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    Unit 1 Biotech Soft Notes

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    Mamta Pant

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    om & % ARYAKUL COLLEGE OF PHARMACY & RESEARCH i NATKUR, P.0.: CHANDRAWAL N yi ARYAKUL COLLEGE ROAD, ADJACENT TO CRPF BASCAMP LUCKNOW - 226002, UTTAR PRADESH (INDIA). UNIT 1 BIOTECHNOLOGY “Biotechnology is a set of powerful tools that uses live organisms (or part of these organisms) in order to obtain or modify products, improve plant and animal species or to develop micro- organisms for specific uses”. “Biotechnology is the technique of manipulating life forms (organisms) in order to obtain pro- ducts that are useful for humanity”: “Biotechnology is the industrial use of live organisms or biological techniques developed in basic research. Biotechnological products include: antibiotics, insulin, interferon, recombinant DNA and monocional antibodies. Biotechnological techniques include: genetic engineering, cell cultures, tissue cultures, bioprocesses, protein engineering, biocatalysis, biosensors and bioengineering”. APPLICATION OF BIOTECHNOLOGY + Recombinant DNA (genetic engineering). This technique is the basis of the processes for obtai- ning enzymes, hormones, antibodies, vaccines, ete. = The cultivation of vegetable cells and unicellular proteins. This technique is used in the produc tion of chemical substances such as steroids, alkaloids, unicellular proteins for the production of biomass, etc. + Industrial fermentation. This is an ancient technique, but nowadays, we are able to control it and even steer it in the direction that most interests us. By means of fermentation we obtain foods, antibiotics and chemical products. = Biocatalysis. This technique is becoming more common and has a broad range of applications; for example, with biocatalysts, food and chemical substances can be obtained. Biosensors and some diagnostic equipment also use biocatalysts. Furthermore, nowadays biocatalysts are applied in order to achieve cleaner technology in sectors such as the textiles, paper and tanning industries, etc. * Bioremediation. Biotechnology is increasingly applied in the treatment and reuse of waste. In fact, it is the field that presents the broadest range of applications. Thus, biotechnological methods are used to detoxity land polluted by herbicides, in the treatment of wastewater, in recovering industrial waste —for example, whey from cheese-making or cellulose waste—, etc. = Process engineering. An industry has developed around biotechnological applications that applies chemical engineering methods to biotechnological processes. For example, we find process engineering in the filtering and pre-treatment of effluents, the recycling of water, the 1 extraction of products, the recovery of catalysts and microorganisms, etc. Industrial application on product based twe look at a more industrial application, we may define the fields of biotechnology in relation with the products obtained. + The production of microbial biomass for animal food += The microbial production of chemical substances, such as citric acid, glutamic acid, aminoacids, etc. + The enzymatic production of special chemical substances, such as certain optical isomers, etc + The microbial or enzymatic production of antibiotics and + The large-scale production of chemical substances p1 as ethanol, butanol, acetone, acetic acid, etc. = Production, based on animal or vegetable cells or the cells of genetically modified micro- organisms, of antigens, antibodies, therapeutic and diagnostic agents that were previously ‘manufactured from superior organisms. mins. usly produced from crude oil, such + Products for agriculture and livestock. This method means the improvement of plant and ani- mal species through genetic engineering and is far quicker and effective than the methods Used to date (cuttings or the selection and crossing of species). + Products for the food industry, for example: enzymes, food coadjuvants and, above all, greater knowledge of the processes of fermentation which have always been used with the possibilty of better selecting the micro-organisms and even genetically improving them. * Cleaner, or less pollutant technologies. The obtaining of environmentally risk-free —or minimal risk— technology, as a result of the application of the different areas of biotechnology, may also be considered as a product obtained from biotechnology and be applicable to different industrial sectors. Recombinant DNA and genetic engineering Molecular biology has made the most important finding in biotechnology possible: today we can separate the gene which is responsible for codifying the production of certain substances and transfer it to another host organism thus achieving the more effective production of certain useful proteins, Thanks to this breakthrough, hormones, vaccines, blood coagulation factors and enzymes are biotechnologically produced today on a large scale. On the other hand, with the biotechnolo- ical production of proteins, the difficulties involved in obtaining them from superior organisms is thus avoided += Itis not practical to cultivate cells of superior organisms on a large scale because they grow slowly and are easily contaminated. It is more practical to cultivate microorganisms. + The cost of a cell culture is higher than that of microbial cultures. += The source of cells of superior organisms is far more limited than the source of unicellular orga- nisms, which, moreover, reproduce easily and quickly. This area of biotechnology also offers the possibility of obtaining new proteins. For example, the production of enzymes as biocatalysts. In the case of biocatalysts, their specific capacity is, governed by molecular structure and by means of the recombinant DNA technique, the genes that codify the cellular synthesis of enzymes may be selectively modified. Then, when transferring the new DNA to a host microorganism, @ new productive strain of the desired enzyme may be obtained. ENZYME IMMOBILIZATION ‘The term immobilized enzymes refers to “enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously.” Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. Methods of Immobilization In the last decades, thousands of protocols have been reported in the literature and various immobilization strategies can be envisioned. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. One way of classifying the various approaches to immobilizing enzymes is in two broad catego- ries: irreversible and reversible methods x2YME IMMOBIUZATION METHODS fowornne) [acre] [avn] Fate] i & @& & i Methods of Reversible Immobilization Because of the type of the enzyme-support binding, reversibly immobilized enzymes can be detached from the support under gentle conditions. The use of reversible methods for enzyme immobilization is highly attractive, mostly for economic reasons simply because when the enzymatic activity decays the support can be regenerated and re-loaded with fresh enzyme. Indeed, the cost of the support is often a primary factor in the overall cost of immobilized catalyst. The reversible immobilization of enzymes is particularly important for immobilizing labile enzymes and for applications in bioanalytical systems. 1.ADSORPTION METHOD The simplest immobilization method is nonspecifi c adsorption which is mainly based on physical adsorption or ionic binding . In physical adsorption the enzymes are attached to the matrix through hydrogen bonding, van der Waals forces, or hydrophobic interactions, whereas in ionic bonding the ‘enzymes are bound through salt linkages. The nature of the forces involved in noncovalent immobilization results in a process which can be reversed by changing the conditions that infl uence the strength of the interaction (pH, ionic strength, temperature, or polarity of the solvent). Immobilization by adsorption is a mild, easy to perform process, and usually preserves the catalytic activity of the enzyme. Such methods are therefore economically attractive, but may suffer from problems such as enzyme leakage from matrix when the interactions are relatively weak. 2. CHELATION OR METAL BINDING Metal Linked immobilization: In metal linked enzyme immobilization, the metal salts are precipitated over the surface of the carriers and it has the potential to bind with the nucleophilic groups on the matrix. ‘The precipitation of the ion on the carrier can be achieved by heating. This method is simple and the activity of the immobilized enzymes is relatively high (30- 80%). The carrier and the enzyme can be separated by decreasing the pH, hence itis a reversible process. The matrix and the enzyme can be regenerated, by the process. IRREVERSIBLE ENZYME IMMOBILIZATION Entrapment: Enzymes are occluded in the synthetic or natural polymeric networks, itis a permeable membrane which allows the substrates and the products to pass, but it retains the enzyme inside the network, the entrapment can be achieved by the gel, fibre entrapping and microencapsulation. The advantage of entrapment of enzyme immobilization is fast, cheap and mild conditions required for reaction process The disadvantage is that limitation in mass transfer. The support matrix protects the enzymes from microbial contamination, proteins and enzymes in the micro Environment, Microencapsulation method is, that the enzyme molecules are capsulated within spherical semipermeable membranes with a selective controlled permeability. This method provides the large surface area between polymeric material and the enzyme. The drawback of this method is inactivation of enzyme during encapsulation. 2.Covalent Binding: Covalent binding is conventional method for immobilization; it can be achieved by direct attachment with the enzyme and the material through the covalent linkage (Wong et al., 2008). The covalent linkage is strong and stable and the support material of enzymes includes polyacrylamide, porous glass, agarose and porous silica. Covalent method of immobilization is mainly used when a reaction process does not require enzyme in the product, this is the criteria to choose covalent immobilization method. The covalent binding is normally formed between the functional group in the support matrix and the enzyme surface that contains the amino acid residues. The amino acid residues involved in the covalent binding are the sulfhydryl group of cysteine, hydroxyl group of serine and threonine.The attachment between the enzyme and the Support material can be achieved either through direct linkage or through the spacer arm, The potentiality of using the spacer arm is that it provides the greater degree of the mobility to the enzymes hence the enzymes show the higher activity when compared to the direct attachment 3.Cross-linking Enzyme immobilization by cross-linking is an irreversible method performed by the formation of intermolecular crosslinkages between the enzyme molecules by covalent bonds. The process is carried out with the assistance of a multifunctional reagent which acts as linkers to connect enzyme molecules into three dimensional cross linked aggregates. The immobilized enzyme is present in the reaction mixture and not bound to any support. There are two approaches in cross linking immobilization which are the uses of cross linking enzyme aggregate (CLEA), and cross linking enzyme crystal (CLEC). Both methods require the use of a cross linking agent such as glutaraldehyde to cross-links enzyme molecules via the reactions of the free amino groups of lysine residues on the reactive site of neighboring enzyme molecules. In CLEC-based method, glutaraldehyde is added to cross-link enzyme crystals after crystallization. Enzymes immobilized by CLEC usually possesses significant improvements in ‘mechanical properties thus immobilized enzyme in CLECs are usually stable and having higher efficiency than the untreated forms. CLEA is an improved version of CLEC production which could ‘work in aqueous solutions while CLEC requires the formation of crystals. In CLEA-based method, the addition of salts, organic solvents or non-ionic polymers results in the formation of enzyme aggregates which retain enzyme catalytic properties. Immobilization by crosslinking is a simple method which based on the strong chemical binding of enzyme biomolecules thus enzyme leakage is minimal. APPLICATION OF THE IMMOBILIZED ENZYMES Biomedical Application Immobilized enzymes are used in medi immobilized ne from 1990 (Ofagain et al., 1992; Tischer et al., 1992)., ‘enzymes are used for diagnosis and treatment of diseases in the medical field. The inborn metabolic deficiency can be overcome by replacing the encapsulated enzymes (ie, enzymes encapsulated by erythrocytes) instead of waste metabolites, the RBC acts as a carrier for the exogenous enzyme drugs and the enzymes are biocompatable in nature, hence there is no immune response (Johnson et al.,1998),The enzyme encapsulation through the electroporation is a easiest way of immobilization in the biomedical field and it is a reversible process for which enzyme can be regenerated (Lizano et al., 1998).The enzymes when combined with the biomaterials it provides biological and functional systems, Food industry Application: In food industry, the purified énzyimes are used but during the purification the enzymes will denature. Hence the immobilization technique makes the enzymes stable. The immobilized enzymes are used for the production of syrups. Immobilized beta-galactosidase used for lactose hydrolysis in whey for the production of bakers yeast. ‘The enzyme is linked to porous silica matrix through covalent linkage. This method is not preferably used due to its cost and the other technique developed by Valio in 1980, the enzyme galactosidase was linked to resin (food grade) through cross linking. This method was used forthe various purposes such as confectionaries and icecreams Biodiesel Production: Biodiesel is monoalky| esters of long chain fatty acids. Biodiesel is produced through triglycerides (vegetable oil animal fat) with esterification of alcohol (methanol, ethanol) in the presence of the catalyst. ‘The production of catalyst is a drawback of high energy requirements, recovery of glycerol and side reaction which may affect the pollution. Hence the biological production of liquid fuel with lipases nowadays has a great consideration with a rapid improvement. Lipase catalyses the reaction with less energy requirements and mild conditions required. But the production of lipase is of high cost, hence the immobilization of lipase which results in repeated use and stability. The immobilization of lipase includes several methods entrapment, encapsulation, cross linking, adsorption and covalent bonding. Adsortion method of immobilization is widely used in recent years, ‘when compared to covalent bond, entrapment and cross linking .In the biological production of biodiesel the methanol inactivates the the lipase, hence the immobilization method is an advantage for the biodiesel production, The low cost of lipase, candida sp as origin is of more industrial use. Textile Industry: ‘The enzymes derived from microbial origin are of great interest in textile industry. The enzymes such as cellulase, amylase, liccase, pectinase, cutinase etc and these are used for various textile applications such asscouring,biopolishing, desizing, denim finishing, treating wools etc. Among these enzymes cellulase has been widely used from the older period to till now. The textile industries now turned to enzyme process instead of using harsh chemical which affects the pollution and ‘cause damage tothe fabrics. The processing of fabrics with enzymes requires high temperatures and, increased pH, the free enzymes does not able to withstand the extreme conditions. Hence, enzyme immobilization for this process able to withstand at extreme and able to maintains its activity for more than 5-6 cycles. PolyMethyl Methacrylateis linked with cellulose covalently. In this method the nanoparticle is synthesized with cellulase as core particle Endoglucanase is a component of Cellulase enzyme, Endoglucanase is microencapsulated with Arabic Gum is a natural polymer with the biodegradable property is used as a matrix for encapsulation of endoglucanase. Encapsulation of endoglucanase prevented it to retain its activity in the presence of detergents. SUPPORTS USED IN ENZYME IMMOBILIZATION Organic Natural polymers ++ Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate ++ Proteins: collagen, albumin ++ Carbon Synthetic polymers += Polystyrene + Ober polymers: polyacrylat, polymethacrylaes, polyacrylamide, polyamides, vinyl and allyl-polymers Inorganic Natural minerals Bentonite, silica Processed materials {Glass (g0n-porous and controlled pore), metals, controlled pore metal oxides Genetic engineering Genetic engineering, also called genetic modification, is the direct manipulation of an organism’s genome using biotechnology. Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is ‘cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA afier direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Genetic engineering has produced a variety of drugs and hormones for medical use. For example, one of its earliest uses in pharmaceuticals was gene splicing to manufacture large amounts of insulin, made using cells of E. coli bacteria, Interferon, which is used to eliminate certain viruses and kill cancer cells, also is a product of genetic engineering, as are tissue plasminogen activator and urokinase, which are used to dissolve blood clots. APPLICATION genetic engineering in producing GM cropsitransgenic crops: It has become possible to produce the crop or animal with the desirable qualities. The combination of GE and traditional breeding can yield better results than any of the techniques applied in isolation. In GE, the genes can be transferred from one organism to another be it of the same kind or not and express these genes through the anti-sense technology. Green revolution is a fine example of conventional breeding, whereby, the production mainly that of wheat increased manifold, thus addressing the issue of hunger very effectively in India, Pakistan and countries like Mexico. Producing crops with enhanced nutritional qualities is another very important area of GM technology. Applications in producing transgenic animals: In 1981, the primary successful experiment was done on the mice genome by modifying its genetic material through genetic engineering technique. Soon afier, in 1982, Richard Palmiter and his fellows revealed that the growth of those mice increased which were produced from a GM egg which was the combination of growth hormone gene of rat and metallothionein gene of the mouse which leads to growth hormone production. This fusion transgene is very helpful in the study of the composition of growth hormone, also, it enhanced the growth in animals and was also helpful in the analysis of over production disease model to consider various diseases in pathobiology as well as human diseases ie. Alzheimer’ . Steps of producing transgenic animals 1. In in-vitro culture environment, these eggs are matured, 2. Transfection with transgene practice needs artificial fertilization of the eggs. 3. After transfection embryo is cultured artificially. 4. Then embryo is transmitted to substitute block. Knock-out animals are produced by selectively or entirely removing the gene actions. Applications in medical science: GE has made some remarkable achievements in medical science dealing with the diseases and reducing the chances of it being passed to the next generation here are different approaches that can be used in the process of gene therapy; Lastly, the expression of a gene can be altereGenetic Engineering for Biopharmaceuticals: With the ever changing dynamics of biotechnology, more and more drugs are being developed by the biological process. To be precise, about one third of the drugs being developed, fall under the category of Biopharmaceuticals. These are those drugs that are being produced by the Applications in environmental biotechnology: Environmental biotechnology is defined as the use of natural elements like bacteria, plants, animals, molds and mushrooms in order to generate sustainable energy and in order to produce of earning methods the food and nutrients use in the synergistic corporate cycle in which each route waste material is used by another process as raw material . ‘There are several applications regarding gene therapy which include the analysis and execution of actions related to microbes and ecology through complex examination techniques, new classes of noxious waste are identified through gene therapy, contaminants reduction methods are introduced, depending upon biochemical active degraders. In some countries, there is tropical and subtropical climate and biological diversity is present ina wide range and it is very helpful in solving environmental issues by accomplishing solut regarding biotechnology ‘Some of the key drugs produced by recombinant DNA technology Active substance Drug Name Diabetes Factor VIII Factor VIII from Bayer Males suffering from Haemophilia Erythropoetin Eprex, Epogen PROTEIN ENGINEERING Protein engineering is the design of new enzymes or proteins with new or desirable functions. Itis based on the use of recombinant DNA technology to change amino acid sequences Protein engineering can be considered a sub-discipline within the broader category of genetic engineering. The distinguishing feature of protein engineering is that the final product is a protein with a modified amino acid sequence, rather than a new (or modified) living organism. Since proteins do not reproduce, many of the concerns (legitimate and otherwise) found in the broader field of genetic engineering (e.g. the current controversy over genetically modified organisms) are not an issue in protein engineering. In this regard, engineered proteins more closely resemble new chemical compounds from non-biological sources, for which concerns around safety and toxicity apply, but which by their very nature are readily biodegradable. ‘Two strategies have emerged to design proteins to work better or under unusual conditions. The first employs site-directed mutagenesis along with protein structural information to rationally design new or improved function. More recently, this rational approach attempted with some success to create functioning “proteins” de novo ("from scratch’ Ja non-rational approach called directed evolution employs recombinant DNA techniques to create thousands of possible variants, and then uses high throughput screening methods to rapidly search for the one that offers the best solution. This approach has emerged as a powerful alternative to rational methods, particularly when the 10 relationship between structure and desired function is obscure, but where a representative screen can be developed. 2. Strategies for Protein Engineering Rational Design Rational design is a particular strategy in protein engineering, which attempts to create improved protein molecules based on the three-dimensional structure and the relationship between structure and function, which has developed over the years as part of protein science. The earliest discussions of rational design describe an iterative process in which x-ray crystallography provides a three-dimensional structure, which can be represented graphically, and mathematically on a computer. The computer model allows predictions to be made, particularly in the realm of the effect of mutations on structure-based properties The simplest modeling task, but perhaps the most widely used, is to employ the computer to visualize the protein in all its spatial detail (by rotation, zooming in on particular residues, highlighting interaction etc) so that important "what if" questions can be asked and intelligent! answered Rational design has been used to create hybrid proteins that are fusions of pre-existing, but unrelated protein domains. Since most protein domains that confer catalytic or other properties can fold independently, they are easy to manipulate, and fusion of proteins with specific binding or catalytic domains has now become a standard tool for protein engineers. 2. De Novo protein synthesis In designing proteins “from scratch,” one strategy is to employ a “four a-helix bundle” motif-a favorite because of its simplicity and functional diversity. Hydrophobic amino acids, such as leucine are introduced on one side of a a-helix to drive association. By incorporating certain functional groups, such as a heme group, itis possible to attain spectroscopic and electrochemical properties closely resembling those of native heme proteins. second major strategy in de novo design uses known protein structures as natural scaffolds to present a new property such as catalysis, inhibition, or metal binding sites. Examples of this approach include the introduction of an elastase-binding loop into the structure of interleukin-16. ‘The inability to predict how 2 protein will fold has led to the use of molecular templates to provide some control in the position of different structural regions. In this approach peptide structural elements are covalently linked to chemical templates with precisely 3. Directed Evolution Directed evolution (DE; also termed in vitro evolution, directed molecular evolution, and accelerated evolution) encompasses several molecular techniques that mimic the processes of Darwinian evolution in vitro, by combining random mutagenesis and/or recombination of DNA (and possibly RNA) with high-throughput screening or selection for corresponding protein variants that have incorporated the desired properties. Because this approach is not based on any “rational” structure- function knowledge of the protein of interest, other than its functional properties and its corresponding DNA sequence, DE is sometimes referred to as “irrational” or “semi-rational” design, Applications of protein engineering uu Food and detergent industry applications Early reports on the importance of protein engineering methods to design new enzymes for enzyme biotechnological industries date back to 1993 (Wiseman, 1993). Particularly, the enzymes used in food industry were emphasized as an important group of enzymes, the industrially important properties of ‘which could be further improved by protein engineering. Those properties include thermostability, specificity and catalytic efficiency. Additionally, the design and production of new enzymes for food industry by using protein engineering. the design and production of new enzymes for food industry by Using protein engineering was discussed to produce new food ingredients. Example- An important application area of protein engineering regarding food industry is the wheat gluten proteins. Their heterologous expression and protein engineering has been studied using a variety of expression systems, such as E.coli, yeasts or cultured insect cells. Wildtype and mutant wheat gluten proteins were produced to compare them to each other for protein structure-function studies, 2.DETERGENT INDUSTRIES. ‘Among different proteases, bacterial alkaline proteases are a commercially important group. They are particularly important for detergent industry and commercial products. The use of protein engineering techniques resulted in improvement of their catalytic efficiency, stability against high temperatures, oxidation and changes in washing conditions. Some large groups of enzymes like proteases, amylases and lipases are important for both food and detergent industries, as they have a broad range of industrial applications. Proteases, for example, are used in several applications of food industry regarding low allergenic infant formulas, milk clotting and flavors. They are also important for detergent industry for removing protein stains. ‘Amylases are also important for both food and detergent industries. In food industry, they are used for liquefaction and saccharification of starch, as well as in adjustment of flour and bread softness and volume in baking, The detergent industry makes use of amylases in removal of starch stains. Another major group of enzymes utilized by food and detergent industries is constituted by lipases. They are used in many applications of food industry such as for the stability and conditioning of dough (as an in situ emulsifier), and in cheese flavor applications. Lipases are also crucial for the detergent industry, as they are used in removal of lipid stains. 3, Environmental applications Environmental applications of enzyme and protein engineering are also another important field. Early reports on enzyme and cell applications in industry and in environmental monitoring, such as environmental biosensors. the importance of microbial strains and their enzymes in bioremediation and biotransformation applications was discussed, pointing out the utilization of modern strategies such as protein engineering or pathway engineering to improve microbial processes. Protein engineering of oxygenases, an important group of enzymes with high selectivity and specificity, which enable the microbial utilization and biodegradation of organic, toxic compounds. Another important environmental application of protein engineering involves fungal enzymes. Particularly peroxidases isolated from fungi can transform xenobiotics and many pollutants. For the development of applications, the enzyme stability and availability need to be improved. Thus, many protein engineering strategies were identified such as improvement of hydrogen peroxide stability, increasing the redox potential to broaden the substrate range, heterologous expression and industrial production developmen 2 4. Petroleum Industries Petroleum biorefining is also an important environmental application area, where new biocatalysts are required. Protein engineering, isolation and study of new extremophilic microorganisms, genetic engineering developments are all promising advances to develop new biocatalysts for petroleum refining. Petroleum biorefining applications such as fuel biodesulfurization, denitrogenation of fuels, heavy metal removal, depolymerisation of asphaltenes. 5. Medical applications ‘The use of protein engineering for cancer treatment studies is a major area of interest. Pretargeted radioimmunotherapy has been discussed as a potential cancer treatment. By pretargeting, radiation toxicity is minimized by separating the rapidly cleared radionuclide and the long-circulating antibody. ‘Advances in protein engineering and recombinant DNA technology were expected to increase the use of pretargeted radioimmunotherapy. Protein engineering applications for therapeutic protein production is an important area, particularly for medicine. in 1996, recombinant protein production for therapeutic purposes was reviewed. It was stated that protein engineering resulted in a second generation of therapeutic protein products with application-specific properties obtained by mutation, deletion of fusion. ‘Owing to advances in recombinant DNA technology, “antibody engineering” is possible. Improvements such as minimal recognition units and antigenized antibodies were described. Combinational approaches such as bacteriophage display libraries have been introduced as a strong alternative to hybridoma technology for antibody production with desired antigen binding characteristics. 6. Applications for biopolymer production Protein engineering applications for biopolymer production are also promising. Particularly, peptides are becoming increasingly important as biomaterials because of their specific physical, chemical and biological properties. Protein engineering and macromolecular selfassembly are utilized to produce peptide-based biomaterials, such as elastin-like polypeptides, silk-ike polymers, etc. 7. Nanobiotechnology applications Nanobiotechnology applications of protein engineering are becoming increasingly important. The synthesis and assembly of nanotechnological systems into functional structures and devices has been difficult and limiting their potential applications for a long time. However, when biomaterials are investigated, it can be realized that they are highly organized from molecular to the nano- and macroscales, hierarchically. Biological macromolecules, such as proteins, carbohydrates and lipids are Used in the synthesis of biological tissues in aqueous environments and mild physiological conditions, where this biosynthetic process is under genetic regulation. 8. Applications with various industrially important enzymes B Ex-Aldolases are also important enzymes for stereoselective synthesis reactions regarding carbon- carbon bond formation in synthetic organic chemistry. Protein engineering or screening methods improved aldolases for such synthesis reactions. De novo computational design of aldolases, aldolase ribozymes etc. are promising applications. Biosensors A Biosensor is an analytical device that detects changes in Biological processes and converts them into an electrical signal. The term Biological process can be any biological element or material like ‘enzymes, tissues, microorganisms, cells, acids, etc. So, a Biosensor is a combination of a Biological sensing element and a transducer, which converts the data into electrical signals. Additionally, there will be an electronic circuit which consists of a Signal Conditioning Unit, a Processor or Microcontroller and a Display Unit. ‘The following is a simplified block diagram showing the important components of a Biosensor. In the above block diagram, the Signal Conditioning unit comprises of an Amplifier and a Filter (usually a Low Pass Filter) circuitry. This block diagram will be clearer when we take a look at an example in the coming sections, Principle of a Biosensor ‘The desired biological material is usually in the form of an enzyme. By a process known as Electroenzymatic approach, which is a chemical process of converting the enzymes into corresponding electrical signals (usually current) with the help of a transducer. One of the commonly used Biological response is the oxidation of the enzyme. Oxidation acts as a catalyst and alters the pH of the biological material. The change in pH will directly affect the current carrying capacity of the enzyme, which is once again, in direct relation to the enzyme being measured. 4 Output of the transducer ice. the current, is a direct representation of the enzyme being measured. The current is generally converted into voltage so that it can be properly analyzed and represented. Working of Biosensors ‘The combination of biological sensitive element and a transducer will convert the biological material into a corresponding electrical signal. Depending on the type of enzyme, the output of the transducer will be either current or voltage. If the output is voltage, then well and good. But if the output is current, then this current should be converted into equivalent voltage (using an Op-Amp based current to voltage converter) before proceeding further. The output voltage signal is usually very low in amplitude and superimposed on a high frequency noise signal. So, the signal is amplified (using an Op-Amp based Amplifier) and then passed through a Low Pass RC Filter. ‘This process of amplifying and filtering the signal is the job of a Signal Processing Unit or a Signal Conditioning Unit. The output of the signal processing unit is an analog signal that is equivalent to the biological quantity being measured. ‘The analog signal can be displayed directly on an LCD display but usually, this analog signal is passed toa Microcontroller, where the analog signal is converted into digital signal, since it is easy to analyze, process or store a digital signal. Example of Biosensor Before proceeding further with different types of Biosensors and applications of Biosensor, let us quickly take a look at a simple example of a Biosensor: The Glacometer, which is one of the most ‘common applications Diabetes is a disease characterized by the levels of glucose in the blood, Regularly checking the blood glucose levels is very important for diabetes patients. Glucometers are a type Biosensors, which ‘measure the concentration of glucose in blood. Usually, they consists of a test strip, which collect a small sample of blood to analyze the glucose levels. This particular sensor implements the Electroenzymatic approach i.e. oxidation of glucose. Slucometer Test Strio ‘The test strip consists of a trigger electrode and a reference electrode. When blood is placed on the test strip, a simple chemical reaction takes place and an electrical current is generated, which is directly proportional to the concentration of glucose. Internally, the Glucometer consists of a powerful processor like a Cortex-M3 or Cortex-M4 along with current to voltage converter, amplifier, filter and a display unit Different Types of Biosensors Biosensors are classified into two groups ie. either based on the Biological Element used in the analysis ‘or the method of transduction implemented. As mentioned already, some of the commonly used iological elements or bio-recognition elements are DNA, enzymes, antibodies, microorganisms, tissues, cell receptors ete. The next and most commonly used classification of Biosensors is based on the type of transduction used in the sensor ie. type of physiochemical resulting from the sensing event. Further, the biosensors based on method of transduction are again divided into three types. They are: ‘Mass based Biosensors Optical based Biosensors Electrochemical Biosensors 15 ‘There are again few subclasses in each of these types. The following image shows a comprehensive list of different types of Biosensors. Biosensors iological Element Electrochemical Mass-based Antibody (Optical Biosensors esac Biss ONA | Fiber Optics Potentiometric Magnetoelectric Piezoelectric Enzyme Surface Plasmon deomroneice Resonance (SPR) Quarts Crystal Biomimetic Microbalace QCM) Raman and FTIR Conductometric Surface Acoustic ave Fs (saw) Others Impedimetric Piezoelectric Biosensors ‘They are a subdivision of Mass based Biosensors. Piezoelectric Biosensors are also known as Acoustic Biosensors as they are based on the principle of sound vibrations ie. acoustics. When a mechanical force is applied on a piezoelectric biosensor, they produce an electrical signal. ‘The biological elements are attached to the surface of the piezoelectric biosensor. The piezoelectric biosensor, which is essentially a mass to frequency converter, converts the mechanical vibrations of the sensing molecules into proportional electrical signals. Electrochemical Biosensors In electrochemical biosensors, the biological molecules are coated onto a probing surface. The sensing molecules are held in place with the help of no: interfering membrane. Then, the sensing molecules react appropriately to the compound to be detected! and produces an electrical signal proportional to the ‘quantity being measured, Electrochemical Biosensors can employ various types of transducers like Potentiometric, Amperometric, Impedimetric etc. converting the chemical information into a measurable electrical signal Optical Biosensors Optical Fibers play an important role in Optical Biosensors. The optical fibers allow detection of the sensing elements based on the different properties of light like absorption, scattering and fluorescence. ‘The reaction causes changes in either of the above mentioned properties as a result of the change in the refractive index of the interacting surface. For example, ifthe biological elements are antibodies and are bound with a metal layer, the refractive index of the medium which comes in contact with this layer will be varied One of the main advantages of using optical biosensors is their non-electrical nature. This allows them to analyze multiple elements on a single layer just by varying the wavelength of the light, Applications of Biosensors 16 Since their development in the early 1950’s, Biosensors have become very important in the fields of medicine, clinical analysis and in general health monitoring. The advantages of biosensors over lab ‘based equipment are as follows: Small size.Low cost, Quick results, Very easy to use Apart from the desired medicine and health based applications, Biosensors have also found critical applications in several other fields like industrial processing, agriculture, food processing, pollution control etc.So, the following is a small list of the potential fields where Biosensors are frequently used. Medicine, Clinical and Diagnostic Applications, Environmental Monitoring Industrial Applications Food Industry Agriculture Industry Let us briefly take a look at these areas of application of Biosensors individually. ‘Medicine, Clinical and Diagnostic Applications The main area of interest of Biosensor is the Medicine, Clinical and Diagnostics applications. Electrochemical based Biosensors are commonly used in biochemical labs and clinies to monitor and measure glucose levels as well as lactic acid. ‘Commercial Biosensor in the field of personal health care are becoming quite popular, especially, monitoring of blood glucose, The main advantage of this method is the blood samples cannot be contaminated and also itis undiluted for more accurate results. Earlier self-monitoring devices are one-time use applications i.e. test can be performed for a single time and the sensor must be disposed after that. But advances in this field allows, reusable sensors for improved patient care. Environmental Monitoring One of the major application of Biosensor is in the field of Environmental Pollution Monitoring. Especially, water pollution monitoring is an area where Biosensors have substantial advantage. There are numbering pollutants that are contaminating ground water and as a result the quality drinking water is getting worse, Biosensors with sensing elements for nitrates and phosphates are becoming common for battling water pollutants Another important application is for the mi specimens that can be used a bio-weapons. Industrial Applications Fermentation is a large industrial operation used in dairy, alcohol and other similar products. Large scale Bacteria and cell culture must be maintained for this purpose. In order to minimize the cost of production and risk free fermentation, it is essential to monitor these delicate yet expensive processes. Biosensors are designed to monitor and measure the generation of a fermented product. Food Industry ‘Commercial Biosensors that can measure carbohydrates, acids, alcohol, etc. are already available in the ‘market. Biosensors are used in food industry for food quality control for measurement of amino acids, carbohydrates, alcohols, gases, ete Some of the common food industries are Wine, Beer, Yogurt, soft drinks ete. Agriculture Industry Biosensors in the field of agriculture are generally used for detection of pesticides, f= ary to detect chemicals and hazardous biological v7 ROLE OF MICROORGANISM FOR PHARMACEUTICAL PRODUCT The important products that manufactured by microorganism in pharmaceutical industry is described below. VACCINE A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and “remember” it, so that the immune system can more easily recognize and destroy any of these ‘microorganisms that it later encounters.There are several types of vaccines in use. These represent different strategies used to try to reduce risk of illness, while retaining the ability to induce a beneficial immune response, ‘Types of Vaccine Killed:Some vaccines contain killed, but previously virulent, micro-organisms that have been destroyed with chemicals, beat, radioactivity or antibiotics. Examples are the influenza vaccine, cholera vaccine, bubonic plague vaccine, polio vaccine, hepatitis A vaccine, and rabies vaccine. Attenuated:Some vaccines contain live, attenuated microorganisms. Many of these are live viruses that have been cultivated under conditions that disable their virulent properties, or which use closely related but less dangerous organisms to produce a broad immune response. Although most attenuated vaccines are viral, some are bacterial in nature. They typically provoke more durable immunological responses and are the preferred type for healthy adults. Examples include the viral diseases yellow fever, measles, rubella, and mumps and the bacterial disease typhoid. The live Mycobacterium tuberculosis vaccine developed by Calmette and Guérin is not made of a contagious strain, but contains a virulently modified strain called "BCG" used to elicit an immune response to the vaccine, 18 Toxoi than the micro-organism, Examples of toxoid-based vaccines include tetanus and diphtheria, Toxoid vaccines are known for their efficacy. Not all toxoids are for micro-organisms; for example, Crotalus atrox toxoid is used to vaccinate dogs against rattlesnake bites. :Toxoid vaccines are made from inactivated toxic compounds that cause illness rather Subunit:Protein subunit— rather than introducing an inactivated or attenuated micro-organism to an immune system (which would constitute a "whole-agent” vaccine), a fragment of it can create an immune response, Examples include the subunit vaccine against Hepatitis B virus that is composed of only the surface proteins of the virus (previously extracted from the blood serum of chronically infected patients, but now produced by recombination of the viral genes into yeast), the virus-like particle (VLP) vaccine against human papillomavirus (HPV) that is composed of the viral major capsid protein, and the hemagglutinin and neuraminidase subunits of the influenza virus. Subunit vaccine is being used for plague immunization Conjugate:Conjugate ~ certain bacteria have polysaccharide outer coats that are poorly immunogenic, By linking these outer coats to proteins (e.g. toxins), the immune system can be led to recognize the polysaccharide as if it were a protein antigen. This approach is used in the Haemophilus influenzae type B vaccine. Valence: Vaccines may be monovalent (also called wnivalent) or multivalent (also called polyvalent). A monovalent vaccine is designed to immunize against a single antigen or single microorganism, A multivalent or polyvalent vaccine is designed to immunize against two or more strains of the same microorganism, or against two or more microorganisms. In certain cases a monovalent. vaccine may be preferable for rapidly developing a strong immune response. ANTIBODY :An antibody (Ab), also known as an immunoglobulin (Ig), is a large Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign ‘objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, called an antigen Each tip of the "Y" of an antibody contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing 19 these two structures to bind together with precision, Using this binding mechanism, an antibody can fag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize its target directly (for example, by blocking a part of a microbe that is essential for its invasion and survival). The production of antibodies is the main function of the humoral immune system, Antibodies are glycoproteins belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably. Antibodies are typically made of basic structural units—each with two large heavy chains and two small ight chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. ANTIBIOTICS :An antibacterial is an agent that inhibits bacterial growth or kills bacteria, The term is often used synonymously with the term antibiotic(s). Today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic(s) has come to denote a broader range of antimicrobial compounds, including anti-fungal and other compounds. 20 With advances in medicinal chemistry, most of today's antibacterials chemically are semisynthetic modifications of various natural compounds. These include, for example, the beta-lactam. antibacterials, which include the penicillins (produced by fungi in the genus Penicillium), the cephalosporins, and the carbapenems. Compounds that are still isolated from living organisms are the aminoglycosides, whereas other antibacterials for example, the sulfonamides, the quinolones, and the oxazolidinones—are produced solely by chemical synthesis. In accordance with this, many antibacterial compounds are classified on the basis of chemicaVbiosymthetic origin into ‘natural, semisynthetic, and synthetic. Another classification system is based on biological activity; in this classification, antibacterials are divided into two broad groups according to their biological effect ‘on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth. PROBIOTICS:Probiotics are live bacteria that may confer a health benefit on the host. in the past, there were other definitions of probiotics. The frst use of the word “Probiotic” as microorganisms that have effects on other microorganism was accredited to Lilly and Stilwell (1965), expressed as follows: Substances secreted by one microorganism that stimulate another microorganism. The probiotics are describing as “Organisms and substances that have a beneficial effect on the host animal by contributing to its intestinal microbial balance”, two important facts of probiotics: the viable nature of probiotics and the capacity to help with intestinal balance. Alternative expert review indicates there is insufficient scientific evidence for supplemental probiotics having a benefit. Lactic acid bacteria (LAB) and bifidobacteria are the most common types of microbes. 21 used as probiotics, but certain yeasts and bacilli may also be used. Probiotics are commonly consumed as part of fermented foods with specially added active live cultures, such as in yogurt, soy yogurt, or as dietary supplements. Probiotics are also delivered in fecal transplants, in which stool from a healthy donor is delivered like a suppository to an infected patient. ENZYME PRODUCTION :There is a large number of microorganisms which produce a variety of enzymes. Enzymes differ with respect to substrates. Some of the microorganisms producing enzymes are listed in Table- ‘Microorganisms producing enzymes Vlicroorganisms [Enzymes jacillus cereus [Penicilinase IB. coagulans amylase |B. licheniformis amylase, protease ‘megaterium [Penicillin acylase |Gitrobacter spp. IC-asparaginase Bacteria [Escherichia coli PPenicilfin acylase, b- Tebsiella pneumoniae Tulanase |Netinomyeetes|Mcrinoplanes sp. [Glucose isomerase spergilus flavus [Urate oxidase niger [Amylases, protease pectinase, glucose ormae [Amylases, lipases, protease iureobasidium pullulans [Esterase, invertase ‘andila lipolytica [Lipase [Mucor micheli and M___[Bennet Neurospora crassa [Trysinase enicillium funiculosum — [Dextranase Tota [Ghucose oxidase izopus SP [Cipase faccharomyces cerevisiae [invertase Fungi: IS. fragilis linvertase Frrichoderma reesei [Celiutase fr viride [Celiulase 2 VITAMIN PRODUCTION ‘Tab. 3, Microbial and Enzymatic Processes for the Production of Fat-Soluble Vitamins Vitamin Enzyme (Microorganism) Method Vitamin E and K, side chains multiple enzyme system (Geotrichum candidum) {(5)-2-methyl-y-butyrolactone] reductase bakers’ yeast, [(S)-3-methyl-y-butyrolactone] _ (Geotrichum sp.etc.) ‘enzymatic conversion from ()-3-(1’,3"-dioxolane-2’-yl)-2-butene-1-01 asymmetric reduction of ethyl-4.4- dimethoxy-3-methylerotonate [(5)- of (R}-B-hydroxy- multiple enzyme system —_ stereoselective oxidation of isobutyric acid ‘sobutyric acid) (Candida sp..etc.) Vitamin K> multiple enzyme system conversion of quinone- and side chain- (Flavobacterium sp.) precursors to the vitamin Arachidonic acid fermentation fermentative production from glucose (Mortierlla alpina) Dihomo-y-linolenic acid fermentation fermentative production from glucose by (Mortierella alpina) a AS-desaturase-defective mutant Mead acid fermentation fermentative production from glucose by (Mortierella alpina) a A12-desaturase-defective mutant Eicosapentaenoic acid multiple enzyme system —_A17-desaturation of arachidonic acid or (Mortirella alpina) conversion from e-linolenic acid 23 ‘Tab. 2. Microbial and Enzymatic Processes for the Production of Water-Soluble Vitamins and Coonzymes ‘Vitamin, Cooazyme Enzyme (Microorganism) ‘Method Vitamin C (@-Keto-t-gulonic acid) 2,S-diketo-D-gulonic acid reductase (Corynebacterium sp.) ‘enzymatic conversion of 25 diketo-D-gluconate obtained {through fermentative process to 2- keto-t-gulonic. followed by cher (Bacillus sphaericus) acid using the biotin biosynthesis enzyme system of a mutant of B sphaericus Pantothenic acid lactonohydrolase resolution of D.L-pantolactone to (BPantoic acid) (Fusarium oxysporum) D-pantoic acid and L-pantolactone by stereoscloctive hydrolysis (Coenzyme A ‘multiple enzyme system ‘conversion by enzymatic coupling (Brevibacteriuum ammontagenes) ‘of ATP-generating system and cosazyme A biosynthesis system of BR. ammoniagenes (pareat strain ‘or mutant) with D-pantothenic ‘acid, L-cysteine, and AMP (or ‘adenosine, adenine, etc.) as ‘substrates Nicotinamide nitrile hydratase hydration of S

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