eChapters

Pollution is the introduction of contaminants into an environment that causes instability, disorder, harm or discomfort to the physical systems or living organisms they are in.  
Pollution can take the form of chemical substances, or energy, such as noise, heat, or light energy.

Pollutants, the elements of pollution, can be foreign substances or energies, or naturally occurring; when naturally occurring, they are considered contaminants when they exceed natural levels. Sometimes the term pollution is extended to include any substance when it occurs at such unnaturally high concentration within a system that it endangers the stability of that system. For example, water is innocuous and essential for life, and yet at very high concentration, it could be considered a pollutant.

(Method of Joseph Mac Gee, 1959)
Principle

A simple and rapid enzymatic method for the quantitative estimation of total cis-methylene- interrupted polyenoic acids has been devised. Linoleic, linolenic and arachidonic acids, the more common acids of this group were used to calibrate the method. The potassium salts of the fatty acids are oxidised by atmospheric oxygen in the presence of the enzyme lipo-oxidase, and the absorption of the conjugated diene hydroperoxide is measured at 234 mµ. As little as 5 µg of linoleic acid can be quantitatively measured with good accuracy and precision. The total content of polyunsaturated fatty acids containing the cismethylene-interrupted diene structure of fats, oils, hydrogenated oils, fatty acids, esters blood plasma, microorganisms and plant seeds has been measured directly by this method.

1. The Aflatoxin Molecule

Aflatoxins are chemically difuro-coumarin derivatives. Presently, 18 different types of aflatoxins have been identified with aflatoxin B₁, B₂, G₁, G₂, M₁ and M₂ being the most common. Of these Aflatoxin B₁ (AFB₁) and G₁ occur most frequently, with AFB₁ being the most potent. Physicochemical and biochemical characteristics of the AFB₁ molecule reveal two important sites for toxicological activity (Heathcote and Hibbert, 1978). The first site is the double bond in position 8,9 of the furofuran ring (Figure 1). The interactions of aflatoxin, DNA and proteins, which occur at this site alter the normal biochemical functions of these macromolecules and leads to deleterious effects at the cellular level.Monteiro et al. (1966) studied the in vitro interaction of groundnut proteins namely arachin and conarachins with AFB₁ molecule. The second reactive group is the lactone ring in the coumarin moiety (Lee et al., 1981). The lactone ring is easily hydrolyzed and is therefore vulnerable to degradation.

Different enzymatic methods have been proposed as potential alternatives to rumen inoculum studies to determine dry matter digestibility and protein degradation. However, the type of enzyme used and the procedure adopted differ (Marten and Barnes, 1979). The enzyme methods offer distinct advantage under circumstances of the difficulties in maintaining rumen fistulated animals as a source of rumen inoculum. Since, the enzymes used in these techniques are commercially available enzyme preparations, and are not of rumen microbial origin, the digestibility obtained from these techniques are related to in vivo digestibility by means of mathematical equations. Therefore, equations developed to predict energy content using enzyme techniques differ with the type of feedstuff and type of enzymes used in the technique (Table 5.1.) (Martens and Barnes, 1979). The enzyme methods are popular among the feed compounding industries to evaluate raw materials and compound feeds.

The method used by DeBoever et al. (1986) to determine digestibility of mixed feeds is given below.

Introduction
Enzymatic regulation refers to the mechanisms that control the activity of enzymes, ensuring that they function optimally and in accordance with the needs of the cell or organism. These mechanisms are crucial for maintaining homeostasis and regulating metabolic pathways. Here are the primary methods of enzymatic regulation:
1. Allosteric Regulation: Allosteric regulation involves the binding of regulatory molecules (allosteric effectors) at sites other than the active site (allosteric sites) on the enzyme. This binding can either enhance (activators) or inhibit (inhibitors) enzyme activity. Allosteric enzymes typically exhibit a sigmoidal (S-shaped) kinetics curve.
2. Covalent Modification: Covalent modification involves the addition or removal of specific chemical groups to an enzyme, altering its activity. The most common form is phosphorylation, where a phosphate group is added by kinases and removed by phosphatases. Other modifications include acetylation, methylation, and ubiquitination.
3. Feedback Inhibition: Feedback inhibition occurs when the end product of a metabolic pathway inhibits an enzyme involved earlier in the pathway. This prevents the overproduction of the end product and helps maintain balance within the cell.
4. Proteolytic Activation: Some enzymes are synthesized as inactive precursors (zymogens or proenzymes) that require proteolytic cleavage to become active. This mechanism is common in digestive enzymes and blood clotting factors.
5. Enzyme Induction and Repression: The synthesis of enzymes can be regulated at the genetic level. Induction increases the production of an enzyme in response to a substrate or other signal, while repression decreases enzyme synthesis. This regulation is often mediated by transcription factors and other regulatory proteins.
6. Substrate Availability: The availability of substrate can directly affect enzyme activity. High substrate concentrations can increase the rate of reaction up to a certain point (saturation), beyond which the enzyme operates at its maximum rate (Vmax).
7. pH and Temperature: Enzymes have optimal pH and temperature ranges within which they function most efficiently. Deviations from these optimal conditions can lead to decreased enzyme activity or denaturation.

Summary

Substance that acts as a catalyst in living organisms, regulating the rate at which life’s chemical reactions proceed without being altered in the process. Enzymes reduce the activation energy needed to start these reactions, without them, most such reactions would not take place at a useful rate. Because enzymes are not consumed, only tiny amounts of them are needed. Enzymes catalyze all aspects of cell metabolism, including the digestion of food, in which large nutrient molecules (including proteins, carbohydrates, and fats) are broken down into smaller molecules; the conservation and transformation of chemical energy and the construction of cellular materials and components. Almost all enzymes are proteins; many depend on a nonprotein cofactor, either a loosely associated organic compound (e.g., a vitamin) or a tightly bound metal ion (e.g., iron, zinc) or organic (often metal-containing) group.

The main groups of some diagnostically important enzymes are as below:

1. Oxido Reductases
Glucose 6-phosphate dehydrogenase, Isocitrate Dehydrogenase, Lactate dehydrogenase, Malate dehydrogenase, Sorbitol dehydrogenase.

2. Transferases
Alanine transaminase (GPT)    
Aspartate transaminase (GOT)
Creatin binase
Hexose-1 phosphate uridyltransferase

3. Hydrolases
Acetylcholinesterase
phosphatases
Amylases
Pepsin
Trypsin
Lipase

Carbohydrates (glucose) are the primary energy source for a body; when digested, glucose is the end product in human beings and simple stomach animals, but it yields volatile fatty acids in ruminants. Precise regulation of levels of blood sugars/glucose in the body is the net result of various processes like digestion, absorption, and metabolism operating under the interaction of various a) enzymes and b) hormones secreted by the different organs of the body. An abnormal secretion of any of the enzymes or hormones associated with the metabolism of carbohydrates disrupts the precise regulation of blood sugar, leading to various disorders.
14.1 Enzyme Deficiency and Disorders in Carbohydrate Metabolism
Usually, various enzymes are involved in the breakdown of carbohydrates, and if their secretion is insufficient, missing, or does not work correctly, it leads to different disorders.
I. Glycogen storage diseases (GSDs, glycogenosis, and dextrinosis)
Glucose is needed by the cells for the generation of energy and other cellular activities. If glucose happens to be in excess, it is stored as glycogen, mainly in the liver and muscles, for future needs. The stored glycogen can be converted back to glucose when the need arises. Many enzymes are related to this interconversion of glucose and glycogen. Deficiencies of one or more enzymes may lead to abnormal glycogen metabolism, leading to glycogen accumulation of normal or abnormal chemical structure in tissue, collectively called glycogen storage diseases (GSDs). Roman numerals identify different types of glycogen storage conditions. They are also classified under various categories like inherited metabolic diseases, autosomal recessive type, and X-linked type (VIII/IX type).

Enzymes are biological catalysts that regulate biochemical reactions and control metabolic pathways. This chapter explains how metabolomics can contribute to the discovery and functional characterization of enzymes by identifying changes in metabolite concentrations associated with enzymatic activity. Comparative metabolomics, gene knockout experiments, stable-isotope labeling, and multi-omics integration are discussed as approaches for linking metabolites with enzyme functions. Analytical platforms such as GC–MS, LC–MS, and NMR, together with databases including KEGG, BRENDA, HMDB, and METLIN, support metabolite identification and pathway analysis. The chapter also considers experimental validation, bioinformatics, challenges associated with unknown metabolites, and future applications involving artificial intelligence and computational biology.

Objective: To extract and perform enzyme assay of Superoxide dismutase from plant.

Background information:

Superoxide dismutase (SOD, EC 1.15.1.1) is the enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide (O₂^-) radical into either ordinary molecular oxygen (O₂) or hydrogen peroxide (H₂O₂). Superoxide is produced as a by-product of oxygen metabolism and, if not regulated, may cause cell damage (Hayyan, et al. 2016). Hydrogen peroxide is also responsible for cell damage and may be degraded by other enzymes like catalase. Thus, SOD is an important antioxidant defense in nearly all living cells exposed to oxygen. One exception is Lactobacillus plantarum and related lactobacilli, which use a different mechanism to prevent damage from reactive O₂^-.

Objective: To extract and perform enzyme assay of catalase from plant.

Background information

Catalase is a commonly distributed enzyme and present in approximately all organisms exposed to oxygen (such as bacteria, plants and animals). Catalysis of H₂O₂ into H₂O and O₂ is done by catalase enzyme (Goodsell, 2004).

Objective: To extract and perform enzyme assay of guaiacol peroxidase from plant.

Background information:

Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:

Objective: To extract and perform enzyme assay of polyphenol oxidase from plant.

Background information:

Polyphenol oxidase (PPO or monophenol monooxygenase or polyphenol oxidase I, chloroplastic) is a tetramer that contains four atoms of copper per molecule, and binding sites for two aromatic compounds and oxygen (www.worthington-biochem.com). The enzyme catalyses the o-hydroxylation of monophenol molecules in which the benzene ring contains a single hydroxyl substituent to o-diphenols (phenol molecules containing two hydroxyl substituents). It can also further catalyse the oxidation of o-diphenols to produce o-quinones.

PPO causes the rapid polymerization of o-quinones to produce black, brown or red pigments (polyphenols) that cause fruit browning. The amino acid tyrosine contains a single phenolic ring that may be oxidised by the action of PPOs to form o-quinone. Hence, PPOs are some time referred as tyrosinases (Mayer, 2006).

Jolley et al. (1974) referred to PPO as 4 electron-transferring phenol oxidase and oxygen. It is responsible for browning reactions throughout the phylogenetic scale.

Enzyme histochemistry constitutes a link between biochemistry and morphology (Hardonk et al., 1976) and provides important information complementary to conventional histology, immunohistochemistry and molecular pathology (Meier Ruge and Bruder 2008). It helps to detect any metabolic changes in tissues and thus can aid in disease diagnosis. It is based on the principal that, the substrates added to tissue are metabolized by enzyme and the reaction is visualized by an insoluble dye product. It is a very sensitive technique that detects even an early metabolic imbalance in the tissue. For example, alkaline phosphatase activity represents tissue barrier functions in brain capillaries, duodenal enterocyte and proximal kidney tubule brush borders and a decrease in enzyme activity will indicate some functional impairment. Similarly, an increase in lysosomal acid phosphatase activity is an early marker of ischemic tissue lesions and acetylcholinesterase enzyme histochemistry has been used for the diagnosis of Hirschsprung disease (Meier Ruge and Bruder, 2008). ATPase methods are used in combination to distinguish between type1 and type 2 muscle fibres and NADH diaphorase is used to detect very minor or early structural abnormality in the sarcoplasmic reticulum network of the fiber, as well as mitochondrial abnormalities eg. Mitochondrial myopathies (Sargaiyan and Bansal, 2014). It has been clearly shown that tumor tissue with high glycolytic activity is particularly resistant to radiotherapy (Mathupala et al., 2007). Enzyme histochemistry for dehydrogenase provides a quick, efficient and inexpensive orientation on the actual energy metabolism of a particular tumor and thus aid in therapeutic strategy (Nakagawa et al., 2002).
For the histoenzymic localization, fresh frozen unfixed tissues preferably 2 to 5 mm square size should be collected immediately after death/slaughtering of animals. Fixation in formalin will inactivate most of enzymes but formalin fixation is also employed to stop enzyme activity of lactic dehydrogenase and

Among various immunodiagnostic tools enzyme immunoassay becomes increasingly popular for the detection of humoral immune response (seroconversion) or microbial antigens for the purpose of diagnosis. Enzyme Linked Immunosorbent Assay (ELISA) is widely used in studies of infectious diseases.Despite several merits of ELISA, it has some limitations such as requirement of ELISA reader to obtain accurate measurements. To overcome this, the dot immunobinding assay was developed .In Dot Immunoassay nitrocellulose membrane is used as an adsorbent matrix instead of plastic surface.The antigen is dotted onto the nitrocellulose membrane and the membrane then incubated with test antibody, and secondly with enzyme conjugated antibody directed against the first antibody.In case of positve
reaction, (specific antigen –antibody reaction) color dot is developed against the white background at the site of antigen deposition.In case of negative reaction, no color development takes place at the site of antigen deposition .
Principle
• In direct ELISA antibody is directly attached to solid phase and targeted by added antigen (detecting antigen).
• These added antigens are targeted by antispecies antibodies linked to enzyme termed as conjugates.
• The bound enzyme is developed by the addition of substrate /chromogen, then stopped and finally read using a spectrophotometer

Early and accurate diagnosis of disease is of prime importance for both the successful treatment of the patient and in limiting the spread of disease in the community. It is in this context that immunodiagnostic methods are now being used extensively since they can be extremely sensitive and highly specific. Among the most successful methods are those utilizing labelled antibodies or antigens. Examples of these are immunofluorescence in which antibodies labelled with fluorescent dyes are utilized and radioimmunoassay in which antigens or antibodies are labeled with isotopes. Both of these procedures have proved to be of great value for particular purposes.

Introduction

The entire living organism from the smaller creatures viruses to the advanced human beings are composed of a set up of biochemical reactions which form the metabolism. These set of metabolic reactions are responsible for the life of organism. The rate of these reactions is a major concern for effective working of organism which is governed by biocatalyst. These biocatalysts are known as enzymes, which accelerates rate of biochemical reaction. These are not utilized in reaction. In 1877, Wilhelm Friedrich Kuhne (Kuhne, 1877) proposed the term enzyme which means ‘in yeast’ and is derived from the Greek word; inzume “leavened” or “in yeast”. At the molecular level of study it is observed that enzymes are proteins which are functional as tertiary and quaratanary structures. Enzymes have active site for the binding to substrate. The active site has two positions; binding and catalytic site (Ogeston, 1948). The former help to bind the substrate with the enzyme followed by the catalysis of reaction by the catalytic site.

Introduction

During last four decade poultry has taken giant step and become corporate sector with double digit growth. Especially in India poultry play pivotal role in fight against malnutrition, unemployment and also contributing in women empowerment. In poultry production the biggest single expense is of feed, which accounting for 70% of total production cost per bird (Kumar et al., 2011). So, obviously more Research and Development focus and new innovative technology need to be implemented in poultry nutrition area. Since last 40 years, feed enzymes have played a major part in helping radically to improve the efficiency of meat and egg production by changing the nutritional profile of feed ingredients. 

Introduction

Billions of chemical reactions are happening inside human body and that too with lightning speed. However, the same reactions if carried out in laboratory, they take a very long time. The reasons behind it are the biocatalysts, also known as enzymes, which are synthesized by living cells. Chemically, they are proteins(except ribonucleic acids: RNA), colloidal in nature, thermo-labile in character and very specific in their action. For example, hydrolysis of proteins by a strong acid at 100°C takes a couple of days; but same proteins can be digested within couple of hours inside human body at 37°C. Similarly, carbonic anhydrase is used as a catalyst by living cells to transfer carbon-di-oxide from cells to blood. This process is 107 times quicker than the same process if occurs without the help of enzymes. Therefore, enzymes can be defined as the biomolecules or biocatalysts which can speed up the rate of the chemical reactions. The substance upon which an enzyme acts is called as substrate and the one which is produced as the result of enzyme-substrate reaction is known as product.

Enzymes are remarkable biological macromolecules that catalyze chemical reactions with extraordinary efficiency, playing an essential role in sustaining life. These large protein molecules and certain catalytic RNA molecules known as ribozymes accelerate reactions by lowering activation energy, enabling them to occur at rates necessary for biological processes. Their specificity is just as impressive as their speed as enzymes are often highly regioselective and, in many cases, absolutely stereospecific, traits that synthetic chemists strive to replicate. They are vital in metabolism, DNA replication, signal transduction, and cellular respiration, operating under mild physiological conditions. Factors such as temperature, pH, substrate concentration, and the presence of inhibitors or activators influence their activity. Given their significance, enzymes have garnered attention in fields like drug design, disease treatment, and industrial applications. The modern study of enzymes continues to provide groundbreaking insights into chemistry and biology, advancing both scientific understanding and human welfare. Brief History of Enzymes The existence of enzymes was first inferred when specific biochemical reactions were observed to occur only after the addition of cell extracts, even though the enzymes themselves could not be directly identified like typical molecules. In the early days, enzymes within these extracts were highly impure and present in low concentrations, as advanced protein