophotometry, fluorescence spectroscopy, x-ray crystallography, and mass spectrometry.

The solubility techniques are the precipitation of molecules with salts and organic solvents.

6.2

TECHNIQUES BASED ON MOLECULAR WEIGHT AND SIZE

Centrifugation

A centrifuge is a device for separating particles from a solution according to their sedimentation rate, which depends on factors like size, shape, density, viscosity of the medium, and centrifugal force (rotor speed). This process of separation of particles based on its sedimentation rate is called centrifugation. In biology, the particles are usually cells, sub-cellular organelles, viruses, and large molecules such as proteins and nucleic acids. The rate of sedimentation will be directly proportional to the molecular weight or size, if all other factors are constant. To simplify mathematical terminology we will refer to all biological material as spherical particles. There are many ways to classify centrifugation.

 

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PRINCIPLES

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UV-Visible Spectrophotometry

The technique of spectrophotometry is generally used for the qualitative and quantitative estimation of biomolecules such as proteins, sugars, carbohydrates, amino acids, nucleic acids, vitamins, etc.

This technique is also based on the Beer-Lambert law and the instrument is known as a spectrophotometer.

A spectrophotometer is employed to measure the amount of light that a sample absorbs.

The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The UV-visible spectrophotometer is used to measure the absorbance in the UV and visible regions of the spectrum. This instrument is an advanced form of colorimeter in which it can provide a monochromatic light. A prism or a grating will split the light into its component colors and can direct the monochromatic light of our choice to the sample solution to be analyzed.

The beam of light consists of a stream of photons. When a photon encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam.

 

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3

THE CELL AND DEVELOPMENT

A

cell is the basic unit of life, the unit of structure and function for all organisms. Part 3 discusses the various aspects of the cell. There are three chapters in this part. In the first chapter we shall discuss the cell and its organization at different levels. We shall study the structure and function of the cell and its various organelles, organization of cell into tissues, and tissues into organs. We further discuss various organs and organ systems and how these organ systems are coordinated to make up a functional organism. A group of organisms forms a population, which is affected by a number of genetical and environmental factors. It is also discussed how various other factors such as adaptation and natural selection contribute to the evolution of population and biodiversity. The last part of the chapter is for discussing the interaction of organisms among themselves and with the ecosystem and how this can affect environmental and climatic conditions, and finally the biosphere.

 

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PRINCIPLES

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GENETIC ENGINEERING

The cells with the same function and structure are arranged together to form tissues. Each tissue carries out a specific function for which the cells are specialized. Different types of tissues are organized together and form specific structures called organs, which cooperate with other similar organs to carry out specific functions of the body. In animals, different organs cooperate to form a system which carries out a specific function of the body. For example, the circulatory system, digestive system, nervous system, excretory system, etc.

Animal Tissues

In animals, there are four basic types of tissues: epithelial or linings, connective or supporting, muscular, and nervous. An organ of the body may have all the four types of tissues. For example, the stomach, an organ of the digestive system has all the four types of tissues. (See Appendix)

Epithelial Tissue

The cells are arranged in single or multilayered sheets. They basically form the covering on the external and internal surfaces of the organs and body parts. Epithelial cells are not supplied with blood vessels. They protect the internal tissues from physical injury and infection. The free surface of the epithelial tissue may be of different types depending on its special function such as secretory, absorption, or excretory functions. Epithelial cells are basically classified according to their shapes.

 

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PRINCIPLES

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generation of cells developing from that will be entirely dependent on the genetic material that they receive from the parent cell. Mitosis is the cell division that occurs in the somatic cells (body cells) and meiosis takes place in the sex organs for the production of gametes. The main features and the process of mitosis and meiosis are discussed below.

Mitosis

Mitosis is the process that facilitates the equal partitioning of replicated chromosomes into two identical groups. Two new daughter cells arise from one original cell. All the cells created through mitosis are genetically identical to one another and to the cell from which they came. The main purpose of mitosis in eukaryotic cells is: n Growth of the individual, n To repair tissue, and n To reproduce asexually.

Mitosis is a nuclear division in which the daughter cells receive the same number of chromosomes as that of the parent cell. The nuclear division is sometimes referred to as karyokinesis, which is followed by the cytoplasmic division known as cytokinesis. The daughter cells resulting from mitosis are identical to each other and also to the parent cell in the quantity and quality of genetic material. The genetic information, which the cell is copying and distributing during mitosis, is contained in the form of chromosomes.

 

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CELL GROWTH

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DEVELOPMENT

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Gas Exchange in Plants

In plants gas exchange usually takes place through stomata, small openings present on the epidermis of the leaves. The stomata open into spongy parenchyma and the gas exchange takes place between the cells and the gas filled in the air space. Gas exchange is needed for both respiration and photosynthesis.

8.7

INTERNAL TRANSPORT

Living things must be capable of transporting nutrients, wastes, and gases to and from cells. Singlecelled organisms use their cell surface as a point of exchange with the outside environment.

Multicellular organisms have developed transport and circulatory systems to deliver oxygen and food to cells and remove carbon dioxide and metabolic wastes. Simple multicellular organisms such as sponges, multicellular fungi, and algae have a transport system. Sea water is the medium of transport and is propelled in and out of the sponge by ciliary action. Simple animals, such as hydra and planaria, lack specialized organs such as hearts and blood vessels, and instead use their skin as an exchange point for materials. This, however, limits the size an animal can attain. To become larger, they need specialized organs and organ systems. In lower plants such as algae and fungi, transport of material takes place through the body surface and cytoplasmic streaming movements.

 

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