Enzymes Involvement in Microbial Technology: Biotechnological Operations - Literature review Example

Introduction Within the past few decades the diagnostic enzymology has made significant developments. This improvement in the use of enzymes in the diagnosis is the fruit of elaborate researches since 1940s. However, the diagnostic potential of enzymes is still to be widened with medical researches covering large areas. Life in a living being is assumed to be characterized by a number of activities, such as turning food into energy, building new tissues up and replacing them with the old, disposing toxin and so on. Most of these actions are not occurred on its own, where as, chemicals, which itself do not under go changes, boost up this biochemical processes. These catalysts of biochemical reactions are termed as enzymes. Metabolism in a living creature can not achieve the pace of biochemical activities as required without enzymes. In fact, examining extensively the types, categories and functions of enzymes have promoted many findings in medical and life science. Enzymes have separate tasks of their own according to their nature, as each of it fit along with on special subtract to modify it in a particular way. This understanding about enzymes led to an important new facet of processing enzymes as drugs by pharmaceutical companies (Cassileth, 1998). Therapeutic enzymes are specifically used as a replacement for metabolic insufficiencies and in divers need as anticoagulants, oncolytics and thrombolytics. Proteinaceouse waste and the soft fibrin found in inflammatory exudates can be effectively terminated by using protolytic enzymes as anti-inflammatory agents. A Broader Reflection Elevated Serum amylase was first associated with pancreatitis in 1908. Since then diagnosis enzymology has grown rapidly (Wohlgemuth, 1908). Yet several considerations are needed to make a sensible interpretation of serum enzyme determination. Increased serum enzyme activity is mostly related with enzymes released from injured cells. The elevated activity occurs to the same enzyme which is abundantly present in the injured organ (Karmen et al., 1954). The time course of increase in activity of the enzyme in serum is analogous to the time course of decrease in enzyme activity from a damaged organ (Jennings et al., 1957). However, the highest elevation of serum enzyme activity does not have to be proportional with the magnitude of cell damage. While, almost all biochemical reactions are catalysed by enzymes, it is essential to be aware of the fact that enzymatic reactions in rare cases proceed in isolation. For instance, a common scenario is in the case of gluconeogenes, glycolysis or the synthesis of fatty acid, in which the enzymes catalyses each steps of the multi-step metabolic pathways. Such a sequence of proceedings would cause any given enzyme to be dependant on the activity of preceding reaction step for its substrate (Abir et al., 2005). This substrate concentration in human is regulated by intake of food, thus it has little to do with the psychologically significant mechanism for the scheduled regulation of enzyme activity. At the same time, enzyme concentration is regularly modulated in accordance with physiological requirements. The concentration of active enzyme in tissue is known to be normally controlled by three key mechanisms; A) the enormity and rate of enzyme synthesis is controlled by the regulation of gene expression B) enzyme dilapidation is regulated by proteolytic enzyme activity C) Active enzymes are generated from pre-existing pools of inactive pro enzymes through covalent modification (Raja et al., 2011). Early Discoveries of Enzyme The existence of enzymes has been acknowledged by scholars since two centuries. It was the Swedish chemist Jon Jakob Berzelius who performed some of the earliest investigations. Jacob termed those chemical actions catalytic as early as in 1835. Nevertheless, the first enzyme in pure form was attained by James B Sumner of Cornell University in 1926. Sumner was able to acquire crystallised enzyme urease from jack bean. In 1947 Sumner shared Nobel Prize with Wendell M Stanley and John H Northrop for initiating a complex procedure for separating pepsin. Later on, this technique developed by Stanley and Northrop has become helpful to crystallize many enzymes (Bennet and Freiden, 1969). Age, sex and activities could result in the variation of enzyme values (Cohen et al., 1967). For example, normal ranges based on the samples of a 20 year old would not be applicable while interpreting the values of serum samples of a 65 year old male patient in a coronary unit. Hence, specificity of enzymes is considered to be one of the key properties of enzymes that make them so significant in diagnostics and research. The specificity they demonstrate is associated with the reactions they catalyse. This could also be categorised into four different specificities as a few enzymes are specified to catalyse only one particular reaction, while other enzymes would be specifically functional for particular chemical group. 1) Absolute specificity in which only one reaction is catalyzed by an enzyme. 2) Group Specificity which means molecules with specific functional groups are catalysed by these enzymes. Amino, phosphate and methyl groups are examples. 3) Linkage Specificity, where the enzyme will act on certain type of chemical bond. The rest of the molecule structure does not affect the reaction. 4) Stereochemichal Specificity in which a particular steric or an optical isomer gets enacted with enzyme. In spite of their specificity, the cofactors might deal with many apoenzymes. For instance nicotinamide adenine dinucleotide (NAD), which acts as a hydrogen acceptor in a number of dehydrogenase reactions, is a coenzyme. Such reactions include lactate dehydrogenase, alcohol dehydrogenase and malate dehydrogenase (Martinek, 1969). Naming of Enzymes and Coenzymes The classification and naming of the enzymes along with definition of the mathematical constants prevails in enzymology are recommended by a special commission called International Union of Biochemistry (IUB). The IUB had brought out the first recommendations in 1964. The updated revisions were also published in 1972, 1978 and 1984. Though some of the originally studied enzymes are named trypsin, rennin and pepsin, most enzyme names end in ‘ase’. The IUB recommended that enzyme names need to indicate both the substrate acted upon and the type of reaction catalyzed (Martinek, 1969).following are the six classes of enzymes: Oxidoreductases Oxidations and reductions of their substrates are the main functions of these enzymes. Examples to these are glucose-6-phosphate dehydrogenase, lactate dehydrogenase, glutathione reductase, alcohol dehydrogenase and xanthine oxidase. Transferases A particular group of substrates are transferred to another by the involvement of these enzymes. For example, omithine carbamoyal transferase, hexokinase, phosphoglucomutase, hexose-1-phosphate uridyletransferase, alanine aminotransferase (ALT), aspartate amino transferase (AST) and so on. Hydrolases Hydrolysis is generated by these enzymes. Examples are pepsin, trypsin, esterases, glucose-6-phosphatase and so on. Lyases Removal of small molecules from a large substrate is catalysed by these enzymes. Histidine decarboxylase, argino succinase and fumarase are some of the examples of lyases. Isomerases Isomerization of substrate is fuelled by these enzymes. UDP-glucose, triose phosphate isomerase, epimerase, racemases and retinal isomerase are the examples to it. Ligases Joining two substrates together is the function of these enzymes. Examples are DNA ligases, alanyl-t-RNA synthetise, glutamine synthetase (Stenesh, 1989). Many enzymes functions with the help of a coenzyme that help as reagent in a group transfer. Without a second organic molecule known as a coenzyme, many enzymes would be inactive in their reactions and group transfers. Coenzyme helps to increase the catalytic capabilities of enzyme to an increased pace. Some of the coenzymes are closely associated with enzymes through covalent union or non covalent forces. Such coenzymes are termed as prosthetic group. The freely diffusible co enzymes are considered to be second substrate as regularly functions as the recycled carriers of hydrogen shuttling it from the point of generation to consumption (Stenesh, 1989). Enzymes that catalyse oxidoreductions, isomerisation reactions and group transfers require coenzymes, while lytic reactions do not require coenzymes. Hydrolytic reactions catalysed by digestive enzymes also do not require coenzymes as they can also be termed into lytic reactions. As far as the IUB recommendations are concerned, coenzymes could be classified as follows. Coenzymes associated with transfer of groups apart from hydrogen: thiamine pyrophosphate, sugar phosphates, lipoic acid, pyridoxel phosphate, folate coenzymes, cobamaide(B12) coenzymes, biotin, CoA-SH and coenzymes concerned with the transfer of hydrogen. Lipoic acid, coenzyme Q, Flavin Adenine Dinucleotide (FAD), Flavin mononucleotide (FMN), Nicotinamide adenine dinucleotide phosphate (oxidised) (NADP+), Nicotinamide adenine Dinucleotide (oxidised) (NAD+) (Murray et al., 2000). Enzymes Involvement in Microbial Technology Following are the enzymes mostly used in biotechnological operations: SI Endonuclease: As a product of Aspergilus Cryzae, This enzyme works on ssDNA or RNA. It carries ss bubbles which help to break super coiled DNA. Since non-supercoiled and covalent circles and nicked circular DNAs are resistant this enzyme, it could also be used to make a distinction between the DNAs. Restriction Endonucleases: DNA molecules are cut at specific position by the enzymes known as restriction endonucleases. It also recognises and degrades unmodified DNA by internal cleavage. Hence, type II restriction enzymes, which have the ability to cut at the recognition site, are the ones used most commonly. DNA polymerase I: it acts on the fixing the gaps formed on the lagging strand during replication of cells. Therefore, it is aptly known as a ‘repair’ polymerase. Klenow fragment of E.coli DNA polymerase I: “this enzyme is used for sequencing DNA using the Sanger Dideoxy System, filling the three recessed termini of restriction enzyme treated DNA and also used for labelling the termini of DNA fragments. The enzyme is also used for second standard cDNA synthesis in the cDNA procedure” (Raja et al 2011). DNA polymerase III: this enzyme is the major replication polymerase. Helicase: In actions like conjugal plasmid transfer, this enzyme unwinds DNA. S1 Nuclease: Single stranded DNA is degraded by this enzyme. Terminal Deoxynucleotidyl Transferase: Being isolated from calf thymus, these enzyme catalyses the joining of dNTP with 3 –OH of DNA. Mending of Vectors and cDNA with complementary bases is one of the primary outcomes of terminal trnsferase. Hence it also let the cloning of cDNA fragments. Transposase: initial steps of transpositions are boosted by this enzyme. There are a number of coenzymes which would be mentioned in the course of this paper. Role of Enzymes in Diagnosis The diagnosis significance of enzymes is shown by the measurement of serum levels of numerous enzymes. Damage in the tissue or cell would result the release of intracellular component to the blood, which causes the presence enzymes in the serum (Machetti et al., 1998). So when a person is assayed for liver enzyme, the aim of the assay would be to find the potential for liver sell damage. Mainly assayed enzymes include the amino transferases such as alanine transaminase (ALT) which some time termed as serum glutamate-pyruvate aminotransferase (SGPT); lactate dehydrogenase and so on. Under various clinical situations almost all the enzymes are assayed. Many enzymes, therefore, have been helpful in the clinical diagnosis of a range of diseases in both human and veterinary medicine (Neilson et al., 1996). Following are the classifications of such enzymes with significant value in diagnosis of pathology. Alkaline Phosphetase: These enzymes are credited as the first group pf enzymes to have acknowledged as clinically significant. The increase of these enzymes in bone and liver diseases was found in 1920. There has been plenty of literature published on these enzymes since the initial investigation (Lone et al., 2003). The best possible Alkaline pH of this class of phosphetase in vitro is denoted by the term “Alkaline”. The main source of Alkaline Phosphatase in human and animals are liver, bone, kidney and placenta. This enzyme is involved with bone and hepatobiliary diseases in human. While the range of serum ALP value is different in different animals, serum ALP values between different individuals are almost constant except for the age difference of the individuals. Creatine Kinase: Creatine Kinase helps to generate creatine phosphate which is the high energy phosphate required by muscles. Therefore it is also considered as the most organ specific serum enzyme for clinical purpose. Though creatine kinases are present in many parts of the body including heart, brain and smooth muscles, their highest specific concentration is found in the skeletal muscles (Aksenova et al., 2000). Myocardial infraction and muscles diseases are associated with creatine kinase in humans. At the same time, large changes in the serum activity are clinically significant, since these enzymes are very sensitive indicators of mild muscle damage. Alanine Aminotransferase: This enzyme was previously termed as Glutamic Puruvate Transaminase (GPT). 2-oxoglutarate and lalanine involve in a reversible transamination to pyruvate and glutamate in the cytoplasm of the cell, catalysed by alanine aminotransferase. Heart, skeletal muscles and liver have ALT concentrations. Though pigs, horses, cattle, sheep or goats have too little ALT in the tissues to have any diagnostic value, the specific activity of ALT in dogs, cats, rabbits and rats have been recognized as liver specific indicator of damage (Kikuchi et al., 1999). Aspartate Aminotransferase: This enzyme, which was earlier called Glutamic Oxaloacetic Transaminase (GOT), enables the trnsamination of 2-oxoglutarate and L-aspertate to oxaloacetate and glutamate. This is an enzyme associated with muscle, myocardial, parenchymal and hepatic disease in humans and animals, it is mostly found in heart, liver, kidney, skeletal muscles and erythrocytes. However, it is not used as an organic specific enzyme because its pre-sense in so many tissues make its serum level a good determinant (Bittinger et al., 2003). Sorbitol Dehydrogenase (SDH): This enzyme is also known as L-iditol dehydrgenase (IDH). The reversible oxidation of D-sorbitol to D-fructose with the factor of NAD is catalysed by it.while dogs and horses have very low plasma activity, the cattle, sheep and goat serum have considerably greater plasma activity. It is found considerably in testes and hepatocytes, thus a consistent increase in plasm SDH is experienced hepatocyte damage (El-Kabbani et al., 2004). Apparently, hepatic injury is the only source of SDH activity and, both in humans and I all species of animals, it is considered a liver specific. Following are the serum patterns in different liver diseases Reference: Schmidt E, Schmidt F.W. 1977. Brief guide to practical enzyme diagnosis. Houston, Boehringer Mann-Heim Diagnostics. Lactate dehydrogenases (LDH): With the factor NAD, the reversible oxidation of pyruvate to (L+) lactate is catalysed by LDH. In spite of the favourable equilibrium it has on lactate formation, the assay direction is in the direction of pyruvate which exercises an inhibitory effect on LDH. Isoenzymes also are present in LDH. Limph nods, skeletal muscles, heart, liver, erythrocyte and platelets are the organs found with LDH concentration. It relates with myocardial infraction, haemolysis and liver diseases in humans (Murray et al., 2000). Cholinesterase (ChE): Two distinct cholinesterases comprises in the ChE activity. The neurotransmitter found at the myoneural junction called acetylcholine is the major substrate in it. The real ChE is the acetylcholinesterase present in the myoneural junction. It happens to vital in re-establishing and preparing the junction to receive additional signals by hydrolysing acetylcholine (Ellis, 2005). “Decreases in ButChe have been reported in humans with acute infection, muscular dystrophy, chronic renal diseases and pregnancy, as well as insecticide intoxification” (Raja et al., 2011). Lipase: The hydrolysis of triglycerides, generally 1 and 3 positions, is catalysed by serum pancreatic lipases. The positions relate to the releasing of two fatty acids and a two monoglyceride. Lipases are involved with pancreatitis and hepatobiliary diseases as it can be found in the pancreases and hepatobiliary tract (Nduka, 1999). Amylase: Being a metalloenzyme amylase has need of an activator ion like Cl- or Br-. They randomly catalyse the hydrolysis of complex carbohydrates. Amylase helps in diagnosing pancreatitis and it is present in salivary glands, ovaries and pancrease (Gupta et al., 2001). Glutamyltransferase: It is termed as a carboxypeptidase which slashes the C-terminal glutamyl groups to turn them into peptides or other proper acceptors. This enzyme is assumed to be related with glutathione metabolism (Kaneko, 1989). It is said to be related with hepatobiliary diseases and alcoholism. The main sources of these enzymes are liver and kidney. Trypsin: “Trypsins are serum proteases which hydrolyse the peptide bonds formed by lysine or arginine with other amino acids. The pancreas as the zymogen trypsinogen, which is converted to tyrosine by intestinal enterokinase or trypsin itself, secretes them” (Raja et al., 2011). Glutathione Peroxidases: The structure of this metalloenzyme is four atoms of selenium per a molecule of enzyme. It enacts the oxidation of reduced glutathione to generate water and oxidised glutathione. A straight correlation between the amount of red blood cell GPx activity and the selenium concentration of other organs is maintained due to the increased concentration of selenium glutathione peroxidases (Chatterjee and Shinde, 2002). Over all, enzymes are in great demand, since it has been conveniently used as a therapeutic agent against many fatal diseases. In-depth studies and investigations are needed to make complete use of the huge microbial resources. This could prove to be a source of significant therapeutic enzymes. When diseases resurge after being restricted with antibiotics, clinical enzymes could offer a potential of treatment. References Abir, F., S Alva, L Kminaski, L Donald and E Walter, 2005. The Role of arachidonic acid regulatory enzymes colorectal diseases. Dis. Colon Rectum 48: 257-303 Aksenova, M, D.A Butterfield and W.R Markesberry, 2000. Oxidative modification of creatine Kinase BB in Alzheimer’s disease brain. 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